Functional Study Of A Key Virulence Factor NLP In Gray Leaf Spot Disease Caused By Stemphylium Lycopersici

The fungus Stemphylium lycopersici（S.lycopersici）is an important plant pathogen causing gray leaf spot disease in tomato.However,due to lack of functional genomics studies,the factors influencing S.lycopersici pathogenicity remain largely unknown.In this study,we present the first example of genetic transformation and targeted gene replacement in S.lycopersici.We functionally analyzed a necrosis-and ethylene-inducing peptide 1（Nep1）-like protein（NLP）in S.lycopersici and showed its role in the pathogenicity,fungal development,and asexual reproduction.The main results are as follows:1.Transcription of a S.lycopersici NLP is induced during disease development on tomato.We identified only one copy of an NLP protein in the released genome.RT-qPCR data showed that NLP transcript abundance became significantly elevated when incubated on tomato leaves but did not change on control medium.It indicates that NLP is involved in the pathogenic process of S.lycopersici.2.NLP affected the production of conidia of S.lycopersici.To reveal the molecular function of NLP in S.lycopersici,we established a gene transformation method.Knockout of NLP mutant（（?）nlp）was generated by targeted gene replacement through a CRISPR/Cas9 enhancement approach.The m Cherry-tagged NLP was constructed under the regulation of the actin promoter（pACTIN:NLP）.The results showed that the （?）nlp mutants produced significantly less conidia compared with the control strain while showed uninfluenced mycelial growth.The results suggest that NLP plays an important role in asexual reproduction in S.lycopersici.3.NLP affected the osmotic stress and oxidative stress adaptation of S.lycopersici.The stress response of fungi to stress can be used as an indicator of pathogen virulence.The results showed that compared with the control strain,the （?）nlp mutant showed stronger growth inhibition under osmotic stress and oxidative stress.4.NLP is an important virulence factor of gray leaf spot disease.Comparison of virulence among the control,（?）nlp mutant and pACTIN:NLP strain shows that deletion of NLP significantly reduced the virulence of （?）nlp mutant on tomato leaves,while higher expression of NLP significantly enhanced the virulence of pACTIN:NLP.It indicates that NLP is a key pathogenic factor in S.lycopersici.5.NLP suppresses the production of ROS in tomato leaves induced by S.lycopersici infection.RT-qPCR results showed that （?）nlp induced significantly higher transcriptional levels of ROS synthesis genes RbohA and RbohB in tomato,while the pACTIN:NLP strain induced a lower level compared to the wild type strain.The results of ROS staining showed that compared with the wild-type strain,the （?）nlp mutant induced more ROS accumulation in plants,while the level of ROS accumulation induced by pACTIN:NLP strain in tomato decreased significantly.6.The S.lycopersici NLP triggers plant immune responses and inhibits plant growth in tomato.Application of the nlp20 peptide to tomato leaves induced cell death and immune response gene expression in tomato leaves.In addition,overexpression of the NLP gene in tomato resulted in constitutive transcription of immune response genes and reduced plant growth.In conclusion,we carried out a functional genomics study in S.lycopersici for the first time.Using the genetically manipulated strains,we studied the function of NLP in pathogen virulence,fungal asexual reproduction,vegetative growth,and stress adaption.It suggests that NLP contributes to pathogen virulence by suppressing the ROS accumulation induced by pathogen infections in tomato.This work provides a paradigm for functional genomics study in the emergent fungal pathogens.The genetically manipulated S.lycopersici strains and transgenic tomato generated in this study are powerful tools to study the interaction between tomato and S.lycopersici.