UBC Theses and Dissertations

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UBC Theses and Dissertations

Biochemical changes in compatible and incompatible flax/flax rust interactions Sutton, Benjamin C. S. 1982

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BIOCHEMICAL CHANGES IN COMPATIBLE AND INCOMPATIBLE FLAX/FLAX RUST INTERACTIONS by BENJAMIN C.S. SUTTON B.Sc. (Hon.), University of Reading, 1976 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Plant Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October 1982 @ Benjamin C.S. Sutton In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of / LO^uf' ^  CU.J2A^CJL The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date DE-6 (3/81) Frontispiece: Flax Seedlings One Week After Seeding. i i i ABSTRACT The possible biochemical mechanisms of v a r i e t a l and species s p e c i f i c i t y of obligate fungal parasites are considered in r e l a t i o n to the established genetics and biochemistry of host-parasite systems. The two general theories of s p e c i f i c i t y which have been put forward are i d e n t i f i e d . One invokes the induction of successful pathogenesis as a primary determinant in s p e c i f i c i t y while the other predicts that recognition of avirulent pathogens by the host leads to induced resistance which acts as the determinant i n s p e c i f i c i t y . The former theory i s supported by evidence for the appearance of novel host enzymes during disease development. The l a t t e r i s supported by observations of host gene derepression and phytoalexin accumulation which occur i n resistance responses at times p r i o r to any s i g n i f i c a n t response in susceptible combinations. These theories are examined experimentally i n the f l a x / f l a x rust system. The molecular o r i g i n of ribonuclease with altered c a t a l y t i c properties, which arises during disease development, i s examined. Rigorous p u r i f i c a t i o n reveals that the altered c a t a l y t i c properties can be i v accounted f o r by a l t e r e d p r o p o r t i o n s of r i b o n u c l e a s e I isozymes. These changes are s i m i l a r d u r i n g r e s i s t a n t and s u s c e p t i b l e r e a c t i o n s u n t i l s p o r u l a t i o n o c c u r s . Induced r e s i s t a n c e i s c h a r a c t e r i z e d and compared to primary changes d u r i n g s u s c e p t i b i l i t y through a d e t a i l e d study of RNA and p r o t e i n s y n t h e s i s . Enhanced RNA s y n t h e s i s occurs i n the r e s i s t a n t r e a c t i o n at times p r i o r to any measurable response i n the s u s c e p t i b l e combination, although both combinations e x h i b i t h i g h e r r a t e s o f RNA s y n t h e s i s a t l a t e r times. RNA was f r a c t i o n a t e d by e l e c t r o p h o r e s i s and a f f i n i t y chromatography and messenger a c t i v i t y assessed by i n v i t r o t r a n s l a t i o n . Enhanced RNA s y n t h e s i s i s c h a r a c t e r i z e d by decreased messenger p o l y a d e n y l a t i o n . However, po l y a d e n y l a t e d and non-polyadenylated messenger RNA were shown to encode many common p o l y p e p t i d e s ; t h i s p r o v i d e s an e x p l a n a t i o n f o r the f a c t t h a t few changes i n iri v i v o p r o t e i n s y n t h e s i s can be de t e c t e d by one or two dimensional e l e c t r o p h o r e s i s . The onl y marked changes i n p r o t e i n s y n t h e s i s occur i n the s u s c e p t i b l e combination and i n c l u d e a dramatic d e c l i n e i n the s y n t h e s i s of the l a r g e subunit of rib u l o s e - 1 , 5 - b i s p h o s p h a t e c a r b o x y l a s e . I t i s concluded that i n c o m p a t i b i l i t y of an a v i r u l e n t race of r u s t with the f l a x v a r i e t y Bombay i s determined by induced r e s i s t a n c e r e s u l t i n g from r e c o g n i t i o n of the a v i r u l e n t pathogen p r i o r to the i n i t i a t i o n of pathogenesis. However, s p e c i f i c b i o c h e m i c a l events i n the host, p a r t i c u l a r l y those i n v o l v i n g c h l o r o p l a s t f u n c t i o n , may be necessary f o r s u c c e s s f u l pathogen development. v i TABLE OF CONTENTS Page F r o n t i s p i e c e i i A b s t r a c t i i i Table of Contents v i L i s t of Tables i x L i s t of F i g u r e s x L i s t of A b b r e v i a t i o n s x i i G l o s s a r y of Terras x i i i Acknowledgements xv Chapter 1. I n t r o d u c t i o n 1 Chapter 2. Review of L i t e r a t u r e 4 2.1 A T h e o r e t i c a l C o n s i d e r a t i o n of the Genetics and Mo l e c u l a r B a s i s of S p e c i f i c i t y 4 2.2 Metabolism and Disease 9 2.2.1 Non-Host Re s i s t a n c e 10 2.2.2 Host P h y s i o l o g y and Pathogen 10 N u t r i t i o n 2.2.3 Induced S u s c e p t i b i l i t y 14 2.2.4 Induced Re s i s t a n c e 17 2.3 Ribonuclease and Disease 25 2.4 RNA and P r o t e i n S y n t h e s i s 36 Chapter 3. Changes i n Ribonuclease Isozyme During Rust I n f e c t i o n of Flax 45 3.1 I n t r o d u c t i o n 45 3.2 M a t e r i a l s and Methods 46 3.2.1 P l a n t M a t e r i a l 46 3.2.2 E x t r a c t i o n and P u r i f i c a t i o n of RNase 46 3.2.3 E l e c t r o p h o r e t i c A n a l y s i s 48 3.2.4 Column I s o e l e c t r i c Focusing 49 3.2.5 Enzyme Assays 49 3.2.6 A n a l y s i s of H y d r o l y s i s Products 50 3.2.7 P r o t e i n E s t i m a t i o n 50 3.3 R e s u l t s 51 3.3.1 P u r i f i c a t i o n of RNase from Flax Cotyledons 51 3.3.1.1 S p e c i f i c A c t i v i t y and Y i e l d s 51 3.3.1.2 H y d r o x y l a p a t i t e Chromatography 52 v i i Page 3.3.1.3 E l e c t r o p h o r e t i c A n a l y s i s 56 3.3.1.4 Sephadex G100 Gel F i l t r a t i o n 58 3.3.2 The In f l u e n c e of Rust I n f e c -t i o n Upon the S p e c i f i c A c t i v i t y of RNase P i and P2 60 3.3.3 P r o p e r t i e s of RNases 62 3.3.3.1 Substrate S p e c i f i c i t y 64 3.3.3.2 K m Values 64 3.3.3.3 Endonuclease A c t i v i t y 64 3.3.3.4 Other Nuclease A c t i v i t i e s 65 3.3.3.5 H y d r o l y s i s Products 65 3.3.3.6 I s o e l e c t r i c Focusing 65 3.3.4 E f f e c t s of Wounding 66 3.4 D i s c u s s i o n 67 Chapter 4. RNA and P r o t e i n S y n t h e s i s f o l l o w i n g I n o c u l a t i o n o f Flax with the Flax Rust Fungus 69 4.1 I n t r o d u c t i o n 69 4.2 M a t e r i a l s and Methods 70 4.2.1 I n o c u l a t i o n and L a b e l l i n g of P l a n t M a t e r i a l 70 4.2.2 E s t i m a t i o n of RNA Synt h e s i s i n Crude E x t r a c t s 71 4.2.3 E x t r a c t i o n and P u r i f i c a t i o n of RNA 72 4.2.4 Poly U and Poly A Sepharose Chromatography 73 4.2.5 E l e c t r o p h o r e s i s of RNA 74 4.2.6 Iji v i t r o T r a n s l a t i o n 75 4.2.7 E x t r a c t i o n o f P r o t e i n 76 4.2.8 One and Two Dimensional E l e c t r o p h o r e s i s of P r o t e i n 76 4.3 R e s u l t s 78 4.3.1 Rates of RNA Sy n t h e s i s 78 4.3.2 C h a r a c t e r i z a t i o n of P u r i f i e d RNA by E l e c t r o p h o r e s i s 81 4.3.3 Pol y U B i n d i n g RNA (A+RNA) 86 4.3.4 Poly A B i n d i n g RNA (U+RNA) and Small RNA 8 8 4.3.5 S i g n i f i c a n c e of the A l t e r e d P a t t e r n s of RNA Synt h e s i s i n Disease 90 v i i i Page 4 .3 .6 In v i t r o T r a n s l a t i o n s of RNA from U n i n f e c t e d P l a n t s 93 4 . 3 . 6 . 1 T r a n s l a t i o n a l A c t i v i t y 93 4 . 3 . 6 . 2 Two Dimensional A n a l y s i s of T r a n s l a -t i o n Products 95 4 .3 .7 P r o t e i n S y n t h e s i s i n v i v o f o l l o w i n g I n o c u l a t i o n 101 4 .3 .8 Two Dimensional A n a l y s i s of i n v i v o L a b e l l e d P r o t e i n f o l l o w i n g I n o c u l a t i o n 108 4.4 D i s c u s s i o n 111 Chapter 5 . General D i s c u s s i o n 117 Summary 125 B i b l i o g r a p h y 127 Appendix: Met h o d o l o g i c a l D e t a i l s and Pre c a u t i o n s 136 i x LIST OF TABLES Page Table I. Table I I . Tabl e I I I . Table IV. Table V. Tabl e V I . Table V I I . Tabl e V I I I . T a b l e IX. Changes i n RNase A c t i v i t y Following Rust I n f e c t i o n . 27 P u r i f i c a t i o n of RNase from Healthy and Ru s t - I n f e c t e d F l a x (var. Bison) Cotyledons. 53 P r o p e r t i e s of Ribonucleases from F l a x . 63 Gross Rates of RNA Sy n t h e s i s at 13 to 16 Hours a f t e r I n o c u l a t i o n . 78 RNA Species found i n P u r i f i e d Flax RNA by Formamide E l e c t r o p h o r e s i s . 83 The R e l a t i v e Rates of A +RNA Synt h e s i s i n Healthy and I n f e c t e d Seedlings a t 18 to 21 Hours a f t e r I n o c u l a t i o n . 92 T r a n s l a t i o n a l A c t i v i t y of RNA F r a c t i o n s Separated by Poly U Sepharose Chromatography. 9 4 Rates of P r o t e i n S y n t h e s i s at 18 hours a f t e r I n o c u l a t i o n . 102 Summary of the Main E a r l y Events i n Resistance and S u s c e p t i b i l i t y d u r i n g Rust I n f e c t i o n of F l a x . 123 X LIST OF FIGURES Page Fi g u r e 1. The Outcome of Host/Rust Combinations with Complementary Genotypes. 2 F i g u r e 2. H y p o t h e t i c a l Host-Pathogen I n t e r a c t i o n Leading to Induced R e s i s t a n c e . 8 F i g u r e 3. Reaction Types i n a Gene-for-gene I n t e r a c t i o n . 16 F i g u r e 4. The Subunit Complementation Hypothesis f o r Flax RNase. 30 F i g u r e 5. G100 Sephadex Gel F i l t r a t i o n P r o f i l e s of Homogenates from One and Two Week Old B i s o n Flax and F o l l o w i n g pH 5 P r e c i p i t a t i o n . 54 F i g u r e s 6. H y d r o x y l a p a t i t e Eluograms of RNase. 55 F i g u r e 7. P o l y n u c l e o t i d e P o l y a c r y l a m i d e Gel E l e c t r o p h o r e s i s . 57 F i g u r e 8. G100 Sephadex Gel F i l t r a t i o n P r o f i l e s of RNase P i and P2 and F r a c t i o n I I I . 59 F i g u r e 9. The S p e c i f i c A c t i v i t y R a t i o s of RNases P i and P2 F o l l o w i n g I n o c u l a t i o n of B i s o n and Bombay Flax Cotyledons with Flax Rust Race No. 3. 61 F i g u r e 10. The Response of RNA S y n t h e s i s and the S y n t h e s i s of A+ RNA to I n f e c t i o n with F l a x Rust. 80 F i g u r e 11. E l e c t r o p h o r e s i s of [32p] RNA from Uninoculated Seedlings i n 4% P o l y a c r y l a m i d e Formamide G e l s . 82 F i g u r e 12. E l e c t r o p h o r e t i c A n a l y s i s of RNA Synthesized 18 to 21 hr a f t e r I n o c u l a t i o n . 85 F i g u r e 13. E l e c t r o p h o r e t i c C h a r a c t e r i z a t i o n of F r a c t i o n a t e d RNA on Formamide G e l s . 87 x i Page F i g u r e 14. A n a l y s i s of U + RNA by E l e c t r o p h o r e s i s . 89 F i g u r e 15. Two Dimensional A n a l y s i s of P o l y p e p t i d e s . 96 F i g u r e 16. A n a l y s i s of in v i v o L a b e l l e d P o l y p e p t i d e s by SDS E l e c t r o p h o r e s i s , 18 hr a f t e r I n o c u l a t i o n . 104 F i g u r e 17. Two Dimensional E l e c t r o p h o r e s i s of Soluble i n v i v o L a b e l l e d P r o t e i n . 109 x i i LIST OF ABBREVIATIONS A + Polyadenylated RNA A - Non-polyadenylated RNA PMSF P h e n y l m e t h y l s u l f o n y l f l u o r i d e kD K i l o d a l t o n K m M i c h a e l i s constant LHC L i g h t h a r v e s t i n g complex MAK Methylated albumin K i e s e l g u h r 3 2P Phosphorus - 32 ( r a d i o a c t i v e isotope) Rf M i g r a t i o n , r a t i o to s o l v e n t f r o n t RNA R i b o n u c l e i c a c i d mRNA Messenger RNA rRNA Ribosomal RNA tRNA T r a n s f e r RNA tcRNA T r a n s l a t i o n a l c o n t r o l RNA RNase Ribonuclease RuBPCase Ri b u l o s e bisphosphate carboxylase S Svedberg, u n i t of sedimentation 35s Sulphur - 3 5 ( r a d i o a c t i v e isotope) SDS Sodium dodecyl sulphate TCA T r i c h l o r o - a c e t i c a c i d U + P o l y - or o l i g o u r i d y l a t e c o n t a i n i n g RNA U~ Po l y - or o l i g o u r i d y l a t e l a c k i n g RNA GLOSSARY OF TERMS Compatible combination Host and pathogen genotypes which enable pathogenesis. Haustorium Fungal i n f e c t i o n s t r u c t u r e which i s found w i t h i n the host c e l l w a l l and i n a p o s i t i o n to the host c e l l membrane. H o r i z o n t a l r e s i s t a n c e R e s i s t a n c e which i s constant r e g a r d l e s s of the race (or genotype) of pathogen a g a i n s t which i t i s t e s t e d . O f t e n i n h e r i t e d p o l y g e n i c a l l y . H y p e r s e n s i t i v i t y An a c t i v e r e s i s t a n c e mechanism i n which the r a p i d death of host c e l l s around the p o i n t of i n f e c t i o n prevents c o l o n i z a t i o n . Incompatible combination Host and pathogen genotypes which le a d to r e s i s t a n c e . Non-host r e s i s t a n c e The q u a l i t y of a p l a n t which completely prevents pathogenesis w i t h a l l races of a pathogen. I t i s , by d e f i n i t i o n , a p a r t i c u l a r type of h o r i z o n t a l r e s i s t a n c e i n which the r e s i s t a n c e i s absolute among a l l genotypes of the p l a n t group ( u s u a l l y r e f e r s to a s p e c i e s ) and the p l a n t s are t h e r e f o r e considered non-hosts. Pathogenesis The development of a pathogen a t the expense of a host producing an accompanying d i s e a s e syndrome. P h y t o a l e x i n F u n g i t o x i c compound which accumulates i n a p l a n t i n response t o i n f e c t i o n by a pathogen. R e s i s t a n c e The a b i l i t y of a host to l i m i t the development of a pathogen. Opposite i s s u s c e p t i b i l i t y . xiv V e r t i c a l resistance Resistance in which there i s a d i f f e r e n t i a l interaction between a variety of host genotypes and a variety of races of pathogen. This d i f f e r e n t i a l interaction i s determined by major genes and i s never polygenically inherited. Virulence A b i l i t y of a pathogen to overcome one or more major resistance genes. Opposite is avirulence. The term has also been used elsewhere in r e l a t i o n to horizontal resistance but i s r e s t r i c t e d to v e r t i c a l resistance here. X V ACKNOWLEDGEMENTS I would l i k e to thank the members of my graduate committee, Drs. Runeckles, Tener, Person and Copeman, and most e s p e c i a l l y my s u p e r v i s o r Dr. Michael Shaw, f o r v a l u a b l e support and c r i t i c i s m . Thanks are a l s o due to the t e c h n i c a l s t a f f of the P l a n t Science Department, U.B.C, f o r t e c h n i c a l a s s i s t a n c e and photography. I am deeply g r a t e f u l t o Mr. Leroy Scrubb f o r h e l p with the experiments on r i b o n u c l e a s e and f o r c o n t i n u i n g i n t e r e s t i n my work. I would a l s o l i k e to acknowledge the b e n e f i t of many s t i m u l a t i n g d i s c u s s i o n s with Dr. Andrew Greenland. F i n a l l y I wish to thank Mrs. Gayle Smith f o r her e x c e l l e n t work i n t y p i n g the t h e s i s . T h i s work was supported by grants from the N a t u r a l Science and Engineering Research C o u n c i l of Canada to P r o f e s s o r Shaw. 1 CHAPTER I INTRODUCTION The r u s t f u n g i , U r e d i n a l e s , r e p r e s e n t one of the l a r g e s t and most e c o n o m i c a l l y important groups of p l a n t pathogens. Among the most s t u d i e d of the d i s e a s e s which they cause are the r u s t s of c e r e a l crops and of f l a x . A s i d e from t h e i r a g r i c u l t u r a l s i g n i f i c a n c e these d i s e a s e s hold g r e a t promise as t o o l s f o r e l u c i d a t i n g the mechanisms of pathogenesis and r e s i s t a n c e i n a l l o b l i g a t e b i o t r o p h i c f u n g a l p a r a s i t e s , of which they are r e p r e s e n t a t i v e . Such p a r a s i t e s have three important f e a t u r e s . F i r s t l y , they are dependent on l i v i n g host c e l l s 'for t h e i r development under n a t u r a l c i r c u m s t a n c es. Secondly, they have a r e s t r i c t e d host range, each formae s p e c i a l e s normally being r e s t r i c t e d to a s i n g l e host s p e c i e s . T h i r d l y , v a r i e t a l r e s i s t a n c e i s c h a r a c t e r i z e d by a h i g h l y s p e c i f i c gene-for-gene system i n which complementary a l l e l e s of host and p a r a s i t e i n t e r a c t to determine r e s i s t a n c e or s u s c e p t i b i l i t y . These f e a t u r e s are c e n t r a l to any understanding of the mechanisms i n v o l v e d i n pathogenesis and r e s i s t a n c e . The gene-for-gene theory of v a r i e t a l r e s i s t a n c e p o s t u l a t e d f o r f l a x by F l o r (1956) has the f o l l o w i n g f e a t u r e s : p l a n t r e s i s t a n c e genes are dominant and r u s t v i r u l e n c e genes are r e c e s s i v e ; a l l r e s i s t a n c e genes must be 2 overcome by t h e i r corresponding v i r u l e n c e genes i n order f o r s u c c e s s f u l d i s e a s e development to occur. Thus the e x p r e s s i o n of r e s i s t a n c e i s c o n d i t i o n a l upon the presence of the corresponding a v i r u l e n c e and r e s i s t a n c e a l l e l e s . T h i s i s i l l u s t r a t e d i n Fi g u r e 1, below. HOST GENOTYPES RR/Rr r r Rust Genotypes AA/Aa - + aa + + FIGURE 1. The outcome of h o s t / r u s t combinations with complementary genotypes. A = a v i r u l e n c e a l l e l e , a = v i r u l e n c e a l l e l e ; R = r e s i s t a n c e a l l e l e , r = s u s c e p t i b i l i t y a l l e l e . A s u s c e p t i b l e combination i s denoted '+' and a r e s i s t a n t one For s i m p l i c i t y the homozygous-dominant and heterozygous genotypes are not d i s t i n g u i s h e d . The f l a x / f l a x r u s t i n t e r a c t i o n s exemplify t h i s system perhaps b e t t e r than any other h o s t - p a r a s i t e i n t e r a c t i o n . R e s i s t a n c e i s expressed at f i v e l o c i (K, L, M, N, P) wit h 1, 11, 6, 3 and 4 a l l e l e s ( r e s p e c t i v e l y ) and tends to be an a l l or nothing response ( F l o r 1971). These f a c t o r s have been i n f l u e n t i a l i n choosing the f l a x / f l a x r u s t system f o r the re s e a r c h presented i n l a t e r c h a p t e r s . Two ge n e r a l t h e o r i e s e x i s t f o r the molecular b a s i s of s p e c i f i c i t y i n gene-for-gene i n t e r a c t i o n s : (1) th a t complementary a l l e l e s of host and pathogen i n t e r a c t to form 3 p r o t e i n s with novel p r o p e r t i e s which enable pathogen development (Chakravorty and Shaw 1977a, Vanderplank 1978); (2) t h a t the products of a v i r u l e n c e and r e s i s t a n c e genes i n t e r a c t to e l i c i t an a c t i v e host r e s i s t a n c e response (Albersheim and Anderson-Prouty 1975). The former may be thought of as the i n d u c t i o n of s u s c e p t i b i l i t y and the l a t t e r as the i n d u c t i o n of r e s i s t a n c e . The o b j e c t i v e of t h i s t h e s i s i s to attempt to r e s o l v e which of the two g e n e r a l t h e o r i e s l i s t e d above may e x p l a i n the molecular b a s i s of v a r i e t a l s p e c i f i c i t y i n which gene-for-gene i n t e r a c t i o n s are i n v o l v e d . These t h e o r i e s are assessed i n r e l a t i o n to the e s t a b l i s h e d g e n e t i c s of the f l a x / f l a x r u s t system and are a l s o i n v e s t i g a t e d e x p e r i m e n t a l l y . The p u r i f i c a t i o n of host r i b o n u c l e a s e with a l t e r e d c a t a l y t i c p r o p e r t i e s from i n f e c t e d p l a n t s has r e p r e s e n t e d a key p i e c e of evidence f o r the idea t h a t the i n d u c t i o n of novel p r o t e i n s i s necessary f o r d i s e a s e development (Chakravorty e t a l . 1974c). Thus a d e t a i l e d examination of r i b o n u c l e a s e has been c a r r i e d out i n order to determine the molecular b a s i s of i t s a l t e r e d p r o p e r t i e s . Research on host RNA and p r o t e i n s y n t h e s i s f o l l o w i n g i n o c u l a t i o n has a l s o been c a r r i e d out i n order to determine t h e i r r o l e i n the r e s i s t a n c e response. In t h i s way i t i s hoped t h a t i t may be p o s s i b l e to g a i n i n s i g h t i n t o the i n d u c t i o n of the r e s i s t a n c e response. 4 CHAPTER 2 REVIEW OF LITERATURE The f i r s t two s e c t i o n s of t h i s review are intended to pr o v i d e a g e n e r a l overview and i n t e r p r e t a t i o n of the s u b j e c t matter. They are not intended as an exhaustive review of a l l the o r i g i n a l r e s e a r c h papers as many such reviews have been w r i t t e n r e c e n t l y and are r e f e r r e d to here. 2.1 A T h e o r e t i c a l C o n s i d e r a t i o n of the Genetics and  Molecular B a s i s of S p e c i f i c i t y The u s e f u l n e s s o f an understanding o f gene-for-gene systems must be assessed i n regard to t h e i r g e n e r a l o c c u r r e n c e . Person (1959) has suggested t h a t such a r e l a t i o n s h i p should occur as a g e n e r a l r u l e i n h o s t - p a r a s i t e systems. Since that time gene-for-gene r e l a t i o n s h i p s have been demonstrated i n nine f u r t h e r p l a n t p a r a s i t e systems and suggested i n seventeen o t h e r s (Sidhu, 1975). Gene-for-gene systems are c l e a r l y important a g r i c u l t u r a l l y . However, students of pathology should be cognizant of other types o f r e s i s t a n c e such as h o r i z o n t a l r e s i s t a n c e and a l s o of the behaviour of g e n e t i c p o p u l a t i o n s w i t h i n an ecosystem or a g r i c u l t u r a l system. P l a n t pathology at t h i s l e v e l has been c a l l e d the pathosystem, a concept which i s developed i n a very p r o v o c a t i v e book by Robinson (1976). 5 The o b j e c t i v e of the t h e s i s i s to determine the bi o c h e m i c a l b a s i s o f s p e c i f i c i t y i n gene-for-gene i n t e r a c t i o n s . B r i e f l y , what determines whether so c a l l e d near i s o g e n i c l i n e s o f a p l a n t a l l o w s u c c e s s f u l development of a g i v e n i s o l a t e of the pathogen? Conversely, why do d i f f e r e n t i s o l a t e s succeed o r f a i l to develop on a s i n g l e p l a n t v a r i e t y ? A d d i t i o n a l l y the b i o c h e m i c a l b a s i s of o b l i g a t e p a r a s i t i s m and r e s t r i c t e d host range are c o n s i d e r e d . I t i s g e n e r a l l y accepted t h a t s p e c i f i c changes i n host metabolism are necessary f o r pathogen development. However, there i s no g e n e t i c framework on which to b u i l d an hyp o t h e s i s of the way i n which such changes are induced. Many of these metabolic changes have been s t u d i e d and are d i s c u s s e d i n S e c t i o n 2.2. The hypotheses presented below are c o n f i n e d to a c o n s i d e r a t i o n of d i f f e r e n c e s between p l a n t v a r i e t i e s and r u s t r a c e s . R e s i s t a n c e i n such systems has been c a l l e d v a r i e t a l or v e r t i c a l r e s i s t a n c e and i s known to be i n h e r i t e d by s i n g l e genes. P o l y g e n i c a l l y i n h e r i t e d r e s i s t a n c e , a l s o r e f e r r e d t o as h o r i z o n t a l r e s i s t a n c e , i s not d i r e c t l y d i s c u s s e d ; s i m i l a r l y , non-host r e s i s t a n c e i s not co n s i d e r e d . T h i s approach appears r a t i o n a l because the i n h e r i t a n c e of v e r t i c a l r e s i s t a n c e i s w e l l understood. The author has concluded from h i s c o n s i d e r a t i o n of the l i t e r a t u r e reviewed i n t h i s chapter t h a t the v a r i o u s 6 t h e o r i e s put forward to e x p l a i n v a r i e t a l s p e c i f i c i t y may be c a t e g o r i z e d w i t h i n three g e n e r a l hypotheses: (1) The p r e - e x i s t i n g n u t r i t i o n a l s t a t u s of the host d e t e r -mines whether s u i t a b l e s p e c i f i c m e t a b o l i t e s are a v a i l -a ble f o r f u n g a l development ( S e c t i o n 2.2.2). (2) S u s c e p t i b i l i t y i s an induced phenomenon which r e l i e s upon complementary gene products of host and pathogen i n t e r a c t i n g to b r i n g about m e t a b o l i c changes i n the host c e l l which enable s u c c e s s f u l pathogen development. ( S e c t i o n 2.2.3). (3) R e s i s t a n c e i s an induced phenomenon i n which a gene product of the pathogen i n t e r a c t s (or i s recognized) by a gene product of the host and thus a r e s i s t a n c e response i s i n i t i a t e d ( S e c t i o n 2.2 .4 ) . The f i r s t or s o - c a l l e d n u t r i t i o n a l hypothesis seems u n l i k e l y to be important i n v a r i e t a l s p e c i f i c i t y as many r u s t s have now been grown on simple media ( S c o t t 1976; and see S e c t i o n 2.2). The o t h e r hypotheses must c l e a r l y be s c r u t i n i z e d i n r e l a t i o n to the gene-for-gene theory. Hypothesis 2 r e q u i r e s t h a t an inducer gene product from the pathogen i n t e r a c t s with a gene or i t s product i n the host i n order to b r i n g about s u s c e p t i b i l i t y . Such an h y p o t h e s i s i s very hard to e x p l a i n i n terms of the gene-for-gene theory. The requirement f o r every r e s i s t a n c e 7 gene to be overcome by a corresponding v i r u l e n c e gene would seem to pre c l u d e t h i s type of mechanism. T h i s i s true even i f a v i r u l e n c e and r e s i s t a n c e genes are assumed to be r e p r e s s o r s of pathogen inducers and host r e c e p t o r s r e s p e c t i v e l y . Furthermore i t can be seen t h a t the v i r u l e n c e a l l e l e i s not f u n c t i o n a l i n pathogenesis; indeed the AA genotype ( F i g . 1) s t i l l l eads to a s u s c e p t i b l e r e a c t i o n i n the absence of the corresponding R a l l e l e . Hypothesis 3 can r e a d i l y be e x p l a i n e d i n terms of the gene-for-gene theory. The scheme o u t l i n e d i n F i g u r e 2 shows how a v i r u l e n c e gene products may be r e s p o n s i b l e f o r i n d u c i n g a r e s i s t a n c e response through i n t e r a c t i o n with a c o r r e s p o n d i n g r e s i s t a n c e a l l e l e i n the host. C l e a r l y some k i n d of r e c o g n i t i o n phenomenon i s i m p l i e d ; f o r the sake of s i m p l i c i t y the mechanism of t h i s r e c o g n i t i o n w i l l not be d i s c u s s e d i n t h i s S e c t i o n . In such a system a v i r u l e n c e genes must be seen as c o n s t i t u t i v e and hence as having a f u n c t i o n a l r o l e i n the pathogen; otherwise they would be expected to have been l o s t d u r i n g e v o l u t i o n because they would c o n f e r no s e l e c t i v e advantage. T h i s i s supported f i r s t l y by t h e i r occurrence at many l o c i ( F l o r 1971) and secondly by the f a c t t h a t v i r u l e n c e a l l e l e s c o n f e r a disadvantage to the pathogen i n the absence of the corresponding r e s i s t a n c e a l l e l e s (Day 1974). So i t can be seen t h a t the theory of induced r e s i s t a n c e p r o v i d e s a s t r o n g m e c h a n i s t i c i n t e r p r e t a t i o n of 8 A- — Jj ^R > Resistance Response Pathogen Host-Pathogen Host Interface FIGURE 2. Hypothetical host-pathogen interaction leading to induced resistance; the product of the avirulence a l l e l e (A) i s seen as interacting with the resistance gene (R) or i t s product in order to induce the resistance response. The location of th i s interaction i n r e l a t i o n to the host-pathogen interface i s unknown but may occur, for example, at the surfaces of the haustoria (fungal i n f e c t i o n structures which invaginate the host c e l l membrane). the gene-for-gene theory on purely l o g i c a l grounds, a conclusion reached by Person and Mayo (1974). A body of evidence as to the way in which such induced resistance occurs i s beginning to emerge. Some of the e a r l i e s t biochemical events in res i s t a n t reactions involve enhanced host RNA and protein synthesis. Examples of such events which are s p e c i f i c to resistance rather than s u s c e p t i b i l i t y support the proposition that resistance i s induced. For this reason these changes in host gene expression are reviewed in Section 2.4. Clearly physiological changes are induced in the host as a r e s u l t of inf e c t i o n with a v i r u l e n t pathogen (Section 2.2). Some of these changes may be esse n t i a l for pathogen development. The successful induction of these changes determines whether the plant i s a host or a non-host for the pathogen. However, as explained above, this induction i s unlikely to be controlled by gene-for-gene interactions of the type described by Flor (1971). Therefore two separate interactions can be i d e n t i f i e d for biotrophic fungal parasites. 9 a) The i n d u c t i o n of s u c c e s s f u l d i s e a s e development, which may be regarded as the breakdown of non-host r e s i s t a n c e . b) The breakdown or e x p r e s s i o n of v e r t i c a l r e s i s t a n c e , i n v o l v i n g gene-for-gene i n t e r a c t i o n s . In e v o l u t i o n a r y terms, once non-host r e s i s t a n c e i s overcome s e l e c t i v e p r e s s u r e w i l l almost i n e v i t a b l y l e a d t o gene-for-gene i n t e r a c t i o n s . Person (1959) has suggested t h a t gene-for-gene systems are a p r e d i c t a b l e e v o l u t i o n a r y outcome of s e l e c t i o n pressure f o r and a g a i n s t r e s i s t a n c e genes. The m etabolic changes a s s o c i a t e d with these l e v e l s o f . i n t e r a c t i o n s are reviewed i n the f o l l o w i n g s e c t i o n . 2 .2 Metabolism and Disease Much of the l i t e r a t u r e which o u t l i n e s m e tabolic changes d u r i n g d i s e a s e i s concerned with changes t h a t occur d u r i n g d i s e a s e development i n s u s c e p t i b l e combinations. In many cases i t i s s t r o n g l y i m p l i e d t h a t these changes are s p e c i f i c to d i s e a s e and cannot be induced by other f a c t o r s . Evidence that d i f f e r e n t types of events occur dur i n g s u s c e p t i b l e r e a c t i o n s i n c l o s e l y r e l a t e d race and v a r i e t a l combinations i s n o n - e x i s t e n t . Indeed, as we have seen, one would not expect the b a s i s of v a r i e t a l s p e c i f i c i t y to r e s i d e i n the s u s c e p t i b l e response. 10 2.2.1 Non-Host R e s i s t a n c e Many workers have suggested t h a t the i n d u c t i o n of a s u i t a b l e m e tabolic environment i n the host i s the b a s i s of host s p e c i e s s p e c i f i c i t y (reviewed by Heath 1980). However, any such assumption should be questioned because i n most i n s t a n c e s non-host r e s i s t a n c e i s expressed p r i o r to h a u s t o r i a l formation (Heath 1980). H a u s t o r i a l s t r u c t u r e s , which i n v a g i n a t e the host c e l l s , are the morphological b a s i s through which a metabolic r e l a t i o n s h i p i s e s t a b l i s h e d . Heath (1981a) has summarized i n f o r m a t i o n which i n d i c a t e s t h at f u n g a l s u p p r e s s i o n of non-host r e s i s t a n c e mechanisms may be the primary determinant of host range. E x t r a c t s of bean r u s t i n f e c t e d French beans are capable o f promoting h a u s t o r i a l f o r m a t i o n of both cowpea r u s t on beans and bean r u s t on cowpeas. Such promotion was not e f f e c t e d by e x t r a c t s from h e a l t h y bean p l a n t s (Heath 1980). Furthermore the i n c r e a s e d h a u s t o r i a l formation was a s s o c i a t e d with a suppression of s i l i c o n d e p o s i t i o n which occurs d u r i n g non-host r e s i s t a n c e (Heath 1981b). 2.2.2 Host P h y s i o l o g y and Pathogen N u t r i t i o n There i s s t i l l much r e s e a r c h t o be done before a c l e a r understanding of non-host r e s i s t a n c e i s reached. For example, there i s a p o s s i b i l i t y t h a t the metabolic a s s o c i a -t i o n between host and p a r a s i t e i s to some degree e s t a b l i s h e d p r i o r to h a u s t o r i a l f o r m a t i o n . In view of the long h i s t o r i -c a l b a s i s of data concerned with the s i g n i f i c a n c e of host metabolism dur i n g s u s c e p t i b i l i t y , the p o s s i b i l i t y of i t s 11 involvement i n host s p e c i e s s p e c i f i c i t y must s t i l l be c o n s i d e r e d . In c h a r a c t e r i z i n g the e s s e n t i a l elements of symptom development i t i s u s e f u l to s p e c u l a t e what metabolic environment favours the pathogen's development. In r e c e n t years there have been many r e p o r t s on the n u t r i t i o n a l requirements of r u s t f u n g i i n axenic c u l t u r e ( S c o t t 1976), which might be expected to shed l i g h t on t h i s matter. The n u t r i t i o n a l requirements are, however, r a t h e r a s p e c i f i c ; i n g e n e r a l , m i n e r a l s , a sugar carbon source, an amino a c i d n i t r o g e n source and a sulphur c o n t a i n i n g amino a c i d are r e q u i r e d . A wide range of sugars w i l l support growth as w i l l s e v e r a l amino a c i d s , although, i n f l a x r u s t , a s p a r t a t e or glutamate are p a r t i c u l a r l y e f f e c t i v e . I n t e r e s t i n g l y , r u s t f u n g i cannot u t i l i z e s u l p h a t e , being d e f i c i e n t i n sulphate r e d u c t i o n , but i n c o r p o r a t e s u l p h i t e which may be taken up through the y-glutamyl c y c l e ( S c o t t 1972). Thus, i t can be a p p r e c i a t e d t h a t the n u t r i t i o n a l requirements of r u s t f u n g i c o u l d , a t l e a s t p o t e n t i a l l y , be s u p p l i e d by any p l a n t . However, the a v a i l a b i l i t y of the a p p r o p r i a t e n u t r i e n t balance i n l i v i n g host t i s s u e s c o u l d be s u b j e c t to the c o r r e c t m e t a b o l i c environment. At p r e s e n t there are i n s u f f i c i e n t d a ta r e l a t i n g to l o c a l i z e d in s i t u carbohydrate, amino a c i d and m i n e r a l balance i n h o s t - p a r a s i t e i n t e r a c t i o n s to assess t h i s . 12 A l l the changes i n host metabolism seen duri n g the development of o b l i g a t e p a r a s i t e s are i n essence b i o t r o p h i c ; enhanced metabolism i s the r u l e . T h i s i s not so f o r n e c r o t r o p h i c p a r a s i t e s which should t h e r e f o r e be regarded s e p a r a t e l y , both p h y s i o l o g i c a l l y and g e n e t i c a l l y . Large i n c r e a s e s are seen i n r e s p i r a t i o n , which appears q u a l i t a t i v e l y d i f f e r e n t from r e s p i r a t i o n i n he a l t h y t i s s u e (Shaw 1963). Much of the i n c r e a s e can be accounted f o r i n terms of increased a c t i v i t y of the pentose phosphate pathway. Concurrent i n c r e a s e s i n b i o s y n t h e t i c a c t i v i t y are a l s o c h a r a c t e r i s t i c ( i n c l u d i n g RNA and p r o t e i n s y n t h e s i s ) . T h i s i n f o r m a t i o n and the apparent l i n k between the r e s p i r a t o r y r i s e and c h l o r o p l a s t c o l l a p s e have prompted the idea t h a t the pentose phosphate pathway i s s t i m u l a t e d by high e r NADP co n c e n t r a t i o n s ( S c o t t 1972). Together with the in c r e a s e d u t i l i z a t i o n of NADH and ATP t h i s evidence i s c o n s i s t e n t w i t h g e n e r a l enhanced metabolic and b i o s y n t h e t i c a c t i v i t y . One p o i n t which does not seem c l e a r i s the involvement of p h o t o s y n t h e s i s . P h o t o s y n t h e s i s normally d e c l i n e s d u r i n g d i s e a s e but i t s maintenance i s a s s o c i a t e d with r e s i s t a n c e . I n f e c t i o n i s not e s t a b l i s h e d i n leaves l a c k i n g c h l o r o p h y l l u n l e s s there i s an exogenous sucrose supply. Thus i t appears t h a t photosynthate i s dr a i n e d to the i n f e c t i o n s i t e s and i s e s s e n t i a l f o r pathogen n u t r i t i o n . There are v a r i o u s p i e c e s of evidence which may pro v i d e c l u e s to the s i g n i f i c a n c e of these metabolic changes 13 to pathogen n u t r i t i o n . Shaw (1963) c i t e s i n c r e a s e s i n f r e e amino a c i d s as w e l l as p r o t e i n i n r u s t i n f e c t e d wheat. Increases i n c e r t a i n amino a c i d s were noted as e a r l y as 2 days a f t e r i n o c u l a t i o n . Enzymes i n v o l v e d i n the s y n t h e s i s and u t i l i z a t i o n of glutamate and i n the s y n t h e s i s of glutamine have a l s o been shown t o i n c r e a s e i n v a r i o u s r u s t i n f e c t e d p l a n t s . Such changes do not occur i n c l o s e l y r e l a t e d r e s i s t a n t p l a n t s . T h i s e a r l y work has been extended by S a d l e r and Shaw (1979a) i n the f l a x / f l a x - r u s t system. These workers have shown t h a t h i g h l y p u r i f i e d glutamate dehydrogenase e x h i b i t s changes i n c a t a l y t i c p r o p e r t i e s d u r i n g d i s e a s e . The glutamate dehydrogenases were examined from h e a l t h y , 1 day i n f e c t e d and 7 day i n f e c t e d cotyledons; the enzymes d i f f e r e d from one another i n the nature of i n h i b i t i o n by ATP and p y r i d o x a l phosphate. Subsequent isotope p u l s e -chase experiments demonstrated t h a t the glutamate synthase-glutamine synthase pathway i s the major pathway of n i t r o g e n a s s i m i l a t i o n ( S a d l e r and Shaw 1979b). Isotope t r a p p i n g experiments were a l s o performed t o e l u c i d a t e the r o l e o f glutamate dehydrogenase (GDH). GDH appeared to be an enzyme of glutamate d e g r a d a t i o n i n 1 d a y - i n f e c t e d cotyledons but pla y e d a minor r o l e i n glutamate s y n t h e s i s i n 7-day i n f e c t e d c o t y l e d o n s . The l a t t e r might be accounted f o r by the e l e v a t e d l e v e l of ammonium ions i n these t i s s u e s . These r e s u l t s are i n t e r e s t i n g i n the l i g h t of a l t e r e d p r o p e r t i e s of GDH i n i n f e c t e d t i s s u e which may be s i g n i f i c a n t i n i t s a l t e r e d f u n c t i o n i n glutamate metabolism. The relev a n c e o f 14 these f i n d i n g s to f u n g a l n u t r i t i o n i s as y e t u n c l e a r but the s p e c i f i c i n d u c t i o n of novel GDH enzymes by the fungus cannot be r u l e d out. 2.2.3 Induced S u s c e p t i b i l i t y There i s a l a r g e body of evidence f o r enhanced metabolism i n d i s e a s e d p l a n t s at l e a s t some of which can be r a t i o n a l i z e d as d i r e c t l y b e n e f i c i a l to pathogen n u t r i t i o n . The c e n t r a l q u e s t i o n remains: to what extent are these changes s p e c i f i c a l l y induced by the pathogen? One most obvious way i n which to t e s t the e x i s t e n c e of induced s u s c e p t i b i l i t y i s t o i n f e c t a non-host p l a n t which has undergone p r i o r i n o c u l a t i o n with an ag g r e s s i v e pathogen. C l e a r l y , there are in h e r e n t problems with t h i s approach. F i r s t l y , important induced metabolic changes may be very l o c a l i z e d and secondly i n d u c t i o n by one pathogen may not generate a s u i t a b l e c e l l u l a r environment f o r another. A l s o , as has alre a d y been mentioned, i n d u c t i o n may merely r e p r e s e n t the supp r e s s i o n of non-host r e s i s t a n c e . However, a few examples of such induced s u s c e p t i b i l i t y do e x i s t . P r e i n o c u l a t i o n of b a r l e y with powdery mildew of b a r l e y ( E r i s y p h e graminis) allows melon mildew (Sphaerotheca f u l i g e n a ) l i m i t e d development on t h i s , normally non-host, p l a n t s p e c i e s ( B e l l 1981). Other examples of induced s u s c e p t i b i l i t y i n v o l v e the breakdown of v e r t i c a l r e s i s t a n c e and are as such a separate i s s u e . Many s t u d i e s have i n v o l v e d the use of RNA and p r o t e i n s y n t h e s i s i n h i b i t o r s i n d e s t r o y i n g v a r i e t a l r e s i s t a n c e (reviewed by B e l l 1981). C l e a r l y no a c t i v e i n d u c t i o n of s u s c e p t i b i l i t y takes p l a c e 15 i n t h i s case; t h i s i n f o r m a t i o n w i l l be d i s c u s s e d i n r e l a t i o n to v e r t i c a l r e s i s t a n c e ( S e c t i o n 2.4). Chakravorty and Shaw (1977a) have reviewed p o s s i b l e ways i n which s u s c e p t i b i l i t y may be induced at the mo l e c u l a r l e v e l . These authors suggest t h a t the l a r g e body of evidence f o r the appearance of enzymes with a l t e r e d p r o p e r t i e s d u r i n g s u s c e p t i b l e r e a c t i o n s i n d i c a t e s s p e c i f i c i n d u c t i o n e i t h e r a t the genomic l e v e l o r mediated through complementation of h o s t and p a r a s i t e DNA, RNA and p o l y p e p t i d e s . Q u a l i t a t i v e changes i n r i b o n u c l e a s e (RNase) seen i n many r u s t and powdery mildew d i s e a s e s p r o v i d e support f o r t h i s i d e a . Increases i n RNase and o t h e r enzymes are p a r a l l e l e d i n s u s c e p t i b l e and r e s i s t a n t combinations e a r l y a f t e r i n f e c t i o n . That other metabolic events such as the r e s p i r a t o r y r i s e occur c o n c u r r e n t l y i n both combinations at these e a r l y stages i s w e l l known (Shaw 1963, S c o t t 1972). Such evidence prompted Chakravorty and Shaw (1977a) t o suggest t h a t r e s i s t a n c e i s not determined d u r i n g these events which they have r e f e r r e d to as 'stage 1' i n t e r a c t i o n s but that s u s c e p t i b i l i t y or r e s i s t a n c e i s determined by the q u a l i t a t i v e changes brought about d u r i n g l a t e r events termed "stage 2' i n t e r a c t i o n s . That such 'stage 2' events are o b l i g a t o r y f o r s u c c e s s f u l pathogenesis i s a p l a u s i b l e and important concept. Vanderplank (1978) has proposed that co-p o l y m e r i z a t i o n of host and pathogen p r o t e i n s i s r e s p o n s i b l e f o r s u s c e p t i b i l i t y . Taken as such, t h i s i d e a i s r a t h e r s i m i l a r to t h a t of Chakravorty and Shaw (1977a), being complementation of a p a r t i c u l a r type. However, he invokes 16 the host and pathogen proteins as being the products of s u s c e p t i b i l i t y and avirulence a l l e l e s respectively. He suggests that i n t h i s way the host protein i s prevented from functioning in autoregulation. The resu l t would be that abnormally large amounts of thi s protein would be synthesized and he surmizes that t h i s extra protein provides an ideal food for the pathogen. By this theory when the corresponding resistance a l l e l e i s present there i s no longer copolymerization, the pathogen protein i s then recognized by the host and a resistance response i s e l i c i t e d . In support of his theory Vanderplank c i t e s the enhanced RNA and protein synthesis observed during disease and the existence of antigens common to host and pathogen of susceptible combin-ations. He also claims that temperature sensitive resistance a l l e l e s become i n e f f e c t i v e at higher temperatures because the proposed hydrophobic copolymerization i s more stable at higher temperatures. Vanderplank's theory has an inconsistency which i s worth mentioning here. It i s useful in t h i s regard to consider a single resistance locus and i t s corresponding avirulence locus (Fig. 3). Host Genotypes RR Rr r r Pathogen AA Genotypes Aa aa + + + + + Figure 3. Reaction types in a gene-for-gene interaction; +, susceptible; -, res i s t a n t . 17 F i r s t l y c o n s i d e r the i n t e r a c t i o n between the heterozygous genotypes (Aa/Rr). In t h i s case one must assume t h a t the non-matching a l l e l e s 'A' and 'R' produce a r e s i s t a n c e response i n s p i t e of the c o p o l y m e r i z a t i o n of the products of 'A' and ' r ' and 'R' and 'a'. In o t h e r words r e s i s t a n c e i s dominant. However, i n the i n t e r a c t i o n between the two homozygous genotypes 'aa' and ' r r ' a s u s c e p t i b l e response r e s u l t s , and hence one must assume t h a t the product of 'a' i s a l s o capable of c o p o l y m e r i z i n g with the ' r ' gene product. Thus i t can be seen t h a t f o r t h i s h ypothesis to be workable (on p u r e l y g e n e t i c grounds) c e r t a i n elements of s p e c i f i c i t y between gene products must be overlooked. As has been p o i n t e d out i n S e c t i o n 2.1, the gene-for-gene theory can only be r a t i o n a l i z e d e f f e c t i v e l y i f the v i r u l e n c e a l l e l e i s assumed t o be n o n - f u n c t i o n a l i n the i n d u c t i o n of s u s c e p t i b i l i t y . 2.2.4 Induced R e s i s t a n c e Thus f a r I have d e a l t with the evidence r e l a t i n g t o s u c c e s s f u l d i s e a s e development. I w i l l now d e a l with t h a t which i s concerned s o l e l y with r e s i s t a n c e mechanisms i n gene-for-gene type i n t e r a c t i o n s . The g e n e t i c theory which p r e d i c t s t h a t such r e s i s t a n c e i s induced as the r e s u l t of the i n t e r a c t i o n of s i n g l e genes has been presented i n S e c t i o n 2.1. On a h i s t o l o g i c a l l e v e l r a c e - s p e c i f i c v a r i e t a l r e s i s t a n c e i s c h a r a c t e r i z e d by r a p i d n e c r o s i s (or h y p e r s e n s i t i v e c e l l death) of the approached or penetrated 18 host c e l l s accompanied by the accumulation of p h y t o a l e x i n s and the i n h i b i t i o n of f u n g a l growth (Heath 1980). A few s t u d i e s have shown t h a t p r o t e i n s y n t h e s i s and RNA s y n t h e s i s ( i n c l u d i n g mRNA) are enhanced p r i o r to t h i s stage i n incompatible but not i n compatible combinations (Heath 1980, B e l l 1981, see a l s o S e c t i o n 2.4). Such r e s u l t s have been demonstrated i n the f l a x / f l a x r u s t , oat/crown r u s t and soybean/Phytophthora megasperma var. sojae systems (reviewed by Heath 1980, B e l l 1981). Thus there i s an i n c r e a s i n g body of evidence demonstrating induced r e s i s t a n c e . The important q u e s t i o n remains as to how t h i s r e s i s t a n c e i s induced at the m o l e c u l a r l e v e l , and how the products of r e s i s t a n c e and a v i r u l e n c e genes are i n v o l v e d . The most c o n c e p t u a l l y a t t r a c t i v e hypothesis f o r the mechanism of i n d u c t i o n was put forward by Albersheim and Anderson-Prouty (1975). These workers p o s t u l a t e t h a t c e l l s u r f a c e s are the primary s i t e of r e c o g n i t i o n which leads t o s p e c i f i c i n d u c t i o n of the r e s i s t a n c e response. They propose t h a t pathogenic a v i r u l e n c e genes encode g l y c o t r a n s f e r a s e s t h a t g i v e r i s e to race s p e c i f i c f u n g a l wall-bound carbohydrate r e s i d u e s . Host r e s i s t a n c e genes are seen as coding f o r s p e c i f i c membrane p r o t e i n s which are capable of b i n d i n g ( r e c o g n i z i n g ) these r e s i d u e s . The r e s u l t i n g complex may then t r i g g e r a r e s i s t a n c e response. Lack of the s p e c i f i c carbohydrate r e s i d u e i n the pathogen would enable i t to escape r e c o g n i t i o n and hence would c o n f e r v i r u l e n c e . T h i s might e x p l a i n why v i r u l e n c e i s a r e c e s s i v e c h a r a c t e r and furthermore why each r e s i s t a n c e gene must be overcome by a r e l a t e d v i r u l e n c e gene i n order f o r s u s c e p t i b i l i t y to occur. The evidence which i s reviewed by Albersheim and Anderson-Prouty (1975) and supports f u n g a l w a l l sugar r e s i d u e s as s p e c i f i c determinants comes from many sources i n c l u d i n g other r e c o g n i t i o n phenomena such as mating types i n y e a s t . A d d i t i o n a l l y i t has been shown t h a t f u n g a l w a l l s have s u f f i c i e n t s t r u c t u r a l v a r i a b i l i t y t o f u l f i l l t h i s r o l e . P l a n t c e l l w a l l s , however, do not meet t h i s c r i t e r i o n . In p l a n t c e l l s , membrane p r o t e i n s r e p r e s e n t a b e t t e r candidate f o r involvement i n r e c o g n i t i o n . Such p r o t e i n s are l i k e l y t o be l e c t i n s ( g l y c o p r o t e i n s i n v o l v e d i n r e c o g n i t i o n ) ; much of the evidence f o r t h i s i s reviewed by Callow (1977) and by S e q u e i r a (1978). Day (1974) has d i s c u s s e d a scheme by which r e s i s t a n c e responses may be induced at the m o l e c u l a r l e v e l f o l l o w i n g r e c o g n i t i o n . He proposes t h a t host r e s i s t a n c e genes are analogous to sensor genes ( e q u i v a l e n t to r e g u l a t o r genes known to e x i s t i n b a c t e r i a ) capable .of r e c o g n i z i n g secondary messengers from membrane r e c e p t o r s . One or more i n t e g r a t o r genes adjacent to each sensor are capable of producing an a c t i v a t o r (perhaps RNA). Each a c t i v a t o r may i n t u r n induce an operon: a t r a n s c r i p t i v e u n i t c o n t r o l l e d by a r e c e p t o r gene s e n s i t i v e t o the a c t i v a t o r . In t h i s way a complex s e r i e s of b i o c h e m i c a l changes co u l d be i n i t i a t e d at the genomic l e v e l . Many of Day's ideas might be r e i n t e r p r e t e d i n the l i g h t of r e c e n t advances i n the understanding of the c o n t r o l of gene e x p r e s s i o n i n eucaryotes, i n p a r t i c u l a r the r o l e of p o s t - t r a n s c r i p t i o n a l c o n t r o l . These advances w i l l be d e t a i l e d i n S e c t i o n 2.4. 20 Whatever the precise mechanism of the induction phenomenon in resistance many workers f e e l that phytoalexin synthesis i s the eventual outcome (B e l l 1981, Heath 1980). Thus phytoalexin accumulation and, i n some cases, hypersensitivity have been used to gauge the resistance response. There are several examples showing that phytoalexins accumulate more rapidly and to a greater extent i n incompatible interactions (Bell 1981, Heath 1980). Phytoalexin production can be e l i c i t e d by a number of chemical agents, including heavy metals, and also by physical agents, such as u l t r a - v i o l e t l i g h t and drought stress. However, there are a few examples of highly active e l i c i t o r s that can be extracted from fungal mycelia. Only one clear instance of a r a c e - s p e c i f i c fungal phytoalexin e l i c i t o r e x i s t s . In the Phytophthora megasperma, var. sojae/soybean system, Keen and Legrand (1980) have shown that glycoproteins isolated from fungal walls e l i c i t g l y c e o l l i n production to a greater extent i n r e s i s t a n t v a r i e t i e s of soybean than i n susceptible ones. The e l i c i t o r a c t i v i t y appears to reside i n a high molecular weight glycoprotein f r a c t i o n of which the carbohydrate moiety i s obligatory for a c t i v i t y . The glycoprotein f r a c t i o n was the only wall constituent bound by concanavalin A. Fluorescein-labelled concanavalin A was found to bind to l i v i n g hyphal walls but not to those from which the glycoprotein had been extracted. Thus, i t seems that the e l i c i t o r i s borne on hyphal surfaces. 21 Wade and Albersheim (1979) have shown t h a t e x t r a c e l l u l a r g l y c o p r o t e i n (ECGP) from P. megasperma v a r . sojae i s capable of p r o t e c t i n g incompatible soybean s e e d l i n g s from i n f e c t i o n f o l l o w i n g subsequent i n o c u l a t i o n with compatible r a c e s . Treatment of s e e d l i n g s with ECGP from v i r u l e n t races d i d not l e a d to s u s c e p t i b i l i t y f o l l o w i n g i n o c u l a t i o n with incompatible r a c e s , nor were compatible combinations p r o t e c t e d by p r i o r treatment with ECGP from v i r u l e n t r a c e s . S u r p r i s i n g l y , ECGP had very low p h y t o a l e x i n e l i c i t o r a c t i v i t y and t h a t which d i d e x i s t was not race s p e c i f i c . Thus i t appears t h a t the p r o d u c t i o n of p h y t o a l e x i n s , although a s s o c i a t e d with r e s i s t a n c e responses, i s not a primary determinant of the outcome of the h o s t -p a r a s i t e i n t e r a c t i o n . These r e s u l t s appear to c o n f l i c t with those of Keen and Legrand i n regard to the l o c a t i o n and e l i c i t o r a c t i v i t y of the g l y c o p r o t e i n . Whether the same g l y c o p r o t e i n i s i n v o l v e d i n both i n s t a n c e s and whether other f a c t o r s a c t i n c o n j u n c t i o n with i t to i n i t i a t e p h y t o a l e x i n s y n t h e s i s are q u e s t i o n s which await f u r t h e r r e s e a r c h i n order to be answered. An e l i c i t o r of p h y t o a l e x i n p r o d u c t i o n i n potato has been i s o l a t e d from hyphal w a l l s of Phytophthora i n f e s t a n s . The e l i c i t o r i s not race s p e c i f i c but has nonetheless been the s u b j e c t of s e v e r a l r e c e n t papers (see Kurantz and Zacharius 1981). Doke and Tomiyama (1980a) have r e p o r t e d t h a t hyphal w a l l components cause r a p i d aggregation and l y s i s 22 of p o t a t o p r o t o p l a s t s . P r o t o p l a s t response was not c o r r e l a t e d with the presence of major r e s i s t a n c e genes but was a s s o c i a t e d with the degree of f i e l d r e s i s t a n c e ( h o r i z o n t a l r e s i s t a n c e ) . However s o l u b l e f u n g a l glucans were capable of suppressing the h y p e r s e n s i t i v e response of p r o t o p l a s t s and t h i s e f f e c t was p a r t i a l l y c o r r e l a t e d with compatible combinations of v e r t i c a l v i r u l e n c e and r e s i s t a n c e a l l e l e s (Doke and Tomiyama 1980b). These r e s u l t s are p a r t i c u l a r l y i n t e r e s t i n g because they are at odds with our understanding of the mechanisms u n d e r l y i n g gene- for-gene systems. However, they are f a r from complete and may merely serve to i l l u s t r a t e the problems of s t u d y i n g the e f f e c t s of r e s i s t a n c e genes i n p l a n t s with g r e a t l y d i f f e r i n g g e n e t i c backgrounds. Hence, a c o r r e l a t i o n with major gene r e s i s t a n c e may be m i s i n t e r p r e t e d i n terms of the true cause and e f f e c t r e l a t i o n s h i p . Furthermore, i t i s c l e a r that e l i c i t o r s of p h y t o a l e x i n s and h y p e r s e n s i t i v i t y are not n e c e s s a r i l y s p e c i f i c t r i g g e r s of the r e s i s t a n t response. The Phytophthora systems are w e l l s t u d i e d and i l l u s t r a t e the d i r e c t i o n s f o r f u t u r e r e s e a r c h . They undoubtedly have s i g n i f i c a n c e f o r workers studying f l a x and c e r e a l r u s t d i s e a s e s . However, Phytophthora d i f f e r s s i g n i f i c a n t l y i n that i t i s a hemibiotroph, causing t i s s u e n e c r o s i s f a i r l y e a r l y i n s u c c e s s f u l d i s e a s e development. L i t t l e f i e l d and Aronson (1969) have undertaken a d e t a i l e d h i s t o l o g i c a l study of r e s i s t a n c e mechanisms i n f l a x 23 f o l l o w i n g i n o c u l a t i o n with f l a x r u s t . Fungal growth i n s u s c e p t i b l e and r e s i s t a n t r e a c t i o n s was s i m i l a r up to 36 to 48 hours f o l l o w i n g i n o c u l a t i o n . A f t e r t h i s time r e s i s t a n c e was c h a r a c t e r i z e d by a r e d u c t i o n i n l e n g t h and number of hyphae and number of h a u s t o r i a per i n f e c t i o n s i t e as compared to s u s c e p t i b i l i t y . L i t t l e f i e l d (1973) l a t e r showed t h a t "L" and 'N' l o c u s genes c o n d i t i o n e d the most r a p i d and l o c a l i z e d r e s i s t a n c e response. At 169 hours a f t e r i n o c u l a t i o n 'M' and 'P' genes c o n d i t i o n e d a h y p e r s e n s i t i v e response over a l a r g e r area and the 'K' gene conveyed moderate r e s i s t a n c e which was accompanied by e x t e n s i v e n e c r o s i s . However n e c r o t i c l e s i o n s i n the f a s t - a c t i n g r e s i s t a n c e c o n f e r r e d by the 'L' and 'N' genes were l a r g e r than w i t h the s l o w e r - a c t i n g 'M' and 'P' genes at 24 hours a f t e r i n o c u l a t i o n . L a t e r work by Keen and L i t t l e f i e l d (1979) showed t h a t two a n t i f u n g a l compounds, c o n i f e r y l a l c o h o l and c o n i f e r y l aldehyde, accumulate more r a p i d l y i n incompatible r e a c t i o n s . Furthermore, these compounds accumulate more r a p i d l y and to a g r e a t e r extent i n r e s i s t a n c e responses c o n d i t i o n e d by f a s t - a c t i n g r e s i s t a n c e genes. These o b s e r v a t i o n s p r o v i d e an e x p l a n a t i o n f o r the p a r t i a l r e s i s t a n c e to v i r u l e n t races which i s observed f o l l o w i n g p r i o r i n o c u l a t i o n with an a v i r u l e n t race ( L i t t l e f i e l d 1969). In summary, the evidence f o r induced v a r i e t a l r e s i s t a n c e i s now very s u b s t a n t i a l . The important areas to 24 be e l u c i d a t e d are, f i r s t l y , the involvement of a v i r u l e n c e and r e s i s t a n c e genes i n t r i g g e r i n g the response and secondly the mechanisms by which t h i s t r i g g e r b r i n g s about the response. The l a t t e r i s l i k e l y to have i t s b a s i s i n the c o n t r o l of gene e x p r e s s i o n . There i s a l r e a d y evidence which shows t h a t RNA and p r o t e i n s y n t h e s i s are enhanced duri n g r e s i s t a n c e a t times p r i o r to h y p e r s e n s i t i v e c e l l death and p h y t o a l e x i n accumulation. Furthermore, i t has been suggested t h a t the h y p e r s e n s i t i v e response i s not the cause of r e s i s t a n c e but merely a consequence o f i t . K i r a l y e t a l . (1972) have presented evidence t h a t h y p e r s e n s i t i v e responses are induced i n compatible i n t e r a c t i o n s i f the f u n g a l mycelium i s k i l l e d by treatment of the i n f e c t e d t i s s u e with s p e c i f i c i n h i b i t o r s . H y p e r s e n s i t i v i t y was not observed when p l a n t s were t r e a t e d w i t h the i n h i b i t o r s alone nor when they were i n f e c t e d i n the absence of i n h i b i t o r s . These workers came to the c o n c l u s i o n t h a t events i n host metabolism p r i o r to the onset of h y p e r s e n s i t i v i t y were r e s p o n s i b l e f o r f u n g a l i n h i b i t i o n d u r i n g r e s i s t a n c e . Once i n h i b i t i o n has occurred they p o s t u l a t e t h a t the damaged f u n g a l mycelium induces h y p e r s e n s i t i v i t y . For these reasons events i n host gene e x p r e s s i o n which occur p r i o r t o h y p e r s e n s i t i v i t y are reviewed i n a f o l l o w i n g s e c t i o n . 25 2.3 Ribonuclease and Disease Rohringer e_t al. (1961) were the f i r s t researchers to investigate the influence of rust i n f e c t i o n on ribonuclease (RNase) a c t i v i t y . E a r l i e r work had shown that RNA synthesis increased markedly i n response to i n f e c t i o n and i t was therefore of interest to gain an insight into RNA degradation (turnover). It was revealed that a bimodal increase i n RNase a c t i v i t y occurred, peaking at 1 to 2 and 6 to 7 days after inoculation in a susceptible wheat/leaf rust combination. In a r e s i s t a n t combination s i m i l a r changes occurred but to a lesser degree. Several l a t e r reports from Shaw's group showed that s i m i l a r bimodal increases in RNase a c t i v i t y could be demonstrated i n other host/rust combinations. Where both r e s i s t a n t and susceptible combinations were studied, the l a t e r increase occurred only in susceptible plants and i t s onset coincided with flecking and the i n i t i a t i o n of sporula-t i o n . This c o r r e l a t i o n could be conveniently studied using near isogenic l i n e s of wheat carrying the dominant or recessive a l l e l e s of the Sr6 gene which i s a temperature sensi t i v e gene for resistance to wheat stem rust (Puccinia  graminis t r i t i c i ) (Chakravorty et a l . 1974a). U t i l i z i n g races of rust carrying the avirulence a l l e l e corresponding to 26 the Sr6 a l l e l e , i t was shown t h a t the l a t e r i n c r e a s e i n RNase oc c u r r e d o n l y at temperatures p e r m i s s i v e to r u s t development. Furthermore p l a n t s p r e v i o u s l y i n o c u l a t e d with these races and he l d at a non-permissive temperature only showed the l a t e i n c r e a s e i n RNase f o l l o w i n g t r a n s f e r to the p e r m i s s i v e temperature. Thus s p o r u l a t i o n and i n c r e a s e d RNase a c t i v i t y c o u l d be delayed up to 14 days a f t e r i n o c u l a t i o n at the non-permissive temperature, but once i n i t i a t e d , o c c u r r e d i n a s i m i l a r f a s h i o n to t h a t i n p l a n t s c a r r y i n g the r e c e s s i v e (sr6) a l l e l e o r i n those i n f e c t e d with a race v i r u l e n t on Sr6. In t h i s and oth e r papers emanating from Shaw's group c a t a l y t i c p r o p e r t i e s of RNase were a l s o s t u d i e d . In the f o u r host r u s t combinations s t u d i e d the e a r l y i n c r e a s e i n RNase a c t i v i t y , r e f e r r e d to as ' e a r l y RNase', was not always accompanied by changes i n the p r o p e r t i e s of the enzyme whereas the l a t e i n c r e a s e , ' l a t e RNase', was accompanied by a v a r i e t y of changes. These f i n d i n g s are summarized i n Table I. The changes i n the c a t a l y t i c p r o p e r t i e s of RNase were shown not to be due to f u n g a l c o n t r i b u t i o n , as the s u b s t r a t e p r e f e r e n c e s f o r the m y c e l i a l enzymes were determined i n each case. The e f f e c t s of wounding were a l s o assessed as i t i s known t h a t wounding induces l a r g e i n c r e a s e s i n RNase a c t i v i t y (Dove 1973). However, the c a t a l y t i c p r o p e r t i e s of RNase from wounded p l a n t s appeared s i m i l a r to t h a t from healthy p l a n t s TABLE I CHANGES IN RNase ACTIVITY FOLLOWING RUST INFECTION PHASE OF SUBSTRATE3 OTHER HOST RUST RUST CYCLE INFECTION PREFERENCE CHANGES REFERENCES Ribes Cronartium Healthy ^ pA>pU>pI>pC Heat s t a b i l i t y Harvey et a l . nigrum r i b i c o l a D i ploid 16 days p . i . pA>pU>plEpC diethylpyrocar- 1974 ( B l i s t e r rust) (Late RNase) bonate s e n s i t i v i t y Pine t i s s u e Cronartium Healthy pU>pA>pC Heat s t a b i l i t y , Harvey et a l . c u l t u r e r i b i c o l a Haploid 64 days p . i . pA=pC diethylpryocar- 1974 ( B l i s t e r rust) (Late RNase) bonate s e n s i t i v i t y Flax Melampsora Healthy pU>pA>RNA>pC Heat s t a b i l i t y . Scrubb et a]. (cotyledons) l i n i D i p l oid 8 days p . i . RNA>pC>pU>pA Detailed study of 1972 (Flax rust) (Late RNase) differences i n Chakravorty p u r i f i e d enzymes et a l . 1974c Wheat Puccinis graminis Healthy pU>pA>pC Heat s t a b i l i t y Chakravorty (Sr6, sr6) t r i t i c i D iploid 3 days p . i . pC>pAEplJ and substrate et a l . 1974a (Wheat stem rust) (Early RNase) preference d i f f e r 10 days p . i . pC>pA=pU between e a r l y and (Late RNase) la t e RNase Substrate preference i s determined with respect to the r e l a t i v e rates of hydrolysis of the ribonucleotide homopolymers polyadenylate (pA), p o l y c y t i d y l a t e (pC), polyuridylate (pU), and polyinosinate (pi) by RNase from the host/rust combinations shown. p . i . post-inoculation. 28 i n each case. These f i n d i n g s i m p l i e d t h a t novel RNase molecules had a r i s e n s p e c i f i c a l l y i n response to i n f e c t i o n ; furthermore, i t was p o s s i b l e t h a t these novel molecules were e s s e n t i a l f o r s u c c e s s f u l d i s e a s e development. These ideas l e d Chakravorty e t a l . (1974c) t o attempt p u r i f i c a t i o n of i n f e c t e d f l a x RNase to homogeneity i n order to determine whether a novel enzyme p r o t e i n had a r i s e n . P u r i f i c a t i o n showed t h a t the m a j o r i t y of the s o l u b l e RNase a c t i v i t y i n f l a x cotyledons e l u t e d as a s i n g l e peak with a mo l e c u l a r weight of approximately 40,000 duri n g g e l f i l t r a t i o n . The enzymes thus p u r i f i e d from healthy or 8-day i n f e c t e d cotyledons had the same molecular weight but markedly d i f f e r e n t c a t a l y t i c p r o p e r t i e s . Among the p r o p e r t i e s which d i f f e r e d were M i c h a e l i s Constant ( K m ) , pH optimum, temperature s t a b i l i t y and the a b i l i t y to degrade v a r i o u s n u c l e o t i d e polymers. Other s t u d i e s on RNase from the f l a x r u s t mycelium grown i n i s o l a t o n from the host showed the changes i n c a t a l y t i c p r o p e r t i e s d u r i n g d i s e a s e could not be accounted f o r i n terms of f u n g a l c o n t r i b u t i o n (Chakravorty e_t a l . 1974b). The onl y f u n g a l enzyme with a molecular weight s i m i l a r to t h a t of the enzyme from f l a x had p r o p e r t i e s q u i t e u n l i k e the enzyme from i n f e c t e d f l a x . Chakravorty e t a l . (197 4c) a l s o undertook a d e t a i l e d study of the aggregation and d i s s o c i a t i o n products which 29 were observed a f t e r f r e e z i n g and thawing of the p u r i f i e d p r e p a r a t i o n s from f l a x . F r e e z i n g and thawing y i e l d e d three peaks of RNase a c t i v i t y by g e l f i l t r a t i o n with apparent mo l e c u l a r weights of 90,000, 39,000 and 27,000 d a l t o n s . These aggregation and d i s s o c i a t i o n products were c o n s i s t e n t l y formed from p u r i f i e d e x t r a c t s of h e a l t h y or eight-day i n f e c t e d c o t y l e d o n s . However, the s u b s t r a t e p r e f e r e n c e of each was not onl y d i f f e r e n t from the n a t i v e enzyme but a l s o depended upon whether the enzymes were d e r i v e d from h e a l t h y or i n f e c t e d c o t y l e d o n s . T h i s l e d Chakravorty et a l . (1974b) to suggest t h a t the a l t e r e d p r o p e r t i e s of the RNase d u r i n g d i s e a s e were brought about by the appearance of a novel s u b u n i t i n the ap p a r e n t l y o l i g o m e r i c enzyme. T h i s h y p o t h e s i s i s summarized i n Fig u r e 4. T h i s work a l s o p r o v i d e d a l a r g e p a r t of the evidence f o r an hypothesis concerning the molecular b a s i s of o b l i g a t e host-pathogen i n t e r a c t i o n s . Chakravorty and Shaw (1977a) proposed that enzymes with novel c a t a l y t i c p r o p e r t i e s are e s s e n t i a l f o r pathogen development. Hence, i t was suggested t h a t the i n d u c t i o n of such enzymes was the molecular b a s i s f o r s p e c i f i c i t y determining r e s i s t a n c e or s u s c e p t i b i l i t y . Thus, these authors suggested t h a t the h y p o t h e t i c a l novel s u b u n i t i n Fi g u r e 4 c o u l d a r i s e i n f o u r p o s s i b l e ways: 1) The t r a n s f e r of c i s t r o n s ( o p e r a t i o n a l u n i t s of DNA) from pathogen to host, A summary of the molecular weights, substrate preference and hypothetical structure of the native RNase molecules and their artificially produced variants from healthy and rust-infected flax cotyledons Enzyme Infected native RNase Native RNases (before freezing and thawing) Artificially produced variants (after freezing and thawing) Molecular Hypothetical" Artifactual Molecular Hypothetical weight Substrate preference structure variant weight Substrate preference structure Healthy native 40 000 p(U) > p(A) > RNA > p(C) RNase ( f t (a) 40 000 RNA>p(C)>p(U)>p(A) (b) P-1 90 000 RNA>p(A)sp(U)>p(C) P-2 39 000 RNA > p(A)p(C) P-3 27 000 RNAsp(C) P-1 90 000 RNA>p(C)>p(A)>p(U) P-2 39 000 p(C)>p(A)>RNA P-3 27 000 RNA>p(A) 8 « 8 (c) (0 (g) (h) • Or the several possible combinations of monomers in (c) to (h) only one is shown, for the sake of simplicity. The number of monomers assigned to the native and the derived RNases is also arbitrary. FIGURE 4. The subunit complementation hypothesis for f l a x RNase (reproduced from Chakravorty, A.K., Shaw, M. and Scrubb, L.A. 1974). 31 2) The t r a n s f e r of inducer substances from pathogen to ho s t , or v i c e v e r s a i n the case of novel pathogen enzymes, l e a d i n g to d e r e p r e s s i o n of an i n d u c i b l e gene. 3) The t r a n s f e r of mRNA coding f o r the subunit from patho-gen to host. 4) D i r e c t t r a n s f e r of p o l y p e p t i d e s from pathogen to host. The p o s s i b i l i t y of t r a n s f e r of macromolecules ( i n 1, 3 and 4) from host to pathogen was considered u n l i k e l y i n view of the f a c t t h a t r u s t f u n g i can develop s u c c e s s f u l l y on simple c h e m i c a l l y d e f i n e d media. Chakravorty and S c o t t (1979) i n v e s t i g a t e d RNase a c t i v i t y d u r i n g development of powdery mildew (Erysiphe  graminis f . sp. hordei) on b a r l e y . Both q u a n t i t a t i v e and q u a l i t a t i v e changes occ u r r e d i n the major s o l u b l e RNase a c t i v i t y d u r i n g the l a t e r stages of development. A d d i t i o n a l l y , a minor RNase component which i s i n s o l u b l e at pH 5 was examined. In a l l e a r l i e r s t u d i e s on RNase duri n g r u s t i n f e c t i o n t h i s f r a c t i o n had been d i s c a r d e d d u r i n g p u r i f i c a t i o n . Thus, i t was thought important to determine whether changes i n t h i s f r a c t i o n had gone unnoticed. Indeed i t was shown that changes i n the s u b s t r a t e preference of t h i s enzyme co u l d be de t e c t e d as e a r l y as one day a f t e r i n o c u l a t i o n . T h i s work has t h e r e f o r e demonstrated t h a t changes i n RNase a c t i v i t y may be c h a r a c t e r i s t i c of powdery mildew i n f e c t i o n a s ' w e l l as r u s t i n f e c t i o n and t h a t pH 5 32 insoluble RNase a c t i v i t y should be re-examined. In p a r t i c u l a r , quantitative changes i n RNase a c t i v i t y which occur very soon after inoculation might be considered both more s i g n i f i c a n t in the establishment of the host-parasite int e r a c t i o n and also less l i k e l y the resu l t of a general non-specific disruption i n host metabolism. If indeed the appearance of novel RNase molecules i s an important component of disease development, then c l e a r l y i t would be of interest to know f i r s t l y , whether such changes occur i n other plant responses and secondly, what the function of the RNase a c t i v i t y i s . In answering the f i r s t of these questions i t i s useful to consider how RNase a c t i v i t y responds to wounding and senescence which may be regarded as p a r a l l e l s to some of the events in disease. As has already been mentioned, at least i n the instances where wounding has been studied, although large increases in RNase a c t i v i t y occur, the properties of th i s a c t i v i t y are quite d i f f e r e n t from those of RNase from infected plants. Novel RNase a c t i v i t y has been shown to appear i n senescing leaves but i t i s accompanied by a corresponding decrease in RNA levels (Dove 1 973 ) . This i s quite uncharacteristic of infected plants, which show increased l e v e l s of RNA (Quick and Shaw 1964, Hamilton 1 969 ) . This evidence suggests that the novel RNase a c t i v i t y of infected plants has a role which is s p e c i f i c to the metabolism of infected plants. 33 The p o s s i b l e f u n c t i o n of the RNase s t u d i e d d u r i n g r u s t and mildew i n f e c t i o n can be assessed with a knowledge of the known f u n c t i o n s of RNases with s i m i l a r c a t a l y t i c p r o p e r t i e s and i n t r a c e l l u l a r l o c a l i z a t i o n s . A d d i t i o n a l l y , i t would be of g r e a t value to know what n a t u r a l s u b s t r a t e s these enzymes can h y d r o l y z e . The major s o l u b l e RNase i n h i g h e r p l a n t s , RNase I, has a pH optimum between 5.0 and 6.0 and b r i n g s about p r e f e r e n t i a l r e l e a s e of p u r i n e 2 " - 3 ' c y c l i c n u c l e o t i d e s by e n d o n u c l e o l y t i c cleavage (Wilson 1975). The p u r i f i e d RNase from h e a l t h y or i n f e c t e d f l a x (Chakravorty e_t a l . 1974c) r e p r e s e n t s the major s o l u b l e f r a c t i o n , has a pH optimum of 5.5 and an e n d o n u c l e o l y t i c mode of a c t i o n . Although the n u c l e o t i d e s produced d u r i n g h y d r o l y s i s have not been analyzed i t appears l i k e l y t h a t t h i s enzyme i s of the RNase I type. RNase I enzymes are thought to be i n v o l v e d i n the breakdown and r e u t i l i z a t i o n of RNA (Wilson 1975). Simpson e t a l . (1980) have examined the a c t i o n of pH 5 s o l u b l e and pH 5 i n s o l u b l e enzymes from b a r l e y on b a r l e y polysomal RNA under l i m i t e d d i g e s t i o n c o n d i t i o n s . The pH 5 s o l u b l e enzyme, analogous to the f r a c t i o n s t u d i e d i n r u s t - i n f e c t e d p l a n t s , was incapable of h y d r o l y z i n g messenger RNA. The i n s o l u b l e enzyme, however, hydrolyzed mRNA to generate monosomes and lower order polysomes. Furthermore, t h i s enzyme p r e f e r e n t i a l l y degraded c h l o r o p l a s t polysomes when the enzyme was d e r i v e d from s u s c e p t i b l e p l a n t s i n f e c t e d 34 with powdery mildew (Chakravorty et a l . 1980). Both enzymes were capable of hydrolyzing ribosomal RNA within intact monosomes but the monosomes remained e f f e c t i v e in protein synthesis despite extensive degradation of t h e i r RNA. Chakravorty and Scott (1979) have attempted to assign an i n t r a c e l l u l a r location to these enzymes using d i f f e r e n t i a l centrifugation. The pH 5 soluble RNase appears within the soluble f r a c t i o n whereas the pH 5 insoluble RNase i s microsomal (which would include ribosomes). As rigorous proof i s lacking, any statements concerning the function of ribonucleases in plants are tentative at best. The evidence for the cytoplasmic l o c a t i o n of RNase I (Wilson 1975) although suggestive does not constitute proof. C e l l f r a c t i o n a t i o n i n the i s o l a t i o n of RNase (Chakravorty & Scott 1979, Wilson 1975) has often been crude and thus there has almost c e r t a i n l y been extensive nuclear disruption in such experiments. Chakravorty and Shaw (1977b) have suggested a role for fl a x RNase in the processing of RNA. This idea i s based upon the rate of synthesis and content of RNA, and upon the s p e c i f i c a c t i v i t y of RNase following rust i n f e c t i o n . These data show that RNA accumulates i n spite of le v e l s of RNase which are more greatly elevated than the rate of RNA synthesis. Although i t would appear l o g i c a l to draw the conclusion that t h i s RNase i s not involved in RNA degradation (substrate r e u t i l i z a t i o n ) , t h i s view may be too s i m p l i s t i c . Such a conclusion requires 35 the assumption t h a t RNA turnover i n the l i v i n g c e l l i s r e g u l a t e d merely by the c o n c e n t r a t i o n of RNase. In r e a l i t y , l o c a l i z a t i o n o f enzyme and s u b s t r a t e as w e l l as the a s s o c i a t i o n of r i b o n u c l e o p r o t e i n s are important f a c t o r s . In summary, r u s t and mildew i n f e c t i o n cause marked i n c r e a s e s i n RNase a c t i v i t y which i s c a t a l y t i c a l l y d i f f e r e n t from t h a t found i n h e a l t h y p l a n t s . The novel RNase i s of host o r i g i n and i n the case of f l a x appears to be the r e s u l t of an a l t e r e d enzyme molecule. The f u n c t i o n of t h i s enzyme i s at present unknown. C l e a r l y , a n a l y s i s of the enzyme's h y d r o l y s i s products and d e t e r m i n a t i o n of i t s i n t r a c e l l u l a r l o c a l i z a t i o n would be h e l p f u l i n a s s i g n i n g a f u n c t i o n . Whether subunit complementation a c t u a l l y occurs to b r i n g about novel enzymatic p r o t e i n s i s a l s o not e s t a b l i s h e d . Only r i g o r o u s p u r i f i c a t i o n and the p r e p a r a t i o n of subunits w i l l enable proof of t h i s to be o b t a i n e d . 36 2.4 RNA and Protein Synthesis Enhanced RNA synthesis has been observed i n many host-parasite interactions. Heitefuss and Wolf (1976) have reviewed many studies which have demonstrated stimulation of cytoplasmic rRNA during disease development. This i s usually accompanied by a continuous decline in chloroplast rRNA (Bennett and Scott 1971) and chloroplast polysome content (Dyer and Scott 1972). Most early studies involving extraction and frac t i o n a t i o n of i n vivo 32p_i abelled RNA were unable to provide conclusive evidence that concurrent increases in mRNA synthesis also occurred. This was due to l a b e l l i n g periods which were too long to detect the rapidly turned over messenger f r a c t i o n and also to the rather ambiguous results which can be obtained from fractionation on methylated albumin kieselguhr (MAK) columns. It might seem reasonable to assume that the synthesis of rRNA, mRNA and soluble RNA (tRNA and 5S rRNA) responds concurrently to inf e c t i o n as they a l l p a r t i c i p a t e in protein synthesis. Tani e_t a l . (1971) demonstrated that incorporation of 32p into RNA fractions bound tenaciously to MAK columns (putative mRNA) increased markedly after crown rust i n f e c t i o n of both susceptible and resist a n t oats. A subsequent investigation f a i l e d to demonstrate any increase 37 i n the template a c t i v i t y of RNA e x t r a c t e d from i n f e c t e d oats by i n v i t r o t r a n s l a t i o n (Tani et a l . 1973). However, s i n c e t h i s i s a measure of mRNA mass whereas the former r e s u l t s are a measure of s y n t h e s i s , t h i s merely i n d i c a t e s r a p i d mRNA turn o v e r . T h i s c l e a r l y does not pre c l u d e the p o s s i b i l i t y t h a t the mRNA f r a c t i o n encodes p r o t e i n s s p e c i f i c a l l y a s s o c i a t e d with i n f e c t i o n . Novel s i z e c l a s s e s of p o l y p e p t i d e s appear t o be encoded by polysomes from r u s t i n f e c t e d wheat (Pure e t a l . 1979, 1980). These p o l y p e p t i d e s are d e t e c t e d by in v i t r o t r a n s l a t i o n and appear to be sy n t h e s i z e d on cy t o p l a s m i c ribosomes on the b a s i s of t h e i r i n s e n s i t i v i t y to chloramphenicol (Pure e t a l . 1980). Chakravorty and Shaw (1971) examined the i n c o r p o r a t i o n o f 32p i n t o two RNA f r a c t i o n s from f l a x c o t yledons f o l l o w i n g i n o c u l a t i o n with f l a x r u s t . Both the s a l t - i n s o l u b l e ( c o n t a i n i n g 25S and 18S ribosomal RNA) and the s a l t - s o l u b l e ( c o n t a i n i n g predominantly 5S rRNA and 4S t r a n s f e r RNA) e x h i b i t e d h i g h e r s p e c i f i c r a d i o a c t i v i t y f o l l o w i n g i n o c u l a t i o n . S p e c i f i c a c t i v i t y i n c r e a s e d to more than two f o l d over t h a t found i n the he a l t h y cotyledons i n both f r a c t i o n s at 2 days a f t e r i n o c u l a t i o n . T h i s e a r l y i n c r e a s e was fol l o w e d by lower l e v e l s of i n c o r p o r a t i o n a t 4 and 6 days a f t e r i n o c u l a t i o n but s t i l l h i g h e r than i n h e a l t h y c o t y l e d o n s . Since i t has been demonstrated that there are 38 s i g n i f i c a n t i n c r e a s e s i n the amount of RNA i n i n f e c t e d c o t y l e d o n s from 6 days p o s t - i n o c u l a t i o n (Hamilton 1969), t h i s r e p r e s e n t s a s u b s t a n t i a l i n c r e a s e i n o v e r a l l RNA s y n t h e s i s i n the l a t e r stages of d i s e a s e . A n a l y s i s of the s a l t i n s o l u b l e f r a c t i o n by d e n s i t y g r a d i e n t c e n t r i f u g a t i o n r e v e a l e d t h a t both the 25S and 18S RNA components were more h e a v i l y l a b e l l e d with 32p f o l l o w i n g i n o c u l a t i o n . The base composition of both l a b e l l e d f r a c t i o n s was a l s o determined a t 2 and 6 days a f t e r i n o c u l a t i o n . I t was shown that the s a l t -s o l u b l e RNA had an a l t e r e d base composition; that of the ribosomal RNA ( s a l t - i n s o l u b l e ) remained unchanged. Thus i t has been shown t h a t s o l u b l e RNA i s s y n t h e s i z e d more r a p i d l y d u r i n g r u s t i n f e c t i o n . T h i s i s i n g e n e r a l agreement with other s t u d i e s , with the e x c e p t i o n of a study by Wolf (see H e i t e f u s s and Wolf 1976) who found no s i g n i f i c a n t d i f f e r e n c e i n the 32p s p e c i f i c a c t i v i t y of s o l u b l e RNA i n wheat le a v e s i n f e c t e d with stem r u s t . Whether the i n c r e a s e d s y n t h e s i s of RNA i n the s a l t - s o l u b l e f r a c t i o n from f l a x i s a l s o r e p r e s e n t a t i v e of i n c r e a s e d mRNA s y n t h e s i s i s , as y e t , unknown. The o b s e r v a t i o n t h a t changes i n the base composition of the RNA s y n t h e s i z e d i n i n f e c t e d p l a n t s are not due to f u n g a l c o n t r i b u t i o n and, furthermore, could not be induced by treatment with the auxin i n d o l e a c e t i c a c i d suggest t h a t gene e x p r e s s i o n may have been a l t e r e d 39 s p e c i f i c a l l y i n response t o i n f e c t i o n (Chakravorty e_t a l . 1971). A number of experiments with i n h i b i t o r s of RNA s y n t h e s i s imply that e a r l y i n c r e a s e s i n host RNA s y n t h e s i s are c r i t i c a l to pathogen development. Development of bean r u s t was g r e a t l y impaired by a p p l i c a t i o n of 5 - f l u o r o u r a c i l t o bean leaves (see H e i t e f u s s & Wolf 1976). The supp r e s s i o n was only e f f e c t i v e i f the i n h i b i t o r was a p p l i e d w i t h i n 48 hr of i n o c u l a t i o n . Furthermore the i n c r e a s e i n RNA s y n t h e s i s normally observed was l a r g e l y e l i m i n a t e d . S i m i l a r r e s u l t s have been observed with P u c c i n i a graminis t r i t i c i on wheat and P. coronata on oats (see H e i t e f u s s and Wolf 1976). I n h i b i t i o n of f l a x r u s t on f l a x occurs i f actinomycin D i s a p p l i e d w i t h i n f i v e to s i x hours a f t e r i n o c u l a t i o n (Shaw 1967). Although d i r e c t i n h i b i t i o n of fu n g a l growth can not be r u l e d out, i t has been demonstrated i n many cases t h a t the l e v e l s of i n h i b i t o r s used have no s i g n i f i c a n t e f f e c t on spore g e r m i n a t i o n . E a r l y changes i n host RNA s y n t h e s i s are t h e r e f o r e a promising area of study. V a l u a b l e i n f o r m a t i o n of t h i s type has been gained by microscopy. Bhattacharya, Shaw and co-workers c a r r i e d out a s e r i e s of cytophotometric s t u d i e s on the n u c l e i of r u s t i n f e c t e d wheat. In the s u s c e p t i b l e r e a c t i o n of wheat leaves ( v a r i e t y L i t t l e Club) i n f e c t e d with race 15B of wheat stem r u s t , RNA l e v e l s i n the host n u c l e i i n c r e a s e d markedly from two days a f t e r i n o c u l a t i o n (Bhattacharya e t a l . 1965). T h i s 40 was accompanied by a decrease i n h i s t o n e s and an i n c r e a s e i n the l e v e l of other n u c l e a r p r o t e i n s . These data and the o b s e r v a t i o n t h a t the n u c l e i s w e l l e d d r a m a t i c a l l y are a l l i n d i c a t i v e of gene d e r e p r e s s i o n and in c r e a s e d RNA s y n t h e s i s . T h i s i s supported by the enhanced i n c o r p o r a t i o n of t r i t i a t e d c y t i d i n e and u r i d i n e i n t o RNA i n the n u c l e i of six-d a y i n f e c t e d p l a n t s , determined by microautoradiography (Bhattacharya and Shaw 1967). Concurrent i n c r e a s e s i n the i n c o r p o r a t i o n of t r i t i a t e d l e u c i n e i n t o n u c l e a r p r o t e i n were a l s o observed. L a t e r s t u d i e s on the n u c l e o l i showed t h a t n u c l e o l a r volume i n c r e a s e d very d r a m a t i c a l l y , being f o u r and e i g h t times t h a t i n h e a l t h y p a r t s of the leaves at two and f o u r days a f t e r i n o c u l a t i o n r e s p e c t i v e l y (Bhattacharya et a l . 1968). T h e r e a f t e r n u c l e o l a r volume d e c l i n e d g r a d u a l l y but remained g r e a t e r than t h a t i n h e a l t h y leaves beyond t h i r t e e n days a f t e r i n o c u l a t i o n . S i m i l a r trends were observed with r e s p e c t t o the e l e v a t e d l e v e l s of RNA and p r o t e i n i n the n u c l e o l i . N u c l e o l i are i n v o l v e d i n ribosomal RNA s y n t h e s i s and ribosome assembly. Thus, i t i s i n d i c a t e d that ribosome p r o d u c t i o n i s g r e a t l y s t i m u l a t e d as soon as two days a f t e r i n o c u l a t i o n , which may account f o r the r e l a t i o n s h i p observed between RNA and p r o t e i n content. Bhattacharya and Shaw (1968) a l s o s t u d i e d the l e v e l s of RNA, h i s t o n e and t o t a l p r o t e i n i n the n u c l e i of i n f e c t e d 41 K h a p l i wheat leaves ( r e s i s t a n t ) . RNA and p r o t e i n l e v e l s were a l r e a d y e l e v a t e d and h i s t o n e s had decreased at 3 days a f t e r i n o c u l a t i o n . These i n i t i a l changes were followed by d e c l i n i n g l e v e l s of a l l three substances which culminated i n a g e n e r a l n u c l e a r c o l l a p s e at the i n f e c t i o n s i t e s at 12 days a f t e r i n o c u l a t i o n . These r e s u l t s are of p a r t i c u l a r i n t e r e s t because they i n d i c a t e the a c t i v e nature of the r e s i s t a n c e response which precedes l o c a l i z e d c e l l death. I t i s u n f o r t u n a t e t h a t n u c l e i i n the r e s i s t a n t r e a c t i o n were not examined p r i o r to three days a f t e r i n o c u l a t i o n , as i t i s l i k e l y t h a t the outcome of the h o s t - p a r a s i t e i n t e r a c t i o n i s determined w i t h i n one or two days a f t e r i n o c u l a t i o n (Shaw 1967). A d d i t i o n a l l y , K h a p l i and L i t t l e Club wheats are of w i d e l y d i f f e r i n g g e n e t i c background. I t i s c l e a r that these h i s t o l o g i c a l s t u d i e s p r o v i d e a s t r o n g b a s i s f o r d e t a i l e d b i o c h e m i c a l work. One problem i s apparent however; the n u c l e i of c e l l s more than 100 ^im away from i n f e c t i o n s i t e s i n s u s c e p t i b l e l eaves appear u n a f f e c t e d (Bhattacharya and Shaw 1967). Thus, i n order to d e t e c t a p p r e c i a b l e changes i n p l a n t e x t r a c t s i t may be necessary t o use very high i n o c u l a t i o n d e n s i t i e s . T a n i and co-workers have c a r r i e d out a number of s t u d i e s on both compatible and incompatible combinations of oat and crown r u s t . These s t u d i e s have shown that RNA and 42 p r o t e i n s y n t h e s i s are enhanced between 12 and 16 hours and 14 and 20 h a f t e r i n o c u l a t i o n r e s p e c t i v e l y i n an incompatible combination (Tani and Yamamoto 1978). S i m i l a r changes were not observed f o l l o w i n g i n o c u l a t i o n of the same oat v a r i e t y w i t h a compatible race of crown r u s t . Furthermore i t was demonstrated t h a t c o r d y c e p i n , an i n h i b i t o r of RNA s y n t h e s i s , and puromycin, an i n h i b i t o r of p r o t e i n s y n t h e s i s , could suppress r e s i s t a n c e . T h i s s u p p r e s s i o n was f u l l y expressed when the i n h i b i t o r s were s u p p l i e d w i t h i n 10 hours a f t e r i n o c u l a t i o n , i n the case of c o r d y c e p i n and w i t h i n 12 hours i n the case of puromycin. In another study s i m i l a r r e s u l t s were observed with r e s p e c t to p r o t e i n s y n t h e s i s and the e f f e c t s of the i n h i b i t o r b l a s t i c i d i n S (Yamamoto e t a l . 1976). In these experiments peroxidase isozymes and p h e n y l a l a n i n e ammonia l y a s e a c t i v i t y were a l s o examined. The l a t t e r i n c r e a s e d markedly i n response to i n f e c t i o n with the incompatible race of crown r u s t . However, b l a s t i c i d i n S was not e f f e c t i v e i n s u p r e s s i n g t h i s i n c r e a s e d a c t i v i t y . T h i s i s i n t e r e s t i n g i n the l i g h t of a r e p o r t by Yoshikawa et a l . (1977) who have i n d i c a t e d t h a t i n c r e a s e d s y n t h e s i s of p o l y a d e n y l a t e d messenger RNA i s a s s o c i a t e d with g l y c e o l l i n p r o d u c t i o n and r e s i s t a n c e i n soybeans i n f e c t e d with an incompatible race of Phytophthora megasperma var. s o j a e . Phenylalanine ammonia l y a s e i s one of the key enzymes i n the s y n t h e s i s of g l y c e o l l i n 43 and o t h e r p h e n o l i c compounds. Mayama e t a l . (1981) have r e c e n t l y i s o l a t e d a n t i f u n g a l compounds from o a t s , which accumulate s p e c i f i c a l l y i n response to i n f e c t i o n with a v i r u l e n t races of crown r u s t . Yamamoto e t a l . (1975) have presented evidence that a novel p r o t e i n accumulates i n the incompatible oat/crown r u s t combination. Immunological comparisons between s o l u b l e p r o t e i n s of healthy and 35 hour i n f e c t e d leaves and those from the fungus rev e a l e d a p r o t e i n which was unique t o i n f e c t e d l e a v e s . U n f o r t u n a t e l y , p r o t e i n s from compatible combinations were not s t u d i e d so i t i s impo s s i b l e to say whether the novel p r o t e i n i s a s s o c i a t e d w i t h r e s i s t a n c e . Von Broembsen and Hadwiger (1972) have a l s o presented evidence t h a t e a r l y i n c r e a s e s i n p r o t e i n s y n t h e s i s are a s s o c i a t e d with the r e s i s t a n c e response i n the f l a x / f l a x r u s t system. These workers f ed h e a l t h y or i n f e c t e d p l a n t s w i t h e i t h e r ^H-leucine or l ^ C - l e u c i n e at v a r i o u s times between 6 and 18 hours a f t e r i n o c u l a t i o n . In t h i s way i t was p o s s i b l e t o analyze d i f f e r e n t l y l a b e l l e d e x t r a c t s from h e a l t h y and i n f e c t e d p l a n t s s i m u l t a n e o u s l y by g e l f i l t r a t i o n . Of the f o u r incompatible combinations s t u d i e d i n c o r p o r a t i o n i n c r e a s e d i n t o v a r i o u s s i z e c l a s s e s of p r o t e i n . In the two compatible combinations e i t h e r no change or a s l i g h t decrease i n s y n t h e s i s was noted. 44 These r e p o r t s of enhanced RNA and p r o t e i n s y n t h e s i s are e v o c a t i v e of a s p e c i f i c t r i g g e r a s s o c i a t e d with r e s i s t a n c e and imply that a l t e r e d p a t t e r n s of p r o t e i n s y n t h e s i s might be c h a r a c t e r i s t i c of events e a r l y i n r e s i s t a n c e responses. In t h i s regard i t i s important to c o n s i d e r how e u k a r y o t i c gene e x p r e s s i o n i s c o n t r o l l e d . There are f o u r p o s s i b l e l e v e l s of c o n t r o l which have i d e n t i f i e d : 1) T r a n s c r i p t i o n a l a c t i v i t y , which i s dependent on chromatin o r g a n i z a t i o n and RNA polymerase a c t i v i t y ( i n a s s o c i a t i o n with r e g u l a t o r y p r o t e i n s ) 2) P o s t - t r a n s c r i p t i o n a l RNA p r o c e s s i n g , i n v o l v i n g removal of i n t e r v e n i n g sequences, s p l i c i n g and m o d i f i c a t i o n ( i n c l u d i n g the a d d i t i o n of p o l y a d e n y l a t e to the 3' end of mRNA). 3) T r a n s l a t i o n a l c o n t r o l 4) P o s t - t r a n s l a t i o n a l p r o c e s s i n g of newly s y n t h e s i z e d p r o t e i n . The c u r r e n t understanding of these processes i n p l a n t s has r e c e n t l y been reviewed (Marcus 1981). 45 CHAPTER 3* CHANGES IN RIBONUCLEASE ISOZYMES DURING RUST  INFECTION OF FLAX 3.1 I n t r o d u c t i o n Scrubb e t a l . (1972) demonstrated that major q u a n t i t a t i v e and q u a l i t a t i v e changes i n the RNase a c t i v i t y o f f l a x c o t yledons occur a f t e r i n o c u l a t i o n with uredospores of f l a x r u s t (Melampsora l i n i (Ehrenb.) Lev., race 3 ). A bimodal i n c r e a s e i n the s p e c i f i c a c t i v i t y of a c e l l f r e e e x t r a c t was demonstrated i n s u s c e p t i b l e f l a x (Linum u s i t a t i s s i m u m L. cv. B i s o n ) . Only the e a r l y i n c r e a s e (2 to 4 days a f t e r i n o c u l a t i o n ) occurs i n the r e s i s t a n t v a r i e t y of f l a x (L. u s i t a t i s s i m u m L. cv. Bombay). The major RNase component from h e a l t h y and i n o c u l a t e d cotyledons of B i s o n f l a x was p u r i f i e d (Chakravorty e t a l . 1974c) to homogeneity with r e s p e c t to Sephadex G100 g e l f i l t r a t i o n d u r i n g the l a t e phase (8 days a f t e r i n o c u l a t i o n ) . Q u a l i t a t i v e changes were demonstrated i n t h i s f r a c t i o n i n accordance with changes observed i n the crude e x t r a c t s at t h i s stage. Among these were d i f f e r e n c e s i n s u b s t r a t e s p e c i f i c i t y , pH response, thermal s t a b i l i t y , K m and V m a x . Such changes i n p r o p e r t i e s could not be accounted f o r on the b a s i s of c o n t r i b u t i o n from the r u s t mycelium; m y c e l i a l RNases had been c h a r a c t e r i z e d p r e v i o u s l y (Chakravorty e t a l . 1974b). *Chapter 3 has been p u b l i s h e d i n a form c l o s e to that presented here (Sutton and Shaw 1982). 46 The i n i t i a l o b j e c t i v e of the pr e s e n t study was to determine the molecular b a s i s of these changes. 3.2 M a t e r i a l s and Methods 3.2.1 P l a n t m a t e r i a l F l a x p l a n t s of v a r i e t y B i s o n and Bombay ( s u s c e p t i b l e and r e s i s t a n t to Melampsora l i n i race 3, r e s p e c t i v e l y ) were grown i n growth chambers at an i r r a d i a n c e of 230 p.E m-2 s-l f o r 18 h d a i l y at 25°C with 20°C n i g h t s (see f r o n t i s p i e c e ) . One week a f t e r the seeds were sown, p l a n t s were sprayed w i t h a d i l u t e suspension of uredospores i n water (approximately 50 mg/100 ml). The p l a n t s were p l a c e d i n a growth chamber at 10°C i n the dark f o r 18 h i n a humid atmosphere. The c o n t r o l p l a n t s were sprayed with water alone but otherwise t r e a t e d i d e n t i c a l l y . T h i s procedure gave r i s e t o about 10 p u s t u l e s per cot y l e d o n s c o r e d 8 days a f t e r i n o c u l a t i o n . 3.2.2 E x t r a c t i o n and p u r i f i c a t i o n of RNase Approximately 50 g of cotyledons were harvested at v a r i o u s stages a f t e r i n o c u l a t i o n ; t h e r e a f t e r a l l o p e r a t i o n s were c a r r i e d out a t 0 to 4°C. The cot y l e d o n s were homogenized i n an O s t e r i z e r with 50 g p o l y v i n y l p o l y -p y r r o l i d o n e (Sigma) and 350 ml 40 mM potassium phosphate b u f f e r pH 6.7. The e x t r a c t was then p u r i f i e d by the f o l l o w i n g s t e p s , adapted from Chakravorty et a l . (1974c). 47 I . C e n t r i f u g a t i o n : The homogenate was c e n t r i f u g e d at 10,000 g f o r 30 min to remove c e l l d e b r i s . I I . A c i d i f i c a t i o n to pH 5.0: The supernatant from step I was a d j u s t e d to pH 5.0 w i t h 1 N HC1 and allowed to stand f o r 2 h. The r e s u l t i n g suspension was c e n t r i f u g e d as before and the supernatant saved f o r f u r t h e r p u r i f i c a t i o n . I I I . Ammonium sulphate f r a c t i o n a t i o n : The s o l u t i o n from ste p II was brought to 50% s a t u r a t i o n by adding s o l i d (NH4)2SC>4 slowly with s t i r r i n g and was allowed to stand f o r 30 min. The p r e c i p i t a t e formed was c o l l e c t e d by c e n t r i f u g a t i o n , d i s s o l v e d i n a s m a l l volume of 5 mM phosphate b u f f e r pH 6.7 and d i a l y z e d o v e r n i g h t a g a i n s t 1 1 of the same b u f f e r . IV. H y d r o x y l a p a t i t e chromatography: A small volume of s o l u t i o n from step I I I , c o n t a i n i n g about 5 mg p r o t e i n , was a p p l i e d to a 3 x 2 cm column packed with h y d r o x y l a p a t i t e (Biorad B i o G e l HTP) e q u i l i b r a t e d with 5 mM phosphate b u f f e r pH 6.7. E l u t i o n was r o u t i n e l y c a r r i e d out i n a stepwise manner with two column volumes of 5, 60 and 120 mM phosphate b u f f e r c o n s e c u t i v e l y . T h i s procedure was developed on the b a s i s of g r a d i e n t e l u t i o n s t u d i e s . V. G100 Sephadex g e l f i l t r a t i o n : For d e t a i l e d s t u d i e s of p r o p e r t i e s , the enzymes which were recovered with 60 mM and 120 mM b u f f e r i n step IV (termed P i and P2 r e s p e c t i v e l y ) 48 were f u r t h e r p u r i f i e d by g e l f i l t r a t i o n . A small volume (about 3 ml) of enzyme s o l u t i o n was l a y e r e d onto a 1.5 x 30 cm G100 Sephadex column e q u i l i b r a t e d with 40 mM phosphate b u f f e r pH 6.7; e l u t i o n was c a r r i e d out with the same b u f f e r at a flow r a t e of 0.5 ml/min and 3 ml f r a c t i o n s were c o l l e c t e d . 3.2.3 E l e c t r o p h o r e t i c a n a l y s i s The v a r i o u s r i b o n u c l e a s e s p r e s e n t at each stage of p u r i f i c a t i o n were v i s u a l i z e d by p o l y n u c l e o t i d e - a c r y l a m i d e g e l e l e c t r o p h o r e s i s according to Van Loon (1975). A l i q u o t s of f r a c t i o n s c o n t a i n i n g 2-100 jig p r o t e i n were a p p l i e d to 7.5% po l y a c r y l a m i d e g e l s c o n t a i n i n g 0.05% p o l y u r i d y l i c a c i d (poly U; Calbiochem, K s a l t , A grade) which was i n c l u d e d w i t h i n the g e l matrix. A f t e r e l e c t r o p h o r e s i s g e l s were s t a i n e d o v e r n i g h t with 1% p y r o n i n Y i n 7% a c e t i c a c i d . In t h i s way enzymes capable of h y d r o l y z i n g p o l y U are v i s u a l i z e d as c l e a r bands a f t e r destain.ing i n 7% a c e t i c a c i d . Comparative experiments were run without p o l y U but i n s t e a d by i n c u b a t i n g g e l s w i t h RNA a c c o r d i n g t o the method of W i l s o n (1971). 49 3.2.4 Column i s o e l e c t r i c f o c u s i n g The homogeneity of p u r i f i e d p r e p a r a t i o n s was assessed by column i s o e l e c t r i c f o c u s i n g i n an LKB 110 ml column c o n t a i n i n g 1% Ampholine (LKB 8100) pH 2.5-6.0 by the method o u t l i n e d i n the Ampholine LKB i n s t r u c t i o n manual ( a l s o see Appendix). 3.2.5 Enzyme assays RNase was assayed r o u t i n e l y with yeast RNA (BDH, Poole, England) which had been r e - p r e c i p i t a t e d i n 3 v o l 95% e t h a n o l , washed and d i a l y z e d e x h a u s t i v e l y a g a i n s t EDTA and subsequently a g a i n s t d i s t i l l e d water. Otherwise, the RNase assay was c a r r i e d out as d e s c r i b e d by Chakravorty e t a l . (1974c). One u n i t of a c t i v i t y i s d e f i n e d as the amount of enzyme y i e l d i n g an i n c r e a s e of 1.0 absorbance u n i t at 260 nm (A260) 0 f a c i a s o l u b l e product i n 30 min at 37°C. Su b s t r a t e p r e f e r e n c e was determined u s i n g t r i t i a t e d homoribopolymers of u r i d y l i c , a d e n y l i c and c y t i d y l i c a c i d (poly U, p o l y A, p o l y C, r e s p e c t i v e l y , M i l e s L a b o r a t o r i e s Inc. , E l k h a r t , Indiana) as d e s c r i b e d p r e v i o u s l y (Scrubb e_t a l . 1972). DNase and phosphodiesterase were determined using heat-denatured c a l f thymus DNA and Ca b i s - p - n i t r o p h e n y l phosphate r e s p e c t i v e l y as s u b s t r a t e s (Scrubb e t a l . 1972). Relevant d e t a i l s are a l s o g i v e n i n the Appendix. 50 3.2.6 A n a l y s i s of h y d r o l y s i s products The mononucleotides r e l e a s e d i n time course s t u d i e s under standard assay c o n d i t i o n s were analyzed by t h i n l a y e r chromatography by the method o u t l i n e d by Tang and Ma r e t z k i (1970). Incubation mixtures were a p p l i e d to c e l l u l o s e t h i n - l a y e r p l a t e s 0.2 mm t h i c k n e s s ) which were run i n two separ a t e s o l v e n t s : Solvent I, (NH4)2S04 (45 g ) , water (20 ml), is o p r o p a n o l (2 ml); Solvent I I , (NH4)2CC>3 (0.96 g ) , water (25 ml), is o p r o p a n o l (75 ml). Spots were observed under U.V. l i g h t and compared with 2'-3' c y c l i c r i b o n u c l e o t i d e standards (2'-3' cUMP, 2'-3' cAMP, 2'-3' cCMP, 2'-3' cGMP; Sigma). 3.2.7 P r o t e i n e s t i m a t i o n P r o t e i n c o n c e n t r a t i o n was estimated by the method of Lowry e t a l . (1951) using bovine serum albumin as a standard. 51 3.3 R e s u l t s 3.3.1 P u r i f i c a t i o n of RNase from f l a x cotyledons 3.3.1.1 S p e c i f i c a c t i v i t y and y i e l d s : P u r i f i c a t i o n was s i m i l a r to t h a t obtained p r e v i o u s l y by Chakravorty e t a l . (1974c). T o t a l u n i t s and hence s p e c i f i c a c t i v i t y were lower from a l l sources than those o b t a i n e d by the e a r l i e r workers. T h i s i s l i k e l y to be due t o the d i f f e r e n t source of y e a s t RNA used i n t h i s present work. T y p i c a l p u r i f i c a t i o n s f o r the v a r i e t y B i s o n are presented i n T a b l e I I . P u r i f i c a t i o n and y i e l d f o r the v a r i e t y Bombay were s i m i l a r . The h i g h e r s p e c i f i c a c t i v i t y i n the o l d e r p l a n t s was l a r g e l y due to apparent d i f f e r e n c e s i n p r o t e i n , the t o t a l enzyme u n i t s on a f r e s h weight b a s i s being s i m i l a r . I n f e c t i o n d i d not s i g n i f i c a n t l y a f f e c t t o t a l p r o t e i n however. The c o n s i s t e n t l y lower y i e l d a f t e r pH 5 p r e c i p i t a t i o n (step I I , Table II) i n younger p l a n t s was i n v e s t i g a t e d by Sephadex G100 g e l f i l t r a t i o n of c e l l f r e e homogenates. The p a t t e r n s from 1 and 2 week o l d f l a x (var. Bison) are p r esented i n F i g . 5. These r e s u l t s show t h a t an RNase component which e l u t e s with the v o i d volume ( F r a c t i o n s 16 t o 24) i s more p r e v a l e n t i n the younger p l a n t s . Gel f i l t r a t i o n of the pH 5 supernatant demonstrates t h a t most of t h i s component i s l o s t d u r i n g step I I . Unpublished data have 52 r e v e a l e d t h a t when pH 5 p r e c i p i t a t e d m a t e r i a l i s s u b j e c t e d to G100 Sephadex g e l f i l t r a t i o n a t pH 6.7 the RNase a c t i v i t y now e l u t e s as a major peak w i t h i n the i n c l u d e d volume ( F r a c t i o n s 30 to 44). Thus, i t i s i n d i c a t e d t h a t some RNase a c t i v i t y becomes weakly bound to l a r g e molecules or p a r t i c u l a t e matter d u r i n g pH 5 p r e c i p i t a t i o n ; t h i s phenomenon i s enhanced i n e x t r a c t s from younger c o t y l e d o n s . 3.3.1.2 H y d r o x y l a p a t i t e chromatography: Gra d i e n t e l u t i o n s t u d i e s using g r a d i e n t s from 5 to 200 mM phosphate b u f f e r pH 6.7 r e v e a l e d that a s m a l l amount of RNase a c t i v i t y was e l u t e d a t 5 mM; two major components were e l u t e d at 35 and 110 mM (termed P i and P2 r e s p e c t i v e l y ) . The amount of a c t i v i t y e l u t e d at 5 mM was h i g h l y i n c o n s i s t e n t and t h i s component appeared to be very u n s t a b l e . The r e l a t i v e amounts of a c t i v i t y e l u t e d at 60 (Pi) and 120 (P2) mM i n r o u t i n e stepwise f r a c t i o n a t i o n s of p a r t i a l l y p u r i f i e d e x t r a c t s from s u s c e p t i b l e (var. Bison) and r e s i s t a n t (var. Bombay) combinations are i l l u s t r a t e d i n F i g . 6. These r e s u l t s show the f o l l o w i n g : a) h e a l t h y p l a n t s c o n t a i n the same r e l a t i v e amounts of P i and P2 r e g a r d l e s s of the v a r i e t y or age of the p l a n t s d u r i n g the experimental p e r i o d ; b) the e a r l y i n c r e a s e s i n RNase are c h a r a c t e r i z e d by an i n c r e a s e i n 53 TABLE II. Purifications of RNase from Healthy (H) and Rust-Infected (I) Flax (Var. Bison) Cotyledons 3 days after inoculation RNase Protein Specific Yield (Units) (mg) Activity (%) Step Fraction (Units/mg) H I H I H I H I I Homogenate 2650 3830 1300 1500 2.0 2.5 100 100 II pH 5 Supernatant 1590 2220 530 554 3.0 4.0 60 58 III 50-80% (NH 4) 2S0 4 1060 1600 53 57 20 28 40 42 PI 183 214 18 18 10 12 IV Hydroxylapatite P2 605 1111 6.5 7.3 92 152 30 35 8 days after inoculation I Homogenate 3140 6900 950 945 3.3 7.3 100 100 II pH 5 Supernatant 2420 5730 480 440 5 13 77 83 III 50-80% (NH4)2S04 1950 4480 65 58 30 78 62 65 PI 425 2077 13 14 33 139 IV Hydroxylapatite P2 849 890 3.3 3.5 255 263 41 43 54 RNase ACTIVITY (units/ml) 7 DAY OLD FLAX, HOMOGENATE R Nate ACTIVITY W DAY OLD FLAX, HOMOGENATE 7 DAY OLD FLAX, pH 5 SUPERNATANT 20 40 FRACTION NUMBER 60 15 1.0 0.5 *ZBO 1.5 0.5 1.0 0.5 BO F i g u r e 5. G100 Sephadex g e l f i l t r a t i o n p r o f i l e s (2.5 x 45 cm column) of homogenates a f t e r c e n t r i f u g a t i o n (Step I) from one and two week o l d B i s o n f l a x and f o l l o w i n g pH 5 p r e c i p i t a t i o n (Step I I ) . C o n s i d e r a b l y more RNase a c t i v i t y e l u t e s w i t h the v o i d volume i n the case of younger c o t y l e d o n s , t h i s a c t i v i t y i s s e l e c t i v e l y p r e c i p i t a t e d a t pH 5. 55 Figure 6. Hydroxylapatite (2 x 3 cm column) eluograms of RNase i n step IV, e l u t i o n was with 5mM, 60 mM and 120 mM potassium phosphate buffer pH 6.7 (fractions 1 to 10, 11 to 20, 21 to 30, r e s p e c t i v e l y ) . Fractions 11 to 20 correspond to Pi and fractions 21 to 30 correspond to P2. The results presented are representative of three experiments performed at each time period for each host-pathogen combination. 56 the p r o p o r t i o n of P2 and t h i s change p e r s i s t s at fo u r days a f t e r i n o c u l a t i o n i n r e s i s t a n t but not i n s u s c e p t i b l e p l a n t s ; c) the l a t e i n c r e a s e i n RNase, found o n l y i n the s u s c e p t i b l e combination, i s c h a r a c t e r i z e d by an i n c r e a s e d p r o p o r t i o n of PI. The absorbance at 280 nm i n d i c a t e s not only the amount of p r o t e i n p r e s e n t but a l s o the amount of contaminating m a t e r i a l , the absorbance maximum being at about 270 nm. I t i s p o s s i b l e t h a t such i m p u r i t i e s may i n t e r f e r e w i t h p r o t e i n e s t i m a t i o n by the Lowry method. However, the amount of contaminating m a t e r i a l i s not i n f l u e n c e d by i n f e c t i o n and thus absorbance at 280 nm i s used i n F i g . 6 to pr o v i d e a standard by which to compare e l u t e d RNase a c t i v i t y from h e a l t h y and i n f e c t e d p l a n t s . 3.3.1.3 E l e c t r o p h o r e t i c a n a l y s i s : E l e c t r o p h o r e s i s shows that the crude homogenates from h e a l t h y f l a x and i n f e c t e d f l a x c o n t a i n the same nucleases capable of h y d r o l y z i n g p o l y u r i d y l a t e r e g a r d l e s s of the v a r i e t y . F i v e components are v i s u a l i z e d ( F i g . 7); the two major components are s e l e c t e d d u r i n g p u r i f i c a t i o n and separated by h y d r o x y l a p a t i t e chromatography. The f a s t e r moving component i n c r e a s e d at 7 days a f t e r i n o c u l a t i o n 57 | M 1 - - 1 1 r u v» m • 1 — I I — l I—I i—I H I P1 P2 + H I P I P2 Figure 7. P o l y n u c l e o t i d e po lyacry l amide g e l e l e c t r o p h o r e s i s of homogenates from hea l thy (H) and i n f e c t e d (I) B i s o n f l a x 7 days a f t e r i n o c u l a t i o n . P u r i f i e d enzymes P i and P2 from h y d r o x y l a p a t i t e chromatography are a l s o shown; w i t h re spec t to the p u r i f i e d enzymes, on ly the g e l s run w i t h enzymes from h e a l t h y p l a n t s are shown f o r convenience . Gels conta ined 7.5% (w/v) a c r y l a m i d e , 0.05% (w/v) p o l y u r i d y l a t e and were s t a i n e d w i t h 1% (w/v) p y r o n i n Y . 58 although no attempt was made to q u a n t i f y t h i s . E l e c t r o p h o r e s i s a l s o confirmed t h a t p u r i f i c a t i o n of the 50-80% (NH4)2S04 f r a c t i o n by g e l f i l t r a t i o n ( a f t e r Chakravorty e t a l . 1974c) gave r i s e to these two components. Thus the RNase f r a c t i o n i s o l a t e d by these workers has been r e s o l v e d i n t o two f r a c t i o n s by h y d r o x y l a p a t i t e chromatography. These two components were a l s o e v i d e n t by i n c u b a t i o n with RNA a f t e r e l e c t r o p h o r e s i s but t h i s technique appeared to be f a r l e s s s e n s i t i v e . 3.3.1.4 Sephadex G100 g e l f i l t r a t i o n : The f r a c t i o n s PI and P2 from h y d r o x y l a p a t i t e were analyzed by g e l f i l t r a t i o n . A comparison w i t h r e s u l t s y i e l d e d by the 50-80% (NH.4)2SC>4 f r a c t i o n r e v e a l s that the apparent molecular weight of the enzymes remains unchanged before and a f t e r s e p a r a t i o n ( F i g . 8). C a l i b r a t i o n s t u d i e s on a 2.5 x 45 cm G100 Sephadex column using p r o t e i n s of known molecular weight r e v e a l e d a molecular weight of 40,000 f o r the RNases PI and P2 from f l a x . Gel f i l t r a t i o n gave r i s e to s p e c i f i c a c t i v i t i e s of 900 and 4,000 units/mg p r o t e i n f o r P i and P2 r e s p e c t i v e l y from 7 days i n f e c t e d B i s o n f l a x . These h i g h l y p u r i f i e d f r a c t i o n s had i d e n t i c a l s u b s t r a t e s p e c i f i c i t i e s to f r a c t i o n s PI and P2 p r i o r to g e l f i l t r a t i o n i n d i c a t i n g that the p r o p e r t i e s of the enzyme are unchanged by f u r t h e r 59 RNase ACTIVITY (units/ml) FRACTION NUMBER F i g u r e 8. G100 Sephadex g e l f i l t r a t i o n p r o f i l e s (1.5 x 30 cm column) of RNases P i and P2 and f r a c t i o n I I I from B i s o n f l a x p r i o r t o s e p a r a t i o n by h y d r o x y l a p a t i t e chromatography. 60 p u r i f i c a t i o n . The f r a c t i o n s from Sephadex G100 were used i n d e t a i l e d s t u d i e s of the p r o p e r t i e s of these enzymes. 3.3.2 The I n f l u e n c e of Rust I n f e c t i o n Upon the S p e c i f i c  A c t i v i t y of RNases PI and P2 The s p e c i f i c a c t i v i t y r a t i o s ( i n f e c t e d / h e a l t h y ) f o r the s u s c e p t i b l e and r e s i s t a n t r e a c t i o n s at v a r i o u s times a f t e r i n o c u l a t i o n are i l l u s t r a t e d f o r RNases P i and P2 i n F i g u r e 9. In both combinations ' e a r l y RNase", d e s c r i b e d p r e v i o u s l y (Scrubb e_t a l . 1972), i s c h a r a c t e r i z e d by a predominance of P2. 'Late RNase' which on l y occurs i n the s u s c e p t i b l e r e a c t i o n i s c h a r a c t e r i z e d by l a r g e i n c r e a s e s i n P i , amounting to approximately 6 - f o l d at 9 days a f t e r i n o c u l a t i o n . A notable and r e p r o d u c i b l e d i f f e r e n c e between s u s c e p t i b l e and r e s i s t a n t r e a c t i o n s i s the p e r s i s t e n c e of e a r l y RNase at 4 days a f t e r i n o c u l a t i o n i n the r e s i s t a n t r e a c t i o n . There i s a notable d e c l i n e i n P2 a c t i v i t y i n the s u s c e p t i b l e r e a c t i o n at t h i s time. The p r o p e r t i e s of the RNases P i and P2 were monitored w i t h r e s p e c t to s u b s t r a t e s p e c i f i c i t y , K m value and pH p r o f i l e at each stage of i n f e c t i o n . No d i f f e r e n c e s were found between h e a l t h y and i n f e c t e d cotyledons or between the two v a r i e t i e s . T h i s i n f o r m a t i o n and the low i n f e c t i o n d e n s i t y i n these experiments would seem to make i t u n l i k e l y 61 9. The s p e c i f i c a c t i v i t y r a t i o s of RNases PI and P2 f o l l o w i n g i n o c u l a t i o n of B i s o n ( s u s c e p t i b l e - S ) and Bombay ( r e s i s t a n t - R ) f l a x cotyledons w i t h f l a x r u s t race no. 3. Data presented are averages f o r three separate experiments. 62 that the increases in Pi can be accounted for by fungal contribution. Although our r e s u l t s indicate that substrate s p e c i f i c i t y may not be a r e l i a b l e indicator of c a t a l y t i c properties, i t would be surprising i f K m and pH optimum remained unaltered by fungal contribution. Chakravorty e_t a l . (1974b) have characterized mycelial RNases extensively. The mycelial enzyme M i l , which has the same molecular weight as Pi and P2, has a pH optimum of 6.2 as opposed to that of pH 5.5 for the plant RNases. 3.3.3 Properties of RNases Although both enzymes appear to be of the RNase I type, as defined by Wilson (1975), they are c l e a r l y quite d i f f e r e n t from one another in t h e i r c a t a l y t i c and k i n e t i c properties (Table I I I ) . That Pi and P2 are not a r t i f a c t u a l variants and do e x i s t iri vivo i s supported by the following observations: (a) Incubation of the crude extracts does not change the r e l a t i v e amounts of Pi and P2, negating changes due to protease a c t i v i t y ; (b) separated fractions of Pi and P2 are not interconvertible with long period of storage. 63 TABLE I I I . P r o p e r t i e s of Ribonucleases from Flax P r o p e r t y Enzyme Mo l e c u l a r weight K m Value pg/ml RNA E l e c t r o p h o r e t i c M o b i l i t y ( a t pH 8.3, towards p o s i t i v e e l e c t r o d e ) pH Optimum pH P r o f i l e I s o e l e c t r i c P o i n t S u b s t r a t e S p e c i f i c i t y P oly U; A; C ( r e l a t i v e ) H y d r o l y s i s Products Mononucleotides r e l e a s e d from RNA at 80 min. Mode of A c t i o n . PI 40,000 105 0.85 5.5 Broader 4.0 100, 62, 60 2' - 3' cGMP Some n o n - c y c l i c n u c l e o t i d e s Endonuclease s p e c i f i c f o r RNA P2  40,000 200 0.66 5.5 Narrower 4.75 86, 62, 20 2' - 3' cGMP Some n o n - c y c l i c n u c l e o t i d e s Endonuclease s p e c i f i c f o r RNA 64 3.3.3.1 Substrate s p e c i f i c i t y : S u bstrate s p e c i f i c i t i e s are g i v e n i n terms of the r e l a t i v e r a t e s of h y d r o l y s i s of t r i t i a t e d p o l y U, p o l y A and p o l y C measured i n counts per minute s o l u b i l i z e d under standard assay c o n d i t i o n s . N e i t h e r these r e s u l t s nor those f o r f r a c t i o n s c o n t a i n i n g both enzymes corresponded c l o s e l y with those obtained p r e v i o u s l y with r e s p e c t to p o l y C h y d r o l y s i s . However, the trends were s i m i l a r , showing a tendency f o r i n c r e a s e d h y d r o l y s i s of p o l y C i n the l a t e r stages of a s u s c e p t i b l e r e a c t i o n . The reason f o r the apparent d i s c r e p a n c y i s u n c l e a r but s i m i l a r r e s u l t s were ob t a i n e d using s e v e r a l batch numbers of t r i t i a t e d p o l y n u c l e o t i d e s . 3.3.3.2 K m Values: K m v a l u e s were obtained from Lineweaver-Burk p l o t s (See Appendix). The lower K m value f o r P i i s c o n s i s t e n t w i t h e a r l i e r o b s e r v a t i o n s t h a t K m decreases i n the l a t e r stages of i n f e c t i o n . 3.3.3.3 Endonuclease a c t i v i t y : Endonuclease a c t i v i t y was demonstrated f o r both enzymes by time course assays with d i f f e r e n t p r e c i p i t a t i n g reagents a f t e r Chakravorty e t a l . (1974c). C o n s i s t e n t l y more A260 m a t e r i a l s o l u b l e i n 10% (w/v) p e r c h l o r i c a c i d was hydr o l y z e d as compared with m a t e r i a l s o l u b l e i n the standard p r e c i p i t a t i n g reagent. P e r c h l o r i c a c i d s o l u b i l i z e s s h o r t o l i g o n u c l e o t i d e s whereas o n l y mononucleotides are s o l u b l e i n the standard p r e c i p i t a t i n g reagent. T h e r e f o r e , the r e s u l t s i n d i c a t e t h a t e n d o n u c l e o l y t i c cleavage takes p l a c e . 3.3.3.4 Other nuclease a c t i v i t i e s : No deoxyribonuclease or phosphodiesterase a c t i v i t y was d e t e c t a b l e i n the p u r i f i e d f r a c t i o n s . 3.3.3.5 H y d r o l y s i s p r o d u c t s : 2'-3' c y c l i c GMP was the onl y c y c l i c n u c l e o t i d e d e t e c t e d a f t e r 80 min of i n c u b a t i o n with y e a s t RNA but a much l e s s e r amount of n o n - c y c l i c n u c l e o t i d e was a l s o d e t e c t e d . The l a t t e r was ev i d e n t as a s i n g l e spot (Rf 0.05) i n s o l v e n t I I , i n which c y c l i c n u c l e o t i d e s have an Rf g r e a t e r than 0.25. 3.3.3.6 I s o e l e c t r i c f o c u s i n g : Column i s o e l e c t r i c f o c u s i n g r e v e a l e d PI and P2 to be homogeneous with r e s p e c t to i s o e l e c t r i c p o i n t but c l e a r l y d i f f e r e n t from one another. Y i e l d of enzyme u n i t s d u r i n g t h i s procedure was low (approximately 40%) due to a 66 tendency f o r p r o t e i n to p r e c i p i t a t e . However, the p r o p e r t i e s of the enzymes remained unchanged by the process suggesting t h a t separate components had not been l o s t s e l e c t i v e l y . 3.3.4 E f f e c t s of Wounding Ex c i s e d cotyledons wounded by s e c t i o n i n g were incubated i n the growth chamber on moist f i l t e r paper i n p e t r i d i s h e s f o r 6 h. S i x - to 7 - f o l d i n c r e a s e s i n the s p e c i f i c a c t i v i t y of a component e l u t i n g with PI from h y d r o x y l a p a t i t e were observed. However, the p r o p e r t i e s of t h i s enzyme were q u i t e d i f f e r e n t from PI or P2. T h i s RNase component e x h i b i t e d a pH optimum of 5.0, K m value of 1000 ug/ml and a s u b s t r a t e p r e f e r e n c e p o l y C > p o l y U = p o l y A. I t co u l d be p a r t i a l l y separated from P i by Sephadex G100 g e l f i l t r a t i o n , had an apparent molecular weight of 25,000 and demonstrated a r e l a t i v e l y unchanged p r o p o r t i o n of P i . Thus the e f f e c t s of wounding appear to be q u i t e d i f f e r e n t from those of r u s t i n f e c t i o n . 67 3.4 D i s c u s s i o n T h i s work c l e a r l y demonstrates that the q u a l i t a t i v e changes i n RNase observed i n p r e v i o u s work can be accounted f o r by the observed s e l e c t i v e enhancement of one of the two components r e s o l v e d here. D e t a i l e d c h a r a c t e r i s t i c s of the enzymes P i and P2 c a t e g o r i z e them as RNase I. RNase I has been i d e n t i f i e d as the major s o l u b l e RNase i n many p l a n t s (see S e c t i o n 2.3). The g e n e r a l f e a t u r e s of RNase I are a pH optimum between 5.0 and 6.0 and p r e f e r e n t i a l r e l e a s e of 2'-3' cGMP du r i n g h y d r o l y s i s of RNA, c o n d i t i o n s f u l f i l l e d by the enzymes from f l a x . The i n c r e a s e s i n RNase c l o s e l y p a r a l l e l q u a n t i t a t i v e and q u a l i t a t i v e changes i n RNA s y n t h e s i s and the accumulation of RNA i n l a t e r stages of d i s e a s e development (see S e c t i o n 2.3). T h i s i n f o r m a t i o n should be viewed i n r e l a t i o n t o the f u n c t i o n of RNase I, which i s thought to e x i s t predominantly i n the cytoplasm and to be r e s p o n s i b l e f o r s u b s t r a t e r e u t i l i z a t i o n ( r a t h e r than p r o c e s s i n g , f o r example). I t i s of i n t e r e s t t h a t e a r l y RNase p e r s i s t s at 4 days a f t e r i n o c u l a t i o n i n the r e s i s t a n t v a r i e t y Bombay whereas i t does not i n the s u s c e p t i b l e v a r i e t y B i s o n . T h i s c o i n c i d e s with the f i r s t appearance of symptoms and with the i n i t i a t i o n of u r e d i a l development i n the fungus. Tani e t a l . (1975) have 68 i n d i c a t e d t h a t t h i s stage of development i s accompanied by s p e c i f i c changes i n host RNA metabolism i n crown r u s t i n f e c t e d o a t s . The subunit complementation hypothesis d e s c r i b e d by Chakravorty and Shaw (1977a) to e x p l a i n the changed p r o p e r t i e s of enzymes i n r u s t - i n f e c t e d p l a n t s i s not supported by the r e s u l t s presented here. The hypothesis i s based on changes i n the c a t a l y t i c p r o p e r t i e s of a s i n g l e RNase s p e c i e s . Here we have shown t h a t no such change takes p l a c e . Rather changes i n the r e l a t i v e amounts of two c a t a l y t i c a l l y d i f f e r e n t RNase isozymes p r o v i d e an e x p l a n a t i o n f o r the q u a l i t a t i v e changes observed p r e v i o u s l y . Observations u t i l i z i n g p o l y n u c l e o t i d e acrylamide g e l e l e c t r o p h o r e s i s (Van Loon 1975 and unpublished) suggest t h a t a m u l t i p l i c i t y of r i b o n u c l e a s e s i s presen t i n a l l p l a n t s as i n E s c h e r i c h i a c o l i (Datta e_t a_l. 1974). Changes i n the r e l a t i v e amounts of such enzymes may w e l l account f o r the q u a l i t a t i v e changes observed i n many r u s t and mildew i n f e c t e d p l a n t s (see S e c t i o n 2.3). Sachse e t a l . (1971) have g i v e n evidence f o r p r e f e r e n t i a l enhancement of three of ten nuclease f r a c t i o n s f o l l o w i n g i n f e c t i o n of wheat wi t h P u c c i n i a  graminis t r i t i c i . 69 CHAPTER 4 RNA AND PROTEIN SYNTHESIS FOLLOWING INOCULATION  WITH THE FLAX RUST FUNGUS 4.1 I n t r o d u c t i o n S e v e r a l r e p o r t s (Yamamoto et a l . 1976, T a n i and Yamamoto 1978, Yoshikawa e t a l . 1977, Von Broembsen and Hadwiger 1972) have i n d i c a t e d t h a t enhanced l e v e l s of RNA and p r o t e i n s y n t h e s i s are important i n determining r e s i s t a n c e i n the gene-for-gene i n t e r a c t i o n s of r u s t and downy mildew d i s e a s e s . These workers have g e n e r a l l y shown th a t e l e v a t e d l e v e l s of s y n t h e s i s occur i n incompatible combinations at times when those i n compatible combinations are e i t h e r unchanged or show a d e c l i n e . In t h i s r e s e a r c h a d e t a i l e d a n a l y s i s of gene e x p r e s s i o n d u r i n g these e a r l y stages of d i s e a s e development has been c a r r i e d out i n the f l a x / f l a x r u s t system (Linum  u s i t a t i s s i m u m L./Melampsora l i n i (Ehrenb) Lev. ) . In these s t u d i e s a s i n g l e v a r i e t y (Bombay) of f l a x c a r r y i n g the N r e s i s t a n c e gene has been u t i l i z e d to generate r e s i s t a n t o r s u s c e p t i b l e combinations with race 3 or race 41 of f l a x r u s t , r e s p e c t i v e l y . In t h i s way changes i n RNA or p r o t e i n which are unique to e i t h e r combination can be assigned to a l t e r a t i o n s i n host gene e x p r e s s i o n , as both races of fungus 70 grow i n the same way durin g the experimental p e r i o d ( L i t t l e f i e l d and Aronson 1969). 4.2 M a t e r i a l s and Methods 4.2.1 I n o c u l a t i o n and l a b e l l i n g of p l a n t m a t e r i a l Flax was grown as d e s c r i b e d i n S e c t i o n 3.2.1. Se e d l i n g s (var. Bombay) were h e a v i l y i n o c u l a t e d (2 mg spores/ml) one week a f t e r seeding with e i t h e r race 3 ( r e s i s t a n t r e a c t i o n ) or race 41 ( s u s c e p t i b l e r e a c t i o n ) of f l a x r u s t . Healthy c o n t r o l p l a n t s were sprayed w i t h water alone but otherwise t r e a t e d i d e n t i c a l l y . The p l a n t s were then p l a c e d i n the dark at 17°C u n t i l 30 minutes p r i o r to the f e e d i n g p e r i o d when they were ret u r n e d to the l i g h t (230 yuE m~2s~^) a t 25°C. For d e t a i l e d s t u d i e s on RNA s y n t h e s i s 10 g s e e d l i n g s were fed v i a the stems with 500 juCi/ml Na[32p] orthho- phosphate (Amersham, c a r r i e r f r e e ) and 6 pq/ml g r a m i c i d i n D f o r three hours. No s i g n i f i c a n t i n c o r p o r a t i o n i n t o RNA was found i f t r i t i a t e d u r i d i n e was fed i n t h i s way. For s t u d i e s on p r o t e i n s y n t h e s i s r e p l i c a t e s of three s e e d l i n g s each were fed i n the same way with [35s]-L-methionine (Amersham or New England Nuclear, s p e c i f i c a c t i v i t y > 1000 Ci/mmole) at a c o n c e n t r a t i o n of 1 pCi/pl f o r one hour. 71 4.2.2 E s t i m a t i o n of RNA s y n t h e s i s i n crude e x t r a c t s Gross r a t e s of RNA s y n t h e s i s were estimated by d etermining c o l d TCA i n s o l u b l e [32p] phosphate. A f t e r 3 h f e e d i n g with r a d i o a c t i v e phosphate (about 5 juCi taken up by three s e e d l i n g s ) , cotyledons were ground i n l i q u i d n i t r o g e n and e x t r a c t e d i n RNA e x t r a c t i o n b u f f e r (0.1 M T r i s s u c c i n a t e , 1% SDS, 0.6% d i e t h y l p y r o c a r b o n a t e pH 7.8). P a r t i c u l a t e m a t e r i a l was removed by c e n t r i f u g a t i o n and 5 jul a l i q u o t s of supernatant s p o t t e d onto f i l t e r paper (Whatman 3 MM). F i l t e r paper d i s c s were p l a c e d i n 10% TCA at 4°C f o r 10 min and then washed with 10% TCA f o l l o w e d by 95% e t h a n o l . A f t e r d r y i n g the f i l t e r paper was p l a c e d i n a s c i n t i l l a t i o n v i a l and 10 ml of E conofluor (Amersham) was added. R a d i o a c t i v i t y was measured by s c i n t i l l a t i o n counting. T o t a l uptake was estimated i n the same way but without TCA p r e c i p i t a t i o n and washing. The p r o p o r t i o n of 32p c o n tained i n RNA was determined by d i g e s t i o n with RNase b u f f e r (50 /ag/ml RNase A i n 0.1 M T r i s HC1, 5 mM MgCl 2) f o r 1 h. An a l i q u o t of e x t r a c t was added to 3 v o l . of 95% e t h a n o l and p l a c e d at -20°C o v e r n i g h t . The r e s u l t i n g p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n and suspended i n RNase b u f f e r . 72 4.2.3 E x t r a c t i o n and P u r i f i c a t i o n of RNA The l e v e l s of r a d i o a c t i v i t y necessary to c h a r a c t e r i z e v a r i o u s f r a c t i o n s of RNA made i t u n d e s i r a b l e to separate c o t y l e d o n s and a p i c e s from s e e d l i n g s a f t e r f e e d i n g with 3 2P. Thus, the stems were removed and the r e s t of the s e e d l i n g s used i n RNA e x t r a c t i o n . The s e e d l i n g s were f r o z e n i n l i q u i d n i t r o g e n and ground with a mortar and p e s t l e . RNA was then e x t r a c t e d w i t h phenol e s s e n t i a l l y as d e s c r i b e d by Chakravorty and Shaw (1971; a l s o see Appendix). However, the f o l l o w i n g m o d i f i c a t i o n s were made f o r r o u t i n e p u r i f i c a t i o n s of smal l amounts of RNA: eth a n o l and cetyltrimethylammonium bromide p r e c i p i t a t e s were c o l l e c t e d and washed on g l a s s f i b e r f i l t e r s (GFA Whatman) and subsequently r e d i s s o l v e d by pa s s i n g aqueous b u f f e r s through the ether d r i e d p r e c i p i t a t e s ; small molecules were removed from the p u r i f i e d RNA by a p p l y i n g to G25 Sephadex (PD10 column, Pharmacia) and e l u t i n g with s t e r i l e d i s t i l l e d water. C o n c e n t r a t i o n and p u r i t y of RNA were assessed s p e c t r o p h o t o m e t r i c a l l y ; the A26u/ A280 r a t i o was t y p i c a l l y 2.3. 73 A p u t a t i v e n u c l e a r RNA f r a c t i o n was prepared by the hot phenol SDS method at pH 5.5, adapted from At h e r t o n and Darby (1974). F o l l o w i n g e x t r a c t i o n of the phenol phase with 2 volumes T r i s s u c c i n a t e , 1% SDS at pH 7.8, an equal volume o f 0.05 M NH4 acetate pH 5.0, 1% SDS was added and the mixture s t i r r e d v i g o r o u s l y f o r 5 min at 60°C. The aqueous phase was c o l l e c t e d a f t e r c e n t r i g f u g a t i o n at 4°C and p u r i f i e d i n the same way as the c y t o p l a s m i c RNA. 4.2.4 Poly U and Poly A Sepharose Chromatography Ethanol p r e c i p i t a t e d RNA was r e d i s s o l v e d i n 500 u l of formamide and heated at 60°C f o r 3 min to minimize ag g r e g a t i o n . An a p p r o p r i a t e volume of the RNA s o l u t i o n was then added immediately to a s l u r r y of 0.2 g p o l y U or p o l y A Sepharose 4B i n 5 ml b i n d i n g b u f f e r (25% formamide, 0.7 M NaCl, 50 mM T r i s HC1, 10 mM EDTA, 0.2% l a u r o y l s a r c o s i n e , pH 7.5). A f t e r g e n t l e s t i r r i n g f o r 15 min the s l u r r y was c o l l e c t e d and washed with 25 ml of b i n d i n g b u f f e r on a g l a s s f i b e r f i l t e r (GFA). The bound RNA was then e l u t e d with e l u t i o n b u f f e r (90% formamide, 10 mM potassium phosphate, 10 mM EDTA, 0.2% l a u r o y l s a r c o s i n e , pH 7.5). RNA b i n d i n g to p o l y U Sepharose or poly A Sepharose i s termed A + and U + RNA 74 r e s p e c t i v e l y ; RNA e l u t e d with b i n d i n g b u f f e r i s termed A~ and U~RNA. The percentage of l a b e l l e d A + and U + RNA was assessed by s c i n t i l l a t i o n counting and f r a c t i o n s were p r e c i p i t a t e d with 0.1 v o l of 2.5 M NH4 a c e t a t e , 50 jig of c o l d y e a s t RNA and 3 v o l of ethanol f o r e l e c t r o p h o r e t i c a n a l y s i s . 4.2.5 E l e c t r o p h o r e s i s of RNA E l e c t r o p h o r e s i s was r o u t i n e l y c a r r i e d out i n a non-aqueous system using formamide as a denaturing s o l v e n t , e s s e n t i a l l y as d e s c r i b e d by Sphor et a l . (1976). Tube g e l s (7 mm x 150 mm) c o n t a i n i n g 4% acrylamide were e l e c t r o p h o r e s e d f o r 12 hr at 100 V and scanned d i r e c t l y at 265 nm i n a quartz c u v e t t e using a G i l f o r d 2400S spectrophotometer with l i n e a r t r a n s p o r t attachment. A l t e r n a t i v e l y s l a b g e l s were run i n the same way and p l a c e d i n c o n t a c t with No-Screen X-ray f i l m (Kodak) to estimate the d i s t r i b u t i o n of 32p f o l l o w i n g e l e c t r o p h o r e s i s . For the a n a l y s i s of s m a l l RNA s p e c i e s RNA was d i s s o l v e d i n 98% formamide (d e i o n i z e d ) c o n t a i n i n g 20 mM sodium phosphate (pH 7.6), 20 mM EDTA, 0.1% bromophenol blue . The samples were then run e i t h e r on 7.5 to 22% e x p o n e n t i a l g r a d i e n t p o l y a c r y l a m i d e SDS g e l s ( S e c t i o n 4.2.8) or on urea g e l s . Urea g e l s contained a 2.5% to 20% e x p o n e n t i a l g r a d i e n t p o l y a c r y l a m i d e (acrylamide: b i s - a c r y l a m i d e 20:1), 7M urea, 75 90 mM t r i s b o r a t e , 4 mM EDTA, pH 8.3; the e l e c t r o d e b u f f e r d i d not c o n t a i n urea. 4.2.6 In v i t r o t r a n s l a t i o n Both A + and A" RNA f r a c t i o n s e l u t e d from p o l y U Sepharose were p r e c i p i t a t e d with 2.5 M ammonium a c e t a t e , d r i e d and r e d i s s o l v e d i n s t e r i l e d i s t i l l e d water. The s o l u t i o n s were then passed through Sephadex G25 e q u i l i b r a t e d w i t h s t e r i l e d i s t i l l e d water and the e l u a t e s l y o p h i l i z e d . The f r a c t i o n s were r e p r e c i p i t a t e d i n e t h a n o l as b e f o r e , d r i e d and taken up i n a s m a l l volume of 100 mM potassium a c e t a t e , 2.5 mM magnesium a c e t a t e . T r a n s l a t i o n was c a r r i e d out i n the m i c r o c o c c a l nuclease t r e a t e d r a b b i t r e t i c u l o c y t e system (Amersham or New England N u c l e a r ) . Assays contained 30 }il t r a n s l a t i o n c o c k t a i l , 33-40 ^ i C i [35g] -L-methionine ( s p e c i f i c a c t i v i t y >1000 Ci/mmol), and e i t h e r 3 fig A + RNA or 16 ^ g A" RNA i n a f i n a l volume of 35 yul. I ncubation was f o r 60 min a t 30°C, a f t e r which SDS e l e c t r o p h o r e s i s sample b u f f e r was added and the samples heated at 100°C f o r 2 min. P r o t e i n s y n t h e s i s was determined from hot TCA i n s o l u b l e r a d i o a c t i v i t y as d e s c r i b e d i n s e c t i o n 4.2.7. 76 4.2.7 E x t r a c t i o n of p r o t e i n Three p a i r s of cotyledons were ground i n l i q u i d n i t r o g e n and e x t r a c t e d i n 500 fil 10 mM T r i s g l y c i n e pH 8.3, 1% 2-mercaptoethanol, 1 mM p h e n y l m e t h y l - s u l f o n y l f l u o r i d e (PMSF, Sigma). E x t r a c t s were c e n t r i f u g e d at 25,000 g f o r 30 min at 4°C. The supernatant and p e l l e t were designated s o l u b l e and i n s o l u b l e p r o t e i n r e s p e c t i v e l y . I n c o r p o r a t i o n o f [35s] methionine i n t o p r o t e i n was assessed on the b a s i s of hot TCA i n s o l u b l e counts. Two to 5 p i of e x t r a c t was spo t t e d on a f i l t e r paper d i s c (Whatmann 3 MM) and p l a c e d i n c o l d 10% TCA, 1 mg/ml methionine f o r 10 min. The d i s c s were then p l a c e d i n 10% TCA at 100°C f o r 10 min and subsequently washed with 10% TCA and e t h a n o l . A f t e r d r y i n g the p r o t e i n was s o l u b i l i z e d from each d i s c with 0.5 ml of P r o t o s o l (New England Nuclear) f o r 30 min at 60°C i n a capped s c i n t i l l a t i o n v i a l . The r a d i o a c t i v i t y i n the v i a l s was then counted a f t e r the a d d i t i o n of 50 yul g l a c i a l a c e t i c a c i d and 10 ml Econ o f l u o r (Amersham). 4.2.8 One- and 2-Dimensional E l e c t r o p h o r e s i s of P r o t e i n One-dimensional sodium dodecyl sulphate (SDS) g e l e l e c t r o p h o r e s i s was c a r r i e d out by the method of Laemmli (1970) using s l a b g e l s (180 x 200 x 0.7 mm) with an e x p o n e n t i a l g r a d i e n t s e p a r a t i n g g e l from 7.5 to 22% 77 p o l y a c r y l a m i d e . P r o t e i n s (5 /ag each) of known molecular weight were e l e c t r o p h o r e s e d a l o n g s i d e the samples; these were phosphorylase b, bovine serum albumin, ovalbumin, chymotrypsinogen, RNase A and cytochrome c (Pharmacia). Two dimensional g e l e l e c t r o p h o r e s i s was c a r r i e d out u s i n g n o n - e q u i l i b r i u m pH g r a d i e n t g e l e l e c t r o p h o r e s i s i n the f i r s t dimension as d e s c r i b e d by O ' F a r r e l l e t a l . (1977). P r o t e i n s were su b j e c t e d to e l e c t r o p h o r e s i s from the p o s i t i v e , a c i d i c , e l e c t r o d e to the negat i v e , b a s i c , e l e c t r o d e f o r 2000 Vhr. The tube g e l s were then a p p l i e d to 7.5 - 22% e x p o n e n t i a l g r a d i e n t SDS acrylamide s l a b g e l s f o r the second dimension and run as d e s c r i b e d f o r one dimensional g e l s . S t a i n i n g was c a r r i e d out i n 50% methanol, 7% a c e t i c a c i d and 0.5% Coomassie B r i l l i a n t Blue G250, fo l l o w e d by d i f f u s i o n d e s t a i n i n g i n 20% methanol, 7% a c e t i c a c i d . R a d i o a c t i v e p o l y p e p t i d e s were d e t e c t e d by fluorography a c c o r d i n g to Bonner and Laskey (1974) using p r e f l a s h e d X-OMAT f i l m (Kodak) e n a b l i n g q u a n t i t a t i o n o f r a d i o a c t i v e p o l y p e p t i d e s by subsequent scanning of the f i l m (Laskey and M i l l s 1975). Scanning was c a r r i e d out at 550 nm with a G i l f o r d 2400-S spectrophotometer with l i n e a r t r a n s p o r t attachment. Q u a n t i f i c a t i o n was c a r r i e d out using g e l s f i x e d without s t a i n i n order t o prevent quenching of r a d i o a c t i v e bands d u r i n g f l u o r o g r a p h y . 78 4.3 R e s u l t s 4.3.1 Rates of RNA S y n t h e s i s When gross r a t e s of RNA s y n t h e s i s were fo l l o w e d at v a r i o u s time p e r i o d s a f t e r i n o c u l a t i o n from e s t i m a t i o n s of the f r a c t i o n of TCA i n s o l u b l e [32p] phosphate i n crude homogenates of c o t y l e d o n s , a high degree of r e p l i c a t i o n was p o s s i b l e i n each experiment. Three r e p l i c a t e s were performed f o r each treatment at each time p e r i o d a f t e r i n o c u l a t i o n . Each r e p l i c a t e c o n s i s t e d of three s e e d l i n g s , the cotyledons of which were e x t r a c t e d together. The r a t i o s of TCA p r e c i p i t a b l e counts to t o t a l uptake are presented f o r the fe e d i n g p e r i o d 13 t o 16 hours a f t e r i n o c u l a t i o n i n Table IV. A h i g h l y s i g n i f i c a n t Table IV. Gross r a t e s of RNA s y n t h e s i s at 13 to 16 hr post  i n o c u l a t i o n R a t i o TCA i n s o l u b l e : t o t a l uptake 3 2 P Treatment R e p l i c a t e s Average Healthy 0.0211 0.0202 0.0186 0.0200 a R e s i s t a n t 0.0225 0.0231 0.0225 0.0227 b S u s c e p t i b l e 0.0202 0.0191 0.0185 0.0193 a F value f o r treatments = 12.122 a V a l u e s marked with the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t by a n a l y s i s of v a r i a n c e . b S i g n i f i c a n t i n c r e a s e i n r e s i s t a n t at the 1% l e v e l by l e a s t square d i f f e r e n c e . 79 i n c r e a s e i n the r a t e of s y n t h e s i s was found i n the r e s i s t a n t combination at t h i s time when the data were assessed by a n a l y s i s of v a r i a n c e . No s i g n i f i c a n t i n c r e a s e was found i n the s u s c e p t i b l e combination at t h i s time by t h i s s t a t i s t i c a l c r i t e r i o n . However, by 18 to 21 hr a f t e r i n o c u l a t i o n both combinations showed g r e a t l y enhanced s y n t h e s i s (not s i g n i f i c a n t l y d i f f e r e n t from one another). R e s i s t a n t p l a n t s showed a smal l but s t a t i s t i c a l l y i n s i g n i f i c a n t i n c r e a s e i n s y n t h e s i s as e a r l y as 5 to 8 hr a f t e r i n o c u l a t i o n . The r e l a t i v e r a t e s of RNA s y n t h e s i s i n the r e s i s t a n t and s u s c e p t i b l e cotyledons at v a r i o u s times a f t e r i n o c u l a t i o n are i l l u s t r a t e d i n F i g . 10. I t i s i n t e r e s t i n g t h a t a r e d u c t i o n i n l a b e l l e d A + RNA occurs concurrent with the i n c r e a s e s i n RNA s y n t h e s i s ( F i g . 10 and S e c t i o n 4.3.3). The r e l a t i v e r a t e s of RNA s y n t h e s i s at 18 to 21 hr a f t e r i n o c u l a t i o n were a l s o estimated on the b a s i s of 32p i n c o r p o r a t e d per A 2 6 0 u n i t of p u r i f i e d RNA from both a p i c e s and c o t y l e d o n s . These r e s u l t s were i n c l o s e agreement with those obtained by measurement of TCA p r e c i p i t a b l e r a d i o a c t i v i t y . Treatment wi t h RNase r e v e a l e d t h a t about 10% (depending on sample t r e a t e d ) of the TCA p r e c i p i t a b l e counts were r e s i s t a n t to RNase treatment. The r a t i o of these counts to t o t a l uptake d i d not respond to i n f e c t i o n . The i n c o r p o r a t i o n of 32p i n t o TCA i n s o l u b l e counts was a l s o estimated f o r the a p i c e s alone i n each host-pathogen combination. The t o t a l amount of i n c o r p o r a t i o n 80 RATIO S/H or R/H L l . 8 1.6 1.4 1.2 1.0 X D.8 I JLD- -O.E L, 0.6 0.4 0.2 32? W TOTAL RNA (RATE OF SYNTHESIS) A- RNA (1 OF 3 2 P Hi TOTAL RNA) T 10 Y///////A 20 TIME AFTER INOCULATION (hr) Figure 10. The response of RNA synthesis and the synthesis of A+RNA to in f e c t i o n with flax rust, race 41 (susceptible combination, S) or race 3 (resist a n t combination, R). RNA synthesis was estimated from crude extracts of cotyledons by TCA p r e c i p i t a t i o n . The synthesis of A+RNA was assessed as a percentage of p u r i f i e d labelled RNA from both cotyledons and apices which bound to poly U Sepharose. Both values are expressed f o r resi s t a n t and susceptible combinations as a r a t i o of the values determined for healthy (H) plants. Bars represent standard errors for r a t i o s . Hatched boxes represent feeding period. 81 w i t h i n the a p i c e s was about 10% of t h a t w i t h i n cotyledons and d i d not appear to be i n f l u e n c e d by i n o c u l a t i o n ( r e s u l t s not shown). Thus i t appears t h a t the apex of the s e e d l i n g does not e x h i b i t a g r e a t e r r a t e of RNA s y n t h e s i s i n response to i n f e c t i o n d u r i n g the f i r s t 21 hours f o l l o w i n g i n o c u l a t i o n . I t has not been p o s s i b l e to determine whether A +RNA s y n t h e s i s ( S e c t i o n 4.3.3) i n the apex i s i n f l u e n c e d by i n o c u l a t i o n because both a p i c e s and cotyledons have been used i n RNA p u r i f i c a t i o n i n o r d e r to avoid the r a d i a t i o n exposure which the experimenter would r e c e i v e while e x c i s i n g the a p i c e s . However, as the RNA s y n t h e s i z e d w i t h i n the a p i c e s r e p r e s e n t s o n l y l e s s than 10% of the l a b e l l e d RNA p u r i f i e d from the s e e d l i n g s , i t i s c o n s i d e r e d t h a t changes observed i n p u r i f i e d RNA l a r g e l y r e f l e c t changes w i t h i n the c o t y l e d o n s . T h i s i s f u r t h e r supported by the f a c t t h a t the f i r s t l e a v e s were seldom i n f e c t e d as they had not emerged from the a p i c a l r o s e t t e at the time of i n o c u l a t i o n . 4.3.2 C h a r a c t e r i z a t i o n of p u r i f i e d RNA by E l e c t r o p h o r e s i s P u r i f i e d RNA from u n i n o c u l a t e d s e e d l i n g s was analyzed by formamide e l e c t r o p h o r e s i s ( F i g . 11 and Table V). The i d e n t i t y of v a r i o u s a b s o r p t i o n peaks was e l u c i d a t e d from m o l e c u l a r weight c a l i b r a t i o n s using a s m a l l amount of 1 4 C - l a b e l l e d E. c o l i 16S and 23S ribosomal RNA which was e l e c t r o p h o r e s e d with the sample. The i d e n t i t y of the 25S, 18S, 5.8S and 5S c y t o p l a s m i c rRNA and the 16S c h l o r o p l a s t 82 Figure 1 1 . Electrophoresis of [ 3 2P] RNA from uninoculated seedlings in 4% polyacrylamide formamide gels. Absorbance curve obtained by di r e c t scanning of tube g e l ; r a d i o a c t i v i t y by autoradiography and scanning from slab gels run in the same way; 23S and 16S positions determined with i 4 C E. c o l i ribosomal RNA. 1, 2 , 3 and 4, absorbance peaks of unknown i d e n t i t y . Solid c i r c l e s represent positions of 23S and 16S E. c o l i RNA; crosses represent positions of 25 , 18 , 16 , 5.8 and 5S flax RNA. 83 rRNA was determined by f i t t i n g t h e i r p u b l i s h e d molecular weights (Leaver 1979) to the molecular weight c a l i b r a t i o n curve ( F i g . 11). The 23 S c h l o r o p l a s t rRNA was notably absent. T h i s i s a common f e a t u r e of RNA p r e p a r a t i o n s run under denaturing c o n d i t i o n s (Leaver 1979) and i s a r e s u l t o f hidden breaks which occur iri v i v o . Four u n i d e n t i f i e d high m o l e c u l a r weight RNA s p e c i e s are a l s o seen (Table V and F i g . 11). TABLE V. RNA s p e c i e s found i n p u r i f i e d f l a x RNA by  formamide e l e c t r o p h o r e s i s . Species M.Wt.a Cytoplasmic: 25S 1.3 x 10 6 18S 0.7 x 10 6 5.8S 5.04 x 10 4 5S 3.75 x 10 4 C h l o r o p l a s t 23S 1.1 x 10 6 (not observed) 16S 0.56 x 106 Unknown: 1 18S Shoulder 0.66 x 10& 2 0.42 x 106 3 0.35 x 106 4 0.18 x 106 a V a l u e s g i v e n f o r known s p e c i e s are p r e v i o u s l y (Leaver 1979) and are i n weights determined here. those p u b l i s h e d agreement with molecular 84 I t i s noteworthy that 0.66, 0.41 and 0.17 x 10 6 d a l t o n (D) fragments of 23S c h l o r o p l a s t rRNA have been d e s c r i b e d p r e v i o u s l y (Leaver 1979). The sum of the 0.66 x 10 6 D fragment and the u n i d e n t i f i e d s p e c i e s of 0.42 x 10 6 D found here i s 1.08 x 10 6 D, very c l o s e to the p u b l i s h e d v alue f o r the 23S c h l o r o p l a s t ribosomal RNA. Thus i t seems t h a t both these s p e c i e s may be fragments of the 23S c h l o r o p l a s t rRNA r e s u l t i n g from a s i n g l e cleavage of the i n t a c t molecule. Whether or not the observed p a t t e r n of 32p i n c o r p o r a t i o n i s r e p r e s e n t a t i v e of l a b e l l i n g of these fragments i s u n c e r t a i n ( F i g . 11, 2, 3 and 4). I t should be r e a l i z e d t h a t r a p i d l y l a b e l l e d mRNA i n t h i s r e g i o n i s l i k e l y t o c o n t r i b u t e to the o v e r a l l p a t t e r n of 3 2 P i n c o r p o r a t i o n . In p a r t i c u l a r , the l a r g e subunit of r i b u l o s e - b i s p h o s h o p h a t e carboxylase (RuBPCase) i s known to be r a p i d l y s y n t h e s i z e d and have a molecular weight of approximately 0.5 x 10 6 D (Steinback 1981). I f indeed, the 23S rRNA fragments are h e a v i l y l a b e l l e d t h i s would imply r a p i d i n v i v o breakdown of the l a r g e c h l o r o p l a s t rRNA ( i n the apparent absence of g e n e r a l d e g r a d a t i o n during e x t r a c t i o n ) . T h i s has not been r e p o r t e d i n the l i t e r a t u r e however. L a b e l l e d RNA has been c h a r a c t e r i z e d f o r the h o s t -pathogen combinations at 18 to 21 hours a f t e r i n o c u l a t i o n . T o t a l p u r i f i e d 3 2 P l a b e l l e d RNA from h e a l t h y or i n f e c t e d p l a n t s was a p p l i e d to s l a b g e l s c o n t a i n i n g 4% p o l y a c r y l a m i d e 85 p o l y a c r y l a m i d e i n formamide. 86 i n formamide. F o l l o w i n g e l e c t r o p h o r e s i s and autoradiography the X-ray f i l m was scanned i n o r d e r t o assess the d i s t r i b u t i o n of 3 2 P l a b e l . The r e s u l t s of one such experiment are presented i n F i g u r e 12 and show that the 25S, 18S and 5S rRNA s p e c i e s are s e l e c t i v e l y enhanced i n both r e s i s t a n t and s u s c e p t i b l e combinations. T h i s r e s u l t i s r e p r e s e n t a t i v e of a t o t a l of f o u r such experiments performed. Minor RNA s p e c i e s were not always s u c c e s s f u l l y r e s o l v e d i n formamide s l a b g e l s . Thus, i t has not been p o s s i b l e to determine with c e r t a i n t y whether the changes seen i n s p e c i e s m i g r a t i n g f a s t e r than 18S rRNA ( F i g . 12) are a c o n s i s t e n t c h a r a c t e r i s t i c of d i s e a s e d p l a n t s . I t i s p o s s i b l e more i n f o r m a t i o n about the e f f e c t s of d i s e a s e upon the s y n t h e s i s of v a r i o u s RNA s p e c i e s might be ob t a i n e d by using a more r e l i a b l e e l e c t r o p h o r e t i c technique. 4.3.3 Poly U b i n d i n g RNA (A+RNA) Poly a d e n y l a t e d (A +) RNA was assessed as a percentage of 32p l a b e l l e d RNA b i n d i n g t o p o l y U Sepharose. T h i s f r a c t i o n was c h a r a c t e r i z e d by a hig h s p e c i f i c r a d i o a c t i v i t y , r e p r e s e n t i n g 0.5% of the t o t a l RNA mass and 7.3% + 1.3 of the r a d i o a c t i v i t y . I t has a l s o been c h a r a c t e r i z e d e l e c t r o p h o r e t i c a l l y ( F i g . 13) and shows enrichment i n heterogeneous high molecular weight s p e c i e s w i t h a s i z e d i s t r i b u t i o n c h a r a c t e r i s t i c of mRNA and a smal l amount of rRNA contamination. 87 MIGRATION (cm) F i q u r e 13. E l e c t r o p h o r e t i c c h a r a c t e r i z a t i o n of f r a c t i o n a t e d F i g u r e ^ ^ formamide g e l s . 25S and IBS p o s i t i o n s determined w i t h t o t a l RNA run on the same s l a b g e l . 88 I n f e c t i o n l e d to marked decreases i n the p r o p o r t i o n of A + RNA s y n t h e s i z e d ( F i g . 10). T h i s decrease was c o r r e l a t e d with the g e n e r a l i n c r e a s e i n t o t a l RNA s y n t h e s i s and o c c u r r e d a t e a r l i e r times a f t e r i n o c u l a t i o n i n the r e s i s t a n t combination than i n the s u s c e p t i b l e combination. 4.3.4 Poly A b i n d i n g (U +) RNA and smal l RNA RNA c o n t a i n i n g p o l y or o l i g o U sequences and b i n d i n g to p o l y A Sepharose c o n s t i t u t e d about 1% of the t o t a l r a d i o a c t i v i t y . Attempts to p u r i f y measurable q u a n t i t i e s (on an absorbance b a s i s ) of t h i s f r a c t i o n were u n s u c c e s s f u l as i t r e p r e s e n t s l e s s than 0.05% of the t o t a l RNA mass. E l e c t r o p h o r e t i c a n a l y s i s ( F i g . 14) showed t h a t t h i s f r a c t i o n was e n r i c h e d i n low molecular mass RNA with an average mol e c u l a r mass of 15 k i l o d a l t o n s (kD). E l e c t r o p h o r e s i s on SDS or urea g e l s (7.5 - 22% or 2.5 - 20% e x p o n e n t i a l p o l y a c r y l a m i d e g r a d i e n t s r e s p e c t i v e l y , see " M a t e r i a l s and Methods") f u r t h e r r e s o l v e d t o t a l RNA i n t h i s r e g i o n i n t o three s p e c i e s ( l a b e l l e d a, b, and c i n F i g . 14). These s p e c i e s were absent from both A + and U~ RNA and would t h e r e f o r e appear to be s e l e c t i v e l y bound by p o l y A Sepharose. M o l e c u l a r masses were c a l c u l a t e d from urea g e l s , as 7M urea would be expected t o y i e l d RNA with conformation-independent m i g r a t i o n d u r i n g e l e c t r o p h o r e s i s . The molecular mass curve was c o n s t r u c t e d from the known value s f o r 5.8S, 5S and 4S f l a x RNA (see S e c t i o n 4.3.2) and e x t r a p o l a t e d to g i v e F i g u r e 14. A n a l y s i s of U+RNA from u n i n f e c t e d p l a n t s by e l e c t r o p h o r e s i s and autoradiography. i ) L a b e l l e d U + and A" f r a c t i o n s separated on 4% p o l y a c r y l a m i d e formamide g e l s . 25S, 18S, 5S and 4S s p e c i e s i d e n t i f i e d as d e s c r i b e d i n S e c t i o n 4.3.2. i i ) R e s o l u t i o n of small RNA s p e c i e s w i t h i n t o t a l l a b e l l e d RNA:1, on SDS g e l s (7.5 t o 22% e x p o n e n t i a l p o l y a c r y l a m i d e g r a d i e n t ) and 2, on urea g e l s (2.5 t o 20% e x p o n e n t i a l p o l y a c r y l a m i d e g r a d i e n t ) . The p o s i t i o n s of the 5.8S, 5S and 4S sp e c i e s were observed a f t e r s t a i n i n g w i t h methylene blue . Species marked a, b and c and >5.8S are d i s c u s s e d i n the t e x t . 90 estimated molecular masses f o r a, b, and c of 14.5, 12.5 and 10.0 kD r e s p e c t i v e l y . When m i g r a t i o n on SDS g e l s was used t o c a l c u l a t e m olecular masses f o r a, b and c i n the same way value s of 14.0, 11.7 and 10.5 kD were o b t a i n e d . V a r i o u s s p e c i e s of RNA l a r g e r than 5.8S rRNA were a l s o found ( >5.8S i n F i g . 14). These had estimated molecular masses ranging from 75 kD to 180 kD. The i n f l u e n c e of i n f e c t i o n upon both the smal l U + RNA s p e c i e s and those l a r g e r than 5.8S RNA has been i n v e s t i g a t e d between 5 and 21 hr a f t e r i n o c u l a t i o n . These experiments showed no s i g n i f i c a n t d i f f e r e n c e s f o l l o w i n g i n o c u l a t i o n . The percentage of U + RNA (as a f r a c t i o n of the t o t a l l a b e l l e d RNA) was not s i g n i f i c a n t l y a l t e r e d by i n f e c t i o n at 18 to 21 hr a f t e r i n o c u l a t i o n . 4.3.5 S i g n i f i c a n c e of the a l t e r e d p a t t e r n s of RNA  s y n t h e s i s i n d i s e a s e The d e c l i n e i n A + RNA as a percentage of the t o t a l RNA i s i n f a c t r e p r e s e n t a t i v e of a d e c l i n e i n the amount o f A + RNA s y n t h e s i z e d . T h i s f o l l o w s from the demonstration t h a t the percentage d e c l i n e i s g r e a t e r than the corresponding i n c r e a s e i n t o t a l RNA s y n t h e s i s , as shown i n Table VI. I t would be s u r p r i s i n g i f t h i s r epresented an almost t o t a l c e s s a t i o n of mRNA s y n t h e s i s as i t has been shown t h a t only i n the s u s c e p t i b l e combination i s there any a p p r e c i a b l e d e c l i n e 91 i n iri vivo protein synthesis and thi s can largely be accounted for by decreased synthesis of the A" encoded large subunit of RuBPCase (Section 4 . 3 . 7 ) . Furthermore, in v i t r o t r a n s l a t i o n (Section 4 .3 .6 ) has shown that about 65% of the t o t a l mRNA a c t i v i t y i n healthy plants i s contained i n the A" f r a c t i o n . Thus the decline in A + RNA could be due to: a) decreased nuclear polyadenylaton of precursor mRNA or b) enhanced poly A removal in the cytoplasm (or possibly a decline i n cytoplasmic polyadenylation) (Hall 1979 ) . In order to gain insight into t h i s , nuclear RNA from healthy and diseased plants has been compared at 18 to 21 hours after inoculation (Table VI). The putative nuclear f r a c t i o n has been assessed in r e l a t i o n to the expected composition of authentic nuclear RNA by formamide electrophoresis (Fig. 1 3 ) . It is enriched in high molecular weight RNA, lacks tRNA and contains enhanced incorporation into a 2.2 x 10^ dalton precursor rRNA. Furthermore, i t i s indicated that the decline in A + mRNA during disease i s due to decreased nuclear polyadenylation of precursor mRNA in the nucleus. 92 TABLE V I . The R e l a t i v e Rates of A+RNA Synthesis i n Healthy and I n f e c t e d Seedlings at 18 to 21 h r a f t e r I n o c u l a t i o n . Healthy R e s i s t a n t S u s c e p t i b l e (H) (R) (S) T o t a l RNA: I n c o r p o r a t i o n (cpm/A 260 u n i t ) 96,900 163,800 149,100 R a t i o , R/H o r S/H 1.0 1.7 1.5 % Poly A+ 9.8 2.5 2.3 R a t i o , R/H or S/H 1.0 0.26 0.23 R e l a t i v e 3 Amount of Poly A + Synt h e s i s 100% 43% 35% P u t a t i v e n u c l e a r RNA: % Poly A+ 5.1 1.4 1.3 R a t i o , R/H or S/H 1_10 0.27 0.25 a R e l a t i v e amount of poly A+ synthesis = % Poly A+ x Ratio of incorporation X 100 % Poly A + i n Healthy 93 4.3.6 In v i t r o t r a n s l a t i o n of RNA from u n i n f e c t e d p l a n t s 4.3.6.1 T r a n s l a t i o n a l a c t i v i t y : The t r a n s l a t i o n a l a c t i v i t y of A + and A~ RNA f r a c t i o n s p u r i f i e d from h e a l t h y s e e d l i n g s (apices and cotyledons) were assessed i n the r a b b i t r e t i c u l o c y t e system. Maximal i n c o r p o r a t i o n of [ 3 5 S ] methionine i n t o hot TCA i n s o l u b l e counts was seven and f i f t e e n times background f o r the A" and A + f r a c t i o n s r e s p e c t i v e l y . I n c o r p o r a t i o n i n c r e a s e d l i n e a r l y with r e s p e c t to i n c r e a s i n g RNA c o n c e n t r a t i o n over the range 0 to 1.5 fig/jdl f o r A~ RNA and over the range 0 to 0.1 yjg/ul f o r A +. T r a n s l a t i o n a l a c t i v i t y was assessed from i n c o r p o r a t i o n i n the l i n e a r range. In t h i s way an estimate of the t o t a l t r a n s l a t i o n a l a c t i v i t y was d e r i v e d (Table V I I ) . However, upon a n a l y s i s of A" encoded p o l y p e p t i d e s by e l e c t r o p h o r e s i s i t was found t h a t about h a l f the i n c o r p o r a t e d r a d i o a c t i v i t y was not v i s u a l i z e d as l a b e l l e d p o l y p e p t i d e s ; approximately twice as much r a d i o a c t i v i t y had to be a p p l i e d to the g e l i n order to o b t a i n a s i m i l a r d e n s i t y on autoradiograms to the A + encoded f r a c t i o n under standard c o n d i t i o n s of exposure. S i m i l a r s t i m u l a t i o n of n o n - s p e c i f i c i n c o r p o r a t i o n was observed upon the a d d i t i o n of tRNA (E. c o l i ) t o t r a n s l a t i o n assays and i t i s t h e r e f o r e concluded t h a t the h i g h c o n c e n t r a t i o n of non-messenger RNA i n the A~ f r a c t i o n i s r e s p o n s i b l e f o r the 94 hot TCA p r e c i p i t a b l e r a d i o a c t i v i t y which does not appear i n l a b e l l e d p o l y p e p t i d e s . For t h i s reason a r e v i s e d estimate of 66% was determined f o r the t r a n s l a t i o n a l a c t i v i t y of the A" f r a c t i o n (Table V I I ) . A remarkably s i m i l a r d i s t r i b u t i o n o f a c t i v i t y was obtained f o r A + and A" RNA from pea s e e d l i n g s (Gray and Cashmore 1976 ) . TABLE V I I . T r a n s l a t i o n a l A c t i v i t y of RNA F r a c t i o n s Separated by Poly U Sepharose Chromatography RNA F r a c t i o n A+ A~ I n c o r p o r a t i o n (dpm 35s/)ug/RNA) 2.22 x 10$ 6.0 x 1 0 4 I n c o r p o r a t i o n c o r r e c t e d f o r n o n - s p e c i f i c counts (dpm 3 5S/ug RNA) 2.22 x 1 0 6 3.0 x 1 0 4 % of t o t a l RNA mass 0.67 99.33 % of t o t a l mRNA a c t i v i t y 34 66 mRNA a c t i v i t y = ( incorporation/^ug RNA) m u l t i p l i e d by jiq RNA i n A + or A" f r a c t i o n 95 4.3.6.2: Two-dimensional a n a l y s i s of t r a n s l a t i o n products The products of t r a n s l a t i o n assays c o n t a i n i n g A + or A~ RNA were compared on n o n - e q u i l i b r i u m two dimensional g e l s ( F i g . 15). I t can be seen t h a t although s e v e r a l p o l y p e p t i d e s are unique t o the A + f r a c t i o n very few are encoded on l y by po l y A l a c k i n g mRNA (A~RNA). C l e a r l y c h l o r o p l a s t mRNA would be expected to be contained w i t h i n the A~ f r a c t i o n (Steinback 1981). T h i s i s demonstrated by the occurrence of a major p o l y p e p t i d e with a molecular mass of 55 kD (L i n F i g . 15.2) which appears i n the same p o s i t i o n as the major i j i v i v o s t a i n e d p r o t e i n i n t h i s s i z e range ( F i g . 15.4). T h i s p o l y p e p t i d e i s probably the l a r g e subunit of r i b u l o s e bisphosphate c a r b o x y l a s e (RuBPCase) and i s unique t o the A" f r a c t i o n . A few other p o l y p e p t i d e s are unique to the A" f r a c t i o n ( F i g . 15.2), however the va s t m a j o r i t y are a l s o found i n the A + f r a c t i o n . Included among t h i s group i s a major p o l y p e p t i d e with a molecular mass of 19 kD (S i n F i g . 15.1 and 15.2) which on the b a s i s of i t s molecular weight and b a s i c i s o e l e c t r i c p o i n t i s probably the p r e c u r s o r to the s m a l l subunit of RuBPCase ( I s h i y e e t a l . 1981). These r e s u l t s i n d i c a t e t h a t many of the cy t o p l a s m i c mRNAs (which comprise the g r e a t e s t degree of mRNA complexity) appear as po l y a d e n y l a t e d and non-polyadenylated s p e c i e s . Assuming equal e f f i c i e n c y of t r a n s l a t i o n of A + and A~ 96 F i g u r e 15.1, 15.2, 15.3 and 15.4. Two dimensional e l e c t r o p h o r e s i s of p o l y p e p t i d e s . F i r s t dimension i s n o n - e q u i l i b r i u m pH g r a d i e n t e l e c t r o p h o r e s i s , second dimension SDS e l e c t r o p h o r e s i s (7.5 to 22% e x p o n e n t i a l g r a d i e n t ) . L, l a r g e s u b u n i t of RuBPCase; S, p u t a t i v e p r e c u r s o r to the s m a l l subunit of RuBPCase; B, l a b e l l e d m a t e r i a l found i n t r a n s l a t i o n blank (no added RNA). Mo l e c u l a r weight markers, phosphorylase b 92 kD, bovine serum albumin 67 kD, ovalbumin 43 kD, chymotrypsinogen 25 kD, RNase A 13.7 kD, cytochrome c 12.8 kD. 97 15.1 In v i t r o products of A + R N A . P o l y p e p t i d e s marked ' V are a l s o encoded by A " " R N A but i n d i f f e r e n t r e l a t i v e amounts. 15.2 In v i t r o products of A"RNA. P o l y p e p t i d e s marked with arrows are not found i n the A + f r a c t i o n ; those marked 'V are examples of those a l s o found i n the A + f r a c t i o n but i n d i f f e r e n t r e l a t i v e amounts. 99 15.3 In v i v o l a b e l l e d s o l u b l e p o l y p e p t i d e s from c o t y l e d o n s . Note the l a r g e subunit of RuBPCase (L) i s quenched due to s t a i n i n g . 2nd D kD 92 67 43 100 1st D L 25 13.7 12.8 15.4 Stained s o l u b l e po lypept ide s from c o t y l e d o n s . 101 messages,the mRNA that codes f o r the s m a l l subunit RuBPCase i s about e q u a l l y d i s t r i b u t e d between the two f r a c t i o n s . 4.3.7 P r o t e i n S y n t h e s i s i n v i v o f o l l o w i n g i n o c u l a t i o n R a d i o l a b e l l e d p r o t e i n s from h e a l t h y and d i s e a s e d cotyledons were examined by SDS e l e c t r o p h o r e s i s and 2-d i m ensional e l e c t r o p h o r e s i s at 8 and 18 hours a f t e r i n o c u l a t i o n . No d i f f e r e n c e s i n the p a t t e r n of s y n t h e s i s were found by these c r i t e r i a u n t i l 18 hours a f t e r i n o c u l a t i o n . At t h i s time a marked d e c l i n e i n the gross r a t e of s y n t h e s i s of s o l u b l e p r o t e i n i n the s u s c e p t i b l e combination was apparent. Gross r a t e s of s y n t h e s i s could be r e p r o d u c i b l y determined from the r a t i o of hot TCA i n s o l u b l e r a d i o a c t i v i t y t o t o t a l uptake a f t e r a 1 hour f e e d i n g with [35s] methionine (Table V I I I ) . The d e c l i n e observed i n the s u s c e p t i b l e combination could be accounted f o r by a d e c l i n e i n the s y n t h e s i s of the l a r g e subunit of RuBPCase; t h i s appeared as the major s t a i n e d and l a b e l l e d s o l u b l e p o l y p e p t i d e with a molecular mass of 55 kD ( F i g . 16.1 and 16.2). T h i s major change was accompanied by i n c r e a s e d s y n t h e s i s of an unknown p o l y p e p t i d e of 50 kD (LSu+1 i n F i g . 16.2), and a d e c l i n e i n a 30 kD p o l y p e p t i d e ( F i g . 16.1 and 16.2). The r e s u l t s of three a d d i t i o n a l experiments i n which s o l u b l e in v i v o l a b e l l e d p r o t e i n s have been analyzed have 102 TABLE V I I I . Rates of P r o t e i n S y n t h e s i s at 18 Hours A f t e r I n o c u l a t i o n R a t i o of To T o t a l Hot TCA i n s o l u b l e 3 5 S 35s Uptake R e p l i c a t e s Healthy R e s i s t a n t S u s c e p t i b l e 1 0.173 0.183 0.160 2 0.189 0.179 0.120 3 0.165 0.176 0.135 4 0.175 0.187 0.133 Average 0 . 1 7 5 5 a 0 . 1 8 1 3 a 0 . 1 370 b F treatments = 16.9 aAverages marked with the same l e t t e r , not s i g n i f i c a n t l y d i f f e r e n t . b S i g n i f i c a n t l y d i f f e r e n t at 0.01 l e v e l by l e a s t square d i f f e r e n c e . 103 shown r e p r o d u c i b l e i n c r e a s e s i n both the RuBPCase l a r g e s u b u n i t ( F i g . 16.2, i n s e t ) and the unknown 30 kD p o l y p e p t i d e ( F i g . 16.2) i n the r e s i s t a n t combination. These i n c r e a s e s are s m a l l (10% i n the case of the l a r g e subunit) but are i n t e r e s t i n g because they r e p r e s e n t a converse r e a c t i o n to the s u s c e p t i b l e response. Attempts to i d e n t i f y p o s i t i v e l y the small subunit of RuBPCase were f r u s t r a t e d by the presence of two i r r e p r o d u c i b l y s t a i n e d bands i n the 13 kD r e g i o n . However, the major r a d i o a c t i v e p o l y p e p t i d e i n t h i s r e g i o n c o i n c i d e d w i t h the l a r g e r of these two s t a i n e d p o l y p e p t i d e s . An i n c o n s i s t e n t l y r e s o l v e d s e r i e s of m u l t i p l e r a d i o a c t i v e bands corresponded with the s m a l l e r s t a i n e d p o l y p e p t i d e . T h i s may i n d i c a t e t h a t the l a r g e r c o n s i s t e n t l y r e s o l v e d band ( l a b e l l e d X i n F i g . 16.2) i s the a u t h e n t i c s m a l l s u b u n i t . I f t h i s i s the case then i t i s i m p l i e d t h a t there i s no d e c l i n e i n s m a l l s u b u n i t s y n t h e s i s i n the s u s c e p t i b l e combination. No r e p r o d u c i b l e decrease occ u r r e d i n the s m a l l e r p o l y p e p t i d e . The major membrane bound c h l o r o p l a s t p r o t e i n s , are c l e a r l y represented i n the i n s o l u b l e f r a c t i o n ( F i g . 16.3). These are the 32 kD t h y l a k o i d membrane p r o t e i n and the major a p o p r o t e i n of the l i g h t h a r v e s t i n g complex (LHC) with a m o l e c u l a r weight of 25 kD. I t i s of i n t e r e s t t h a t the s y n t h e s i s of n e i t h e r of these p o l y p e p t i d e s decreases s i g n i f i c a n t l y i n the s u s c e p t i b l e combination, p a r t i c u l a r l y 104 F i g u r e s 16/ 16.1, 16.2 and 16.3. A n a l y s i s of i n v i v o l a b e l l e d p o l y p e p t i d e s by SDS e l e c t r o p h o r e s i s , 18 hr a f t e r i n o u c l a t i o n . P o l y p e p t i d e s of known molecular mass were bovine serum albumin, 67 kD, ovalbumin, 43 kD, chymotrypsinogen, 25 kD, RNase A, 13.7 kD, cytochrome c, 12.8 kD. Other values r e f e r t o l a b e l l e d f l a x p o l y p e p t i d e s d i s c u s s e d i n the t e x t ; LSu, l a r g e subunit of RuBPCase. 105 SOLUBLE INSOLUBLE 16.1 Fluorogram of l a b e l l e d p o l y p e p t i d e s . T r acks 1, 2 and 3: equal amounts of r a d i o a c t i v i t y i n s o l u b l e p r o t e i n s from h e a l t h y , r e s i s t a n t and s u s c e p t i b l e c o t y l e d o n s ; t r a c k s 4, 5 and 6: s o l u b l e p r o t e i n s from h e a l t h y , r e s i s t a n t and s u s c e p t i b l e c o t y l e d o n s , each t r a c k c o n t a i n i n g equal amounts of p r o t e i n ; t r a c k s 7, 8 and 9 i n s o l u b l e p r o t e i n s from h e a l t h y r e s i s t a n t and s u s c e p t i b l e c o t y l e d o n s (equal amounts of r a d i o a c t i v i t y ) . 106 RADIOACTIVITY 35c LSu WGHAT10N (cm) 16.2 Densi t o m e t r i e scans of t r a c k s 1, 2 and 3 from F i g . 16.1 ( s o l u b l e p r o t e i n ) . Inset; p a r t of a scan i n the 60 to 40 kD r e g i o n from another experiment showing r e s o l u t i o n of the l a r g e s u b u n i t of RuBPCase (LSu) and an unknown p o l y p e p t i d e (LSu+1) with a molecular mass of 50 kD. X, i s the p u t a t i v e s m a l l subunit of RuBPCase. The peak marked 30 i s d i s c u s s e d i n the t e x t . 107 16.3 D e n s i t o m e t r i c scans of t r a c k s 7 , 8 and 9 from F i g . 16.1 ( i n s o l u b l e p r o t e i n ) . Peaks marked 32 and 25 are d i s c u s s e d i n the t e x t . 108 as the former i s encoded i n the c h l o r o p l a s t and the l a t t e r i n the nucleus (Steinback 1 981 ) . 4 .3 .8 Two-dimensional a n a l y s i s of i n v i v o l a b e l l e d  s o l u b l e p r o t e i n f o l l o w i n g i n o c u l a t i o n In v i v o l a b e l l e d s o l u b l e e x t r a c t s were analyzed by two-dimensional e l e c t r o p h o r e s i s ( n o n - e q u i l i b r i u m pH g r a d i e n t ) a t 8 and 18 hours a f t e r i n o c u l a t i o n . No changes i n the p a t t e r n of l a b e l l e d p o l y p e p t i d e s was observed u n t i l 18 hr a f t e r i n o c u l a t i o n . At t h i s time s e v e r a l p o l y p e p t i d e s showed enhanced s y n t h e s i s i n the s u s c e p t i b l e combination ( F i g . 1 7 ) . Four of these were not d i s c e r n a b l e on g e l s of e x t r a c t s from the h e a l t h y or r e s i s t a n t combinations. Three (A,B and C i n F i g . 17) were a l s o present i n h e a l t h y and r e s i s t a n t combinations but at a lower l e v e l . No d i f f e r e n c e s were observed between the he a l t h y and r e s i s t a n t combinations throughout the experimental p e r i o d . Other experiments u t i l i z i n g pH g r a d i e n t s of 3.5 to 7.0 i n the f i r s t dimension (adapted from O ' F a r r e l l 1975) have enabled a high degree of r e s o l u t i o n i n the a c i d i c r e g i o n . However, these experiments f a i l e d to r e v e a l any a d d i t i o n a l i n f o r m a t i o n with regard to the e f f e c t s of d i s e a s e . 109 F i g u r e 17. Two-dimensional e l e c t r o p h o r e s i s of in v i v o l a b e l l e d s o l u b l e p o l y p e p t i d e s 18 hr a f t e r i n o c u l a t i o n . P a r t of the fluorograms i s shown here. C i r c l e d spots are enhanced i n the s u s c e p t i b l e combination. The corresponding spots are shown i n the oth e r combinations when present(A, B, C). Spots w i t h i n the box e x h i b i t a l t e r e d r e l a t i v e i n c o r p o r a t i o n i n the s u s c e p t i b l e combination. I l l 4.4 D i s c u s s i o n The r e s u l t s presented here support the n o t i o n of e a r l y host r e c o g n i t i o n of the a v i r u l e n t pathogen. T h i s i s c h a r a c t e r i z e d by i n c r e a s e d RNA s y n t h e s i s and a probable s h i f t i n mRNA s y n t h e s i s to predominantly non-polyadenylated s p e c i e s . These changes occur as e a r l y as 8 hr a f t e r i n o c u l a t i o n and at a time when no s i g n i f i c a n t response i s seen i n the s u s c e p t i b l e combination. I t seems t h a t t h i s race s p e c i f i c r e c o g n i t i o n occurs p r i o r to h a u s t o r i a l development ( L i t t l e f i e l d and Aronson 1969) and i t i s t h e r e f o r e necessary to invoke the involvement of a d i f f u s i b l e substance of f u n g a l o r i g i n . Among the f i r s t o bservable changes i n p r o t e i n s y n t h e s i s i s a marked decrease i n the s y n t h e s i s of the l a r g e s u b u n i t of RuBPCase i n the s u s c e p t i b l e combination. T h i s i s p a r a l l e l e d by a s l i g h t i n c r e a s e i n the r e s i s t a n t combination. I t i s hard to imagine t h a t these phenomena have any e f f e c t on RuBPCase a c t i v i t y i n view of the l a r g e excess of t h i s enzyme (Wildner 1981 ) . Furthermore no s i g n i f i c a n t change i n the amount of enzyme p r o t e i n was observed e l e c t r o p h o r e t i c a l l y d u r i n g the two days f o l l o w i n g i n o c u l a t i o n . However, i t i s w e l l known t h a t p h o t o s y n t h e s i s d e c l i n e s d u r i n g s u c c e s s f u l d i s e a s e development (Daly 1976, S c o t t 1972 ) . A recent 112 r e p o r t by M o n t a l b i n i e t a l . (1981) has shown t h a t i n v i t r o r e d u c t i o n of NADP by c h l o r o p l a s t membranes decreased from 1 day a f t e r i n o c u l a t i o n i n e x t r a c t s d e r i v e d from r u s t i n f e c t e d V i c i a f aba. C l e a r l y t h i s i n f o r m a t i o n i m p l i e s a primary r o l e f o r c h l o r o p l a s t f u n c t i o n i n determining the outcome of the h o s t - p a r a s i t e i n t e r a c t i o n . Of the v a r i o u s c h l o r o p l a s t p o l y p e p t i d e s that can be i d e n t i f i e d , the l a r g e subunit of RuBPCase i s the only one e x h i b i t i n g a dramatic d e c l i n e i n the i n c o r p o r a t i o n of 35 s-methionine i n the s u s c e p t i b l e combination. T h i s i m p l i e s a f a i r l y s p e c i f i c e f f e c t on the s y n t h e s i s of t h i s p o l y p e p t i d e and argues a g a i n s t the p o s s i b i l i t y t h a t the d e c l i n e i s a r e s u l t e i t h e r of reduced uptake of r a d i o - l a b e l by the c h l o r o p l a s t s or of a g e n e r a l d i s r u p t i o n of c h l o r o p l a s t p r o t e i n s y n t h e s i s . The l a t t e r i s f u r t h e r supported by the r e l a t i v e l y unchanged p r o p o r t i o n of the t h y l a k o i d membrane p r o t e i n and the LHC a p o p r o t e i n , i n view of t h e i r d i f f e r e n t s i t e s of s y n t h e s i s (Steinback 1981). Another s t r i k i n g f e a -ture of the a l t e r e d p a t t e r n of i n c o r p o r a t i o n i n the s o l u b l e f r a c t i o n i s the i n v e r s e r e l a t i o n s h i p between the l a r g e sub-u n i t and the 50 kD p o l y p e p t i d e ( F i g . 16.2, LSu+1). T h i s i s i l l u s t r a t e d i n r e s u l t s from ot h e r experiments where the two p o l y p e p t i d e s were more c l e a r l y r e s o l v e d ( F i g . 16.2 - i n s e t ) . 113 These r e s u l t s i n d i c a t e a p o s s i b l e p r e c u r s o r - p r o d u c t r e l a t i o n s h i p between the two p o l y p e p t i d e s ; however, pulse-chase and p e p t i d e mapping experiments would be r e q u i r e d to c o n f i r m t h i s . The r e l a t i o n s h i p between the l a r g e subunit of RuBPCase and the 50 kD p o l y p e p t i d e (LSu+1) should be viewed i n l i g h t of a r e c e n t r e p o r t by M i l l e r and Huffaker (1982). These workers have i s o l a t e d endopeptidases from senescing b a r l e y leaves which c l e a v e RuBPCase to generate d i s c r e t e fragments. Among fragments from the l a r g e subunit are 54.5 and 49 kD p o l y p e p t i d e s . A fragment s m a l l e r than the s m a l l s u b u n i t i s a l s o observed which i s d e r i v e d from the s m a l l s u b u n i t by endopeptidase-3 a c t i v i t y which c o p u r i f i e s with RuBPCase and i s i n s e n s i t i v e to 1 mM PMSF. T h i s may e x p l a i n the presence of two major bands i n the 13 kD r e g i o n here. In f l a x cotyledons the apparent s h i f t of r a d i o l a b e l to LSu+1 was not accompanied by a s h i f t i n s t a i n e d p r o t e i n . Thus, i f indeed t h i s phenomenon i s the r e s u l t of protease a c t i v i t y , i t appears to be s e l e c t i v e f o r newly s y n t h e s i z e d p o l y p e p t i d e s . A p o s s i b l e e x p l a n a t i o n f o r t h i s would be that o n l y non-assembled subunits are a v a i l a b l e f o r p r o t e o l y s i s . Two dimensional a n a l y s i s of in v i v o l a b e l l e d p r o t e i n s from f l a x has shown t h a t although an a l t e r e d p a t t e r n of s y n t h e s i s occurs i n the s u s c e p t i b l e combination at 18 h r a f t e r i n o c u l a t i o n l i t t l e change i s observed i n the r e s i s t a n t 114 combination i n s p i t e of the marked a l t e r a t i o n i n p o l y A + mRNA s y n t h e s i s . T h i s may be e x p l a i n e d by the f a c t t h a t i n v i t r o t r a n s l a t i o n has r e v e a l e d many p o l y p e p t i d e s encoded both by poly A + and by po l y A - mRNA. T h e r e f o r e , i t i s unnecessary to invoke the appearance of novel mRNA s p e c i e s i n the r e s i s t a n t combination but merely decreased p o l y a d e n y l a t i o n of p r e c u r s o r mRNA i n the nucleus. I t has been suggested t h a t 3'poly A t a i l s have a r o l e i n determining mRNA s t a b i l i t y ( H a l l 1979). I t may be th a t t h i s i s r e p r e s e n t a t i v e of a g e n e r a l i z e d s t r e s s response and r e s u l t s i n r a p i d mRNA turnover. W i l t i n g s e e d l i n g s have a l s o been observed to show a marked r e d u c t i o n i n A + mRNA (Author, u n p u b l i s h e d ) . T h i s would enable g r e a t e r f l e x i b i l i t y i n gene e x p r e s s i o n . A f u r t h e r o b s e r v a t i o n of i n t e r e s t i s th a t both s t a i n e d and in v i v o l a b e l l e d p r o t e i n s from f l a x c o t y l e d o n s ( F i g . 15.3 and 15.4) show a f a r more a c i d i c d i s t r i b u t i o n than those t r a n s l a t e d i n v i t r o . The most obvious e x p l a n a t i o n i s th a t newly t r a n s l a t e d p r e c u r s o r p o l y p e p t i d e s c o n t a i n b a s i c amino a c i d s which are subsequently c l e a v e d to y i e l d f u n c t i o n a l p o l y p e p t i d e s . The p r e c u r s o r to the small subunit of RuBPCase i s a w e l l known example of such a p o l y p e p t i d e ( H i g h f i e l d and E l l i s 1978). Thus the ob s e r v a t i o n s presented here i n d i c a t e t h a t such p o s t - t r a n s l a t i o n a l p r o c e s s i n g i s a common f e a t u r e of many p o l y p e p t i d e s . Roberts and Lord (1981) 115 have shown t h a t p o s t - t r a n s l a t i o n a l p r o c e s s i n g i s a s s o c i a t e d w i t h t r a n s p o r t across membranes. I t i s p o s s i b l e t h a t b a s i c p r e c u r s o r s are r e q u i r e d f o r s u c c e s s f u l passage through membranes. The s m a l l RNA s p e c i e s d e s c r i b e d i n S e c t i o n 4.3.4 are of c o n s i d e r a b l e i n t e r e s t as they have no known p h y s i o l o g i c a l f u n c t i o n . A number of sm a l l RNA s p e c i e s from maize t i s s u e have been d e s c r i b e d by Link and Benecke (1980); these have mo l e c u l a r weights i n the range of those from f l a x which are l a r g e r than 5.8S. S i m i l a r s p e c i e s have been found i n Hela c e l l s ( E l i c e i r i 1974). O l i g o U and p o l y U c o n t a i n i n g RNA have been i s o l a t e d from wheat embryos (Dobzanska et a l . 1980). These RNA f r a c t i o n s contained up to 87 mol % UMP, la c k e d p o l y A and were capable o f a c t i n g as a template f o r i n v i t r o t r a n s l a t i o n . T h e i r s i z e d i s t r i b u t i o n was heterogeneous and s i m i l a r t o th a t of mRNA. No enrichment of RNA s m a l l e r than 4S was observed. As f a r as can be determined, the small U + RNA of the type observed i n f l a x has not been d e s c r i b e d i n p l a n t s p r e v i o u s l y . A s i m i l a r RNA s p e c i e s has been i s o l a t e d from the myosin messenger r i b o n u c l e o p r o t e i n of embryonic c h i c k muscle (Heywood and Kennedy 1976). T h i s o l i g o U - c o n t a i n i n g RNA sp e c i e s has a molecular mass of 10 kD and r e a c t s s t o i c h i o m e t r i c a l l y (1:1) with myosin mRNA r e s u l t i n g i n t o t a l i n h i b i t i o n of t r a n s l a t i o n a l a c t i v i t y . T h i s RNA has t h e r e f o r e 116 been d e s c r i b e d as t r a n s l a t i o n a l c o n t r o l RNA (tcRNA) and i s thought to e x e r t i t s e f f e c t p a r t l y through duplex formation of the o l i g o U moiety, with the 3" p o l y A t r a c t of the myosin mRNA (Bester e t a l . 1975). I t can be ap p r e c i a t e d t h a t f l a x U + RNA bears a c l o s e resemblance t o c h i c k myosin tcRNA. Attempts to assay the e f f e c t of f l a x U+RNA on the t r a n s l a t i o n a l a c t i v i t y f l a x mRNA were f r u s t r a t e d by the t e c h n i c a l d i f f i c u l t i e s of p u r i f y i n g the sm a l l amounts of U + RNA pr e s e n t . E s t a b l i s h e d p r o t o c o l s have been developed f o r the assay of tcRNA (Heywood et a l . 1979) and i t i s t h e r e f o r e c o n s i d e r e d t h a t f u r t h e r work on f l a x U+RNA would be warranted. 117 CHAPTER 5 GENERAL DISCUSSION The r e s e a r c h presented here has c l e a r l y demonstrated a primary r o l e f o r changes i n host RNA and p r o t e i n s y n t h e s i s i n determining the outcome of the h o s t - p a r a s i t e i n t e r a c t i o n s s t u d i e d . A n a l y s i s of the major r i b o n u c l e a s e f r a c t i o n from f l a x c o tyledons has r e v e a l e d t h a t a novel enzyme does not a r i s e d u r i n g pathogenesis. Furthermore the enhancement o f the isozyme P2 occurs i n the ' e a r l y ' phase of both the r e s i s t a n t and s u s c e p t i b l e i n t e r a c t i o n . Only at the onset o f p u s t u l e development do d i f f e r e n c e s between the r e s i s t a n t and the s u s c e p t i b l e combination manifest themselves. The q u a l i t a t i v e changes observed by Chakravorty et a l . (1974c) at t h i s stage can be accounted by the s e l e c t i v e enhancement of the isozyme PI. The r i b o n u c l e a s e isozymes have s i m i l a r p r o p e r t i e s . Both have a pH optimum of 5.5 and showed p r e f e r e n t i a l r e l e a s e of 2'-3' cGMP and are t h e r e f o r e of the RNase I type. As RNase I i s g e n e r a l l y thought to be i n v o l v e d i n s u b s t r a t e r e - u t i l i z a t i o n , i t i s now i n a p p r o p r i a t e to invoke them as having a c e n t r a l r o l e i n gene e x p r e s s i o n . I t was suggested p r e v i o u s l y (Chakravorty and Shaw 1977b) t h a t the major f l a x RNase i s i n v o l v e d i n p o s t - t r a n s c r i p t i o n a l p r o c e s s i n g . As f a r as the author i s aware, isozymes of RNase I have not been d e s c r i b e d p r e v i o u s l y . I t i s of great i n t e r e s t , 118 t h e r e f o r e , to s p e c u l a t e what s i g n i f i c a n c e the d i f f e r e n t c a t a l y t i c p r o p e r t i e s of these isozymes may have. Although they both r e l e a s e 2'-3' cGMP p r e f e r e n t i a l l y , they c l e a r l y have d i f f e r e n t a f f i n i t i e s f o r macromolecular RNA; t h i s i s undoubtedly due to the f a c t t h a t a f f i n i t y f o r macromolecular RNA i s dependent on secondary s t r u c t u r e which i s known to depend on RNA sequence. I t i s t h e r e f o r e reasonable to suggest t h a t they may be i n v o l v e d i n h y d r o l y z i n g d i f f e r e n t types of RNA. Both r e s i s t a n t and s u s c e p t i b l e combinations e x h i b i t a marked i n c r e a s e i n RNA s y n t h e s i s i n the e a r l y stages of d i s e a s e . In the absence of an accumulation o f RNA d u r i n g these e a r l y stages (Hamilton 1969), there i s c l e a r l y r a p i d RNA turnover i n which RNase I isozymes have a c e n t r a l r o l e . The l a t e r stages of the s u s c e p t i b l e r e a c t i o n e x h i b i t both e l e v a t e d RNA s y n t h e s i s and enhanced l e v e l s of RNase P i which do not occur i n the r e s i s t a n t r e a c t i o n . I t i s p o s s i b l e t h a t t h i s enzyme may be i n v o l v e d i n the h y d r o l y s i s of s p e c i f i c types of host RNA a s s o c i a t e d with f u n g a l s p o r u l a t i o n . T h i s phenomenon appears to be d i s t i n c t from e i t h e r wounding or senescence (see S e c t i o n s 2.3 and 3.3 .4 ) . I t may t h e r e f o r e be an important element of d i s e a s e development but not, however, the molecular b a s i s of s p e c i f i c i t y . 119 A f u r t h e r q u e s t i o n which should be addressed i s the p o s s i b l e involvement of the pH 5 i n s o l u b l e RNase f r a c t i o n (see S e c t i o n 2.3). Gel f i l t r a t i o n of t h i s f r a c t i o n r e v e a l e d t h a t most of the a c t i v i t y was l o o s e l y a s s o c i a t e d with high m o l e c u l a r weight m a t e r i a l , and was composed of predominantly RNase P2. The s u b s t r a t e s p e c i f i c i t y of t h i s f r a c t i o n was c o n s i s t e n t with t h i s o b s e r v a t i o n and d i d not respond to i n f e c t i o n . The p o s s i b i l i t y t h a t a minor component w i t h i n the pH 5 i n s o l u b l e f r a c t i o n responds to i n f e c t i o n cannot be r u l e d out, however. Although race s p e c i f i c i n d u c t i o n of the r e s i s t a n c e response i s manifested i n an a l t e r e d p a t t e r n of RNA s y n t h e s i s w i t h i n a few hours a f t e r i n o c u l a t i o n , no a s s o c i a t e d novel p r o t e i n s have been i d e n t i f i e d . Increased s y n t h e s i s of the RuBPCase l a r g e subunit has been observed, however. In a d d i t i o n , the s y n t h e s i s of a 30 kD s o l u b l e p o l y p e p t i d e i s s t i m u l a t e d . C l e a r l y the i n c r e a s e d s y n t h e s i s of the c h l o r o p l a s t encoded l a r g e subunit i s u n l i k e l y to be a primary event i n gene e x p r e s s i o n a s s o c i a t e d with the r e s i s t a n c e response as t h i s i s expected to be mediated by Mendelian type r e s i s t a n c e genes l o c a t e d i n the nucleus. Whether the 30 kD p o l y p e p t i d e i s of c y t o p l a s m i c or o r g a n e l l a r o r i g i n i s 120 unknown. I f the former i s the case, then i t does r e p r e s e n t a candidate f o r involvement as a primary t r i g g e r i n the r e s i s t a n c e response. Other minor p r o t e i n s may undergo q u a n t i t a t i v e changes i n s y n t h e s i s . These are hard to observe by two dimensional e l e c t r o p h o r e s i s i n the absence of a s u i t a b l e scanning d e v i c e . I t should be r e a l i z e d t h a t only p r e v a l e n t or moderately p r e v a l e n t mRNA encoded p o l y p e p t i d e s can be observed by two dimensional e l e c t r o p h o r e s i s . P o l y p e p t i d e s encoded by t h i s c l a s s of mRNA repre s e n t a complexity of 10 3 k i l o b a s e s (kB) of a t o t a l mRNA complexity of 10 4 to 10 5 kB i n mammalian c e l l s (Davidson and B r i t t e n 1979 ). In c o n c l u s i o n , one might expect t h a t s u b t l e changes i n gene e x p r e s s i o n are i n v o l v e d i n the i n i t i a t i o n of the r e s i s t a n c e response. Von Broembsen and Hadwiger (1972) observed f a i r l y marked i n c r e a s e s i n the s y n t h e s i s of c e r t a i n s i z e c l a s s e s of p r o t e i n . T h i s i s not i n agreement with the f i n d i n g s presented here. These workers d i d not study responses i n v o l v i n g the "N" l o c u s ( r e s i s t a n c e l o c u s i n Bombay f l a x ) , however, responses i n v o l v i n g the 'L' l o c u s , which were s t u d i e d , are s i m i l a r ( L i t t l e f i e l d 1973) and t h i s i s t h e r e f o r e probably not a s u i t a b l e e x p l a n a t i o n f o r the apparent d i s c r e p a n c y here. One f u r t h e r p o s s i b i l i t y i s that t h e i r r e s u l t s were the consequence of is o t o p e e f f e c t s across 121 i n t r a c e l l u l a r membranes which would undoubtedly i n f l u e n c e the r e s u l t s obtained with t h e i r d u a l l a b e l l i n g technique (see S e c t i o n 2 . 4 ) . The changes i n RNA s y n t h e s i s i n the r e s i s t a n c e response are observed p r i o r to h a u s t o r i a l formation ( L i t t l e f i e l d and Aronson 1969) and as much as 24 hours before s i g n i f i c a n t p h y t o a l e x i n accumulation (Keen and L i t t l e f i e l d 1979 ) . Whether p h y t o a l e x i n accumulation i s r e s p o n s i b l e f o r c e s s a t i o n of fu n g a l growth i s u n c l e a r as both occur s i m u l t a n e o u s l y at about 36 hours a f t e r i n o c u l a t i o n . However, s t a i n i n g with a c i d f u c h s i n i n d i c a t e s host c e l l n e c r o s i s and accompanying h a u s t o r i a l n e c r o s i s as e a r l y as 24 hours a f t e r i n o c u l a t i o n ( L i t t l e f i e l d 1 973 ) . Thus, h y p e r s e n s i t i v i t y i n the case of f l a x may i n f a c t be a cause r a t h e r than a consequence of f u n g a l i n h i b i t i o n as suggested by Prusky e t a l . (1980) f o r the oat/crown r u s t system. I t seems t h e r e f o r e t h a t the r e s i s t a n c e response i n f l a x i s c h a r a c t e r i z e d by d e r e p r e s s i o n of the genome l e a d i n g to an a l t e r e d p a t t e r n of RNA s y n t h e s i s , s u b t l e changes i n p r o t e i n s y n t h e s i s f o l l o w e d by host c e l l and h a u s t o r i a l n e c r o s i s , c u l m i n a t i n g i n the i n h i b i t i o n of fu n g a l growth. A very important p o i n t i s t h a t n e c r o s i s i s l o c a l i z e d to a few c e l l s around i n f e c t i o n s i t e s u n l e s s i t occurs i n the v i c i n i t y of v e i n s which are capable of t r a n s m i t t i n g n e c r o s i n g substances ( L i t t l e f i e l d 1973 ) . 122 On the other hand, as i n c r e a s e d RNA s y n t h e s i s can be observed i n e x t r a c t s and at times p r i o r to h a u s t o r i a l formation, i t i s i m p l i e d t h a t there i s a g e n e r a l i z e d p r e c o n d i t i o n i n g which enables r a p i d n e c r o s i s simultaneous with h a u s t o r i a l f o r m a t i o n . T h i s would p r o v i d e an e x p l a n a t i o n f o r the c r o s s p r o t e c t i o n observed by L i t t l e f i e l d (1969). T h i s scheme of events i s summarized i n Table IX. The o b s e r v a t i o n s ( S e c t i o n 4.3.8) of a l t e r e d RuBPCase s y n t h e s i s might be i n t e r p r e t e d as demonstrating the importance of f u n c t i o n a l c h l o r o p l a s t i n t e g r i t y i n the i n i t i a t i o n of the r e s i s t a n c e response. T h i s i m p l i e s t h a t c h l o r o p l a s t d i s r u p t i o n i s an important e a r l y event i n pathogenesis (see S e c t i o n 2.2.2). Some a d d i t i o n a l work with the f l a x / f l a x r u s t system i s l i k e l y t o y i e l d important r e s u l t s . The in c r e a s e d RNA s y n t h e s i s a s s o c i a t e d with the r e s i s t a n c e response p r o v i d e s a convenient bioassay system f o r f u n g a l e l i c i t o r s of the response. The r e s u l t s presented here have shown that e s t i m a t i o n of a c i d i n s o l u b l e 3 2 P r a d i o a c t i v i t y p r o v i d e s h i g h l y s i g n i f i c a n t r e s u l t s with three treatment r e p l i c a t e s and i n c r e a s e s i n i n c o r p o r a t i o n of as l i t t l e as 10% can be 123 TABLE IX. Summary of the main e a r l y events i n r e s i s t a n c e and s u s c e p t i b i l i t y d u r i n g r u s t i n f e c t i o n of f l a x Time a f t e r I n o c u l a t i o n ( h r s ) Combination  R e s i s t a n t S u s c e p t i b l e  8 Increased RNA s y n t h e s i s S h i f t to A - mRNA 18 G r e a t l y i n c r e a s e d RNA G r e a t l y i n c r e a s e d s y n t h e s i s . Enhanced RNA s y n t h e s i s s y n t h e s i s of l a r g e sub- Marked d e c l i n e i n u n i t RuBPcase. s y n t h e s i s of l a r g e subunit RuBPCase 24 a H a u s t o r i a l formation H a u s t o r i a l formation Host c e l l and haus-t o r i a l n e c r o s i s 3 6 a I n h i b i t i o n of f u n g a l Continued f u n g a l growth. P h y t o a l e x i n growth, accumulation. Events d e s c r i b e d f o r the r e s i s t a n t r e a c t i o n are those i n v o l v i n g the 'N' gene i n Bombay f l a x . a D a t a from L i t t l e f i e l d (1973). 124 d e t e c t e d . In view of the p o s s i b l e e x i s t e n c e of d i f f u s i b l e e l i c i t o r s i t may be p o s s i b l e to i d e n t i f y these by f e e d i n g p l a n t s with exudates from germinating uredospores. Such experiments are simply performed and may provide a more s p e c i f i c measure of e l i c i t a t i o n than p h y t o a l e x i n accumulation (see S e c t i o n 2.2.4). 125 SUMMARY P u r i f i c a t i o n of r i b o n u c l e a s e (RNase) from f l a x has shown t h a t the q u a l i t a t i v e changes which are observed i n the l a t e r stages of d i s e a s e can be accounted f o r by a l t e r e d p r o p o r t i o n s of two RNase I isozymes. The RNase isozymes d i f f e r from one another with r e s p e c t to s u b s t r a t e s p e c i f i c i t y , M i c h a e l i s constant, pH response and i s o e l e c t r i c p o i n t . The p r o p e r t i e s of these isozymes remain unchanged d u r i n g r u s t i n f e c t i o n i n both r e s i s t a n t and s u s c e p t i b l e combinations of f l a x and f l a x r u s t . E a r l y i n c r e a s e s (3 days a f t e r i n o c u l a t i o n ) i n RNase a c t i v i t y f o l l o w i n g i n o c u l a t i o n are c h a r a c t e r i z e d by i n c r e a s e d a c t i v i t y of one isozyme, a f e a t u r e common to both combinations. Late i n c r e a s e s (8 days a f t e r i n o c u l a t i o n ) are c h a r a c t e r i z e d by enhancement of the o t h e r isozyme and are unique to compatible combinations. Studi e s on RNA s y n t h e s i s w i t h i n the f i r s t 21 hours f o l l o w i n g i n o c u l a t i o n show t h a t the r e s i s t a n t r e a c t i o n i s c h a r a c t e r i z e d by enhanced RNA s y n t h e s i s which occurs p r i o r t o any measurable response i n the s u s c e p t i b l e combination. However by 18 to 21 hours a f t e r i n o c u l a t i o n both combinations show g r e a t l y enhanced r a t e s of s y n t h e s i s . Enhanced s y n t h e s i s of RNA i s a l s o accompanied by decreased p o l y a d e n y l a t i o n of messenger RNA which occurs as e a r l y as 8 hours a f t e r i n o c u l a t i o n i n the r e s i s t a n t combination. In v i t r o 126 t r a n s l a t i o n of RNA from u n i n f e c t e d p l a n t s has shown t h a t p o l y a d e n y l a t e d and non-polyadenylated RNA encode many common p o l y p e p t i d e s . One- and two-dimensional e l e c t r o p h o r e s i s of i n  v i v o l a b e l l e d p o l y p e p t i d e s has f a i l e d t o show any changes i n p r o t e i n s y n t h e s i s p r i o r to 18 hours a f t e r i n o c u l a t i o n . At t h i s time s e v e r a l p o l y p e p t i d e s are s y n t h e s i z e d at a l t e r e d r a t e s i n the s u s c e p t i b l e combination; no marked changes were de t e c t e d i n the r e s i s t a n t combination, however. 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O ' F a r r e l l PZ, HM Goodman, PH O ' F a r r e l l 1977 High r e s o l u t i o n two-dimensional e l e c t r o p h o r e s i s of b a s i c as w e l l as a c i d i c p r o t e i n s . C e l l 12: 1133-1142. Person CO 1959 Gene-for-gene r e l a t i o n s h i p s i n h o s t : p a r a s i t e systems. Can J Bot 37: 1101-1130. Person CO, GME Mayo 1974 Genetic l i m i t a t i o n s on models o f s p e c i f i c i n t e r a c t i o n s between a host and i t s p a r a s i t e . Can J Bot 52: 1339-1347. Prusky D, A Dinoor, B Jacoby 1980 The sequence of death of h a u s t o r i a and host c e l l s d u r i n g the h y p e r s e n s i t i v e r e a c t i o n of oat to crown r u s t . P h y s i o l P l a n t P a t h o l 17: 33-40. Pure GA, AK Chakravorty, KJ S c o t t 1979 C e l l - f r e e t r a n s l a t i o n of polysomal messenger RNA i s o l a t e d from h e a l t h y and r u s t - i n f e c t e d wheat l e a v e s . P h y s i o l P l a n t P a t h o l 15: 201-209. Pure GA, AK Chakravorty, KJ S c o t t 1980 Changes i n wheat l e a f polysomal messenger RNA p o p u l a t i o n s d u r i n g the e a r l y stages of r u s t i n f e c t i o n . E f f e c t s of chloramphenicol and l i n c o m y c i n on c e l l - f r e e t r a n s l a t i o n by polysomes from h e a l t h y and i n f e c t e d l e a v e s . P l a n t P h y s i o l 66: 520-524. Quick WA, M Shaw 1964 The p h y s i o l o g y of h o s t - p a r a s i t e r e l a t i o n s . XIV. The e f f e c t of r u s t i n f e c t i o n on the n u c l e i c a c i d content of wheat l e a v e s . Can J Bot 42: 1531-1540. Roberts LM, JM Lord 1981 Sy n t h e s i s and p o s t t r a n s l a t i o n a l s e g r e g a t i o n of glyoxysomal i s o c i t r a t e l y a s e from c a s t o r bean endosperm. Eur J Biochem 119: 43-49. 133 Robinson RA 1976 P l a n t pathosystems. Advanced s e r i e s i n a g r i c u l t u r a l s c i e n c e s V.3. (S p r i n g e r - V e r l a g ) Rohringer R, DJ Samborski, CO Person 1961 Ribonuclease a c t i v i t y i n r u s t e d wheat l e a v e s . Can J Bot 39: 775-784. Sachse B f G Wolf, WH Fuchs 1971 Nubleinsaure abbauenda Enzyme i n B l a t t e r n von T r i t i c u m aestivum nach I n f e k t i o n mit P u c c i n i a graminis t r i t i c i . A c t a Phytopathol Acad S c i Hung 6: 39-49. S a d l e r R, M Shaw 1979a St u d i e s on glutamate dehydrogenase from r u s t i n f e c t e d f l a x c o t y l e d o n s . Z P f l a n z e n p h y s i o l 91: 211-224. Sa d l e r R, M Shaw 1979b Pathways of n i t r o g e n a s s i m i l a t i o n i n r u s t i n f e c t e d f l a x c o t y l e d o n s . Z P f l a n z e n p h y s i o l 93: 105-115. S c o t t KT 1972 O b l i g a t e p a r a s i t i s m by phytopathogenic f u n g i . B i o l Rev 47: 537-572. S c o t t KJ 1976 Growth of b i o t r o p h i c p a r a s i t e s i n axenic c u l t u r e ; i n En c y c l o p e d i a of P l a n t P h y s i o l o g y , New S e r i e s V.4. P h y s i o l o g i c a l P l a n t Pathology. Eds. H e i t e f u s s R, PH W i l l i a m s ( S p r i n g e r V e r l a g ) . Scrubb LA, AK Chakravorty, M Shaw 1972 Changes i n the r i b o n u c l e a s e a c t i v i t y of f l a x cotyledons f o l l o w i n g i n o c u l a t i o n with f l a x r u s t . P l a n t P h y s i o l 50: 73-79. Seq u e i r a L 1978 L e c t i n s and t h e i r r o l e i n host-pathogen s p e c i f i c i t y . Ann Rev Phytopathol 16: 453-481. Shaw M 1963 The p h y s i o l o g y and h o s t - p a r a s i t e r e l a t i o n s of the r u s t s . Ann Rev Phytopathol 1: 259-28 3. Shaw M 1967 C e l l b i o l o g i c a l aspects of h o s t - p a r a s i t e r e l a t i o n s of o b l i g a t e f u n g a l p a r a s i t e s . Can J Bot 45: 1205-1220. Sidhu GS 1975 Gene-for-gene r e l a t i o n s h i p s i n p l a n t p a r a s i t i c systems. S c i Prog Oxf 62: 467-485. Simpson RS, AK Chakravorty, KJ S c o t t 1980 Messenger and ribosomal RNA h y d r o l y s i s by r i b o n u c l e a s e s I . The a c t i o n of two b a r l e y l e a f r i b o n u c l e a s e s on the messenger and ribosomal RNA of i s o l a t e d polysomes. P l a n t C e l l P h y s i o l 21: 413-423. 134 Sphor G, M-E M i r a u l t , T Imaizumi, K Sche r r e r 1976 Molecular-weight d e t e r m i n a t i o n of a n i m a l - c e l l RNA by e l e c t r o p h o r e s i s i n formamide under f u l l y d e n aturing c o n d i t i o n s on e x p o n e n t i a l p o l y a c r y l a m i d e g e l s . Eur J Biochem 62: 313-332. Steinback KE 1981 P r o t e i n s of the c h l o r o p l a s t . In The Bio c h e m i s t r y o f P l a n t s , a comprehensive t r e a t i s e . Eds. Stumpf and Conn. pp. 303-319. Sutton BCS, M Shaw 1982 Changes i n two r i b o n u c l e a s e isozymes d u r i n g r u s t i n f e c t i o n of f l a x c o t y l e d o n s . P l a n t P h y s i o l 69: 205-209. Tang WJ A Maretz k i 1970 P u r i f i c a t i o n and p r o p e r t i e s of l e a f r i b o n u c l e a s e from sugar cane. Biochim Biophys A c t a 212: 300-307. T a n i T, H Yamamoto 1978 N u c l e i c a c i d and p r o t e i n s y n t h e s i s i n a s s o c i a t i o n with the r e s i s t a n c e of oat leaves to crown r u s t . P h y s i o l P l a n t Pathol 12: 113-121. T a n i T, M Yoshikawa, M N a i t o 1971 Changes i n 3 2 p - r i b o n u c l e i c a c i d s i n oat leaves a s s o c i a t e d with s u s c e p t i b l e and r e s i s t a n t r e a c t i o n s to P u c c i n i a c o r o n a t a . Ann Phytopathol Soc Japan 37: 43-51. T a n i T, M Yoshikawa, N Naio 1973 Template a c t i v i t y of r i b o n u c l e i c a c i d e x t r a c t e d from oat leaves i n f e c t e d by P u c c i n i a c o r o n a t a . Ann Phytopath Soc Japan 39: 7-13. T a n i T, M Yoshikawa, M N a i t o 1975 S e l e c t i v e enhancement of ribosomal r i b o n u c l e i c a c i d s y n t h e s i s of crown r u s t - i n f e c t e d oat le a v e s by stem e x c i s i o n . P h y s i o l P l a n t P athol 5: 193-199. T a y l o r JM 1979 The i s o l a t i o n of e u k a r y o t i c messenger RNA. Ann Rev Biochem 48: 681-717. Vanderplank JE 1978 Gen e t i c and molecular b a s i s of p l a n t pathogenesis. Advanced s e r i e s i n a g r i c u l t u r a l s c i e n c e s V.6. (Spr i n g e r V e r l a g ) . Van Loon LS 1975 P o l y n u c l e o t i d e - a c r y l a m i d e g e l e l e c t r o p h o r e s i s of s o l u b l e nucleases from tobacco l e a v e s . FEBS L e t t 51: 266-269. 135 Von Broembsen SL, LA Hadwiger 1972 C h a r a c t e r i z a t i o n o f di s e a s e r e s i s t a n c e responses i n c e r t a i n gene-for-gene i n t e r a c t i o n s between f l a x and Melampsora l i n i . P h y s i o l P l a n t P a t h o l 2: 207-215. Wade M, P Albersheim 1979 R a c e - s p e c i f i c molecules t h a t p r o t e c t soybeans from Phytophthora megasperma v a r . soj a e . Proc Nat Acad S c i USA 76 : 4433-4437. Wildner GF 1981 R i b u l o s e - 1 , 5 - b i s p h o s p h a t e carboxylase-oxygenase: Aspects and p r o s p e c t s . P h y s i o l P l a n t 52: 385-389. W i l s o n CM 1971 P l a n t nucleases I I I . Polyacrylamide e l e c t r o p h o r e s i s of corn r i b o n u c l e a s e isoenzymes. P l a n t P h y s i o l 48: 64 -68 . W i l s o n CM 1975 P l a n t nucleases. Ann Rev P l a n t P h y s i o l 26: 187-208. Yoshikawa M, H Masago, WT Keen 1977 A c t i v a t e d s y n t h e s i s of p o l y ( A ) - c o n t a i n i n g messenger RNA i n soybean hy p o c o t y l s i n o c u l a t e d with Phytophthora megasperma var. s o j a e . P h y s i o l P l a n t Pathol 10 : 125-138. Yamamoto H, T T a n i , N N a i t o 1975 Changes i n p r o t e i n contents of oat leaves d u r i n g the r e s i s t a n t r e a c t i o n a g a i n s t P u c c i n i a coronata avenae. Phytopath Z 82 : 138 -145 . 136 APPENDIX. Met h o d o l o g i c a l D e t a i l s and P r e c a u t i o n s RIBONUCLEASE Ribonuclease e x t r a c t i o n T h i s study was designed to e x p l o r e the mechanisms u n d e r l y i n g the q u a l i t a t i v e changes i n RNase which are observed d u r i n g r u s t i n f e c t i o n . As t h i s study and those of o t h e r workers suggest t h a t a m u l t i p l i c i t y of RNase isozymes may be p r e s e n t p a r t i c u l a r a t t e n t i o n i s p a i d to the y i e l d of t o t a l enzyme u n i t s at each stage of p u r i f i c a t i o n . U s u a l p r e c a u t i o n s , such as low temperature and the avoidance of a e r a t i n g enzyme s o l u t i o n s , have been taken t o m a i n t a i n high y i e l d . In a d d i t i o n , the isozymic composition and p r o p e r t i e s of v a r i o u s f r a c t i o n s have been examined throughout p u r i f i c a t i o n . In t h i s way the r e l a t i o n s h i p between i n d i v i d u a l isozymes and the t o t a l s o l u b l e e x t r a c t can be assessed. ENZYME ASSAYS Ribonuclease assay The r e a c t i o n mixture ( f i n a l volume 1 ml) contained 50 umoles of sodium a c e t a t e b u f f e r pH 5.5, 0.5 mg of y e a s t RNA (BDH, Poole England) which had been r e p r e c i p i t a t e d with 3 v o l . 95% e t h a n o l , washed and d i a l y s e d e x h a u s t i v e l y a g a i n s t EDTA and subsequently a g a i n s t d i s t i l l e d water. V a r y i n g amounts of enzyme s o l u t i o n were added and samples were incubated f o r 30 min. at 37°C. P r e c i p i t a t i n g reagent - 2 ml (1 MHC1, 0.5% LaCl3 i n 76% ethanol) was added and the tubes allowed to stand at 4°C f o r 30 min. R e a c t i o n blanks were i d e n t i c a l except they were not incubated; t h i s gave the most r e a l i s t i c blank as i t accounted f o r i n c r e a s e d RNA p r e c i p i t a t i o n which r e s u l t s from the i n c l u s i o n of p r o t e i n . A f t e r removal of p r e c i p i t a t e s by c e n t r i f u g a t i o n at 4°C the c l e a r supernatant was read a t 260 nm i n a SP800 spectrophotometer; t h i s enabled the s p e c t r a l i d e n t i t y of the product to be checked. Deoxyribonuclease (DNase) assay The i n c u b a t i o n mixture ( f i n a l volume, 2.0 ml) c o n t a i n e d : 50 yumoles sodium a c e t a t e b u f f e r pH 5.8; 10 /amoles MgCl2; 0.5 mg n a t i v e or heat denatured DNA ( c a l f thymus DNA, Calbiochem). D e n a t u r a t i o n was c a r r i e d out by h e a t i n g to 100 °C f o r 10 min and c o o l i n g r a p i d l y on i c e . The i n c u b a t i o n and measurement of DNase a c t i v i t y were c a r r i e d out as d e s c r i b e d f o r RNase. 137 Phosphodiesterase assay The r e a c t i o n mixture ( f i n a l volume 2.5 ml) contained: 30 jumoles K2HPO4; 1.75 mg c a l c i u m b i s ( p - n i t r o p h e n y l ) phosphate with e q u i v a l e n t amounts of enzyme s o l u t i o n to those used i n RNase and DNase assays. A f t e r i n c u b a t i o n f o r 1 hr at 3 7°C, the tubes were read a t 410 nm a g a i n s t a reagent blank. M i c h a e l i s constant The constant K m was c a l c u l a t e d from double r e c i p r o c a l p l o t s of the form: 1 a g a i n s t 1 , React i o n v e l o c i t y Substrate c o n c e n t r a t i o n 1/v vs 1/[S] (see F i g u r e ) . Assays f o r RNase were s e t up as d e s c r i b e d p r e v i o u s l y but with d i f f e r e n t amounts of RNA v a r y i n g from 25 ^ ug t o 150 pq. Before p r e c i p i t a t i o n a d d i t i o n a l RNA was added to g i v e a f i n a l c o n c e n t r a t i o n of 200 jug i n 1.2 ml. T h i s procedure was e s s e n t i a l t o g i v e c o n s i s t e n t p r e c i p i t a t i o n . S u b s t r a t e p r e f e r e n c e Substrate p r e f e r e n c e was determined by s u b s t i t u t i n g t r i t i a t e d homoribopolymers o f p o l y c y t i d y l i c , p o l y a d e n y l i c and p o l y c y t i d y l i c a c i d (20 /iCi/mmole P) (poly U, po l y A, po l y C f M i l e s L a b o r a t o r i e s Inc., E l k h a r t , Indiana) f o r RNA under standard assay c o n d i t i o n s . Assays were c a r r i e d out under c o n d i t i o n s of l i m i t i n g s u b s t r a t e c o n c e n t r a t i o n so th a t 0.125, 0.250 and 0.500 jaCi produced l i n e a r l y i n c r e a s i n g r e a c t i o n v e l o c i t i e s . A f t e r i n c u b a t i o n 0.5 mg of c o l d RNA were added to a i d p r e c i p i t a t i o n . A f t e r removal of the p r e c i p i t a t e by c e n t r i f u g a t i o n 1 ml of the supernatant was added t o 10 ml o f toluene s c i n t i l l a t i o n f l u i d (Aquasol, New England Nuclear) and counted f o r r a d i o a c t i v i t y i n a l i q u i d s c i n t i l l a t i o n system. OTHER PROPERTIES OF RIBONUCLEASE pH P r o f i l e RNase assays were run a t v a r i o u s pH v a l u e s . The f o l l o w i n g b u f f e r s were used: pH 3.5-4.5 Na2HP04 - c i t r i c a c i d 40 mM; pH 5.0-6.0 Na ace t a t e 40 mM; pH 6.5-8.0 KH2PO4 - NaOH 40 mM. 00 Figure: Lineweaver-Burk plots for RNases PI and P2, and p a r t i a l l y pu r i f i ed RNase from wounded cotyledons (W). M o l e c u l a r weight d e t e r m i n a t i o n Molecular weight was determined by Sephadex G100 g e l f i l t r a t i o n on a 2.5 x 40 cm column. The v o i d volume (Vo) was assessed with Blue Dextran and the i n c l u d e d volume (Vi) was assessed with 2 , 4 - d i n i t r o p h e n o l : E l u t e d volumes (Ve) were assessed f o r the f o l l o w i n g p r o t e i n s of known molecular weight: P a n c r e a t i c RNase A; 13,700, Myoglobin, 17,800; Chymotoypsinogen A, 25,000; Ovalbumin, 43,000; Bovine Serum Albumin, 67,000. K a v v a l u e s were c a l c u l a t e d from the e q u a t i o n : K a v = Ve - Vo V i - Vo and a c a l i b r a t i o n curve K a v a g a i n s t l o g molecular weight, c o n s t r u c t e d . Column i s o e l e c t r i c f o c u s i n g P r e p a r a t i v e column e l e c t r o f o c u s i n g was performed i n 110 ml (LKB 810) Ampholine column. The column contained: 1) Anode s o l u t i o n at bottom: Phosphoric a c i d 0.2 ml H 20 14 ml Sucrose 12 g 2) S e p a r a t i o n g r a d i e n t : 0 - 50% sucrose l i n e a r g r a d i e n t 110 ml Ampholine pH 2.5-6.0 1 % Enzyme p r e p a r a t i o n 1 mg p r o t e i n 3) Cathode s o l u t i o n at top: Ethanolamine 0.8 ml H 20 40 ml Focusing was c a r r i e d out f o r 36 hr at 300 v. During t h i s time the c u r r e n t decreased and reached a constant value i n d i c a t i n g t h a t the ampholytes were focused. The column was maintained a t 8°C throughout the run by c o o l i n g with tap water. The power d i d not exceed 2.4 Watts and thus c o n v e c t i o n was minimized. The anode was p l a c e d at the bottom of the column i n o r d e r to reduce i n t e r f e r e n c e from p r o t e i n s p r e c i p i t a t i n g through the g r a d i e n t as i t was c o n s i d e r e d t h a t more p r e c i p i t a t i o n would occur at the a c i d i c end of the pH g r a d i e n t . 140 RNA SYNTHESIS RNA e x t r a c t i o n The RNA e x t r a c t i o n method used i n t h i s r e s e a r c h i n v o l v e s an i n i t i a l phenol treatment of the aqueous e x t r a c t . Since i t has been suggested p o l y a d e n y l a t e d RNA i s s e l e c t i v e l y absorbed to the phenol and i n t e r p h a s e l a y e r s during e x t r a c t i o n i n the absence of c h l o r o f o r m ( T a y l o r 1979), the e f f e c t of c h l o r o f o r m a d d i t i o n has been examined. A sample of f l a x s e e d l i n g s was fed with [ 3 2P] Na phosphate. H a l f of t h i s sample was e x t r a c t e d with phenol i n the o r g a n i c phase and h a l f was e x t r a c t e d with phenol:chloroform, 1:1. The percentage of r a d i o a c t i v i t y which bound to p o l y U Sepharose w i t h i n the p u r i f i e d RNA d e r i v e d from each e x t r a c t was assessed. The percentages were 6.6% and 5.3% f o r RNA d e r i v e d from phenol o r p h e n o l / c h l o r o f o r m e x t r a c t s r e s p e c t i v e l y . Thus i t appears t h a t the i n c l u s i o n of c h l o r o f o r m does not enhance the recovery of p o l y a d e n y l a t e d RNA. 

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