Construction Of A T-DNA Insertion Mutant Library And Screening Of The Pathogenicity-related Genes In Colletotrichum Gloeosporioides

Colletotrichum gloeosporioides is the main pathogenic fungi of rubber anthracnose.In order to find a new prevention and control strategyfor rubber tree anthracnose,it is of great importance to explore the pathogenic mechanism of C.gloeosporioides.Constructing T-DNA insertion mutants is an effective method to research the pathogenesis.In this study,the Agrobacterium tumefaciens-mediated transformation system of C.gloeosporioides was optimized and the T-DNA insertion mutant library of C.gloeosporioides was constructed for screening the pathogenic factors.The results are as follows:1.Based on the facts that C.gloeosporioides is insensitive to Hygromycin B and sensitive to Chlorimuron-ethyl,we constructed the binary vector pCAMBIA1300-SUR with chlorimuron-ethyl resistance gene by using the vector backbone of pCAMBIA1300 and replacing the hygromycin resistance gene?Hyg?with the chlorimuron-ethyl resistance gene?SUR?.2.In order to obtain sufficient mutants of C.gloeosporioides,we optimized the Agrobacterium tumefaciens-mediated transformation system of C.gloeosporioides in the Agrobacterium strain,concentration of acetosyringone?AS?,concentration of C gloeosporioides spores and co-culture time.The results showed that the transformation efficiency of 480 transformants per 106 conidia was obtained by co-culturing OD600=0.8 of A.tumefaciens strain AGL-1 and 106 conidia/mL of C.gloeosporioides conidium for 48h with 100 mmol/L of acetosyringone.3.Based on the optimized system,a T-DNA insert mutant library containing 861 mutants was constructed.Several mutants were randomly selected from the library and identified to be positive by PCR amplification.Two growth-decreasing mutants?T003 and T062?and one aerial hyphae-deficient mutant?T261?were select from the mutant library.Another 8 mutants showed impaired virulence to rubber tree,thereinto,two mutants?T734 and T735?showed significant difference compared to wide type.4.T-DNA insertion site flanking sequences of the T734 and T735 mutant strains were amplified by hiTAIL-PCR.The T-DNA insertion site was determined by sequence alignment with the wild type genome.The results showed that T734 T-DNA insertion site located at 514205bp of scaffold7;T735 T-DNA insertion site located at 21791bp of scaffold242.5.Homologous searches on GeneBank showed that the T-DNA insertion site in T735 was 175 bp upstream of a gene encoding a FAD binding domain protein?XM₀07286226?.XM₀07286226 contains an encoding sequence of 1336bp and named CgFADBP in this study.Semi-quantitative RT-PCR showed that the insertion of T-DNA led to up-regulated of CgFADBP expression in the T735 mutant.6.CgFADBP overexpressing mutant and knockout mutant of C.gloeosporioides will be constructed for further function identification analysis.So far,the construction of CgFADBP knockout mutant has been finished.