Identification And Functional Study Of Two NIS1-like Proteins From Valsa Mali

Apple Valsa canker,caused by the fungus Valsa mali(Cytospora sacculus(Schwein.)Gvrit.),is a serious hazard in our country that causes huge economic losses every year.It is a key target for prevention and control in apple production.Analyzing the pathogenic mechanism of the pathogen has important guiding significance for the scientific prevention and control of apple Valsa canker,and the discovery of key pathogenic substances is one of its important basic tasks.In recent years,although a series of progress have been made in the researches on the pathogenic mechanism of V.mali,the key pathogenic factors still remain to be explored.NIS1 protein(Necrosis-inducing secreted protein 1)is a core effector widely distributed in fungi,and it play many roles in the interaction between pathogen and its host plants.Therefore,in this study,we focused on the NIS1-like proteins in V.mali,and carried out identification and functional analysis of the NIS1 homologous protein.The main results and conclusions are as following:(1)Identifications of VmNIS1 and VmNIS2To identify the NIS1-like proteins in V.mali,based the more in-depth study of Co NIS1(the NIS1 protein in Colletotrichum orbiculare)and Mo NIS1(the NIS1 protein in Magnaporthe oryzae)as the basic protein sequences,two sequences VM1G＿07228 and VM1G＿03758 were successfully identified and named VmNIS1 and VmNIS2 respectively.Conserved domain analysis showed that neither VmNIS1 nor VmNIS2 had known domains,which is similar to Co NIS1 and Mo NIS1.Protein sequence analysis found that the similarity between Co NIS1,Mo NIS1,VmNIS1 and VmNIS2 was between 20% and 45%.Phylogenetic analysis showed that VmNIS1 was close related to Co NIS1,whereas VmNIS2 was close related to Mo NIS1.(2)VmNIS1 is an immune elicitor not virulence factor of V.maliwe transiently expressed VmNIS1 in Nicotiana benthamiana.The results showed that VmNIS1 can induce intense cell death but failed to inhibit INF1-induced cell death in N.benthamiana.To further explore the related functions of VmNIS1 in terms of immunity,we firstly expressed the VmNIS1 recombinant protein in Escherichia coli,then treated N.benthamiana with 1 μM VmNIS1 purified protein.It showed that VmNIS1 can elicit a strong reactive oxygen species(ROS)burst in N.benthamiana;q RT-PCR analysis showed that VmNIS1 activated the expression of multiple immune marker genes,including HR marker genes(Nb HIN1 and Nb HSR203J),salicylic acid pathway marker genes(Nb PR1 a,Nb PR2),jasmonic acid immune pathway marker genes(Nb PR4 and Nb LOX)and PTI marker genes(Nb PTI5,Nb Acre31,Nb WRKY7 and Nb CYP71D20).Among them,Nb PR1 a and Nb CYP71D20 had higher up-regulation times,139.1-fold and 138.8-fold respectively;the pathogen inoculation assays showed that Phytophthora capsici relative biomass was decreased by 72.4% in N.benthamiana pre-treated with VmNIS1 protein.These results indicate that VmNIS1 is an elicitor.In order to explore the effect of VmNIS1 on the pathogenicity of V.mali,we used a PEG-mediated genetic transformation system to obtain a deletion mutant of VmNIS1,ΔVmNIS1-50.Inoculated with apple tree branches,it was found that the infection degree of ΔVmNIS1-50 did not change significantly compared with that of the wild type 03-8,indicating that VmNIS1 is not an pathogenic factor of V.mali.(3)VmNIS2 is an immune suppressor and an important virulence factor of V.malwe transiently expressed VmNIS2 in N.benthamiana,VmNIS2 showed no cell deathinducing activity but it strongly inhibited INF1-induced cell death.Further,we found that VmNIS2 significantly inhibited flg22-induced ROS burst,and markedly attenuated the expression of Nb PR1 a and Nb PR4 in N.benthamiana.In addition,VmNIS2 significantly promoted the infection of P.capsici.Among them,Nb PR1 a and Nb PR4 decreased by 63.4%and 78.3% respectively,and the relative biomass of P.capsici increased by 91.0%.These results indicate that VmNIS2 is an immune suppressor.In order to explore the effect of VmNIS1 on the virulence of V.mali,we successfully obtained VmNIS2 deletion mutantsΔVmNIS2-13 and ΔVmNIS2-87 through a PEG-mediated genetic transformation system.The virulence test showed that ΔVmNIS2-13 and ΔVmNIS2-87 decreased by 13.2% and 16.9%,respectively,compared with the wild type 03-8.After gene supplementation,the pathogenicity of mutants returned to a level equivalent to that of the wild type,indicating that VmNIS2 is an virulence factor of V.mali.In summary,this study identified two NIS1-like proteins VmNIS1 and VmNIS2 from V.mali and have clarified their basic biological functions.VmNIS1 is an immune elicitor and have no obvious influence on the pathogenicity of V.mali;VmNIS2 is an immune suppressor and contributes pathogen virulence.This work elucidated the basic functions of the conserved NIS1 proteins in V.mali,and provided novel insights into the interaction between V.mali and its apple host.