Construction Of A T-DNA Insertional Library Of Colletotrichum Higginsianum And CDNA Cloning Of Two Physiological Characteristics Associated Genes

Colletotrichum higginsianum causes anthracnose disease on cruciferous vegetables. It is an ascomycete fungus which employs a two-stage, hemibiotrophic infection strategy.In this study, A library of 2,000 random insertional transformants of C. higginsianum was generated by Agrobacterium tumefaciens-mediated transformation. The library provides us with necessary materials for further research.And the Arabidopsis thaliana-C. higginsianum pathosystem was used to identify fungal pathogenicity.From this library,thirty-three mutants were screened out, two mutants with slow growth phenotype and colony could not develop radially;Four mutants showing reproducible pathogenicity defects on Arabidopsis;Three mutants could not produce any conidia on PDA plate, Four mutants could produce more conidia on PDA plate, Two mutants induce host defense responses of hypersensitive cell death(HR);Thirteen mutants produced abnormal pigment as compared with Ch-1,Two mutants were significantly different in colony morphology; Three mutants'appressoria germinate abnormally.Slow- growth phenotype mutant Pch-1E393 was studied in more detail. There was not significant difference between Pch-1E393 and Ch-1 on the pathogenicity to Arabidopsis thaliana, It formed melanized appressoria (A) with penetration pores (PP) and successfully established biotrophic primary hyphae (PH) and necrotrophic secondary hyphae in host epidermal cells.Southern blot analysis showed that mutant Pch-1E393 was double-copied. The tagged genes was isolated from this mutant by obtaining the DNA sequence flanking the T-DNA insertions using inverse PCR.Based on the flanking DNA, the sequence of a gene was abtained. Sequence analysis revealed that the T-DNA insertion damaged a possible gene, whose nucleotide sequence contains a 1432bp open reading frame (ORF) with three introns and four exons. The ORF encoded putatively a protein with 352 amino acids, The deduced amino acid sequence shared high identity with meiotic recombination protein DMC1.Pch-1C36 produced abnormal pigment as compared with Ch-1. The colony of Pch-1C36 show white and intermediate hyphae collapsed and then degraded. There was not significant difference between Pch-1C36 and Ch-1 on the pathogenicity and other biology characters. Southern blot analysis showed that mutant Pch-1C36 was double-copied. The tagged genes was isolated from this mutant by obtaining the DNA sequence flanking the T-DNA insertions using inverse PCR.Based on the flanking DNA, the sequence of a gene was abtained. Sequence analysis revealed that the T-DNA insertion damaged a possible gene, whose nucleotide sequence contains a 690bp open reading frame (ORF) with two introns and three exons. The ORF encoded putatively a protein with 174 amino acids, The deduced amino acid sequence shared high identity with endoribonuclease L-PSP.