Functions Of The Extracellular Effector SCR82 Of Phytophthora Capsici

The oomycete pathogen Phytophthora capsici often attacks hundreds of crops including solanaceous,cucurbit and legume,threatening the world's agriculture safety.During the infection,Phytophthora pathogens secrete a variety of effector proteins into plant apoplastic space or inside plant cells to interfere with the normal cellular activities,suppress or evade the immune defense of host plants.Therefore,it is essential to understand the effector functions and to elucidate the pathogenicity mechanism of Phytophthora pathogens in order to design better control strategies for Phytophthora blight diseases.PcF/SCR effectors are one group of oomycete apoplastic effectors.Until now,their roles in the plant infection are still indeterminate.In the present study,(1)to dissect if P.capsici PcF/SCR effector SCR82 has been under the host selection pressure during the evolution,the sequence polymorphism of SCR82 in 50 strains from different regions in China was analyzed.It was found that there are 10 different nucleotide sequences encoding 7 different amino acid sequences of SCR82,indicating that the effector has been subjected to the natural selection pressure during the evolution.These 7 different SCR82 homologs were transiently expressed in hot pepper,tomato and Nicotiana benthamiana through Agrobacterium tumefaciens infiltration and only one can induce visible cell death in hot pepper and tomato;(2)In order to further analyze the functions of SCR82,gene knockout by CRISPR/Cas9 was performed in P.capsici.The results showed that knockout of the scr82 gene significantly impaired the virulence of P.capsici on host plants,and led to increased sensitivity of P.capsici to exogenous active oxygen(Cumene hydroperoxide).However,reduction in virulence of gene knockout transformants in host plants could be restored by gene complementation.The results indicate that SCR82 plays an important role during the infection,which may function through responding to the active oxygen defense in plants;(3)In order to understand if SCR82 affects plant resistance during the infection,real-time RT-PCR were used to analyze the expression of resistance-related genes when SCR82 was expressed in N.benthamiana.The results showed that SCR82 can stimulate the expression of resistance,active oxygen-related and cell death-related genes during the infection process;(4)In order to identify what plant proteins are possibly the targets of SCR82,the gene transcript changes of plant receptor-like kinases were checked when N.benthamiana was inoculated with the gene knockout transformant compared with the inoculation with the wild type isolate.It turned out that two genes encoding LRR receptor-like kinase and RKF3-like receptor-like kinase were responsive to the effector,implying that they may be the potential targets of SCR82.In the study,the functions of PcF/SCR effector SCR82 during the P.capsici infection were analyzed.The primary results provide important experimental data for further understanding the action mode of the PcF/SCR proteins,and lay a foundation for dissection of the mechanism how the extracellular effectors contribute to the pathogen virulence.