Study On The Mechanism Of Effector VmEP1 From Valsa Mali In Interfering The Host Resistance

Apple Valsa canker,caused by the fungus Valsa mali,is a potential factor threatening the development of the apple industry.The pathogen specifically infects the colonized bark and causes necrosis,but there is still a lack of in-depth research and understanding of the pathogenic mechanism of the pathogen,resulting in a lack of efficient control technologies.Previous,in order to gain a radical understanding of the pathogenic mechanism of V.mali in the early stage of our research team,the genome of V.mali was sequenced.A total of 779secreted proteins were predicted in V.mali genome,from which 193 candidate effector proteins were predicted.The pathogenicity of the knockout mutants of the effector protein Vm EP1 was significantly reduced,indicating that it is a very important pathogenic factor for V.mali.However,the mechanism of Vm EP1 has not been clearly understood.In this study,Vm EP1 was used as research object.In order to analyze the pathogenic mechanism of Vm EP1,we screened and verified the target proteins of Vm EP1 by yeast two-hybrid（H2Y）and co-immunoprecipitation combined with mass spectrometry（Co IP-MS）.Pathogenesis related protein 10（Md PR10）and RNA binding protein（Md KRBP4）were screened and identified.The effect of Vm EP1 on the function of the target protein and the relationship between Md PR10 and Md KRBP4 were explored.The main research contents are as follows:Vm EP1 promoted the infection of pathogens by inhibiting plant immunity.Based on the background of Vm EP1 inhibiting BCL-2-associated X protein（BAX）-induced cell death,this study explored the function of Vm EP1.Overexpression of Vm EP1 inhibited the expression of plant immunity-related genes,the production of ROS,and the deposition of callose,indicating that Vm EP1 inhibited the plant immune response.We also discovered that overexpression of Vm EP1 promoted the infection of pathogens including Sclerotinia sclerotiorum,Phytophthora capsici,and V.mali.These results showed Vm EP1 promoted the infection of pathogens by inhibiting plant immunity.The amino acid 346 is essential for Vm EP1 function.A potential glycosylation site was found at N346 in the amino acid sequence of Vm EP1 by online predicting.The mutant of Vm EP1,Vm EP1^N346A,no longer inhibited the cell death induced by BAX,and promoted the infection of V.mali in apple.These results indicated that the N346 is important for Vm EP1 function.Vm EP1 inhibited plant immunity by targeting Md PR10 and affecting the callose deposition induced by Md PR10.For exploring the mechanism of Vm EP1 to inhibit plant immunity,we screened the candidate interacting proteins by H2Y,and identified and confirmed by Bi FC and Co-IP.The results showed that Vm EP1 interacted with Md PR10.Silence of Md PR10 promoted the infection of V.mali,and overexpression of Md PR10enhanced the resistance of apple to V.mali.Overexpression of Md PR10 induced callose deposition in apple.These results indicated that Md PR10 is a positive regulator of plant immunity.The co-expression of Md PR10 and Vm EP1 decreased the deposition of callose,and reduced the resistance of apple to V.mali.Above results suggested that Vm EP1weakened the resistance of apple to V.mali by interfering with the Md PR10-mediated resistance.Vm EP1 weakened the resistance of apple by promoting the degradation of Md KRBP4.For exploring more information about how Vm EP1 inhibits plant immunity,another target of Vm EP1,Md KRBP4,was identified and confirmed by Co-IP.Md KRBP4silencing enhanced the susceptibility of apple to V.mali and compromised reactive oxygen species（ROS）production,whereas transient overexpression of Md KRBP4 stimulated ROS accumulation in apple leaves,indicating that Md KRBP4 involved in the regulation of plant immunity by controlling the production of ROS.The phylogenetic tree analysis and the multiple sequence alignment indicated that Md KRBP4 is homologous to HUA ENHANCER 4（HEN4）,indicating that HEN4-like proteins involve in the regulation of plant immunity.Co-expression of Md KRBP4 with Vm EP1 inhibited cell death and ROS production induced by Md KRBP4 in N.benthamina,and reduced the Md KRBP4 protein accumulation,illustrating that Vm EP1 enhanced the susceptibility of apple by reducing the protein level of Md KRBP4 to inhibit ROS production.Meanwhile,silence of Md KRBP4not only inhibited the expression of SA and JA marker genes but also reduced the expression level of Md PR10 which is a target of Vm EP1.These results indicated that Vm EP1 inhibited the expression of Md PR10 by reducing the protein level of Md KRBP4.In conclusion,not only did Vm EP1 suppress plant immunity by targeting Md PR10 to affect the callose deposition induced by Md PR10,Vm EP1 also suppressed plant immunity by targeting Md KRBP4 to decrease the protein level of Md KRBP4 to inhibit the expression of Md PR10 and immunity related genes.