Screening And Characterization Of Plant Necrosis-inducing Secreted Proteins In Botrytis Cinerea

Plant gray mold disease caused by Botrytis cinerea results in severe crop yieldlos s,and is one of the important diseasesin agricultural production,causing serious harm in the production,storage,and transportation of fruits,vegetables,and flowers.B.cinerea has a wide host range and is a worldwide distributed dead vegetative plant disease fungus.B.cinerea can infect more than 1000 plants,including important economic crops in agricultural production,such as vegetables,fruits,and various flowers.Studying the functions of pathogenic genes related to B.cinerea can further clarify the pathogenic mechanism of B.cinereaand provide theoretical basis for developing new strategies for the prevention and control of plant gray mold.In the previous research work,a small molecular secretory protein BcSSP2 was selected based on the secretory proteome and RNA-Seq data during the infection stage of B.cinerea.Agroinfiltration with A.tumefaciensstrains of this protein in the leaves of N.benthamianacan cause yellowing and chlorosis of plant leaves at the initial stage,and further lead to leaf necrosis at the later stage.In this study,we conducted bioinformatics analysis and protein localization of BcSSP2 protein;In order to further study the function of the BcSSP2 gene during the infection process of B.cinerea,we conducted knockout and overexpression of the BcSSP2 gene,and analyzed the phenotypes of the knockout and overexpression transformants,such as growth speed,colony morphology,spore production,stress toleranceand pathogenicity;In addition,we have also conducted in-depth studies on the thermal stability,toxicity to different plants,induction of plant resistance,and signal regulation of BcSSP2 by BAK1 and SOBIR1.The experimental results are as follows:(1)Bioinformatics analysis shows that BcSSP2 is a single copy gene with a total of 90 amino acids and 10 cysteine residues,of which 8 are highly conserved,and the first 20 N-terminal amino acids of BcSSP2 encode signal peptides.The 3D structure shows that the BcSSP2 protein contains two outer α-Spiral structure and two interiorsβ-Folding structure.The highly similar BcSSP2 homologous protein only exists in a few fungal genera,most of which are plant pathogens,and its homologous protein has not been found in living vegetative plant pathogenic fungi and human pathogens,indicating that this protein may play an important role in inducing plant necrosis;(2)The protein localization analysis of BcSSP2 confirmed the secretion function of the BcSSP2 signal peptide,and confirmed that BcSSP2 is a secreted protein,and its phytotoxicity depends on its localization in the extracellular space of the leaf cytoplasm.The results of subcellular localization of BcSSP2 protein showed that BcSSP2 protein was distributed near granular or hypothetical vesicular structures in the plasma membrane and around the plant cell membrane.In addition to having phytotoxic activity in the apoplast,BcSSP2 protein may also secrete into the apoplast space at the initial stage of infection and then re-enter the plant cell;(3)The results of functional studies on BcSSP2 gene showed that the expression of BcSSP2 gene was upregulated in the late stage of infection,and there was no significant difference in the colony morphology,sclerotia formation,spore formation,mycelial growth rate,conidia yield,stress tolerance,and pathogenicity of BcSSP2 gene knockout transformants and overexpression transformants compared to wild type B.cinerea strains.Knockout or overexpression of the BcSSP2 gene does not significantly affect the final outcome of B.cinereainfection.This may reflect the presence of redundant functions or a large number of similar effectors in fungi,knocking out or overexpressing an effector protein alone will not significantly affect the final pathogenic effect of the pathogen;(4)The pathogenicity study of BcSSP2 protein showed that BcSSP2 was toxic to a variety of dicotyledonous plants,but not to monocotyledonous plants.BcSSP2 protein has thermal stability.Boiling BcSSP2 protein at 100 ℃ for 30 minutes can reduce plant toxicity,but it cannot completely eliminate plant toxicity.By studying the effects of cysteine residues on the toxicity of BcSSP2 protein,it was verified that certain epitopes of BcSSP2 protein are affected by specific cysteine residues,which can mediate the phytotoxicity of BcSSP2 protein;(5)The study of plant resistance induced by BcSSP2 protein showed that BcSSP2 not only has phytotoxicity,but also can induce plant resistance,and the enhanced tobacco resistance induced by BcSSP2 is related to changes in the expression of tobacco resistance related genes,but BcSSP2 cannot induce systemic resistance in plants;(6)Using virus induced gene silencing,the results showed that BAK1 and SOBIR1 negatively regulate the phytotoxicity of BcSSP2 to N.benthamiana.The toxicity of BcSSP2 in silenced plants of BAK1 or SOBIR1 is more severe than that in wild type plants.The results showed that the phytotoxicity signal of BcSSP2 was mediated through an unknown signal transduction pathway and was negatively regulated by BAK1 and SOBIR1.