The Identification Of Strain-specific Effectors With Immunity Suppression In Phytophthora Infestans Super-race CN152

The potato(Solanum tuberosum L.) is the fourth most imporatant food crop after maize, wheat and rice in the globe, also ranks as the fourth largest yield food crop in China, thus playing a particular important role in food and energy security strategy around the world. The potato late blight, regarded as “cancer in potato”, is caused by the oomycete pathogen Phytophthora infestans(Mont.) de Bary, which is the chief factor limiting the production of potato. The pathogen P.infestans possess a highly variable genome with the feature of accelerating adaptation to host plants. Further researches on effectors can provide crucial cues when studying pathogenesis of P.infestans, above all, to acquire a set of effectors with diverse and distinct functions can supply the further functional analysis of candidate effectors with a starting pointcuts.By proceeding the mRNA sequencing and comparative analysis of pre- and post-incubation on a typical super-race isolate(CN152) and universal race 0 isolate(89148-9), we made a comparative analysis on genes expression varied from pre-incubation to post- incubation in two isolates, respectively, to acquire potential virulence factors(effectors), then obtaining CN152 strain-specific effectors by comparison between these effectors of the two isolates, subsequently conducting a functional identification on these candidate effectors.The primary transcriptome analysis revealed both of those two isolates showed highly similarity to sequenced reference strain(T30-4) genome in genome sequences(≥97%). Meanwhile, we assembled the part unmapped to reference genome in a de novo manner, then conducted annotations on novel transcripts. The results suggested there just existed little differential on gene numbers, inferring that the huge gap between two isolates may focus on gene function. In consideration of the majority of pathogenic factors turned to be secretory proteins when studying Phytophthora, here we mainly focused on the differential within sequences and function characteristic in different aggressive isolates. We then found the Pfam families mainly distributed over protein kinase family, phosphoesterase family, cysteine-rich secretory protein family, carbohydrate enzymes family and repeat family by the domain anlysis, suggesting the secreted proteins may be involved in signal transduction during infection. We obtained 301 and 290 potential virulence factors(effectors) in isolate 89148-9 and CN152 on the basis of transcriptional expression of secreted proteins, respectively, within those 154 genes were both appeared in two isolates, hence, totally 437 candidate effectors were screening. And we treated the 136 candidate effectors only appeared in isolate CN152 as its particular candidate effectors. To narrow the scope of candidate effectors in CN152 for further functional identification, we finally acquired 31 candidate effectors that expressed differentially(P<0.05) both in pre- and post-incubation time points in two isolates, while only showed a two-folds upregulation in isolate CN152.A realtime PCR were conducted on candidate effector genes of interest and the upregulated expression results in planta showed these effector genes were involved in pathogenicity.The pEDV recombinant plasmid containing candidate effector genes of interest was constructed and transformed to Burkholderia glumae strain carrying Type III Secretion System(T3SS), and then injected into N. benthamiana leaf panels by high pressure infiltration to perform the transient expression in leaf cells. The results identified that candidate effector genes of interest enable suppress hypersensitive reaction in N. benthamiana, when taking gfp as negative control.