Multiple Candidate Effectors from the Oomycete Pathogen Hyaloperonospora arabidopsidis Suppress Host Plant Immunity

et al. (2011) Multiple Candidate Effectors from the Oomycete Pathogen Hyaloperonospora arabidopsidis Suppress Host Plant Immunity. PLoS Pathog 7(11): e1002348. doi:10.1371/journal.ppat.1002348 Multiple Candidate Effectors from the Oomycete Pathogen Hyaloperonospora arabidopsidis Suppress Host Plant Immunity Georgina Fabro Jens Steinbrenner Mary Coates Naveed Ishaque Laura Baxter David J. Studholme Evelyn Ko rner Rebecca L. Allen Sophie J. M. Piquerez Alejandra Rougon-Cardoso David Greenshields Rita Lei Jorge L. Badel Marie-Cecile Caillaud Kee-Hoon Sohn Guido Van den Ackerveken Jane E. Parker Jim Beynon Jonathan D. G. Jones Frederick M. Ausubel, Massachusetts General Hospital, Harvard Medical School, United States of America Oomycete pathogens cause diverse plant diseases. To successfully colonize their hosts, they deliver a suite of effector proteins that can attenuate plant defenses. In the oomycete downy mildews, effectors carry a signal peptide and an RxLR motif. Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on the model plant Arabidopsis thaliana (Arabidopsis). We investigated if candidate effectors predicted in the genome sequence of Hpa isolate Emoy2 (HaRxLs) were able to manipulate host defenses in different Arabidopsis accessions. We developed a rapid and sensitive screening method to test HaRxLs by delivering them via the bacterial type-three secretion system (TTSS) of Pseudomonas syringae pv tomato DC3000-LUX (Pst-LUX) and assessing changes in Pst-LUX growth in planta on 12 Arabidopsis accessions. The majority (,70%) of the 64 candidates tested positively contributed to Pst-LUX growth on more than one accession indicating that Hpa virulence likely involves multiple effectors with weak accession-specific effects. Further screening with a Pst mutant (DCEL) showed that HaRxLs that allow enhanced Pst-LUX growth usually suppress callose deposition, a hallmark of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). We found that HaRxLs are rarely strong avirulence determinants. Although some decreased Pst-LUX growth in particular accessions, none activated macroscopic cell death. Fewer HaRxLs conferred enhanced Pst growth on turnip, a non-host for Hpa, while several reduced it, consistent with the idea that turnip's non-host resistance against Hpa could involve a combination of recognized HaRxLs and ineffective HaRxLs. We verified our results by constitutively expressing in Arabidopsis a sub-set of HaRxLs. Several transgenic lines showed increased susceptibility to Hpa and attenuation of Arabidopsis PTI responses, confirming the HaRxLs' role in Hpa virulence. This study shows TTSS screening system provides a useful tool to test whether candidate effectors from eukaryotic pathogens can suppress/trigger plant defense mechanisms and to rank their effectiveness prior to subsequent mechanistic investigation. - Funding: This work was supported by the following grants: The ERA-PG Effectoromics project funded by the British Biotechnological and Biological Sciences Research Council (BBSRC), the German Research Foundation (Deutsche Forschungsgemeinshaft, JEP/DFG) and the Germany-Netherlands Genomics Initiative/ Netherlands Organization for Scientific Research (NGI/NOW) to GF, JS, MC, RLA, JEP, GV, JB and JDGJ. The HFSP grant RGP0057/20067-C to DG, JLB and JDGJ; The Gatsby Foundation GAT2545 to SJMP, AR, RL, DJS, EK and GF. The BBSRC grants BB/F0161901, BB/E024882/1 to NI and JDGJ; The BBSRC CASE studentship T12144 to NI and a Marie Curie early stage training program fellowship (019727) to SJMP. The EMBO ALTF 6142009 and Marie Curie FP7-PEOPLE-2009-IEF funded MCC. The BBSRC grants BB/E024815/1, BB/G015066/1, BB/F005806/1 supported JB. The funders had no role in experimental design, data collection and analysis, decision to publish or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Current address: CIQUIBIC-CONICET, Departamento de Qumica Biolo gica, Facultad de Ciencias Qumicas, Universidad Nacional de Co rdoba, C ordoba, Argentina Plants face constant attacks by a wide array of microorganisms including bacteria, fungi and oomycetes. Obligate biotrophic pathogens are particularly interesting because they can effectively evade or suppress host recognition, thus thwarting host defenses and enabling pathogen growth and reproduction [1]. In natural environments, plant disease is rare because plants activate a multilayered defense to most potential pathogens [2]. Relatively conserved molecules, called pathogen (or microbe)associated molecular patterns (PAMPs), are recognized by the plants via pattern recognition receptor proteins (PRRs) [3,4]. This interaction results in pattern-triggered immunity (PTI). Successful pathogens target effector proteins to the host cell cytoplasm to Hyaloperonospora arabidopsidis (Hpa) is an obligate biotroph whose population coevolves with its host, Arabidopsis thaliana. The Hpa isolate Emoy2 genome has been sequenced, allowing the discovery of dozens of secreted candidate effectors. We set out to assign functions to these candidate effectors, investigating if they suppress host defenses. We analyzed a sub-set of Hpa candidate effectors (HaRxLs) that carry the RxLR motif, using a bacterial system for in planta delivery. To our surprise, we found that most of the HaRxLs enhanced plant susceptibility on at least some accessions, while few decreased it. These phenotypes were mostly confirmed on Arabidopsis transgenic lines stably expressing HaRxLs that became more susceptible to compatible Hpa isolates. Furthermore, effectors that conferred enhanced virulence generally suppressed callose deposition, a hallmark of plant defense. This indicates that the effectorome of Hpa comprises multiple distinct effectors that can attenuate Arabidopsis immunity. We found that many HaRxLs did not confer enhanced virulence on all host accessions, and also that only ,50% of the effectors that conferred enhanced Pst growth on Arabidopsis, were able to do so on turnip, a non-host for Hpa. Our data reveal interesting HaRxLs for detailed mechanistic investigation in future experiments. suppress PTI [5]. To counteract this, plants have evolved a second line of defense comprising resistance (R) proteins that recognize particular effectors either directly or through their activities on plant targets. This recognition leads to effector-triggered immunity (ETI) [2,5]. It has been proposed that the effector repertoire of a given pathogen specifies its ability to infect a given host genotype [6,7,8]. Recent publications report many effector candidates predicted in the genomes of filamentous obligate biotrophs [9,10,11]. Comparison of effector sets of phylogenetically related species of obligate biotrophs that grow on different hosts reveals little overlap, suggesting host species-specific adaptation [10]. However, there are few studies about the functionality of obligate biot (...truncated)