Screening And Characterization Of Proteins Interacted With RxLR129113 Effector From Phytophthora Capsici In Capsicum Annuum

Phytophthora capsici is the casual agent of pepper blight,which is one of the most destructive diseases that harms vegetable industry around the world.P.capsici mainly invades the leaves,fruits and stems,causing soft rot and lodging,which may reduce production above50%.P.capsici belongs to oomycota,which deliver a variety of effectors into the host apoplast to promote infection by regulation plant defense system.Bioinformatics analysis of the genome sequence of P.capsici has identified 357 RxLR effectors,we know little about how fungal and oomycete effectors target host proteins to promote susceptibility,yet it is vital to understand plant disease.In this study,we selected Nicotiana benthamiana as the mode host,explored the interaction between the PcRxLR129113 and Capsicum annuum.The results are as follows:1.The agrobacterium-mediated transient expression method was used to analyze pathogenic phenotype of PcRxLR129113.Agrobacterium transient expression of PcRxLR129113 suppressed cell death induced by INF1 and Bax in N.benthamiana.Confocal microscope showed that PcRxLR129113 was localized in the cytoplasm and nucleus.2.Screening C.annuum cDNA library for target protein interacting with PcRxLR129113.First,the pepper cDNA plasmid library was successfully transformed into yeast strain Y187.The qualities of the cDNA libraries were evaluated as the followings:the titers were 1.13×10~6CFU/mL,the capacity of cDNA library was 1.7×10~7 CFU.Second,bait plasmids were successfully constructed and the auto-activation and toxicity of PcRxLR129113 was tested.The pepper cDNA yeast library was screened by the mating bait protein PcRxLR129113.Nine proteins were screened by Y2H,which were aldehyde dehydrogenase,bZIP transcription factor TGA1,V-type proton ATPase subunit,glyceraldehyde-3-phosphate dehydrogenase,actin-depolymerizing factor,ferredoxin,chlorophyll a-b binding protein and fructose-bisphosphate aldolase.Finally,Y2H illustrated that PcRxLR129113 interacted with ALDH and TGA1.3.Co-immunoprecipitation(co-IP)showed that PcRxLR129113 associated with ALDH in planta.The recombinant vectors PcRxLR129113 and ALDH7 were transiently co-expressed respectively in leaves of N.benthamiana.It was confirmed that ALDH7 interacted with PcRxLR129113 in vivo.4.Bimolecular fluorescence complementation(BiFC)illustrated an association between PcRxLR129113 and ALDH or TGA1 at the cytoplasm by confocal microscope.5.The key domain of PcRxLR129113,RxLRW1-3,was interacted with ALDH7 verified by yeast two-hybrid,BiFC,and co-IP assays;the interaction between RxLRW1-3 and TGA1was confirmed by yeast two-hybrid and BiFC assays.In conclusion,screening the host target protein of PcRxLR129113,studying how the target regulates the plant defense response during pathogen infection.This will lay the foundation for further exploration of the pathogenic mechanism,and provide a basis for the cultivation of resistant varieties and screeningof drug targets site.