Study On Pathogenic Function Of Subtilis Protease VmSpm1 Of Valsa Mali

Apple Valsa canker is an apple stem disease caused by the infestation of plant pathogenic fungus Valsa mali(Vm),The serious occurrence of this disease has had an extremely serious impact on the apple industry in East Asian countries,so it is of great significance to deeply analyze the pathogenic molecular mechanism of Vm to formulate green and effective disease prevention and control strategies.Bacillus subtilis is widely distributed and has diverse biological functions,but its mechanism of action in Vm is still not well understood.Previous studies have found that the VmSpm1 is significantly upregulated during Vm infection,and the pathogenicity of VmSpm1 knockout mutant is significantly reduced,suggesting that our VmSpm1 may play a crucial role in decay infection.To this end,the biological function of the effector protein VmSpm1 was studied in this study,and the specific results are as follows:1.The exosome protein VmSpm1 induces plant cell necrosis in an enzyme-dependent mannerBioinformatics analysis found that VmSpm1 has 7 α helixes and 13 β folds,including the S8 domain and the I9 domain.Through sequence alignment,it was found that VmSpm1 had a three-way catalytic active site composed of serine,histidine and aspartic acid.Further yeast heterologous expression and apoplastic protein extraction assays proved that VmSpm1 was an exogenic protein.VmSpm1-GST recombinant protein was obtained by prokaryotic expression and protein purification technology,and after vacuum N.benthamiana injection,it was found that it could cause N.benthamiana leaf necrosis,calloin accumulation and reactive oxygen species burst,indicating that VmSpm1 can stimulate plant immune response.However,after mutation of the enzyme activity site of VmSpm1,the ability of VmSpm1 to cause cell necrosis is lost,indicating that VmSpm1 induces plant cell necrosis in an enzyme-dependent manner.On the other hand,by determining the protease activity of VmSpm1 under different p H conditions,VmSpm1 was found to be an acidic protease with the highest enzyme activity at p H 4.Determination of p H at the site of infection by inoculation of VmSpm1 gene deletion mutants found that VmSpm1 gene has a significant contribution to the acidification host of Vm.2.VmSpm1 interacts with apple abscisic acid receptor MdPYL4,which positively regulates apple resistance to rotting bacteriaIn order to find the interaction target protein of VmSpm1 and explore the specific mechanism of VmSpm1 exerting pathogenic function,we used co-immunoprecipitation combined with mass spectrometry(IP-MS)to screen eight candidate VmSpm1 candidate target proteins.Further through heterologous expression in N.benthamiana,subcellular localization observations found that VmSpm1 and plant abscisic acid receptor PYL4 protein(MdPYL4)were colocalized on the plasma membrane.At the same time,yeast two-hybrid(Y2H),bimolecular fluorescence complementation(BIFC)and Luciferase Complementation Assay(Luc)were used to prove the interaction relationship between VmSpm1 and MdPYL4 protein.In order to explore the biological function of MdPYL4,q RTPCR analysis of the transcription level of MdPYL4 during the infection of Vm showed that MdPYL4 was significantly upregulated during the infection.The instantaneous overexpression of MdPYL4 in apple tissue culture seedlings was carried out by Agrobacterium-mediated transformation system,and the resistance of apple leaves to Vm was significantly enhanced after inoculation with Vm,and the MdPYL4 gene was silenced in apple tissue culture seedlings by RNAi-mediated transient silencing technology,and the silencing efficiency was 80%,and the disease spot area found by inoculation of Vm was significantly increased,and the above evidence showed that MdPYL4 as a disease resistance positively regulated apple resistance to Vm.3.VmSpm1 mediates the protein degradation of MdPYL4,thereby affecting the plant ABA pathwayIn order to further investigate the interaction mechanism between VmSpm1 and MdPYL4,co-incubation of VmSpm1 with MdPYL4 in vitro found that the level of MdPYL4 protein decreased,and further transient co-expression of VmSpm1 and MdPYL4 in N.benthamiana found that the level of MdPYL4 protein was also decreased and inhibited by MG132,indicating that VmSpm1 mediates the degradation of the 26 S proteasome pathway of MdPYL4.Since MdPYL4 is a abscisic acid receptor,we further explored the effect of VmSpm1 on the abscisic acid pathway during the interaction with MdPYL4.By measuring the transcription level of ABA pathway-related genes during the infection of Vm,it was found that the genes Md PYL6,Md ABI2 and Md Sn RK2.6 were significantly upregulated after 24 h inoculation of the VmSpm1 knockout mutant strain,and the co-expression of VmSpm1 and MdPYL4 in N.benthamiana found that the ABA content of plants was significantly lower than that of MdPYL4 alone.The above evidence suggests that the interaction between VmSpm1 and MdPYL4 inhibits the plant ABA pathway.