Characterization Of RXLR Effector Pi23014 Of Phytophthora Infestans In Mediating Plant Susceptibility

Phytophthora infestans belongs to oomycetes,causes late blight of potato and tomato,and threatens sustainable crop production.Oomycetes are highly plastic in terms of genetic variation and virulence evolution,leading to frequent loss of host genotype-specific resistance and difficulties in disease control.The use of resistance genes and disease-resistant varieties is the most effective strategy in prevention and control of crop diseases.P.infestans genome encodes a large number of RXLR effectors（about 600）,which play important roles in both pathogenesis and in activating host genotype-specific disease resistance.Therefore,RXLR effectors have potential to understand the potato late blight resistance and identify important genetic resources for potato disease resistance breeding.In addition,understanding virulence mechanisms of RXLR effectors is the key to dissect the genetic basis of crop susceptibility,which has potential to develop novel resistance strategies.In this study,we analyzed 16 candidate core RXLR effectors of P.infestans on 6potato germplasm materials resistant to late blight,by transient gene expression mediated by Agrobacterium tumefaciens.The virulence function of effector Pi23014,which can activate potato genotype specific resistance response,was analyzed.Its host candidate targets were identified by liquid chromatography-tandem mass spectrometry（LC-MS/MS）.The main results are as follows:1.The RXLR effector Pi23014 of P.infestans promotes plant susceptibility by inhibiting plant PTI immune responses.Subcellular localization analysis revealed that Pi23014 was localized in the nucleus and chloroplasts.Pi23014 promotes susceptibility to disease by inhibiting PTI（Pathogen associated molecular pattern-induced immunity）immune responses in host plants.The results of A.tumefaciens-mediated transient overexpression and infection assays showed that the Pi23014 chloroplast localization deletion mutant _NLSPi23014-CGFP lost its ability to render plant susceptible to P.infestans infection,suggesting that the chloroplast localization was critical for its immune function.The results of gene expression and biochemical analysis showed that Pi23014 induced the down-regulation of photosystem II（PSII）related genes,inhibited PSII activity,and suppressed the photosynthetic efficiency.2.LC-MS/MS analysis led to the identification of Pi23014 target,RNA-binding protein Nb RBP3a.The interaction of Pi23014 with Nb RBP3a were confirmed by multiple assays,including Luciferase complementation imaging,Yeast two-hybrid,Bimolecular fluorescence complementation,and Co-Immunoprecipitation.These results indicate that Nb RBP3a is Pi23014 target.3.The expression of Nb RBP3a in Nicotiana benthamiana was induced by P.infestans as determined by RT-qPCR assay.The results of VIGS assays showed that the expression of WRKY7,WRKY8,PR1 and PR2 were significantly down-regulated in Nb RBP3-silenced plants under flg22 treatment compared with the control TRV-GFP silenced plants.A.tumefaciens-mediated transient overexpression of Nb RBP3a significantly enhanced plant resistance to P.infestans,indicating that Nb RBP3a is a positive regulator of plant immunity.The results of subcellular localization analysis showed that Pi23014 and RNA-binding protein Nb RBP3a co-localized in plant cell nucleus.Mutation analysis showed that the N-terminal RNA-recognition motif（RRM）of Nb RBP3a mediates its interaction with Pi23014.The N-terminal RRM motif deletion mutant Nb RBP3aΔN lost ability to positively regulate plant resistance to P.infestans,suggesting that RRM motif is involved in the immune function of Nb RBP3a.4.Gene silencing analyses showed that Nb RBP3a is required for PSII functionality.Nb RBP3-silenced plants reduced PSII activities compared to the TRV-GFP control plants,as indicated by the fluorescence parameters of photosystem II,the maximum quantum yield,Fv/Fm,electron transport rate（ETR）,non-photochemical quenching（NPQ）,PSII quantum Yield（Y（II））and the state of the QA electron acceptor of PSII（1-qP）.Notably,NbRBP3-silenced plants showed decreased accumulation of photosystem II（PSII）proteins.These results indicate that loss-of-function of Nb RBP3a led to chloroplast dysfunction.In conclusion,in the process of host plant infection by P.infestans,the core effector Pi23014,targets Nb RBP3a to inhibit plant immune function and PSII activity.Our results reveal a new mechanism by which pathogens inhibit the activation of plant defense by targeting host chloroplasts and inhibiting PSII activity during infection.Protecting PSII from the effects of pathogen effectors has the potential to be used to develop new strategies for plant disease resistance.