Functional Characterization And Mechanism Exploration Of Phytophthora Effectors PsCRN63 And PcCRN230 On Ecologically-relevant Bacteria Growth Inhibition

Plant diseases caused by oomycetes are widely distributed throughout the world and seriously threaten the world’s agricultural production.The soybean root rot caused by Phytophthora sojae is a devastating disease for soybean.Phytophthora blight caused by the soil-borne pathogenic oomycete Phytophthora capsici is also highly destructive for a variety of vegetables.Oomycetes secrete an arsenal of effectors during interaction with plant host.In recent years,the role of effectors in oomycetes’ pathogenicity and the mechanism of effectors to manipulate plant immune responses have made many breakthroughs.Some of the effectors function as virulence factors to suppress plant immune response and promote pathogen infection;while other effectors can be recognized by the host R genes and induce host immune response,act as avirulence factor.However,only a small number of effectors have been functional characterized,the function of a large number of effectors are still unknown.The oomycete effectors which are functional characterized all involved in the interaction with the host plant.Interactions between oomycete effectors and ecologically-relevant bacteria are poorly reported.Here,we analyzed the new function of the Phytophthora sojae effector PsCRN63 and the Phytophthora capsici effector PcCRN230 in oomycete and bacteria interaction.The main results and conclusions obtained are as follows:The Phytophthora sojae effector PsCRN63 exhibit strong antibacterial activity on a variety of ecologically-relevant bacteria of Phytophthora.The induced expression of PsCRN63 in E.coli BL21(DE3)cytoplasm and periplasmic space can significantly inhibit cell growth.While its homologous protein PsCRN115 cannot inhibit bacteria.The transiently expressed full-length protein of PsCRN63 in tobacco leaves co-cultured with E.coli DH5α,also caused decreased number of E.coli cells.Moreover,total protein of tobacco leaves overexpressing PsCRN63 co-incubated with E.coli cells also resulted in decreased number of E.coli cells.These results indicate that PsCRN63 can inhibit E.coli by an exogenous contact-dependent manner.In addition,we found PsCRN63 can inhibit several biocontrol bacteria and pathogenic bacteria.The induced expression of PsCRN63 in Bacillus subtilis 168 cytoplasm cannot inhibit bacteria growth.While,exogenous exposure of PsCRN63 to Bacillus subtilis 168 resulted in decreased number of bacteria cells,indicating that PsCRN63 inhibit Bacillus subtilis 168 through an exogenous contact-dependent manner.Using the method of transiently expressed PsCRN63 protein co-incubating with bacteria,we also found that PsCRN63 can inhibit biocontrol bacteria Lysobacter enzymogenes OH11 and pathogenic bacteria Pectobacterium carotovorum subsp.Carotovorum Pcc,but cannot inhibit biocontrol bacteria Bacillus amyloliquefaciens FZB42,pathogenic bacteria Pseudomonas syringae pv.glycinea A1 or plant growth promoting rhizobacteria Pseudomonas protegens Pf-5.The homologous proteins of PsCRN63 in Phytophthora and Pythium can inhibit the growth of E.coli.The CRN effector family is a highly conserved family of effectors in oomycetes.PsCRN63 homologous proteins are widely distributed in Phytophthora and Pythium.In this study,we conducted a preliminary screening of the antibacterial activity of its homologous proteins using the E.coli BL21(DE3)expression system.Results showed that a number of effectors have the ability to inhibit E.coli BL21(DE3).Effectors such as Phytophthora capsici PcCRN4 and PcCRN9;Phytophthora infestans PiCRN2,PiCRN3,PiCRN7,PiCRN37,PiCRN10 and PiCRN10-2;Pythium ultimum Pyul11651-1,Pyul11651-2 and Pyul14996;Pythium aphanidermatum Pyap1465-1 all showed strong inhibition activity on E.coli growth after induction.Agrobacterium-mediated transient transformation expressing these proteins in tobacco leaves revealed that most effectors which are cytotoxic to E.coli also induce programmed cell death in plants,except Pyul14996.Effectors that are non-cytotoxic to E.coli cannot cause programmed cell death in tobacco leaves.The Phytophthora capsici effector PcCRN230 have antibacterial activity on several ecologically-relevant bacteria of Phytophthora.We analyzed the transcriptome data of the interaction between Phytophthora capsici and E.coli,and choose differentially expressed genes for further study.66 effectors,including 30 RxLR effectors,18 CRN effectors,9 Elicitin effectors and 9 NLP effectors,were successfully cloned from Phytophthora capsici.Their antibacterial activity was preliminary tested in E.coli cytoplasmic expression system.The effector PcCRN230 was found to be bactericidal inside the cytoplasm of E.coli.The PcCRN230 expression in E.coli BL21(DE3)periplasmic space also inhibit bacteria growth.Transient expression of PcCRN230 protein in tobacco leaves is toxic to E.coli DH5α,biocontrol bacteria Bacillus subtilis 168 and pathogenic bacteria Pectobacterium carotovorum subsp.Carotovorum Pcc,while has none effect on plant growth promoting rhizobacteria Pseudomonas protegens Pf-5.In addition,we also found that transient expression of PcCRN230 cannot induce cell death in tobacco leaves.