Identification And Function Analysis Of Candidate Effector Gene CgBASP2and CgCFEM3of Colletotrichum Gloeosporioides Hevea Brasiliensis

Anthracnose is one of the important fungal diseases of rubber tree leading to serious loss of rubber production and is mainly caused by Colletotrichum gloeosporioides on leaves, petioles and sheets. Effector is one of the main pathogenic factors of fungi, but the study on effector of C. gloeosporioides is limited. In the previous study, the genome of C. gloeosporioides of Hevea brasiliensis was sequenced and the genes encoding secretory proteins were predicted. Two of these candidate genes, GME3318_g and GME12162_g, were cloned and the functions of these two genes were investigated. The main results were as follows:1. The open reading frame of GME3318_g was amplified by RT-PCR. Bioinformatics analysis results showed that the ORF of GME3318_g was300bp, encoding an100amino acid peptides with a18aa signal peptide at N terminal and six conserved cysteine residues, but without any conservative structure domain. It was named as CgBASP2due to highly homologous with Biotrophy-Associated Secreted Protein2. Alignment analysis showed that amino acid sequence of CgBASP2is100%,90%,87%,87%and67%similar with that of CgBASP2from C. gloeosporioides Nara gc5, C. orbiculare MAFF, C. graminicola, C. higginsiaum and Magnaporthe oryzae70-15, respectively. The CgBASP2gene knockout mutant strain ?CgBASP2was constructed by homologous recombination and used to study the biological function of this gene. Compared with wild type strains, the mycelium growth of ACgBASP2was slower and its aerial hyphae were reduced. ?CgBASP2was barren in conidia production and showed higher utilization rate of maltose and galactose compare with wild type strain. Pathogenicity analysis showed that ?CgBASP2lost pathogenicity on the leaves of rubber tree and the mechanical penetration of ?CgBASP2hyphae loss. Further analysis showed that cellulose and pectin enzyme activity and the oxidative stress tolerance of ?CgBASP2were significantly lower than the wild type strains. Moreover, the accumulation and distribution of reactive oxygen species in ACgBASP2were different from wild type strain. The above results indicated that CgBASP2gene was an important regulation factor of hypha growth, spore development and pathogenicity of C. gloeosporioides.2. The open reading frame of gene GME12162_g was amplified by RT-PCR. Bioinformatics analysis results show that the length of the ORF of GME12162_g is384bp, encoding an127amino acid peptides with a27aa signal peptide at N terminal, eight conserved cysteine residues and a CFEM domain, and was named as CgCFEM3. Alignment analysis showed that amino acid sequence of CgCFEM3is100%,85%,80%and80%similar with that of CgCFEM3from C. gloeosporioides Nara gc5, C. orbiculare MAFF, C. graminicola, C. higginsiaum, respectively. CgCFEM3was confirmed as a extracellular secreted protein by detecting the GUS activity in liquid culture medium of C. gloeosporioides expressing CgCFEM3-GUS fusion protein. Biochemical analysis showed that CgCFEM3can be glycosylated in rubber cells. The CgCFEM3gene knockout mutant strain ACgCFEM3was constructed by homologous recombination and used to study the biological function of this gene. The aerial mycelium and conidia of ACgCFEM3were significantly less than wild type strain and the utilization rate of maltose, sucrose and glucose was higher than wild type strain obviously. ?CgCFEM3showed compromised pathogenicity on the leaves of rubber tree and lost hyphae mechanical penetration. The peroxide level and distribution showed no significant differences between ACgCFEM3and wild type strain, but other types of reactive oxygen species level in ?CgCFEM3were higher than that in wild type strain. The results above showed that CgCFEM3gene encoded a extracellular secreted protein which not only was involved in regulate hypha and conidia production but also played important role in the pathogenicity of C. gloeosporioides.