Multiple candidate effectors from the oomycete pathogen Hyaloperonospora arabidopsidis suppress host plant immunity.

PublishedResearch Support, Non-U.S. Gov'tOomycete 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 (ΔCEL) 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.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 614–2009 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