Cloning And Function Analysis Of Effector Gene CgCP1 Of Colletotrichum Gloeosporioides

Colletotrichum gloeosporioides is the causal agent of rubber tree anthracnose and leads to serious loss of rubber production in Hainan Province.At present,the research on the pathogenic mechanism of Colletotrichum gloeosporioides is still limited.Effector is one of the main pathogenic factors of fungi.Based on the genome sequence of C.gloeosporioides,a gene encoding a candidate effector,named as CgCP1,was predicted by silico analysis and the function was investigated.The main results are as follows:1.The coding region sequence of CgCP1 was cloned by RT-PCR.The sequencing result showed that CgCP1 contained a 678bp open reading frame(ORF),encoding a 226 amino acid peptide.Bioinformatics analysis showed that CgCP1 belong to a branch of the Cerato-platanin protein family containing a signal peptide sequence(1-19aa)at N terminal,a conserved Cerato-platanin domain(36-132aa)and a conserved 13-peptide motif(apopolysialoglycoprotein,ApoPSGP)at C terminal.2.The CgCP1 knockout mutant strain ACgCPl and its complement strain Res-?CgCP1 of C.gloeosporioides were constructed by homologous recombination.3.By analyzing the growth speed and conidiation,it was found that there were significant differences in the conidia production of WT,ACgCPl and Res-ACgCPl.The conidia production of WT and Res-?CgCP1 strains were significantly higher than that of?CgCP1.However,there was no significant difference in the growth speed among the three strains.The results above showed that CgCP1 affected the conidia production of C.gloeosporioides but does not affect the growth of C.gloeosporioides.4.The colony color of WT,?CgCP1,and Res-?CgCP1 strains were found significantly different whether in solid culture or liquid culture.The melanin content in?CgCP1 was significantly lower than that in WT and Res-?CgCP1 strains and There was no significant difference in the melanin content between WT and Res-ACgCPl,indicating that the CgCP1 affected melanin synthesis in C.gloeosporioides.By analyzing the expression level of Cg3HNR and CgLACl,two genes related to melanin synthesis pathway of C.gloeosporioides,we found that the expression level of these two genes in ?CgCP1 was significantly lower than that of WT and Res-?CgCP1,suggesting that the effect of CgCPl on melanin synthesis in C.gloeosporioides is related to Cg3HNR and CgLAC1.5.Complementary mutant strains Res-?CgCP1-m1,Res-?CgCP1-m2 and Res-?CgCP1-m3 with different phosphorylation sites mutation were obtained by point mutations on the four phosphorylation sites,respectively.It was found that the conidiation capacity of Res-?CgCP1-m1,Res-?CgCP1-m2,and Res-?CgCP1-m3 are significantly lower than WT,but they are not significantly different from ?CgCP1.The growth rates of Res-?CgCP1-m1,Res-?CgCP1-m2 and Res-?CgCP1-m3 were not significantly different,but their colony colors were different from that of WT.The experimental results above showed that the phosphorylation of CgCP1 may be related to the conidiation ability of C.gloeosporioides and the regulation of the melanin synthesis,but has little relation with the growth of mycelium.6.Pathogenicity analysis showed that the pathogenicity of ?CgCP1 on rubber trees was significantly lower than that of WT and Res-?CgCP1,indicating that the deletion of the CgCPl of C.gloeosporioides caused a significant decrease in its pathogenicity on rubber tree leaves,suggesting that CgCPl plays a role in the pathogenicity of C.gloeosporioides on rubber trees.Further more,the mechanical penetration analysis showed that the penetrating ability of ?CgCP1 on cellophane was impaired,suggesting that the damage of ?CgCP1 mechanical penetrability may be the one of causees of the decline of?CgCP1 pathogenicity.7.The effect of ACgCPl on the production of reactive oxygen species(ROS)in rubber tree leaves was analyzed by DAB staining.It was found that the content of ROS in rubber tree leaves decreased significantly after inoculation with ?CgCP1 compared with the results of inoculation with WT;It was found that CgCPl can induce the accumulation of ROS in rubber tree mesophyll protoplasts by rubber tree mesophyll protoplasts transient expression system.8.The effects of rubber tree leaves inoculated with WT,?CgCP1,and Res-?CgCP1 on the expression of defense response genes HbPRl and HbPR5 were analyzed by qRT-PCR.The results showed that the expression level of HbPRl and HbPR5 in leaves of rubber trees inoculated with ?CgCP1 were significantly lower than that in leaves inoculated with WT and Res-?CgCP1.9.Through the Agrobacterium-mediated tobacco transient expression system,we found that the expression of CgCP1 in tobacco leaves can cause necrosis in tobacco leaves,accompanied by the occurrence of cell death,suggesting that CgCP1 can activate hypersensitive response(HR)in plants.10.The subcellular localization of the CgCPl was analyzed through the rubber tree mesophyll protoplasts transient expression system.The results showed that the CgCP1 was located in the cell membrane and cytoplasm,but the specific location in cytoplasm still need further co-location detection.In brief,we finished the clone and identification of CgCPl,and preliminarily clarified that CgCP1 is involved in the regulation of conidiation and melanin synthesis and plays an important role in the pathogenic process of C.gloeosporioides on rubber trees.In addition,CgCPl can activate plant innate immune response,including the expression of plant defense response gene,ROS accumulation,and HR in plants.This study provides new evidence for elucidating the pathogenic mechanism of C.gloeosporioides and its application.