Functional Analysis Of The Apoplastic Effector SCR209 Of Phytophthora Capsici

The oomycete Phytophthora capsici Leonian is a significant broad-host-range plant pathogen that often attacks a variety of crops including solanaceous,cucurbit and legume.During the infection,Phytophthora can secret an array of effectors into plant apoplastic space or cells to interfere with the physiological function and suppress the immune defense reaction of plants in favor of the infection.Therefore,understanding the functions of Phytophthora effectors and elucidating the pathogenicity mechanism of Phytophthora is very important to control plant Phytophthora diseases.PcF/SCR effectors are one class of oomycete apoplastic effectors.However,their function during the infection is still unknown.In the present study,the functions of P.capsici SCR209 were analyzed.(1)Semi-quantitative RT-PCR was utilized to analyze the expression level of the PcF/SCR effector encoding gene scr209 during the pathogen development(mycelium,sporangium,zoospore and germinating cyst)and the stage of Nicotiana benthamiana infection(1.5,3,6,12,24,36,72 h after inoculation).It was found that scr209 was up-regulated during the stages of the pathogen development and N.benthamiana infection.(2)The gene was cloned into Potato virus X(PVX)expression vector using high-fidelity DNA polymerase.The Agrobacterium-mediated transient expression was conducted to observe whether the gene could cause plant cell death(PCD).The results revealed that SCR209 can induce cell death on N.benthamiana and tomato cultivar L402.(3)SCR209 was fused with mCherry red fluorescent protein and the localization of it during the infection was observed by.Agrobacterium-mediated transient expression in N.benthamiana.It turned out that SCR209 was found localized on plant plasma membrane.(4)To further analyze the function of the effector,gene silencing and CRISPR/Cas9 gene knockout were conducted in P.capsici to analyze whether the pathogenicity and biological characters of the pathogen were changed.The results showed that both scr209 silencing and knockout compromised the virulence of P.capsici on plants.Moreover,gene-silenced and gene-knockout transformants demonstrated reduced tolerance to exogenous oxidants(Hydrogen peroxide and Cumene hydroperoxide).All together,these results indicated that PcF/SCR effector SCR209 may play an important role during the plant infection by P.capsici and the protein may contribute to the pathogen virulence via responding the reactive oxygen species of the plants.In this study,the role of PcF/SCR effector SCR209 during the P.capsici infection was analyzed.The results provided significant experimental data for understanding the interaction mechanism in intercellular space between P.capcisi and plants,and set the basis for further analyzing the action mechanism of this class of effectors.