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Genetic analysis of anp2/3-mkk6-mpk4 cascade in plant immunity Lian, Kehui

Abstract

Recognition of pathogens through pathogen-associated molecular patterns (PAMPs) or effectors in plants activates a variety of defense responses including MAPKs signaling pathways and defense related genes expression. ANPs (Arabidopsis Nucleus- and Phragmoplast- localized kinase 1 related protein kinases), including ANP1, ANP2, ANP3 are three MAP kinase kinase kinases that form a MAP kinase cascade with downstream MKK6 and MPK4 to regulate cytokinesis process. In this study, we showed that the anp2 anp3 double mutants exhibit constitutive expression of PR (Pathogenesis-Related) genes and enhanced resistance against oomycete pathogen H. a. Noco2, suggesting that ANP2 and ANP3 negatively regulate plant immunity. In addition, loss function of MKK6 causes high levels of PR gene expression, indicating that MKK6 is involved in negative regulation of defense responses. Since MPK4 was previously shown to function as a negative regulator of plant immunity, we tested whether MPK4 functions downstream of ANP2/ANP3 and MKK6 in plant immunity by introducing CA-MPK4 transgene, which expresses a constitutively active (CA) variant of MPK4, to anp2 anp3 and mkk6. Constitutive expression of PR genes and enhanced resistance to H.a. Noco2 in anp2 anp3 and mkk6 were partially suppressed by expressing CA MPK4, suggesting that the ANP2/ANP3, MKK6 and MPK4 function in a MAPK cascade to negatively regulate defense responses. To find out components that function downstream of ANP2/ANP3- MKK6-MPK4 cascade in plant immunity, two mutants summ2-8 (SUPPRESSOR OF MKK1 MKK2 2) and pad4-1 (PHYTOALEXIN DEFICIENT 4) were crossed into anp2 anp3 respectively. The constitutive defense responses in anp2 anp3 were fully suppressed by pad4-1, but not affected by the summ2-8 mutation, suggesting that PAD4 functions downstream of ANP2/ANP3 and that immune responses mediated by certain TIR-NB-LRR R proteins might be activated in the anp2 anp3 mutant.

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