Functional Characterization And Mechanism Exploration Of A Phytophthora Sojae Effector PsCRN63 To Regulate Plant Innate Immunity And Cell Death

Plant innate immunity is activated upon the perception of conserved Pathogen-Associated Molecular Patterns (PAMPs) or diversified pathogen effector proteins.Together, PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI) constitute two kinds of effective defenses against various pathogens infection. Virulent pathogens can produce plenty of effectors, which work either outside the plant cell or inside the intracellular space when delivered into host cell. The major objective of effectors is to facilitate growth and reproduction by inhibiting host immunity, leading to successful infection. But the biochemical function and molecular mechanism remains unknown for the vast majority of these effector proteins. Therefore, identification of function and molecular mechanism of these effectors during pathogenic processes will help us to know more about the virulence mechanisms of pathogens and plant immune mechanism.The genus Phytophthora contains many typical plant pathogens, among which P.infestans for potato late blight and P. sojae for soybean root rot are two of the most notorious species, causing devastating loss to agricultural production every year worldwide.P. sojae is an oomycete pathogen, although it is similar to fungi morphologically,it has a close relationship to diatoms and cyanobacteria by phylogenetic lineage analysis. At this stage, chemical fungicide applied in fungi are usually ineffective in controlling the diseases caused by P.sojae and other oomycete pathogens. Among the oomycete intracellular effectors, the RXLR (R represents arginine, L represents leucine and X is any amino acid)and CRN (crinkler or crinkling- and necrosis-inducing protein) effectors are two utmost important groups. These effectors are modular proteins; their N-terminal are conserved and responsible for delivering proteins into hosts plant cells, while the C-terminal parts are relatively diverse and function inside host cells to manipulate plant immunity responses. It is usually difficult to predict their functions and mechanisms because of a lack of sequence similarity to known proteins.We analyzed the function of a Phytophthora sojae intracellular effector, PsCRN63,then explored its possible toxicity mechanisms, the main results and conclusions obtained are as follows:A Phytophthora sojae effector PsCRN63 regulate plant cell death via interactions with plant catalases. We have shown previously that P. sojae PsCRN63 (for crinkling- and necrosis-inducing proteins) induces programmed cell death (PCD) while PsCRN115 blocks PCD in planta; however, they are jointly required for full pathogenesis. Here, we find that PsCRN63 alone or PsCRN63 and PsCRN115 together might suppress the immune responses of Nicotiana benthamiana and demonstrate that these two cytoplasmic effectors interact with catalases from N. benthamiana and soybean (Glycine max). Also, NbCAT1(for N. benthamiana CATALASE 1) and GmCAT1 are destabilized when PsCRN63 is co-expressed, and PsCRN115 inhibits the processes. Transient expression of PsCRN63 increases hydrogen peroxide (H2O2) accumulation,whereas PsCRN115 suppresses this process. At last, we find that transient overexpression of NbCAT1 or GmCAT1 specifically alleviates PsCRN63-induced PCD. Thus, PsCRN63/115 manipulates plant PCD through interfering with catalases and perturbing H2O2 homeostasis. Taken together, we suggest that P. sojae secretes these two effectors to regulate plant PCD and H2O2 homeostasis through direct interaction with catalases and, therefore, overcome host immune responses.A Phytophthora sojae effector PsCRN63 forms dimers in plant cell to suppress plant immunity. In this study, we find that a P. sojae effector, PsCRN63,suppresses flg22-induced expression of FRK1 gene,a molecular marker in pathogen-associated molecular patterns (PAMP)-triggered immunity (PTI). However, PsCRN63 does not suppress upstream signaling events including flg22-induced MAPK activation and BIK1 phosphorylation,indicating that it acts downstream of MAPK cascades. The PsCRN63-transgenic Arbidopsis plants showed increased susceptibility to bacterial pathogen Pseudomonas syringae pathovar tomato (Pst) DC3000 and oomycete pathogen Phytophthora capsici.Flg22-induced oxidative burst and callose deposition were suppressed in PsCRN63-transgenic plants compared with the wild-type control plants.Genes involved in PAMP-triggered immunity (PTI) were also down-regulated in PsCRN63-transgenic plants. Interestingly, we found that PsCRN63 forms a dimer that is mediated by inter-molecular interactions between N-terminal and C-terminal domains in an inverted association manner. Furthermore, the N-terminal and C-terminal domains required for the dimerization are widely conserved among CRN effectors, suggesting that homo-/hetero-dimerization of Phytophthora CRN effectors is required to exert biological functions. Indeed,the dimerization was required for PTI suppression and cell death-induction activities of PsCRN63. This study will advance our understanding of how oomycete effectors manipulate plant immunity to promote infection.