Identification Of Antifungal Proteins From The Biocontrol Trichoderma Guizhouense NJAU 4742

Trichoderma guizhouense NJAU4742 belonging to the group of T.harzianum,is a kind of beneficial filamentous fungi.This strain is isolated from soil samples by Professor Shen Qirong’s team from Nanjing Agricultural University,which has significant growth-promoting and broad-spectrum biocontrol effects.T.guizhouense NJAU 4742 synthesizes a variety of biocontrol factors,including the secretion of extracellular hydrolase and antifungal secondary metabolites to destroy the cell structure of plant pathogenic fungi,thereby inhibiting the growth of pathogenic fungi and hence protecting plants.At the same time,NJAU 4742 can also secrete effector proteins to promote plant growth and stimulate plant immunity,thereby improving plant disease resistance.Previous studies about the identification and function of fungal effectors mainly focused on animal and plant pathogenic filamentous fungi.It has been found that these pathogenic fungi can transport different kinds of effector proteins to the host cell to interfere with the host’s cellular metabolism and promote the infection of pathogenic fungi.In contrast to these eariler findings,this study uses the biocontrol fungus-T.guizhouense NJAU 4742 to identify effector proteins with antifungal activity,by which providing material support for the development of corresponding antifungal biocontrol products and serving the prevention and control of plant fungal diseases.The main results of this study are described as follows:To identify effector protein candidates from T.guizhouense NJAU 4742,the reference genome of this strain was used.Bioinformatics approaches were adopted to predict such effector candidates based on two following principles:（i）Selection of secretory proteins with homology to those characterized effector proteins from plant pathogenic fungi by BLAST method（Evalue:1E-5）;（ii）Combination of the transcriptome data of T.guizhouense,the effector protein candidate genes in T.guizhouense interacting with pathogenic fungi were selected as the screening criteria with the upregulation ratio of more than 10 folds and/or the enrichment degree of cysteine（Cys）greater than 5%.Via these selections,a total of forty-three effector protein candidates were finally identified from T.guizhouense NJAU 4742.In order to explore whether these predicted effector proteins are toxic to fungal cells,two fungal models were selected in this study.One is the filamentous（multicellular）plant pathogenic fungus,Fusarium graminearum,which is a reported model of filamentous fungi in molecular plant pathology.The other is a widely used single-cell fungal model,Saccharomyces cerevisiae.First,a genetic vector for inducing gene expression in F.graminearum was constructed and validated to be effective in this study.Subsequenlty,each of the predicted effector genes of T.guizhouense NJAU 4742 was cloned into the vector,and 12 effector proteins from T.guizhouense were successfully transferred into F.graminearum.The F.graminearum transformants were selected to test their growth rate,conidia production,as well as the phenotypes of the colony diameter with or without the challenge of T.guizhouense,and the results showed that all test phenotypes of the F.graminearum transformants were similar with that of the untransformed wild-type F.graminearum.Therefore,by using F.graminearum as a working model,no effectors with toxicity（antifungal）to filamentous fungi were identified from 12 effector proteins from T.guizhouense in this study.At the same time,this study used the mature S.cerevisiae gene induction expression system to carry out toxicity screening of 33 effector proteins from T.guizhouense.Through the detection of the colony growth of S.cerevisiae transformants,it was successfully screened that the T.guizhouense effector protein Tg10636 could significantly inhibit the growth of S.cerevisiae under salt stress（0.6 M Na Cl）.Meanwhile,it was found that the effector proteins Tg10636,Tg3425 and Tg8975 of T.guizhouense can significantly inhibit the growth of S.cerevisiae under oxidative stress（1 m M H₂O₂）.Among them,the effector protein Tg10636 of T.guizhouense encodes a metalβ-lactamase family protein,and Tg3425and Tg8975 encode a hypothetical protein and a phospholipase-like protein,respectively.It is noteworthy that all these three T.guizhouense effector proteins（Tg10636,Tg3425 and Tg8975）are not toxic to the filamentous（multicellular）fungus,F.graminearum,revealing that these three T.guizhouense effector proteins may have specific antifungal activity against unicellular fungus,i.e.S.cerevisiae.In summary,this study identified three effector proteins from T.guizhouense NJAU 4742that are toxic to the unicellular fungus,S.cerevisiae under oxidative stress or salt stress conditions.The use of these toxic effector proteins targeting single-cell pathogenic fungi,i.e.Candida albicans for fungal disease control has a promising future.