Screen Of Pathogenicity-Deficient Mutants From A T-DNA Insertional Library Of Stylo Colletotrichum Gloeosporioides And The Cloning Of Pathogenic Genes

Stylosanthes guianensias is the most important commercial forage legume in tropical areas of the world. The fungus Colletotrichum gloeosporioides is the causal agent of Stylosanthes guianensias anthracnose, which causes disease seriously and widespreadly. The genetic transformation system of stylo Colletotrichum gloeosporioides mediated was established by Agrobacterium tumefaciens in laboratory. In this research, the pathogenicity-deficient mutants were screened from a T-DNA insertional library. A T-DNA insertion mutation site believed responsible for loss of pathogenicity was identified by using thermal asymmetric interlaced PCR (TAIL-PCR). Using the methodology of bioinformatics, this research analyses the genes of T-DNA insertional sites marked. The targeting vector ware constructed by Split Marker and the author analysed the characterized function of genes by gene knockout technology. From this research, it helps to explain the pathogenic mechanism by analysis the function of virulence genes at molecular level. Moreover, it provides a theoretical basis for the improvement of disease resistance and prevention strategies of stylo.(1) The genetic transformation system of wild strain CH008 of stylo Colletotrichum gloeosporioides mediated was established by ATMT. The 11 pathogenicity-deficient mutants were screened from a T-DNA insertional library. There were 8 mutants loss their pathogenicity, including 952,994,995,1171,1250, 1338,1869 and 2508. And there were 3 mutants weaken their pathogenicity, including 1681,1980 and 2638. The molecular detection of mutant strain by PCR showed the T-DNA was inserted into genes of 11 pathogenicity-deficient mutants steadily. The genomic Southern blot analysis showed 9 mutants were proved to be single-copy insertion and 2 mutants were double-copied including 1250 and 2508.(2) The 11 pathogenicity-deficient mutants were compared with wild type strain CH008 for phenotypic characteristics. The results showed the phenotypic of mutant strain 952,1980 and 2508 on PDA medium were different from CH008. The growth rates of mutant strains 952,994,1338,1869,1980 and 2508 decreased significantly. The sporulation of strains 1250,1338,1681,1869,1980,2508,2638 and the spore germination of strains 952,995,1681,1980 and 2638 markedly reduced. There were different responses and adaptations between mutant strains 952,994,1171,1250 and 1681 to the wild strain CH008 to salt stress. The growth rates of 952 and 995 were different from CH008 to the high-efficient penetrating agent stress with sorbitol. The inhibition rates of mutant strains 952,1869,1980 and 2638 with different fungal cell wall inhibitors increased obviously. What's more, the mutant strains 994,995,1250, 1338,1869,1980 and 2638 also exerted effects on metal ions.(3) DNA of the mutant strain was extracted, and using TAIL-PCR a T-DNA insertion mutation site believed responsible for loss of pathogenicity was identified. Using the methodology of bioinformatics, this research analyses the genes of T-DNA insertional sites marked. The bioinformatics analysis showed the T-DNA of mutant strain 952 marked the area of CDSf (initial exon). The gene CgDps contained a complete open reading frame (ORF), encoding 259 amino acids. The blast result showed it belonged to DNA polymerase ?,?,? subunit. The T-DNA of mutant strain 994 marked the area of CDSI (ending with stop codon). The gene CgAss contained a complete ORF, encoding 556 amino acids. The blast result showed it belonged to Thiamine pyrophosphate (TPP) family, the catalytic subunit of Acetohydroxyacid synthase (AHAS). The T-DNA of mutant strain 995 marked the area of TSS (transcription start site). The gene CgCgt contained a complete ORF, encoding 444 amino acids. The blast result showed it belonged to ceramide glycosyltransferase. The T-DNA of mutant strain 1171 marked the area of CDSI. The gene CgGgh contained a complete ORF, encoding 114 amino acids. The blast result showed it belonged to gene glycoside hydrolase. The one T-DNA of mutant strain 1250 marked the area of CDSI. This gene CgGpp contained a complete ORF, encoding 314 amino acids. The blast result showed it belonged to glucose-regulated protein precursor. The T-DNA of mutant strain 1338 marked gene CgHpl which contained a complete ORF, encoding 155 amino acids. The blast result showed it belonged to hypothetical protein. The T-DNA of mutant strain 1681 marked the area of CDSf. The gene CgPss contained a complete ORF, encoding 682 amino acids. The blast result showed it belonged to the class II aminoacyl-tRNA synthetases (aaRS) like-core super family based upon its structure. The T-DNA of mutant strain 1869 marked the area of TSS. The gene CgPtrcontains a complete ORF, encoding 418 amino acids. The blast result showed it belonged to pH-Responsive transcription regulator. The one T-DNA of mutant strain 2508 marked the area of CDSI. This gene CgHp2 contained a complete ORF, encoding 313 amino acids. The blast result showed it belonged to hypothetical protein. The T-DNA of mutant strain 2638 marks the area of TSS. The gene CgNsp contained a complete ORF, encoding 198 amino acids. The blast result showed it belonged to NADPH sensor protein (HSCARG). As genes mentioned above, the gene CgPtr shared a homology with known gene Pac1, others were the first reported in this paper. The sequences of gene CgDps and CgNsp were first found.(4) The targeting vector ware constructed by Split Marker. The genes CgDps and CgPtr which the T-DNA of mutant strain 952 and 1869 mark were deleted with homologous recombination. Verified by protoplast transformation and PCR molecular detection, there were 8 gene knock-out transformants of CgDps and 5 gene knock-out transformants of CgPtr of Colletotrichum gloeosporioide. The test of virulence showed that there were 6 gene knock-out transformants of CgDps lost virulence and 2 transformants weakened their pathogenicity. There were 3 gene knock-out transformants of CgPtr lost virulence and 2 transformants weakened their pathogenicity. Biological characteristics of gene knock-out transformants were determined. The results showed the gene CgDps and CgPtr were in relation to the growth rates, sporulation and spore germination. And they involved in salt stress response. The gene CgDps and CgPtr probably had a effect to induce complete responses against the metal ions stress. The gene CgDps involved in high-efficient penetrating agent stress. And the gene CgPtr had a effect to induce complete responses against the variety of pH from the external environment. From that, we knew the CgDps and CgPtr were pathogenic genes and CgDps was a novel one.