Effects On Its Biological Function By Genes Cghn01197 And Cghn13741 Colletotrichum Gloeosporioides Infected Chilli Plants

Colletotrichum gloeosporioides is one of the main pathogenic fungi that cause pepper anthracnose,which can harm the whole growth process of pepper,resulting in a serious decline in yield and quality,and economic losses in severe cases.At present,the control of this disease mainly relies on disease-resistant varieties and chemical control,but there are more than 10 kinds of pathogenic fungi causing pepper anthracnose,and the proportion of compound infection in the field exceeds 60%,leaded to the loss of variety resistance and serious disease in pepper production areas,prevention and control are relatively difficult,so new prevention and control strategies are urgently needed.In the previous study,we taked the genome of Colletotrichum gloeosporioides CSLL-11 as the object,and used the specific extracellular effector protein CFEM of pathogenic fungi as the entry point,our analysis found that 30 CFEM effector proteins were encoded,and there were differences in the number of signal peptides and structural domains;secondly,the combination of pathogenic fungi transcriptome data of artificially inoculated anti-infected peppers were analyzed,and it was found that there were significant differences in the expression patterns of CFEM effector proteins during the infection process,indicated that they play important functions in pathogenicity,growth and development,and interacting with hosts,moreover,function of these proteins was differentiations,which suggested these proteins were potential control target.Based on these results,we selected Cghn01197 and Cghn13741,which is related to plant nutrition and up-regulated in the pathogenic process,respectively,as targets to analyze its biological functions in the growth,development and pathogenic processes,our research lays a foundation for identifying their mechanisms in the interaction between pathogens and their hosts,and provides a new target for the control of pepper anthracnose disease.The specific research results are as follows:1.The Cghn01197 gene and Cghn13741 gene were cloned by RT-PCR,and the subcellular localization vectors p Bin::Cghn01197::GFP and p Bin::Cghn13741::GFP were constructed.Transient expression in Nicotiana benthamiana revealed that Cghn01197 localized in the nucleus and cytoplasmic membrane;Cghn13741 localized in the nucleus and cytoplasmic membrane,while causing host cell necrosis.2.The knockout vectors pCX62::Cghn01197 and pCX62::Cghn13741 were constructed and introduced into the protoplasts of C.gloeosporioides CSLL-11 by PEG-mediated genetic transformation,and 20 corresponding mutant strainsΔCghn01197 and 19 ΔCghn13741 were obtained by PCR screening,and their corresponding transformation efficiency were 71.42% and 63.33%,respectively.Some of the mutants were verified to be single copies by Southern blot,indicated that both the target genes Cghn01197 and Cghn13741 were successfully knocked out.3.The complement vectors pGTN::Cghn01197 and pGTN::Cghn13741 were successfully constructed,and the mutant strains ΔCghn01197-12 and ΔCghn13741-3were selected as test strains.The complement vectors were introduced into the corresponding mutant protoplasts by PEG-mediated genetic transformation,respectively,and 17 complement strains ΔCghn01197/Cg HN01197 and 15 complement strains ΔCghn13741/Cg HN13741 were obtained by PCR screening,and their corresponding complementation efficiency were 60.71% and 53.57%,respectively.4.biological phenotype analysis: compared with the growth differences of the wild-type strain cultured in five media(OM medium,PDA medium,CM medium,V8 medium,SDC medium),we found that the colony diameter of ΔCghn01197-12 was significantly reduced,the polar growth of hyphae was blocked with blunting of the tip,the conidial production decreased by 95%,but the conidial morphological characteristics did not change,the conidial germination time was delayed by 2 h,and the conidial tube length decreased by 63.1% after germination,the tolerance of the fungi to hydrogen peroxide decreased significantly at 6 m M,the integrity of the cytoplasmic membrane was the same as that of the wild-type,there was no difference,the pathogenicity to the host was weakened,while the corresponding biological phenotype of the complement strain was consistent with that of the wild-type strain.The growth diameter of ΔCghn13741-3 decreased on PDA,the polar growth of hyphae was blocked with deformed or blunted tips.The conidial production decreased by 57.14%,but the conidial morphology also did not change yet.Generally the tolerance to hydrogen peroxide was decreased,which have a significant difference was observed in 2 m M.There is no difference for the integrity of the cytoplasmic membrane between the mutant and the wild-type.The pathogenicity of hpyae and conidia from the targert mutant was signifcantly reduced to the host,while the corresponding biological phenotype of the complement strain was consistent with that of the wild-type strain.