Characterization And Functional Analysis Of Grapevine Downy Mildew RXLR Effectors

Plasmopara viticola,the causal agent of grapevine downy mildew,is an obligate brotrophic oomycete which belongs to family Peronosporaceae,order Peronosporales.Downy mildew is the most destructive disease of grapevine,causing tremendous economic loss in the grape and wine industry.Although different approaches such as intervarietal hybridization,induced polyploidy and genetic modification were used to improve the disease resistance of grapevine,and the downy mildew remains epidemic in cultivars.Therefore,elucidating the molecular mechanism of grapevine resistance and P.vitcola virulence could provide effective methods to control this pathogen.Pathogens always secret a series of low-molecular-weight proteins,named effectors,to modulate plant immune system.In previous study,102 RXLR effector genes(PvRXLRs)were identified in P.viticola isolate "JL-7-2" via RNA-Seq analysis and whole-genome sequencing.Here,we cloned 83 effector genes from P.viticola "JL-7-2" genome in total.Of the 83 cloned PvRXLR genes,45 were previously shown to be expressed in infected grapevine leaves by RNA-Seq analysis.To determine whether the remaining 38 candidate PvRXLR genes were transcribed,RT-PCR analysis was carried out on the total RNA extracted from "JL-7-2" infected grapevine leaves.The results confirmed that 33/38 could be amplified from cDNA.We transient expressed all the effectors in N.benthamiana leaves by using Agrobacterium-mediated method.The result showed that 76 effectors could observed informative fluorescence in planta,among which 34 effectors localized in nucleus and cytosol,29 effectors targeted to nucleus specifically,and 9 effectors target membrane system.Intriguingly,there are four effectors(PvRXLR54,61,86,and 161)target to chloroplasts,to the best of our knowledge,this is the first study to provide experimental evidence of oomycete effectors targeted to plant organelles.PvRXLR54,one of the chloroplast-targeting effectors,was selected to further study.Co-localization demonstrated that PvRXLR54 was dually targeted to chloroplasts and mitochondria.Western blotting result showed that PvRXLR54 had no cleavable N-terminal transit peptide,however,the exposed N-terminal is crucial to its trafficking in plant cells.Moreover,we found that PvRXLR54 could decrease the content of chlorophyll and inhibit photochemical reaction,ROS burst,and callose deposition in transgenic seedlings.By using yeast-two hybrid screen and co-precipitation/mass spectrometry,we identified a grapevine chlorophyll a-b binding protein(VvCBP151)interacted with PvRXLR54 directly.Our findings revealed that the effector probably inhibit the light energy absorption and transformation of VvCBP151,resulting in suppressing plant innate immunity.