{"http:\/\/dx.doi.org\/10.14288\/1.0388362":{"http:\/\/vivoweb.org\/ontology\/core#departmentOrSchool":[{"value":"Science, Faculty of","type":"literal","lang":"en"},{"value":"Non UBC","type":"literal","lang":"en"},{"value":"Microbiology and Immunology, Department of","type":"literal","lang":"en"}],"http:\/\/www.europeana.eu\/schemas\/edm\/dataProvider":[{"value":"DSpace","type":"literal","lang":"en"}],"https:\/\/open.library.ubc.ca\/terms#identifierCitation":[{"value":"Pathogens 9 (1): 19 (2019)","type":"literal","lang":"en"}],"http:\/\/purl.org\/dc\/terms\/contributor":[{"value":"Michael Smith Laboratories","type":"literal","lang":"en"}],"http:\/\/purl.org\/dc\/terms\/creator":[{"value":"Kretschmer, Matthias","type":"literal","lang":"en"},{"value":"Damoo, Djihane","type":"literal","lang":"en"},{"value":"Djamei, Armin","type":"literal","lang":"en"},{"value":"Kronstad, James Warren","type":"literal","lang":"en"}],"http:\/\/purl.org\/dc\/terms\/issued":[{"value":"2020-01-24T17:01:29Z","type":"literal","lang":"en"},{"value":"2019-12-24","type":"literal","lang":"en"}],"http:\/\/purl.org\/dc\/terms\/description":[{"value":"Chloroplasts play a central role in plant immunity through the synthesis of secondary metabolites and defense compounds, as well as phytohormones, such as jasmonic acid and salicylic acid. Additionally, chloroplast metabolism results in the production of reactive oxygen species and nitric oxide as defense molecules. The impact of viral and bacterial infections on plastids and chloroplasts has been well documented. In particular, bacterial pathogens are known to introduce effectors specifically into chloroplasts, and many viral proteins interact with chloroplast proteins to influence viral replication and movement, and plant defense. By contrast, clear examples are just now emerging for chloroplast-targeted effectors from fungal and oomycete pathogens. In this review, we first present a brief overview of chloroplast contributions to plant defense and then discuss examples of connections between fungal interactions with plants and chloroplast function. We then briefly consider well-characterized bacterial effectors that target chloroplasts as a prelude to discussing the evidence for fungal effectors that impact chloroplast activities.","type":"literal","lang":"en"}],"http:\/\/www.europeana.eu\/schemas\/edm\/aggregatedCHO":[{"value":"https:\/\/circle.library.ubc.ca\/rest\/handle\/2429\/73381?expand=metadata","type":"literal","lang":"en"}],"http:\/\/www.w3.org\/2009\/08\/skos-reference\/skos.html#note":[{"value":"pathogensReviewChloroplasts and Plant Immunity: Where Are theFungal Effectors?Matthias Kretschmer 1, Djihane Damoo 1, Armin Djamei 2 and James Kronstad 1,*1 Michael Smith Laboratories, Department of Microbiology and Immunology, University of British Columbia,Vancouver, BC V6T 1Z4, Canada; kretschm@msl.ubc.ca (M.K.); djihane.damoo@msl.ubc.ca (D.D.)2 Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) OT Gatersleben Corrensstrasse 3,D-06466 Stadt Seeland, Germany; djamei@ipk-gatersleben.de* Correspondence: kronstad@msl.ubc.ca; Tel.: +604-822-4732Received: 4 December 2019; Accepted: 21 December 2019; Published: 24 December 2019 \u0001\u0002\u0003\u0001\u0004\u0005\u0006\u0007\b\u0001\u0001\u0002\u0003\u0004\u0005\u0006\u0007Abstract: Chloroplasts play a central role in plant immunity through the synthesis of secondarymetabolites and defense compounds, as well as phytohormones, such as jasmonic acid and salicylicacid. Additionally, chloroplast metabolism results in the production of reactive oxygen speciesand nitric oxide as defense molecules. The impact of viral and bacterial infections on plastids andchloroplasts has been well documented. In particular, bacterial pathogens are known to introduceeffectors specifically into chloroplasts, and many viral proteins interact with chloroplast proteins toinfluence viral replication and movement, and plant defense. By contrast, clear examples are just nowemerging for chloroplast-targeted effectors from fungal and oomycete pathogens. In this review, wefirst present a brief overview of chloroplast contributions to plant defense and then discuss examplesof connections between fungal interactions with plants and chloroplast function. We then brieflyconsider well-characterized bacterial effectors that target chloroplasts as a prelude to discussing theevidence for fungal effectors that impact chloroplast activities.Keywords: fungal phytopathogenesis; reactive oxygen species; light-harvesting complex; effector1. IntroductionPlastids are dynamic plant organelles that differentiate during development into photosyntheticchloroplasts as well as leucoplasts, chromoplasts, and additional subtypes [1]. In the context of plantimmunity, most studies focus on chloroplasts, which are responsible for the formation of energyequivalents, such as ATP and NADPH during photosynthesis. These energy equivalents can be usedduring and after carbon fixation to produce primary carbon-containing metabolites, such as sugars,starch, nucleotides, amino acids and fatty acids\/lipids. Plastids, and more specifically chloroplasts,also make important contributions to plant defense against pathogens, including participation inthe pathogen-associated molecular pattern (PAMP), triggered immunity (PTI) and effector-triggeredimmunity (ETI) [2\u20138]. As a basal layer of plant defense, PTI is usually triggered by conserved PAMPson pathogens (e.g., bacterial flagellin and fungal chitin) or by damage-associated molecular patterns(DAMPs, such as oligogalacturonides or cellobiose) that may originate from the plant cell wall [2,4,5].ETI is a second layer of plant immunity that requires the recognition of effectors by resistance (R)proteins leading to a hypersensitive response (HR) [2,4,5]. Key reactions during defense includethe synthesis of secondary metabolites and phytoalexins, the formation of reactive oxygen species(ROS) and nitric oxide (NO), calcium oscillations, stomatal closure, apoplastic alkalization, cell wallstrengthening and the expression of plant defense proteins including pathogenesis-related proteins(PRs) [4,5,9,10]. Chloroplasts are the sites of production of ROS, calcium oscillations, and the synthesisof plant defense molecules, including jasmonic acid (JA) and salicylic acid (SA), that are criticalPathogens 2020, 9, 19; doi:10.3390\/pathogens9010019 www.mdpi.com\/journal\/pathogensPathogens 2020, 9, 19 2 of 16components of the plant defense strategy against necrotrophic (JA) and biotrophic pathogens (SA).Overall, it is clear that chloroplasts are a critical hub connecting plant defense responses to primaryanabolic functions.In this review, we first briefly summarize the contributions of the chloroplast to immunity toset the stage for understanding potential targets of pathogen attack on the plastid. Our focus hereis primarily on fungal pathogens, and readers are directed to several excellent recent reviews onthe role of the chloroplast in interactions with viral and bacterial pathogens [4,5,11]. We initiallydiscuss recent examples of chloroplast functions that are impacted by interactions with fungi andspecifically consider chloroplast contributions to defense against fungi. Information on bacterialinteractions is included to reinforce findings of key chloroplast functions that mediate defense or serveas targets of pathogen attack. We then use the emerging information about bacterial effectors thattarget chloroplast functions as a guide to consider recent studies on fungal effectors. Effectors have alsobeen studied in detail in oomycetes, and we refer readers to recent papers and reviews for coverage ofthese pathogens [12\u201314]. Finally, we discuss future directions, including the prediction that fungaleffectors targeting the chloroplast will be prominent contributors to disease.2. An Overview of Chloroplast Contributions to Plant ImmunityThe phytohormone JA is synthesized in the chloroplast and plays a crucial role in plant immunity [4,5].In particular, JA (along with ethylene (ET)) signaling contributes to defense against necrotrophicpathogens but is considered to be ineffective against biotrophic pathogens [5]. JA is derived fromlinolenic acid that is released from chloroplast membranes and oxidized to form 12-oxo-phytodienoicacid (OPDA) by the action of allene oxide synthases and cyclases. OPDA is then exported from thechloroplast to form JA in the peroxisome [4,5,8]. Exogenous JA is known to reduce the chlorophyllcontent of potato leaves, to reduce electron transport of the photosystems (PSI and PSII), and to impactcarbon fixation by downregulation of chlorophyll biosynthesis and photosynthesis-related genes [5].The phytohormone ET also regulates chlorophyll content, PSI and PSII efficiency, and rubisco activityin Arabidopsis and tobacco in an age-dependent manner [5].As a defense hormone, SA is antagonistic to ET and JA in its activity to induce effective plant defensemainly against hemibiotrophic and biotrophic pathogens. Two SA biosynthesis pathways that involvechloroplast functions are proposed in plants. One pathway converts chorismate to isochorismate viaisochorismate synthase (ICS1) in the chloroplast with subsequent conversion of isochorismate to SA inthe cytosol [15]. A second pathway accounts for ~10% of SA synthesis by converting isochorismate toSA via the phenylalanine ammonia-lyase pathway in the cytosol [3,4,8,15,16]. Phenylalanine is alsosynthesized in the chloroplast from chorismate [5]. Thus, in both cases, chloroplasts play a major rolein SA biosynthesis. SA stimulates photosynthesis at low externally applied concentrations (10\u22125 \u00b5M)but is inhibitory at higher concentrations. Although not the mobile signals, SA or derivatives, such asmethyl-SA, are also involved in systemic acquired resistance after a local primary infection leading toHR [4,5].The production of ROS is also a key defense contribution of chloroplast metabolism. Thephotosynthetic electron transport chains of PSI and II of the chloroplast are major players in providingelectrons for free radical formation (e.g., singlet 1O2 and superoxide O2\u2212). PSI produces O2\u2212 that isconverted to H2O2 by superoxide dismutases, while PSII mainly forms singlet oxygen 1O2 [3]. ROShave several plant defense activities besides the direct killing of pathogen cells. These activities includeinvolvement in both PTI and ETI with contributions to cell wall strengthening, signaling and HRinduction. For PTI, PAMPs are known to trigger the downregulation of PsbS, a thylakoid sensorthat regulates non-photochemical quenching (NPQ); this regulation drives the electrons from thephotosystems to form increased amounts of oxygen radicals rather than dissipation of the free energyas heat [4,17]. The second extended high amplitude ROS burst is important for plant defense responsesin the context of ETI and HR. For example, tobacco with reduced chloroplastic ROS formation showedreduced HR when challenged with Xanthomonas campestris pv. vesicatoria [5]. The activation of thePathogens 2020, 9, 19 3 of 16mitogen-activated protein kinases MPK3 and MPK6 is important for ETI and leads to repressionof photosynthesis-related genes and induces plant defense\/secondary metabolism genes [10]. Thisuncoupling of photosynthesis by inhibiting PSII leads to the overpowering of the NPQ and the rapidaccumulation of ROS under light conditions [10]. MPK3\/6 deletion mutants are compromised in HR,and the expression of a chloroplast-targeted flavodoxin reduced inhibition of photosynthesis, ROSformation and HR [10]. Thus, ETI depends on the MPK3\/6 signaling cascade during light conditionsfor ROS formation, which might also explain why plants are more resistant during daylight comparedto dark phases where no ROS is formed. This information also indicates that the global reprogrammingof chloroplast functions, such as the downregulation of photosynthesis versus the upregulation of plantdefense\/secondary metabolism, is an active plant defense strategy, rather than a pathogen-inducedphenotype [9,10]. However, plant pathogenic microbes are known to secrete effectors that target severalchloroplastic functions, as discussed further below.Chloroplast-to-nucleus retrograde signaling is crucial for the proper functioning and assemblyof the photosynthetic apparatus [18]. Similarly, changes in the developmental or metabolic statesof the chloroplast result in severe changes in the transcript profiles of nuclear genes [19]. Thissuggests that the chloroplast may act as an environmental sensor, mediating environmental stresses toregulate the transcription of certain nuclear genes. In the context of disease, lesion mimic mutantsin plants form spontaneous lesions that resemble HR, and some of these mutations are in nucleargenes that encode chloroplast proteins [20]. Retrograde signaling is thought to involve Ca2+ sensingand a ROS signal that could be transferred to the nucleus via stromule bridges originating from thechloroplasts and observed during pathogen attacks [4\u20136,8,10,17,21\u201325]. Stromules are stroma-filled,tube-like extrusions originating from the chloroplast [21\u201325]. They depend on the chloroplast unusualpositioning 1 protein (CHUP1) that promotes stromule formation along microtubule-guided extensions,with actin microfilaments providing anchoring points [21\u201325]. Ca2+ fluxes are induced during pathogenrecognition and include Ca2+ spikes in the chloroplast after PTI is triggered by flagellin or chitin.Stromal Ca2+ spikes are regulated by a thylakoid membrane-bound Ca2+ sensing protein [4,6,17]. Ca2+sensing in concert with ROS was shown to be responsible for PTI and ETI by regulating Ca2+ fluxes, SAbiosynthesis and the induction of nuclear-encoded defense genes [6]. For example, PTI is compromisedin a mutant defective in Ca2+ sensing with reduced stomatal closure, reduced callose deposition andphytoalexin formation after the plant was challenged with Pseudomonas syringae. ETI, in this situation,was severely delayed [5].Retrograde signaling during plant defense further involves the chloroplast protein N-receptorinteracting protein 1 (NRIP1) and the metabolites 3-phosphoadenosine-5-phosphate (PAP) andC-methyl-D-erythritol-2,4-cyclopyrophosphate (MEcPP) [3,4,8,25,26]. NRIP1 is transferred fromthe chloroplast to the nucleus during plant defense activation by the N resistance protein (againstthe tobacco mosaic virus), which is thought to involve stromules [8,21]. PAP originates in the cytosolbut is degraded in the chloroplast by the phosphatase SAL1. The SAL1-PAP pathway, in turn, wasshown to regulate the SA and JA biosynthesis pathways [26]. For example, sal1 mutants displayeddownregulation of SA and JA signaling pathways and showed enhanced symptom formation uponinfection with both hemibiotrophic and necrotrophic bacterial pathogens [26]. MEcPP is an intermediateof the MEP pathway, which is a precursor for isoprenoids and the hormones GA and ABA. Understress situations, MEcPP accumulates and promotes the expression of ICS1, leading to higher SA levelsand increased SA defense signaling [4]. PAP and MEcPP are redox-regulated, and thus light-dependentROS formation may potentiate these signals [4].In addition to PAP and MEcPP retrograde signaling, targets of pathogen interference may involvethe genome uncoupled (GUN) genes characterized in Arabidopsis thaliana. An unbiased forward geneticscreen led to the identification of six GUN genes [27,28]. GUN1 is a chloroplast-localized protein, anda gun1 mutant shows increased susceptibility to photooxidative stress [29,30]. Similar phenotypescan be observed in plants treated with inhibitors that target de novo protein synthesis in plastidsor carotenoid biosynthesis (e.g., lincomycin and norflurazon, respectively) [28]. In addition, a co-IPPathogens 2020, 9, 19 4 of 16experiment revealed that GUN1 interacts with many proteins involved in the maintenance of thechloroplast proteome as well as chaperones, suggesting it plays a role in coordinating chloroplastprotein import and protein degradation [31]. The other GUN proteins encode enzymes involved intetrapyrrole metabolism, and GUN2, GUN3 and GUN6 are involved in heme metabolism leading tothe suggestion that heme may be a retrograde signal [18].It is clear that retrograde signaling plays a major role in chloroplast biogenesis and plant immunity.However, it is unknown if several individual signals lead to retrograde signaling or if multiplesignals are connected in a network required to control the expression of nuclear-encoded chloroplastproteins [18]. Pathogen effectors that directly target the retrograde signaling pathway in plants havenot been identified so far.3. Impact of Fungi on Chloroplast Functions3.1. Chloroplast Morphology and PositionChanges in chloroplast morphology and position have been observed during disease. For example,the infection of rice by the fungal pathogen Rhizoctonia solani severely disturbs chloroplast morphologyand function [32]. Examination of diseased tissue revealed structural disintegration of chloroplastmembrane structures (grana-, thylakoid and stroma organization) at 3 days post-infection. It was furthershown that the chloroplasts at this time were the main source for ROS formation, with accompanyingreduced photosynthetic performance. Factors such as maximum quantum yield of PSII, electrontransport rate and non-photochemical quenching were markedly reduced [32]. In contrast, genesencoding secondary metabolism and plant defense functions associated with chloroplasts showedincreased expression. Consequently, metabolites, such as sinapic acid (phenylpropanoid pathway) andalpha-linolenic acid (JA biosynthesis pathway), accumulated in higher amounts [32].The activation of plant defense often results in chloroplast repositioning from an optimal positionfor photosynthesis to locations in proximity to the invading pathogen or the nucleus. For example,chloroplasts in Nicotiana benthamiana tend to accumulate close to the nucleus with consequent stromuleformation during PTI and ETI defense responses, during bacterial or viral infection, or during thetransient expression of viral proteins, such as REP or p50 [33]. Exogenous H2O2 is able to triggerchloroplast movement in tobacco, and chloroplast aggregation around nuclei is reduced upon inhibitionof ROS formation by NADPH-oxidase inhibitors or external application of ROS scavengers, such asTiron or dimethylthiourea [33].3.2. Chloroplasts and ROS GenerationROS generated in chloroplasts is a major contributor to plant immunity. For example, theexpression of a cyanobacterial chloroplast-targeted flavodoxin (which reduces ROS accumulation)in transgenic tobacco reduced the symptoms of infection by the necrotrophic fungus Botrytis cinereaor the hemibiotrophic bacterium X. campestris [34\u201336]. The transgenic plants infected with B. cinereashowed less tissue damage and reduced fungal growth as well as changes in photosynthetic activityand plant defense responses [34]. In particular, mycelial growth was reduced by 67\u201390% in thetransgenic plants expressing the cyanobacterial flavodoxin protein compared to wild-type plantsduring a time course of infection [34]. Several photosynthetic parameters, such as maximum quantumyield, photosynthetic performance index and electron transfer efficiency, were inhibited to a lesserdegree in plants expressing flavodoxin. Furthermore, wild-type plants showed fewer intact activereaction centers of the PSII per leaf area compared to the flavodoxin-expressing lines [34]. Phytoalexinaccumulation was also reduced in the infected plants expressing flavodoxin, and the expressionof plant defense genes, such as glucanases, chitinases or PR1, was delayed compared to wild-typeplants. Overall, it appears that chloroplast production of ROS contributes to the virulence of B. cinereaand that decreased ROS accumulation in the transgenic flavodoxin plants provides some protectionagainst a necrotrophic pathogen. This conclusion is supported by a recent study on the contribution ofPathogens 2020, 9, 19 5 of 16chloroplast-produced tocopherols as antioxidants during infection of Arabidopsis with B. cinerea [37].In this case, mutants with T-DNA insertions in VTE1 (encoding tocopherol cyclase) or VTE4 (encoding\u03b3-tocopherol methyltransferase) were used to alter tocopherol composition [37]. In particular, avte1 mutant lacking \u03b1 and \u03b3-tocopherols and a vte4 mutant that accumulates \u03b3-tocopherol but lacks\u03b1-tocopherol both showed delayed resistance to B. cinerea. Lipid peroxidation also increased duringinfection with B. cinerea, which correlated with increased fungal biomass and fungal virulence. Thealtered susceptibility in the vte1 and vte4 mutants may be due to delayed linolenic acid processingleading to reduced JA formation [37].The contribution of chloroplastic ROS for defense against biotrophic pathogens is illustrated by arecent study with the Wheat Kinase START1 (WKS1) gene [38]. WKS1 is a race nonspecific resistancegene that encodes a protein with a serine\/threonine kinase domain and a steroidogenic acute regulatoryprotein-related lipid transfer (START) domain. Partial plant resistance against the rust fungus Pucciniastriiformis f. sp. tritici is seen in wheat transformed with multiple copies of the WKS1 gene originallyidentified in wild tetraploid wheat. The WKS1 protein is located in the chloroplast, and was shown tointeract with and ultimately phosphorylate a thylakoid-associated ascorbate peroxidase, thus limitingascorbate detoxification of ROS leading to a slow cell death response and partial plant resistance [38].The impact of ROS in the defense against hemibiotrophs is illustrated by an analysis of Arabidopsisplants expressing a ferrodoxin that is a major distributor of electrons in plastids [39]. Deletion of thegene encoding the plastid-localized ferredoxin FD2 led to increased virulence of the bacterial pathogenP. syringae, and this was correlated with a reduction in ROS formation during PTI [39]. Plants lackingFD2 showed dramatically induced JA formation, while SA formation was unchanged compared towild type plants. This hormone imbalance induced JA-dependent defense gene expression and furtherled to reduced expression of SA biosynthetic genes (e.g., ICS1) and SA-dependent genes (e.g., PR1).In ETI with P. syringae containing avrRpt2, plants lacking flavodoxin produced ~2 times more H2O2and consequently were more resistant than wild-type plants [39]. Ferredoxin2 was found to be locatedin stromules, which indicates a potential contribution to retrograde signaling [39].Given that ROS functions play an important role during plant immunity, it is not surprising that thelight-harvesting complexes, especially PSII, appear to be an integral part of plant defense. For example,the light-harvesting complex II protein LHCB5 is phosphorylated during infection of rice by therice blast fungus Magnaporthe oryzae in a light-dependent manner [40]. Plant resistance to blast wasincreased in high light conditions for some rice lines, and this was correlated with expression changesin LHCB5 caused by promoter variations in the different rice lines [40]. Plants overexpressing LHCB5were significantly more resistant to M. oryzae, while RNAi knockdown lines were hypersusceptible.The resistance was linked to differences in ROS formation with the overexpression line showingincreased ROS amounts. LHCB5 phosphorylation was both dependent on M. oryzae infection and onlight intensity, and specific phosphorylation at threonine 24 was responsible for the ROS burst andHR formation in a chloroplast-dependent manner [40]. In this context, it is also interesting that thephosphorylation state of the photosynthesis-related chaperonin-60 is important for plant immunity inA. thaliana during infection with the bacterium X. campestris pv. campestris [41]. Chaperonin-60 is thetarget of the chloroplast-localized protein phosphatases PP2C62\/26, and the deletion of genes for thesephosphatases increased plant resistance [41].3.3. Chloroplast Pigments and Carbohydrate MetabolismSeveral studies establish links between fungal disease and carotenoid or chlorophyll biosynthesisor catabolism. For example, virus-induced silencing of carotenoid or chlorophyll biosynthesis at thephytoene desaturase or Mg-chetalase H steps, respectively, led to a faster and stronger appearanceof HR symptoms (likely due to higher ROS accumulation) during an infection of wheat with thehemibiotrophic fungus Zymoseptoria tritici [42]. The fungus Z. tritici switches to a necrotrophic attackat later stages of infection, and sporulation of the pathogen was impaired in plants with reducedchlorophyll biosynthesis [42]. This result indicates that modification of pigment biosynthesis can makePathogens 2020, 9, 19 6 of 16a strong contribution to plant resilience to pathogen attack. Interestingly, earlier studies demonstratedthat loss of the chloroplast protein Les22, a uroporphyrinogen decarboxylase involved in chlorophyllbiosynthesis, results in spontaneous lesions that resemble an HR [43]. This example with the Les22protein is characteristic of the phenomenon observed in lesion mimic mutants of several plant species(e.g., rice, maize and the model plant Arabidopsis) in which spontaneous HR like symptoms, thehallmark of ETI, are observed without a pathogen being present [44]. Lesion mimic mutations have alsobeen identified in the maize (ZmLls1) and wheat (TaLls1) genes encoding pheophorbide a oxygenase(PaO) for chlorophyll breakdown [45]. TaLls1 expression is induced in wheat upon infection withP. striiformis f. sp. tritici or wounding, and silencing of the gene increased tolerance to P. striiformis [45].The biotrophic smut fungus Ustilago maydis also appears to influence chloroplast function, and thisfungus provokes the formation of white tumors surrounded by normal green tissues [46]. Tumors onmaize incited by U. maydis are chlorotic with all major chloroplast pigments severely reduced, especiallychlorophyll b (41%) [47]. Overall, the genes for chlorophyll biosynthesis, photosynthesis complexproteins and CO2 fixation functions showed severely reduced expression in tumors. On average, mostof the genes related to photosynthesis showed a 3\u201310 fold reduction in expression in tumors compared touninfected plants [46]. Other chloroplast functions, such as fatty acid and lipid biosynthesis, amino acidbiosynthesis, and secondary metabolism functions of the shikimate pathway and the phenylalaninebiosynthetic pathway, were also downregulated [47]. Impairment of chloroplast functions in achloroplast biosynthesis mutant or by treatment of plants with herbicides (e.g., glyphosate\u00ae) resultedin increased disease symptom formation upon U. maydis inoculation. This result indicates thatchloroplasts are integral for plant immunity in this pathosystem. In contrast, chloroplast-relatedcarbohydrate functions, including genes for several starch biosynthesis proteins (e.g., sbe1 and ae1),were upregulated 2.5 to 14 fold in tumors. Consequently, more starch was seen in tumor tissuecompared to uninfected stems of maize seedlings. Interestingly, the sugary1 maize line (with alteredstarch biosynthesis) was more resistant than the wild type line during seedling infection with U.maydis [47].Ustilago maydis also exerts a metabolic priming influence on the maize defense response bysecreting the enzyme chorismate mutase (encoded by the effector gene cmu1) into the cytosol of hostcells. The enzyme acts to redirect the shikimate pathway and promote reduced SA production [48].Recently, one of 20 maize-encoded kiwellins, ZmKWL1, was found to disarm the Cmu1 chorismatemutase by hindering the access of the substrate to the enzyme\u2019s active site [49,50]. ZmKWL1 belongsto a subgroup of kiwellins found exclusively in cereal plants, and ZmKWL1 interacts exclusively withU. maydis Cmu1 in vitro and not with the maize version of chorismate mutase [49,51]. It was proposedthat ZmKWL1 is secreted into the apoplast, where it binds Cmu1 and presumably decreases the importof the enzyme into host cells [49].Variegated plants that show green and white sectors also provide an informative approach toexamine the contributions of chloroplast functions to plant immunity. The photosynthetic-activegreen tissue shows normal chloroplast development and function, while the white sectors display asink-like behavior with undeveloped, non-photosynthetically active plastids. The immutans mutationin Arabidopsis causes a variegated phenotype due to variations in the plastid terminal oxidase (PTOX)that disturb carotenoid and chlorophyll biosynthesis during chloroplast biogenesis [52]. Analysis of themutant showed that white sectors experience ROS stress and demonstrated remodeling of the plant cellwall with reduced lignin amounts and cellulose microfibrils as well as changes in galactomannans andxyloglucans amounts\/distribution [52]. Although tests with fungal pathogens have not been reported,white sectors challenged with the bacterial pathogen P. syringae showed reduced plant defense geneactivation (e.g., PR1 and PR5 expression), reduced callose deposition and higher colonization of thetissue by the bacteria compared to green sectors or wt plants [52]. Perhaps the higher bacterial growthreflects greater nutrient availability due to the sink behavior of the white sectors.In the context of the impact of fungi on chloroplast function, a long-standing observation isthat certain biotrophs, such as rust or powdery mildew fungi, and some hemibiotrophic pathogens,Pathogens 2020, 9, 19 7 of 16cause so-called green islands of photosynthetically active tissue around infection sites [53]. Theseislands occur in a background of otherwise yellowing and senescent tissue. Photosynthetic activity ismaintained in green island tissue, although it is generally reduced compared to healthy tissue. Forexample, overall net photosynthesis can be reduced by as much as 32%, and quantum yield can be47% lower in the green islands on barley leaves infected with the powdery mildew pathogen Blumeriagraminis [53,54]. Chloroplast organization and morphology appear to be normal between green islandsor uninfected tissue, and photosynthetic capacity may be reduced by an initial loss of chlorophyllwith subsequent re-biosynthesis of new chlorophyll (the re-greening hypothesis). It has also beenproposed that the chlorophyll in green islands is retained in chloroplasts (the chlorophyll retentionhypothesis) and that cytokinins, possibly produced by the biotrophic fungi, play an important role ingreen island formation [53]. In general, the process of green island formation requires considerableadditional investigation to uncover the underlying molecular mechanisms.3.4. Positive Influences of Fungi on Chloroplast FunctionIn general, the study of beneficial fungi and their interactions with chloroplasts may provideimportant insights for improving plant productivity and for understanding the impact of pathogenicfungi on chloroplasts. It is well established that endophytic and mycorrhizal fungi can increase plantperformance and photosynthetic capacity. For example, the fungal endophyte Epichloe typhina increasedthe abundance of PSI proteins (PsaC, Lhca2) and PSII proteins (D1, Lhcb3) of its host plant orchardgrass by 2\u20133 fold [55]. Chlorophyll accumulation increased by 33%, especially chlorophyll b, andoverall net photosynthesis was increased by ~32% at saturated light conditions [55]. Similarly, theinteraction of arbuscular mycorrhizal fungi with watermelon also triggers increased carotenoid andchlorophyll a and b amounts and improved photosynthesis parameters such as net photosynthesisrate, PSII maximum yield, actual photochemical quantum and photochemical quenching [56]. Abioticsalt stress negatively influences these photosynthetic parameters, but the negative effects can bealleviated by mycorrhizal colonization of the plant roots [56]. Interestingly, it was previously foundthat the plastid-localized proteins CASTOR and POLLUX are essential for symbiotic plant interactionsin lotus with nitrogen-fixing bacteria or arbuscular mycorrhizal fungi [57]. Loss of either proteinleads to the inhibition of cytoplasmic Ca2+ spiking and abortion at early infection stages. During thebacterial interaction, infection threads are not formed, while during the mycorrhizal interaction, thefungus is unable to establish root epidermal cell invasion. The arbuscular mycorrhizal interaction inalfalfa also significantly reduces the negative effects of the herbicide atrazine, a PSII electron transportinhibitor, on chloroplast structure and PSII performance, but does not mitigate the negative effectson chloroplast pigment accumulation [58]. In contrast, secondary metabolites, such as coriloxineor quinone derivatives from the endophytic fungus Xylaria feejeensis, have negative effects on ATPsynthesis of spinach thylakoid preparations, and these compounds can further be chemically modifiedto show even greater effects against photosynthetic functions [59].As mentioned above, plant growth and photosynthetic capacity are generally known to increaseduring interactions with beneficial microorganisms. Therefore, it seems counter-intuitive to observepositive effects on chloroplast functions during a necrotrophic fungal attack, although this phenomenonhas been recently observed for the fungal pathogen Alternaria alternata [60]. Volatile compounds ofbeneficial microorganisms are known to have positive effects on plant growth and photosynthesiscapacity, and the work with A. alternata indicates that volatiles from the necrotrophic fungus alsohave similar effects on plants [60]. The influences of the volatiles included increased photosynthesisparameters, such as PSII operating efficiency and photochemical quenching, and increased chlorophyllcontent in proximity to A. alternata volatiles [60]. These phenotypes depended on the redox state of theplant, and analysis of the thiol redox proteome indicated that volatile compounds lead to a reducedprotein state, especially for photosynthesis-related proteins. These phenotypes are controlled by thechloroplast protein NADPH-dependent thioredoxin reductase [60]. The consequences of improvedchloroplast functions for the necrotrophic plant pathogen require further investigation.Pathogens 2020, 9, 19 8 of 164. Effectors and ChloroplastsGiven that the chloroplast plays a key role in plant immunity, it is reasonable that the organellewould be a prime target of effector proteins introduced by pathogens [8]. Plant pathogens secrete acocktail of effector proteins or virulence factors that are known to act in the apoplast or the cytoplasm,where they may target specific organelles [7,61\u201366] (Figure 1). While effector proteins are very diverse,with different mechanisms of action, ultimately, what they have in common is their ability to facilitatepathogen proliferation in the host in the absence of direct or indirect detection by correspondingcompatible resistance proteins (R) to trigger ETI. In compatible interactions, effectors contribute tovirulence by suppressing the plant immune response, by interfering with the host\u2019s physiology topromote nutrient acquisition, by influencing organelle function and gene expression, or by as yetunknown mechanisms [64,66,67].Pathogens 2020, 9, x FOR PEER REVIEW 8 of 15   of effector proteins o  virulence fac ors that are known to act in the apoplas  or the cytoplasm, where th y may target specific organelles [7,61\u201366] (F gure 1). While effector proteins ar  very iv rse, with different mechanisms of action, ultimately, what they hav  in comm n is their ability to facilitate pathogen proliferation in the host in the abse ce of dir ct or i dire t d tection by rresponding compatible resistance p oteins (R) to trigger ETI. I  compatible interactions, effectors contribute to virulence by su pressing the plant immune response, by in erfering with the host\u2019s hysi logy o promote nutrient acquisition, by influencing organelle function and gene expres ion, or by as yet unknown mechanisms [64,66,67].   Figure 1. Diagram of fungal and bacterial delivery of known and candidate effectors that influence chloroplast function. The left side of the diagram depicts the interaction of a fungal pathogen with a plant cell to deliver effectors by mechanisms that could involve colonization of the apoplast, penetration of host cells or formation of haustorial feeding structures. Known effectors (Cmu1 and ToxA) are shown with ToxA localized to the chloroplast. Candidate effectors (CTP1, 2 and 3) are localized to the chloroplast, but their impact of function is not yet known. The right side of the diagram shows the delivery of effectors by bacterial pathogens via a type III secretion system. Representative effectors delivered by Pseudomonas syringae are listed. 4.1. Examples of Effectors that Target the Chloroplast Several bacterial pathogens secrete effectors that target chloroplast functions [7,68,69]. Much of what is known about these effectors comes from studies with P. syringae pathovars and, more specifically, the interaction of P. syringae pv. tomato (Pst) strain DC3000 with A. thaliana [70,71]. Several P. syringae effectors are known to manipulate chloroplast functions, including the well-characterized protein HopI1, an effector with a J-domain usually found in co-chaperones. HopI1 localizes to the chloroplast using a non-cleavable transit peptide and the protein targets Hsp70, resulting in modification of thylakoid structures and suppression of SA accumulation [72,73]. Expression of HopI1 in transgenic A. thaliana plants expressing high SA levels resulted in a 60% decrease in the level of SA-inducible PR-1 (Pathogenesis related-1) gene transcript, and around 50% lower free and total levels of SA [72]. HopNI is another well-studied P. syringae effector protein that is targeted to the chloroplast using a non-cleavable transit peptide [74]. HopN1 codes for a cysteine protease that cleaves PsbQ, an intrinsic protein of photosystem II in tomato cells, thereby diminishing the photolysis of water [74]. In addition, HopN1 was previously found to suppress cell death associated with HR [75]. It was i . i l t i l li i t ff t t t i fl.t cell to deliver ffectors by mechanisms that could involve colonization of the apoplast, penetrationof host cells or f rmation o haus rial feeding structures. Known effectors (Cmu1 and ToxA) areshown with T xA localized to the chloroplast. Candidate effectors (CTP1, 2 and 3) are localize to thchloroplast, but their impact of function is not yet k ow . The right side of the diagram showselivery of effectors by bacterial pathogens via ype III secretion system. R presentative effectorsd liv r d by Ps udomonas syringae are li te .4.1. Examples of Effectors that Target the ChloroplastSeveral bacterial pathogens secrete effectors that target chloroplast functions [7,68,69]. Much ofwhat is known about these effectors comes from studies with P. syringae pathovars and, more specifically,the interaction of P. syringae pv. tomato (Pst) strain DC3000 with A. thaliana [70,71]. Several P. syringaeeffectors are known to manipulate chloroplast functions, including the well-characterized proteinHopI1, an effector with a J-domain usually found in co-chaperones. HopI1 localizes to the chloroplastusing a non-cleavable transit peptide and the protein targets Hsp70, resulting in modification ofthylakoid structures and suppression of SA accumulation [72,73]. Expression of HopI1 in transgenicA. thaliana plants expressing high SA levels resulted in a 60% decrease in the level of SA-inducible PR-1(Pathogenesis related-1) gene transcript, and around 50% lower free and total levels of SA [72].Pathogens 2020, 9, 19 9 of 16HopNI is another well-studied P. syringae effector protein that is targeted to the chloroplastusing a non-cleavable transit peptide [74]. HopN1 codes for a cysteine protease that cleaves PsbQ,an intrinsic protein of photosystem II in tomato cells, thereby diminishing the photolysis of water [74].In addition, HopN1 was previously found to suppress cell death associated with HR [75]. It wasfurther demonstrated that HopN1 lacking the catalytic activity of the cysteine protease was unable toinhibit ROS production compared with the wild-type protein [74].Two other P. syringae effectors, AvrRps4 and HopK1, target the chloroplast via a proposed cleavabletransit peptide [68]. Specifically, AvrRps4 and HopK1 share sequence similarity in an N-terminalregion that may represent the chloroplast transit peptide. AvrRps4 triggers RPS4-dependent immunityin Arabidopsis, and transgenic expression of AvrRps4 in rps4 plants resulted in enhanced growth of PstDC3000 and suppression of PTI [76]. Similar to AvrRps4, HopK1 has been shown to contribute to PstDC3000 virulence. HopK1 activity was also shown to reduce ROS production and callose deposits,indicating that the effector can suppress PTI [68].Although HopI1, HopN1, AvrRps4 and HopK1 are the best-characterized chloroplast-targetedeffectors from P. syringae, other putative chloroplast-targeted effectors have been identified, includingHopBB1 and HopM1 [discussed in [5]. HopBB1 interacts with nuclear and chloroplast proteins andmay have more than one intracellular location. HopBB1 also interacts with proteins involved in JAsignaling, and with the PTF1 protein that regulates photosynthesis. HopM1 interacts with chloroplastproteins, but some uncertainty remains about its subcellular location [5]. Importantly, HopM1 interactswith MIN7 and MIN10, proteins with known roles in plant immunity.Chloroplast-targeted effectors are also produced by other bacterial species, including theeconomically important pathogen Ralstonia solanacearum, the causative agent of bacterial wilt diseasesin potato, tomato and banana [77]. Similar to P. syringae, R. solanacearum uses a type three secretionsystem to facilitate the delivery of more than 70 effector proteins called Rips (Ralstonia injectedproteins) into plant cells [69]. One of the best-studied chloroplast-targeted effectors in R. solanacearumis RipAL, a protein with a putative lipase domain. RipAL presumably targets chloroplast lipids and isthought to induce JA production and consequently suppress SA-mediated defense responses (PTI) inN. benthamiana [69]. Another R. solanacearum effector, RipG, contains an F-box domain and is thought tobe part of an SCF-type E3 ubiquitin ligase complex, controlling specific protein ubiquitination [78]. TheRipG effector family contains seven members that interact with chloroplast proteins, suggesting thatthese proteins could be targeted for ubiquitination and proteasomal degradation [78]. A chloroplast-targeted effector Las5315 has also been characterized in Candidatus Liberibacter asiaticus [79]. Thisbacterium causes the Huanglongbing (citrus greening) disease of citrus crops typified by chlorosisand starch accumulation. Las5315 acts to upregulate the expression of enzymes involved in starchproduction and to downregulate functions for starch degradation [79]. Finally, we note that theWtsE effector of Pantoea stewartii, a wilt and leaf blight pathogen of maize, has a major impact onchloroplast-associated functions, including secondary metabolism and photosynthesis, although it isnot clear that the effector localizes to chloroplasts [80].4.2. Fungal Effectors Targeting Chloroplast FunctionsPerhaps the clearest example of a chloroplast-targeted fungal effector comes from the study ofPyrenophora tritici-repentis, a fungal pathogen that produces the host-selective toxins ToxA and ToxB tosupport a necrotrophic attack on wheat [81\u201383]. Plant sensitivity to ToxA is governed by the Tsn1 locusthat encodes an NBS-LRR type R protein, and the toxin is thought to enter host cells by endocytosisand to act in chloroplasts. ToxA induces cell death in a light-dependent manner and provokes ROSaccumulation in chloroplasts resulting in a reduction in the levels of PSI and PSII protein complexes.ToxA interacts with the chloroplast protein ToxA Binding Protein 1 (ToxABP1) in wheat, and a homologdesignated Thylakoid formation 1 (Thf1) in A. thaliana. Thf1 is proposed to play a role in PSII biogenesisor degradation. Interestingly, the severity of tissue necrosis provoked by ToxA can be blocked bypreventing the accumulation of ROS or by silencing ToxABP1 in wheat.Pathogens 2020, 9, 19 10 of 16Some effectors from rust fungi show sequence signatures with similarity to host transit peptidesfor translocation into chloroplasts. Examples of these effectors are found in Melampsora larici-populina,the poplar leaf rust fungus that causes annual epidemics and severe damage to poplar plantations(especially in Northern Europe) [84\u201387]. M. larici-populina has been used as a model rust, and itsgenome was one of the first rust genomes to be sequenced, with 16,399 predicted genes, includinggenes for candidate secreted proteins. Candidate effectors with a predicted chloroplast transit peptidewere recently identified in M. larici-populina and designated chloroplast-target proteins (CTP1, CTP2and CTP3) [88]. Subsequent work revealed that the N-terminal transit peptide, which is cleaved inplanta, is sufficient for CTP1, CTP2 and CTP3 accumulation in chloroplasts [89]. Although the impactof the proteins on chloroplast function is just starting to be examined [90], these studies show thatfungi have evolved strategies to direct their effectors to the host\u2019s chloroplasts by mimicking the host\u2019ssorting signals.Compared with the information on bacterial effectors, there is a paucity of documented localizationof fungal effectors in chloroplasts. For fungi, the rarity of chloroplast-targeted effectors is illustrated by arecent review that presented a non-exhaustive list of well-characterized biotrophic and hemibiotrophicfungal effectors [91]. Surprisingly, chloroplast-targeted effectors have not been readily identified insome of the best-characterized fungal pathogens, such as the hemibiotrophic fungus M. oryzae [92]or the biotrophic gall-inducing fungus U. maydis. For the latter, classic disease symptoms, such as asource-to-sink tissue transition during colonization and the loss of chlorophyll in infected maize tissue,suggest that U. maydis produce effectors to target the chloroplast, although these effectors have yet tobe discovered. We should note that a recent study identified four RXLR effectors from the oomycetepathogen Plasmopara viticola that localize to the chloroplast, although a functional impact remains to bedemonstrated [12].One emerging platform to predict fungal effector proteins, especially when combined within planta expression data, is EffectorP (http:\/\/effectorp.csiro.au\/) [93]. Compared to other effectorprediction platforms, EffectorP is focused entirely on fungal phytopathogens. Notably, computationalmethods exist for predicting the subcellular localization of plant proteins, but they perform poorlyfor effector proteins because the bioinformatic recognition of potential plastid localization signals isconfounded by the presence of pro-domains of varying length between the N-terminal signal peptidesand translocation signals. The LOCALIZER platform (http:\/\/localizer.csiro.au\/) was recently developedfor predicting effector localization in plants [94]. This approach predicted the chloroplast locationof the ToxA protein of P. tritici-repentis (discussed above), and the prediction was experimentallyconfirmed. Additionally, LOCALIZER was employed to survey the secreted proteins of several fungalphytopathogens, and this analysis revealed that rust pathogens have an enrichment of candidatechloroplast effectors compared with other fungi. Subsequent localization experiments identifiedchloroplast locations for two effectors from Puccinia graminis f. sp. tritici. Both EffectorP andLOCALIZER have become valuable tools for prioritizing fungal effector candidates for functionalinvestigations. For instance, these two platforms, when used in unison, could provide preliminarypredictions for localization of fungal chloroplast-targeted candidate effectors. While most effectorsuntil now have been discovered by genetic and biochemical strategies, computational predictions ofeffector proteins may provide a faster approach to identify novel candidate effectors.5. Conclusions and Future WorkPlastids and, more specifically, chloroplast-located processes are potentially important targets ofmicrobial effectors. The relevance of plastids and chloroplasts for plant metabolism and immunityis clear and experimentally validated plastid-targeting fungal and oomycete effectors are emerging.Diseases caused by fungal pathogens, or interactions with beneficial fungi, impact specific chloroplastactivities for photosynthesis, chlorophyll biosynthesis and stability, organelle position and the formationof ROS. Although there are well-characterized interactions between viral or bacterial proteins andchloroplast proteins, little is known about the influence of specific fungal effectors that target plastids.Pathogens 2020, 9, 19 11 of 16In addition, the impact of effectors from fungi and other pathogens on retrograde signaling isunder-explored and requires additional investigation. Whereas bioinformatic prediction approachesbased on the known plastid-translocation signals can be the basis for targeted experimental searchesfor plastid-localized effectors, non-classical organelle targeting of effectors should not be excluded.Therefore, non-targeted systematic effector localization screens and biochemical approaches to co-purifyeffectors with organelle-preparations followed by mass spectrometry might lead to exciting discoveries.Although fungal effectors are now being identified in chloroplasts, questions remain about thebiochemical state of these effectors and how they reach their subcellular destination. The translocationof proteins from the plant cytosol to plastids occurs for polypeptides in an unfolded state and is a GTP-and ATP-driven process. Plant precursor proteins are kept in an unfolded, translocation-competentstate by chaperones of the hsp70 family. How fungal plastid-effectors first get secreted from the fungalcells and then passage through the biotrophic interface safely, translocate into the host cytoplasm, andfinally get translocated into plastids is enigmatic and awaits future research. Overall, effector researchwill deepen our understanding of the relevance of plastids in biotic interactions and highlight potentialcentral regulatory hubs located in plastids.Author Contributions: Conceptualization: M.K., D.D.; Literature review, critical analysis, and synthesis: M.K.,D.D., A.D., J.K.; Writing\u2014Original Draft Preparation: M.K., D.D., A.D., J.K.; Writing\u2014Editing: A.D., J.K.;Supervision: A.D., J.K. All authors have read and agreed to the published version of the manuscript.Funding: Research in the Kronstad laboratory on fungal pathogens of plants is supported by a Discovery Grantfrom the Natural Sciences and Engineering Research Council of Canada (NSERC). Additional support comes froman NSERC CREATE award as part of the IRTG 2172 PRoTECT program of the G\u00f6ttingen Graduate School GGNB.Research in the Djamei laboratory on biotrophy and immunity in plants is done with the financial support of theIPK Gatersleben (Germany), the European Research Council under the European Union\u2019s Seventh FrameworkProgram (FP7\/2007-2013)\/ERC grant agreement no [GA335691 \u2018Effectomics\u2019], the Austrian Science Fund (FWF):[I 3033-B22, P27818-B22] and the Austrian Academy of Sciences (OEAW).Acknowledgments: J.K. is a fellow of the CIFAR program: the Fungal Kingdom, Threats & Opportunities, and aBurroughs Wellcome Fund Scholar in Molecular Pathogenic Mycology.Conflicts of Interest: The authors declare no conflict of interest.References1. Sadali, N.M.; Sowden, R.G.; Ling, Q.; Jarvis, R.P. Differentiation of chromoplasts and other plastids in plants.Plant Cell Rep. 2019, 38, 803\u2013818. [CrossRef]2. Jones, J.D.; Dangl, J.L. The plant immune system. Nature 2006, 444, 323\u2013329. [CrossRef] [PubMed]3. Trotta, A.; Rahikainen, M.; Konert, G.; Finazzi, G.; Kangasj\u00e4rvi, S. Signalling crosstalk in light stress andimmune reactions in plants. Philos. Trans. R. Soc. B Biol. Sci. 2014, 369, 20130235. [CrossRef] [PubMed]4. Sowden, R.G.; Watson, S.J.; Jarvis, P. The role of chloroplasts in plant pathology. Essays Biochem. 2018, 62,21\u201339. [PubMed]5. Lu, Y.; Yao, J. Chloroplasts at the crossroad of photosynthesis, pathogen infection and plant defense. Int. J.Mol. Sci. 2018, 19, 3900. [CrossRef] [PubMed]6. Nomura, H.; Komori, T.; Uemura, S.; Kanda, Y.; Shimotani, K.; Nakai, K.; Furuichi, T.; Takebayashi, K.;Sugimoto, T.; Sano, S.; et al. Chloroplast-mediated activation of plant immune signalling in Arabidopsis. Nat.Commun. 2012, 3, 926. [CrossRef] [PubMed]7. De Torres Zabala, M.; Littlejohn, G.; Jayaraman, S.; Studholme, D.; Bailey, T.; Lawson, T.; Tillich, M.; Licht, D.;B\u00f6lter, B.; Delfino, L.; et al. Chloroplasts play a central role in plant defence and are targeted by pathogeneffectors. Nat. Plants 2015, 1, 15074. [CrossRef]8. Serrano, I.; Audran, C.; Rivas, S. Chloroplasts at work during plant innate immunity. J. Exp. Bot. 2016, 67,3845\u20133854. [CrossRef]9. Delprato, M.L.; Krapp, A.R.; Carrillo, N. Green light to plant responses to pathogens: The role of chloroplastlight-dependent signaling in biotic stress. Photochem. Photobiol. 2015, 91, 1004\u20131011. [CrossRef]10. Su, J.; Yang, L.; Zhu, Q.; Wu, H.; He, Y.; Liu, Y.; Xu, J.; Jiang, D.; Zhang, S. Active photosynthetic inhibitionmediated by MPK3\/MPK6 is critical to effector-triggered immunity. PLoS Biol. 2018, 16, e2004122. [CrossRef]Pathogens 2020, 9, 19 12 of 1611. Bhattacharyya, D.; Chakraborty, S. Chloroplast: The trojan horse in plant-virus interaction. Mol. Plant Pathol.2018, 19, 504\u2013518. [CrossRef] [PubMed]12. Liu, Y.; Lan, X.; Song, S.; Yin, L.; Dry, I.B.; Qu, J.; Xiang, J.; Lu, J. In planta functional analysis and subcellularlocalization of the oomycete pathogen Plasmopara viticola candidate RXLR effector repertoire. Front. Plant Sci.2018, 9, 286. [CrossRef] [PubMed]13. Wang, W.; Jiao, F. Effectors of Phytophthora pathogens are powerful weapons for manipulating host immunity.Planta 2019, 250, 413\u2013425. [CrossRef] [PubMed]14. Franceschetti, M.; Maqbool, A.; Jim\u00e9nez-Dalmaroni, M.J.; Pennington, H.G.; Kamoun, S.; Banfield, M.J.Effectors of filamentous plant pathogens: Commonalities amid diversity. Microbiol. Mol. Biol. Rev. 2017, 81,e00066-16. [CrossRef]15. Rekhter, D.; L\u00fcdke, D.; Ding, Y.; Feussner, K.; Zienkiewicz, K.; Lipka, V.; Wiermer, M.; Zhang, Y.; Feussner, I.Isochorismate-derived biosynthesis of the plant stress hormone salicylic acid. Science 2019, 365, 498\u2013502.[CrossRef]16. Qi, G.; Chen, J.; Chang, M.; Chen, H.; Hall, K.; Korin, J.; Liu, F.; Wang, D.; Fu, Z.Q. Pandemonium breaks out:Disruption of salicylic acid-mediated defense by plant pathogens. Mol. Plant 2018, 11, 1427\u20131439. [CrossRef]17. Stael, S.; Kmiecik, P.; Willems, P.; Van Der Kelen, K.; Coll, N.S.; Teige, M.; Van Breusegem, F. Plant innateimmunity\u2014Sunny side up? Trends Plant Sci. 2015, 20, 3\u201311. [CrossRef]18. Chan, K.X.; Phua, S.Y.; Crisp, P.; McQuinn, R.; Pogson, B.J. Learning the languages of the chloroplast:Retrograde signaling and beyond. Annu. Rev. Plant Biol. 2016, 67, 25\u201353. [CrossRef]19. Koussevitzky, S.; Nott, A.; Mockler, T.C.; Hong, F.; Sachetto-Martins, G.; Surpin, M.; Lim, J.; Mittler, R.;Chory, J. Signals from chloroplasts converge to regulate nuclear gene expression. Science 2007, 316, 715\u2013719.[CrossRef]20. Lorrain, S.; Vailleau, F.; Balagu\u00e9, C.; Roby, D. Lesion mimic mutants: Keys for deciphering cell death anddefense pathways in plants? Trends Plant Sci. 2003, 8, 263\u2013271. [CrossRef]21. Caplan, J.L.; Kumar, A.S.; Park, E.; Padmanabhan, M.S.; Hoban, K.; Modla, S.; Czymmek, K.;Dinesh-Kumar, S.P. Chloroplast stromules function during innate immunity. Dev. Cell 2015, 34, 45\u201357.[CrossRef] [PubMed]22. Park, E.; Nedo, A.; Caplan, J.L.; Dinesh-Kumar, S.P. Plant-microbe interactions: Organelles and thecytoskeleton in action. New Phytol. 2018, 217, 1012\u20131028. [CrossRef] [PubMed]23. Park, E.; Caplan, J.L.; Dinesh-Kumar, S.P. Dynamic coordination of plastid morphological change bycytoskeleton for chloroplast-nucleus communication during plant immune responses. Plant Signal Behav.2018, 13, e1500064. [CrossRef]24. Kumar, A.S.; Park, E.; Nedo, A.; Alqarni, A.; Ren, L.; Hoban, K.; Modla, S.; McDonald, J.H.; Kambhamettu, C.;Dinesh-Kumar, S.P.; et al. Stromule extension along microtubules coordinated with actin-mediated anchoringguides perinuclear chloroplast movement during innate immunity. eLife 2018, 7, e23625. [CrossRef]25. Hanson, M.R.; Hines, K.M. Stromules: Probing formation and function. Plant Physiol. 2018, 176, 128\u2013137.[CrossRef] [PubMed]26. Ishiga, Y.; Watanabe, M.; Ishiga, T.; Tohge, T.; Matsuura, T.; Ikeda, Y.; Hoefgen, R.; Fernie, A.R.; Mysore, K.S.The SAL-PAP chloroplast retrograde pathway contributes to plant immunity by regulating glucosinolatepathway and phytohormone signaling. Mol. Plant Microbe Interact. 2017, 30, 829\u2013841. [CrossRef] [PubMed]27. Susek, R.E.; Ausubel, F.M.; Chory, J. Signal transduction mutants of arabidopsis uncouple nuclear CAB andRBCS gene expression from chloroplast development. Cell 1993, 74, 787\u2013799. [CrossRef]28. Pesaresi, P.; Kim, C. Current understanding of GUN1: A key mediator involved in biogenic retrogradesignaling. Plant Cell Rep. 2019, 38, 819\u2013823. [CrossRef]29. Fukui, K.; Kuramitsu, S. Structure and function of the small MutS-related domain. Mol. Biol. Int. 2011, 2011,691735. [CrossRef]30. Mochizuki, N.; Susek, R.; Chory, J. An intracellular signal transduction pathway between the chloroplast andnucleus is involved in de-etiolation. Plant Physiol. 1996, 112, 1465\u20131469. [CrossRef]31. Colombo, M.; Tadini, L.; Peracchio, C.; Ferrari, R.; Pesaresi, P. GUN1, a Jack-Of-All-Trades in ChloroplastProtein Homeostasis and Signaling. Front. Plant Sci. 2016, 7, 1427. [CrossRef] [PubMed]32. Ghosh, S.; Kanwar, P.; Jha, G. Alterations in rice chloroplast integrity, photosynthesis and metabolomeassociated with pathogenesis of Rhizoctonia solani. Sci. Rep. 2017, 7, 41610. [CrossRef] [PubMed]Pathogens 2020, 9, 19 13 of 1633. Ding, X.; Jimenez-Gongora, T.; Krenz, B.; Lozano-Duran, R. Chloroplast clustering around the nucleus is ageneral response to pathogen perception in Nicotiana benthamiana. Mol. Plant Pathol. 2019, 20, 1298\u20131306.[CrossRef] [PubMed]34. Rossi, F.R.; Krapp, A.R.; Bisaro, F.; Maiale, S.J.; Pieckenstain, F.L.; Carrillo, N. Reactive oxygen speciesgenerated in chloroplasts contribute to tobacco leaf infection by the necrotrophic fungus Botrytis cinerea.Plant J. 2017, 92, 761\u2013773. [CrossRef] [PubMed]35. Zurbriggen, M.D.; Carrillo, N.; Tognetti, V.B.; Melzer, M.; Peisker, M.; Hause, B.; Hajirezaei, M.R.Chloroplast-generated reactive oxygen species play a major role in localized cell death during the non-hostinteraction between tobacco and Xanthomonas campestris pv. vesicatoria. Plant J. 2009, 60, 962\u2013973. [CrossRef][PubMed]36. McCormick, S. Chloroplast-targeted antioxidant protein protects against necrotrophic fungal attack. Plant J.2017, 92, 759\u2013760. [CrossRef]37. Cela, J.; Tweed, J.K.S.; Sivakumaran, A.; Lee, M.R.F.; Mur, L.A.J.; Munn\u00e9-Bosch, S. An altered tocopherolcomposition in chloroplasts reduces plant resistance to Botrytis cinerea. Plant Physiol. Biochem. 2018, 127,200\u2013210. [CrossRef]38. Gou, J.Y.; Li, K.; Wu, K.; Wang, X.; Lin, H.; Cantu, D.; Uauy, C.; Dobon-Alonso, A.; Midorikawa, T.; Inoue, K.;et al. Wheat stripe rust resistance protein WKS1 reduces the ability of the thylakoid-associated ascorbateperoxidase to detoxify reactive oxygen species. Plant Cell 2015, 27, 1755\u20131770. [CrossRef]39. Wang, M.; Rui, L.; Yan, H.; Shi, H.; Zhao, W.; Lin, J.E.; Zhang, K.; Blakeslee, J.J.; Mackey, D.; Tang, D.; et al.The major leaf ferredoxin Fd2 regulates plant innate immunity in Arabidopsis. Mol. Plant Pathol. 2018, 19,1377\u20131390. [CrossRef]40. Liu, M.; Zhang, S.; Hu, J.; Sun, W.; Padilla, J.; He, Y.; Li, Y.; Yin, Z.; Liu, X.; Wang, W.; et al.Phosphorylation-guarded light-harvesting complex II contributes to broad-spectrum blast resistance in rice.Proc. Natl. Acad. Sci. USA 2019, 116, 17572\u201317577. [CrossRef]41. Akimoto-Tomiyama, C.; Tanabe, S.; Kajiwara, H.; Minami, E.; Ochiai, H. Loss of chloroplast-localizedprotein phosphatase 2Cs in Arabidopsis thaliana leads to enhancement of plant immunity and resistance toXanthomonas campestris pv. campestris infection. Mol. Plant Pathol. 2018, 19, 1184\u20131195. [CrossRef] [PubMed]42. Lee, W.S.; Devonshire, B.J.; Hammond-Kosack, K.E.; Rudd, J.J.; Kanyuka, K. Deregulation of plant cell deaththrough disruption of chloroplast functionality affects asexual sporulation of Zymoseptoria tritici on wheat.Mol. Plant Microbe Interact. 2015, 28, 590\u2013604. [CrossRef] [PubMed]43. Hu, G.; Yalpani, N.; Briggs, S.P.; Johal, G.S. A porphyrin pathway impairment is responsible for the phenotypeof a dominant disease lesion mimic mutant of maize. Plant Cell 1998, 10, 1095\u20131105. [CrossRef] [PubMed]44. Balint-Kurti, P. The plant hypersensitive response: Concepts, control and consequences. Mol. Plant Pathol.2019, 20, 1163\u20131178. [CrossRef] [PubMed]45. Tang, C.; Wang, X.; Duan, X.; Wang, X.; Huang, L.; Kang, Z. Functions of the lethal leaf-spot 1 gene in wheatcell death and disease tolerance to Puccinia striiformis. J. Exp. Bot. 2013, 64, 2955\u20132969. [CrossRef] [PubMed]46. Kretschmer, M.; Croll, D.; Kronstad, J.W. Maize susceptibility to Ustilago maydis is influenced by genetic andchemical perturbation of carbohydrate allocation. Mol. Plant Pathol. 2017, 18, 1222\u20131237. [CrossRef]47. Kretschmer, M.; Croll, D.; Kronstad, J.W. Chloroplast-associated metabolic functions influence thesusceptibility of maize to Ustilago maydis. Mol. Plant Pathol. 2017, 18, 1210\u20131221. [CrossRef]48. Djamei, A.; Schipper, K.; Rabe, F.; Ghosh, A.; Vincon, V.; Kahnt, J.; Osorio, S.; Tohge, T.; Fernie, A.R.;Feussner, I.; et al. Metabolic priming by a secreted fungal effector. Nature 2011, 478, 395\u2013398. [CrossRef]49. Han, X.; Altegoer, F.; Steinchen, W.; Binnebesel, L.; Schuhmacher, J.; Glatter, T.; Giammarinaro, P.I.; Djamei, A.;Rensing, S.A.; Reissmann, S.; et al. A kiwellin disarms the metabolic activity of a secreted fungal virulencefactor. Nature 2019, 565, 650\u2013653. [CrossRef]50. Bange, G.; Altegoer, F. Plants strike back: Kiwellin proteins as a modular toolbox for plant defense mechanisms.Commun. Integr. Biol. 2019, 12, 31\u201333. [CrossRef]51. Wildermuth, M. Plants fight fungi using kiwellin proteins. Nature 2019, 565, 575\u2013577. [CrossRef] [PubMed]52. Pogorelko, G.V.; Kambakam, S.; Nolan, T.; Foudree, A.; Zabotina, O.A.; Rodermel, S.R. Impaired chloroplastbiogenesis in immutans, an Arabidopsis variegation mutant, modifies developmental programming, cell wallcomposition and resistance to Pseudomonas syringae. PLoS ONE 2016, 11, e0150983. [CrossRef] [PubMed]53. Walters, D.R.; McRoberts, N.; Fitt, B.D. Are green islands red herrings? Significance of green islands in plantinteractions with pathogens and pests. Biol. Rev. 2008, 83, 79\u2013102. [CrossRef] [PubMed]Pathogens 2020, 9, 19 14 of 1654. Coghlan, S.E.; Walters, D.R. Photosynthesis in green islands on powdery mildew infected barley leaves.Phys. Mol. Plant Pathol. 1992, 40, 31\u201338. [CrossRef]55. Rozpa\u02dbdek, P.; We\u02dbz\u02d9owicz, K.; Nosek, M.; Waz\u02d9ny, R.; Tokarz, K.; Lembicz, M.; Miszalski, Z.; Turnau, K. Thefungal endophyte Epichlo\u00eb typhina improves photosynthesis efficiency of its host orchard grass (Dactylisglomerata). Planta 2015, 242, 1025\u20131035. [CrossRef] [PubMed]56. Ye, L.; Zhao, X.; Bao, E.; Cao, K.; Zou, Z. Effects of arbuscular mycorrhizal fungi on watermelon growth,elemental uptake, antioxidant, and photosystem II activities and stress-response gene expressions undersalinity-alkalinity stresses. Front. Plant Sci. 2019, 10, 863. [CrossRef]57. Imaizumi-Anraku, H.; Takeda, N.; Charpentier, M.; Perry, J.; Miwa, H.; Umehara, Y.; Kouchi, H.; Murakami, Y.;Mulder, L.; Vickers, K.; et al. Plastid proteins crucial for symbiotic fungal and bacterial entry into plant roots.Nature 2005, 433, 527\u2013531. [CrossRef]58. Fan, X.; Chang, W.; Feng, F.; Song, F. Responses of photosynthesis-related parameters and chloroplastultrastructure to atrazine in alfalfa (Medicago sativa L.) inoculated with arbuscular mycorrhizal fungi.Ecotoxicol. Environ. Saf. 2018, 166, 102\u2013108. [CrossRef]59. Mac\u00edas-Rubalcava, M.L.; Garc\u00eda-M\u00e9ndez, M.C.; King-D\u00edaz, B.; Mac\u00edas-Ruvalcaba, N.A. Effect of phytotoxicsecondary metabolites and semisynthetic compounds from endophytic fungus Xylaria feejeensis strainSM3e-1b on spinach chloroplast photosynthesis. J. Photochem. Photobiol. B 2017, 166, 35\u201343. [CrossRef]60. Ameztoy, K.; Baslam, M.; S\u00e1nchez-L\u00f3pez, \u00c1.M.; Mu\u00f1oz, F.J.; Bahaji, A.; Almagro, G.; Garc\u00eda-G\u00f3mez, P.;Baroja-Fern\u00e1ndez, E.; De Diego, N.; Humpl\u00edk, J.F.; et al. Plant responses to fungal volatiles involve globalposttranslational thiol redox proteome changes that affect photosynthesis. Plant Cell Environ. 2019, 42,2627\u20132644. [CrossRef]61. Djamei, A.; Kahmann, R. Ustilago maydis: Dissecting the molecular interface between pathogen and plant.PLoS Pathog. 2012, 8, e1002955. [CrossRef]62. Wang, S.; Boevink, P.C.; Welsh, L.; Zhang, R.; Whisson, S.C.; Birch, P.R.J. Delivery of cytoplasmic andapoplastic effectors from Phytophthora infestans haustoria by distinct secretion pathways. New Phytol. 2017,216, 205\u2013215. [CrossRef] [PubMed]63. Lanver, D.; Tollot, M.; Schweizer, G.; Lo Presti, L.; Reissmann, S.; Ma, L.-S.; Ma, L.S.; Schuster, M.; Tanaka, S.;Liang, L.; et al. Ustilago maydis effectors and their impact on virulence. Nat. Rev. Microbiol. 2017, 15, 409\u2013421.[CrossRef] [PubMed]64. Uhse, S.; Djamei, A. Effectors of plant-colonizing fungi and beyond. PLoS Pathog. 2018, 14, e1006992.[CrossRef]65. Han, X.; Kahmann, R. Manipulation of phytohormone pathways by effectors of filamentous plant pathogens.Front. Plant Sci. 2019, 10, 822. [CrossRef]66. Toru\u00f1o, T.; Stergiopoulos, I.; Coaker, G. Plant pathogen effectors: Cellular probes interfering with plantdefenses in spatial and temporal manners. Annu. Rev. Phytopathol. 2016, 54, 419\u2013441. [CrossRef]67. Petit-Houdenot, Y.; Fudal, I. Complex interactions between fungal avirulence genes and their correspondingplant resistance genes and consequences for disease resistance management. Front. Plant Sci. 2017, 8, 1072.[CrossRef] [PubMed]68. Li, G.; Froehlich, J.E.; Elowsky, C.; Msanne, J.; Ostosh, A.C.; Zhang, C.; Awada, T.; Alfano, J.R. DistinctPseudomonas type-III effectors use a cleavable transit peptide to target chloroplasts. Plant J. 2014, 77, 310\u2013321.[CrossRef]69. Nakano, M.; Mukaihara, T. Ralstonia solanacearum type III effector RipAL targets chloroplasts and inducesjasmonic acid production to suppress salicylic acid-mediated defense responses in plants. Plant Cell Physiol.2018, 59, 2576\u20132589. [CrossRef]70. Mansfield, J.; Genin, S.; Magori, S.; Citovsky, V.; Sriariyanun, M.; Ronald, P.; Dow, M.; Verdier, V.; Beer, S.;Machado, M.; et al. Top 10 plant pathogenic bacteria in molecular plant pathology. Mol. Plant Pathol. 2012,13, 614\u2013629. [CrossRef]71. Xin, X.-F.; He, S.Y. Pseudomonas syringae pv. Tomato DC3000: A model pathogen for probing diseasesusceptibility and hormone signaling in plants. Annu. Rev. Phytopathol. 2013, 51, 473\u2013498. [CrossRef][PubMed]72. Jelenska, J.; Yao, N.; Vinatzer, B.A.; Wright, C.M.; Brodsky, J.L.; Greenberg, J.T. A J domain virulence effectorof Pseudomonas syringae remodels host chloroplasts and suppresses defenses. Curr. Biol. 2007, 17, 499\u2013508.[CrossRef] [PubMed]Pathogens 2020, 9, 19 15 of 1673. Jelenska, J.; van Hal, J.A.; Greenberg, J.T. Pseudomonas syringae hijacks plant stress chaperone machinery forvirulence. Proc. Natl. Acad. Sci. USA 2010, 107, 13177\u201313182. [CrossRef] [PubMed]74. Rodr\u00edguez-Herva, J.J.; Gonz\u00e1lez-Melendi, P.; Cuartas-Lanza, R.; Ant\u00fanez-Lamas, M.; R\u00edo-Alvarez, I.; Li, Z.;L\u00f3pez-Torrej\u00f3n, G.; D\u00edaz, I.; Del Pozo, J.C.; Chakravarthy, S.; et al. A bacterial cysteine protease effectorprotein interferes with photosynthesis to suppress plant innate immune responses. Cell. Microbiol. 2012, 14,669\u2013681. [CrossRef]75. L\u00f3pez-Solanilla, E.; Bronstein, P.A.; Schneider, A.R.; Collmer, A. HopPtoN is a Pseudomonas syringae Hrp(type III secretion system) cysteine protease effector that suppresses pathogen-induced necrosis associatedwith both compatible and incompatible plant interactions. Mol. Microbiol. 2004, 54, 353\u2013365. [CrossRef]76. Sohn, K.H.; Zhang, Y.; Jones, J.D.G. The Pseudomonas syringae effector protein, AvrRPS4, requires in plantaprocessing and the KRVY domain to function. Plant J. 2009, 57, 1079\u20131091. [CrossRef]77. Lopes, C.A.; Rossato, M. History and status of selected hosts of the Ralstonia solanacearum species complexcausing bacterial wilt in Brazil. Front. Microbiol. 2018, 9, 1228. [CrossRef]78. Dahal, A.; Chen, L.; Kiba, A.; Hikichi, Y.; Ohnishi, K. Chloroplastic proteins are targets for the RipG effectorsof Ralstonia solanacearum. Int. J. Environ. Technol. Sci. 2018, 5, 147\u2013156.79. Pitino, M.; Allen, V.; Duan, Y. Las\u22065315 effector induces extreme starch accumulation and chlorosis as Ca.Liberibacter asiaticus infection in Nicotiana benthamiana. Front. Plant Sci. 2018, 9, 113. [CrossRef]80. Asselin, J.E.; Lin, J.; Perez-Quintero, A.L.; Gentzel, I.; Majerczak, D.; Opiyo, S.O.; Zhao, W.; Paek, S.M.;Kim, M.G.; Coplin, D.L.; et al. Perturbation of maize phenylpropanoid metabolism by an AvrE family typeIII effector from Pantoea stewartii. Plant Physiol. 2015, 167, 1117\u20131135. [CrossRef]81. Manning, V.A.; Chu, A.L.; Steeves, J.E.; Wolpert, T.J.; Ciuffetti, L.M. A host-selective toxin of Pyrenophoratritici-repentis, Ptr ToxA, induces photosystem changes and reactive oxygen species accumulation in sensitivewheat. Mol. Plant Microbe Interact. 2009, 22, 665\u2013676. [CrossRef] [PubMed]82. Manning, V.A.; Chu, A.L.; Scofield, S.R.; Ciuffetti, L.M. Intracellular expression of a host-selective toxin,ToxA, in diverse plants phenocopies silencing of a ToxA-interacting protein, ToxABP1. New Phytol. 2010, 187,1034\u20131047. [CrossRef] [PubMed]83. Ciuffetti, L.M.; Manning, V.A.; Pandelova, I.; Betts, M.F.; Martinez, J.P. Host-selective toxins, Ptr ToxA andPtr ToxB, as necrotrophic effectors in the Pyrenophora tritici-repentis-wheat interaction. New Phytol. 2010, 187,911\u2013919. [CrossRef] [PubMed]84. Persoons, A.; Morin, E.; Delaruelle, C.; Payen, T.; Halkett, F.; Frey, P.; De Mita, S.; Duplessis, S. Patterns ofgenomic variation in the poplar rust fungus Melampsora larici-populina identify pathogenesis-related factors.Front. Plant Sci. 2014, 5, 450. [CrossRef]85. Lorrain, C.; Hecker, A.; Duplessis, S. Effector-mining in the Poplar rust fungus Melampsora larici-populinasecretome. Front. Plant Sci. 2015, 6, 1051. [CrossRef]86. Lorrain, C.; Petre, B.; Duplessis, S. Show me the way: Rust effector targets in heterologous plant systems.Curr. Opin. Microbiol. 2018, 46, 19\u201325. [CrossRef]87. Lorrain, C.; Dos Santos, K.C.G.; Germain, H.; Hecker, A.; Duplessis, S. Advances in understanding obligatebiotrophy in rust fungi. New Phytol. 2019, 222, 1190\u20131206. [CrossRef]88. Petre, B.; Saunders, D.G.; Sklenar, J.; Lorrain, C.; Win, J.; Duplessis, S.; Kamoun, S. Candidate effector proteinsof the rust pathogen Melampsora larici-populina target diverse plant cell compartments. Mol. Plant MicrobeInteract. 2015, 28, 689\u2013700. [CrossRef]89. Petre, B.; Lorrain, C.; Saunders, D.G.O.; Win, J.; Sklenar, J.; Duplessis, S.; Kamoun, S. Rust fungal effectorsmimic host transit peptides to translocate into chloroplasts. Cell. Microbiol. 2016, 18, 453\u2013465. [CrossRef]90. Germain, H.; Joly, D.L.; Mireault, C.; Plourde, M.B.; Letanneur, C.; Stewart, D.; Morency, M.J.; Petre, B.;Duplessis, S.; S\u00e9guin, A. Infection assays in Arabidopsis reveal candidate effectors from the poplar rustfungus that promote susceptibility to bacteria and oomycete pathogens. Mol. Plant Pathol. 2018, 19, 191\u2013200.[CrossRef]91. Selin, C.; de Kievit, T.R.; Belmonte, M.F.; Fernando, W.G. Elucidating the role of effectors in plant-fungalinteractions: Progress and challenges. Front. Microbiol. 2016, 7, 600. [CrossRef] [PubMed]92. Zhang, S.; Xu, J.-R. Effectors and effector delivery in Magnaporthe oryzae. PLoS Pathog. 2014, 10, e1003826.[CrossRef] [PubMed]Pathogens 2020, 9, 19 16 of 1693. Sperschneider, J.; Catanzariti, A.M.; DeBoer, K.; Petre, B.; Gardiner, D.M.; Singh, K.B.; Dodds, P.N.; Taylor, J.M.LOCALIZER: Subcellular localization prediction of both plant and effector proteins in the plant cell. Sci. Rep.2017, 7, 44598. [CrossRef] [PubMed]94. Sperschneider, J.; Gardiner, D.M.; Dodds, P.N.; Tini, F.; Covarelli, L.; Singh, K.B.; Manners, J.M.; Taylor, J.M.EffectorP: Predicting fungal effector proteins from secretomes using machine learning. New Phytol. 2016, 210,743\u2013761. [CrossRef] [PubMed]\u00a9 2019 by the authors. Licensee MDPI, Basel, Switzerland. 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