Identification Of Plant Targets And Functional Characterization Of Phytophthora Effectors

Phytophthora capsici is a wide host range plant pathogens that can cause a variety of diseases on crops..Phytophthora specie.s are fungus-like eukaryotic microorganisms classified as oomycetes that are phylogenetically distinct from true fungi.a.However,the pathogenicity mechanisms of Phytophthora are quite different from fungi.Genome sequencing showed that Phytophthora encodes alarge number of effectors,among which RxLRs and CRNs(Crinkler)are two kinds of cytoplasmic effectors.RxLR has a conserved N-terminal domain present in the oomycete Avr proteins.RxLR effectors can be divided into three parts based on the protein structure:the signal peptide,N-terminal RxLR-dEER translocation domain and C-terminal functional domain.It has been shown that the RxLR effectors can translocate into host plant cells and contribute to virulence.CRN effectors have high expression levels during infection,and have high levels of variation and control virulence.The CRNs contain a conserved LxLFLAK motif in their N-termini.Similar to the RxLR motif,the LxFLAK motif may becapable of mediating CRN effectors translocation into plant cells.Although the AVR proteins has been shown to cause plant defense responses when the corresponding resistance proteins are present.However,the virulence function and the mechanisms of most effectors are largely unkown.Screening a Y2H library:The Phytophthora effectors sRxLR22,CRN4,RxLR242,RxLR207 and Avrlbwere used as baits to screen the ArabidopsiscDNA libraryusing yeast two-hybrid approach.The prey plasmids of the primary positive clones were extracted and sequenced.The sequences of the preyswere searched in NCBI and annotated.Analyses of the interactions between each tested effectors and disease resistence-related proteinsfrom Arabidopsis:In this study,72 P.capsici effector genes including 34 CRNs and 38 RxLRs were separately inserted into bait vectorpGBKT7,and 11 Arabidospsis resistance-related genes(CMPG1?EDS1?BAK1?BIK1?OSD1?RIN4?PAD4?SGTla?SGTlb?NPR1 and NPR3)were cloned to prey vector pGADT7,repectively.The baits and preys were introduced into yeast using mating approach.The potential interactions were determined based on yeast growth on selection medium plates.The results showed that RxLR103 may interact with EDS 1,BAK1,BIK1 and NPR1 separately;and RxLR172 may interact with EDS1 and SGT1b;and RxLR48 may interact with NPR3 directly.Preparation of transgenic Arabidopsis thaliana:To study the effects of Phytophthora effector RxLR103,RxLR172,RxLR237 on plant cell function and physiology,we cloned these effector genes into the binary vector pART27,and transformed imtoAgrobacterium strain GV3101.The floral dip method was used to transform these effectors into Arabidopsis thaliana.Positive transgenic plants were screened using kanamycin selection.The RxLR103-transgenic Arabidopsis plants were small,and grow up slowly,and finally died.Transgenic plants expressing RxLRl 72 or RxLR237 grew normally.These transgenic plants provide useful materials for future functional characterization.Functional characterization of RxLR103:RxLR103 is an effector of Phytophthora capsici.The PVX expression vector and Agrobacterim-mediated transient expression in N.benthaminan were employed to analyze function of RxLR103.The results showed RxLR103 can cause cell death in plants.Subcellular localization assay showed RxLR103 distributed in both plant cytoplasm and nucleus.RT-PCR analysis showed that the gene was upregulated in the late stage of Phytophthora infection.The inoculation assay showed RxLR103 can promote Phytophthora infection.Taken together,our results indicatethat RxLR103 is a virulence effector,which maybe involved in transition from biotrophy to necrotrophy in the infection process.