Biological Function Of Two CFEM Effect Proteins From Colletotrichum Gloeosporioides Caused Pepper Anthrancose

In peppers production pepper anthracnose is the most serious fungal disease caused by Colletotrichum complex,which caused tissue necrosis symptoms on stems,leaves and fruits of peppers,resulting in serious loss of yield sharply decline and huge economic losses.At present,there is no any better way to control this disease due to Colletotrichum complex in the field,the genetic variation of the pathogenic populations,the development of resistance to chemical fungicides as well as vairety resistance breaking,although the main control measures chemicals and planting resistant vaireties to prevent the damages made by Colletotrichum complex.It is very diffcult to control successfully with increasing occurrence frequence of this disease.Rcently,CFEM effect proteins have been interaction research hotspots between pathogenic fungi and host plants.Although the CFEM domain is relatively conserved for CFEM effect proteins in the pathogenic fungi,there is significantly difference that the number of CFEM domains,transmembrance domains and sceretion signal peptides in different fungi,which indicates differentiate and diverse biological functions of CFEM effect proteins.Colletotrichum gloeosporioides is a dominant population,among of all Colletotrichum complex from infected peppers in pepper produciton areas,by now,there is no any relevant research about its CFEM effect proteins.In the study,CFEM effect proteins Cghn13279 and Cghn13471 as targets from Colletotrichum gloeosporioide CSLL-11 strain,their biological functions of development and pathogencity in the target strain CSLL-11 were elucidated carefully,the relevant results would be opened out interaction mechanism between target pathogens and pepper plants,recongnition as well,simultaneously there will be provide theoretical proofs for new prevention and control technologies of this disease.Take all research results as following:1.Total of 30 CFEM effect proteins are encoded in Colletotrichum gloeosporioides CSLL-11 genome by bioinfromatic analysis.26 among of them with secreted signal peptides could be secreted extracellular;17 of them contain varying numbers of transmembrane domains,other of them without transmembrane domains,2.The transcriptome from inoculated pepper fruits by using conidia of C.gloeosporioides CSLL-11 strain,including three levels post-inoculated 12 hrs,48hrs and 96 hrs.To compare with control,the results showed that there was variation expression levels for all CFEM effect proteins,Cghn01651,Cghn07586,Cghn10235,Cghn12664,Cghn13471 and Cghn14998 with lower expression,on the contrary,Cghn02929,Cghn09727,Cghn12645 and Cghn14146 were maintained high expression in the experiments.The decline trend was observed in the three stages for effectors Cghn07893 with fluctuation variation.In addition,Cghn06262,Cghn09571 and Cghn13279 were observed higher expression in 48 hrs and 96 hrs.3.Two hygromycin-labeled knockout vectors pCX62::Cghn13279 and pCX62::Cghn13471 were successfully constructed,respectively.After PEG-mediated protoplast transformation,there was positive 10.00% and 11.11% by PCR detection,respectively.The mutant strains were confirmed the target genes with successfully knocked out by Southern hybridization.4.Two G418-labeled complementary vectors pGTN::Cghn13279 and pGTN::Cghn13471 were successfully constructed by infusion methods,respectively.Using PEG-mediated protoplast transformation,Cghn13279 and Cghn13471 was complemented successfully for mutant strains ΔCghn13279 and ΔCghn13471 with positive of 94.12% and 93.75%,respectively.Thus complementary strains was named ΔCghn13279/CgHN13279 and ΔCghn13471/CgHN13471,respectively.5.Five different vectors pBin-GFP:Cghn13279,pBin-GFP:Cghn13471,pBin-GFP:Cghn02929,pBin-GFP:Cghn09727 and pBin-GFP:Cghn14146 were successfully constructed,frequently subcellular localization analysis was preformed on Nicotiana benthamiana by Agrobacterium GV3101 infiltration.The results showed that Cghn14146,Cghn09727 and Cghn13279 were all located in cell membrane and cytoplasm;while Cghn02929,Cghn13471 were located in nucleus,cell membrane,cytoplasm and plasmodesmata,induced cell necrosis as well.6.To compare with wild type,the hyphae growth rate of ΔCghn13279 andΔCghn13471 was reduced with their hyphae growth polarity retarded,respectively.However,abnormal hyphae was observed to mutant ΔCghn13471 only.The ability of sporulation was also reduced for ΔCghn13279 and ΔCghn13471,but conidia size and morphology didn’t make any change.In addition,both mutants also took on reduce ability of pathogenicity and tolerance to hydrogen peroxide,while the expand ability of their hyphae was retarded during infecting Nicotiana benthamianan and Capsicum annuum L.leaves,respectively.At the same time,the cell wall integrity broken was observed for both mutants.All related biological characters above-mentioned were recovered for ΔCghn13279/CgHN13279 and ΔCghn13471/CgHN13471,there is in accordance with wild type.Take all,in this study,the CFEM effector proteins composition and differentiation expression level of target C.gloeosporioides CSLL-11 strain were clarified.The subcellular localization was determined for differentiation five CFEM effector proteins.The biological functions of Cghn13279 and Cghn13471 has been elucidated.Expected results is helpful to the interaction research between pathogen and host resistance breeding.