Research on the function of CgCDC2 gene in Colletotrichum gloeosporioides with RNAi and TMT-based proteomic analysis

Abstract: 【Objective】CDC2 is one of the main factors in cell cycle regulation, which plays an important role in the growth and development of plant pathogens. However, there are few studies on the biological function of CDC2 in Colletotrichum gloeosporioides. The purpose of this study was to obtain and analyze the CgCDC2 RNAi mutants of Colletotrichum gloeosporioides and provide some information about the function of CgCDC2.【Methods】The CgCDC2 gene was cloned by RT-PCR. RNA interference and the CgCDC2 RNAi mutants were obtain by using PEG-mediated protoplast transformation system. PCR amplification, agarose gel electrophoresis, along with qRT-PCR were used to determine whether the transformant obtained were RNAi mutants. The biological phenotypes including colony growth rate, spore yield and sensitivity to stress factors were tested to understand the phenotypic differences between mutants and wild-type strain. TMT-based proteomic analysis and enzyme activity assay were performed in order to analyze the differential proteins in the mutants and the wild-type strain. The pathogenicity of the mutants and the wild-type strain were determined by inoculating them on the mango. The diameter of the lesion was observed and measured every other day.【Results】The CgCDC2 gene encoded a protein containing 326 amino acids. It was found that through sequence alignment analysis the protein sequence of CgCDC2 was highly conserved. Compared with other plant pathogenic fungi,CgCDC2 was highly homologous. RNAi mutants were obtained by constructing RNAi expression vector and PEG-mediated protoplast transformation system. After the mutants and the wild-type strain inoculated on PDA and Czapek plates for several days, the colony growth rate of the mutants was found lower than that of the wild-type strain by observing and measuring the diameter of lesion. The collected conidia were counted by the hemocytometer and it was found that the conidia yield of mutants significantly decreased compared with the wild-type strain, it was about 1/8 of that of the wild-type strain. The sensitivity test of the mutants and the wild type strain to stress factors showed that both of them were not inhibited on the medium containing 0.7 mol · mL-1 NaCl or 100 μg · mL-1 Congo Red. However, the growth of the mutants was inhibited to varying degrees, while the growth of the wild-type strain was not inhibited on the medium containing 5 mmol · mL-1 H2O2. TMT-based proteomic analysis showed that 5 420 proteins were identified both in the mutants and the wild-type strain. In pSilent-1: CgCDC2-1 vs WT, 86 significant differential proteins were identified, of which 47 were up-regulated and 39 were down-regulated. In pSilent-1:CgCDC2-2 vs WT, 73 significant differential proteins were identified, of which 37 were up-regulated and 36 were down-regulated. GO enrichment analysis showed that in pSilent-1:CgCDC2-1 vs WT, the biological processes involved in differential proteins were mainly concentrated in the carbohydrate metabolic process, the regulation of transcription from RNA polymerase III promoter, nucleosome assembly and amino acid transmembrane transport. The molecular function were mainly concentrated in lyase activity, substrate-specific transmembrane transporter activity. The cellular components were mainly concentrated in chromatin. In pSilent-1:CgCDC2-2 vs WT, the biological processes involved in differential proteins were mainly concentrated in the aromatic amino acid family metabolic process. The molecular functions were mainly concentrated on oxidoreductase activity, carbon-sulfur lyase activity and antioxidant activity. The expression levels of PG and PL genes in the mutants the wildtype strain were determined by qRT-PCR. The results showed that the expression levels of PG and PL genes were significantly down-regulated in the mutants compared with the wild-type strain. The results of enzyme activity assay showed that the enzyme activities of PG and PL in mutants were significantly lower than those of the wild-type strain, which was consistent with the results of TMT-based proteomic analysis. The result of pathogenicity test showed that after inoculating mango for 5 days, the diameter of lesion caused by mutants was significantly smaller than that of the wild-type strain. This indicated that the pathogenicity of the mutants was weakened.【Conclusion】Sequence alignment results showed that the CgCDC2 protein was highly conserved. The biological phenotypic determination of the mutants indicated that the CgCDC2 gene was involved in the regulation of the colony growth rate, conidia yield, oxidative stress response, the expression levels of PG and PL genes, the expression levels of PG and PL proteins and enzyme activities in Colletotrichum gloeosporioides, thereby reducing the pathogenicity.