Identification And Functional Research Of Nep1-like Proteins From Valsa Mali

Apple Valsa canker caused by the pathogenic fungus Valsa mali（Vm）,can lead to bark ulceration and severe necrosis,resulting in death of trees and even destroying the whole orchard.During the past several decades,though much progress has been made on the pathogen biology of Vm,the major pathogenic factors of this fungus remain to be fully explored.Given that Vm can cause necrosis of apple host,we speculated that mycotoxins and phytotoxic proteins play a pivotal role during Vm infection.Thus far,whilst a few mycotoxins have been identified in Vm,no any phytotoxic proteins have been reported yet.Nep1（Necrosis and ethylene-inducing peptide 1）-like protein（NLP）has long been demonstrated to be a family of phytotoxic protein widely spread in bacteria,fungi and oomycetes.NLPs act as cytolytic phytotoxins that cause cell necrosis on multiple dicots,and they frequently function as important virulence factors in phytopathogens.In this study,we identified two NLPs in Vm and investigated their biological functions.The main results obtained are as follows:（1）Vm possesses two NLP proteins（Vm NLP1 and Vm NLP2）,and Vm NLP2 exhibits cytotoxic activityA blast search of the Vm genome was performed with the Fo Nep1 from Fusarium oxysporum in NCBI database and two NLP genes were identified,which we respectively designated as Vm NLP1 and Vm NLP2.Transient expression revealed that only Vm NLP2displayed cytotoxic activity in N.benthamiana.Next,Vm NLP2 recombinant protein was produced in Escherichia coli and was used for activity test in apple host.We found that Vm NLP2 can induce cell necrosis in apple leaves as well.To be noted,NLP has a highly conserved heptapeptide motif（GHRHDWE）.However,the second histidine among the heptapeptide is mutated to tyrosine for Vm NLP2（GHRYDWE）.After replacing this tyrosine with the conserved histidine(Vm NLP2^Y137H),the variant exhibited enhanced cytotoxic activity than that of Vm NLP2.These results demonstrated that Vm NLP2 is a phytotoxic protein,and the tyrosine site in its heptapeptide motif affects its cytotoxicity.（2）Vm NLP2 is an important virulence factorTo explore whether Vm NLP1 and Vm NLP2 are involved in the Vm virulence,we generated gene knockout mutants of Vm NLP1 and Vm NLP2 using PEG-mediated protoplast transformation and then inoculated into apple leaves and twigs for pathogenicity determination.The results showed that,compared with WT,the virulence of Vm NLP1knockout mutant displayed no apparent alterations,however,the virulence of Vm NLP2knockout mutant（ΔVm NLP2-36）decreased about 16%and 27%in detached leaves and twigs,respectively.To explore the potential relationship between the virulence and cytotoxicity of Vm NLP2,site-directed mutation transformants(ΔVm NLP2-C^Y137H)were generated by introducing Vm NLP2^Y137H fragments intoΔVm NLP2-36 strain and virulence tests were performed.The results showed thatΔVm NLP2-C^Y137H transformants displayed apparent increased virulence than Vm NLP2 complementation transformant.These results altogether demonstrated that Vm NLP2 is required for Vm full virulence and its contribution to virulence is probably correlated to its cytotoxicity.（3）Vm NLP1 and Vm NLP2 differentially contribute to salt,osmotic and membrane stressesTo explore whether Vm NLP1 and Vm NLP2 are involved in Vm responsiveness to abiotic stresses,Vm wild-type strain,gene deletion mutants,and corresponding complementation transformants were cultured in PDA plates supplemented with 0.3 M KCl（salt stress）,1 M sorbitol（osmotic stress）,0.01%SDS（cell membrane damaging agent）,6 m M H₂O₂（oxidative stress）or 400μg m L^-1 CR（congo red,cell wall inhibitor）.The colony diameter was observed and measured after 3 days of culturing.The results illustrated that the vegetative growth of Vm NLP1 or Vm NLP2 knockout mutants on plates containing SDS,congo red and sorbitol were inhibited or promoted to varying degrees compared to WT,indicating that Vm NLP1 and Vm NLP2 are involved in maintenance of Vm cell membrane integrity and they differentially participate in Vm response to salt and osmotic stress.（4）A peptide nlp25 among Vm NLP2 can be recognized by Arabidopsis thalianaMultiple studies have revealed that nlp20,a conserved polypeptide in NLP protein,can be recognized by A.thaliana.In order to explore whether the corresponding conserved peptides in Vm NLP1 and Vm NLP2 display immunogenic activity,two corresponding peptides nlp20（Vm NLP1）and nlp25（Vm NLP2）were synthesized and used for assays in Arabidopsis.The results showed that only 1μM nlp25（Vm NLP2）but not nlp20（Vm NLP1）can induce reactive oxygen species（ROS）burst and the expression of immune-related marker genes（At PR1,At WRKY33,At FRK1）.In accordance,we found that pretreatment of A.thaliana with1μM nlp25（Vm NLP2）significantly promoted Arabidopsis resistance to Sclerotinia sclerotiorum.Further studies showed that nlp25（Vm NLP2）failed to induce ROS burst and expression of immune-related marker genes in apple host.These results suggest that the conserved peptide in Vm NLP2 can be recognized by Arabidopsis but not its apple host.In summary,we identified two NLP proteins from Vm（Vm NLP1 and Vm NLP2）,and revealed that Vm NLP2 serves as a phytotoxic protein as well as an important virulence factor.In addition,we clarified the role of Vm NLP1 and Vm NLP2 in regulating Vm response to abiotic stresses,and explored the immunogenic activity of a conserved peptide among Vm NLP2 in Arabidopsis.This study provides evidence for functional diversification of NLP proteins.More importantly,as an important virulence factor,Vm NLP2 can be exploited as a promising target in controlling of apple Valsa canker,thereby providing scientific basis for development of targeted agents.