Screening Of A T-DNA Insertional Library Of Colletotrichum Higginsianum And The Identification Of Pathogenicity And Physiological Genes

Colletotrichum higginsianum causes anthracnose disease on a wide range of cruciferous plants., and is widely distributed in the world. Typical anthracnose lesions were observed on the leaves, petioles and stems of cruciferous plants.In this study,4500hygromycin B-resistant C. higginsianum transformants were obtained using ATMT. After subjecting638transformants to two rounds of testing on detached and attached leaves of Arabidopsis thaliana,58transformants were screened out including two transformants causing reduced symptoms, one transformant with slow growth phenotype, one transformant generating bubble hyphae, one transformant with chlamydospore-like structure and fifty-two transformants showing no pigment on PDA.No symptom was observed on healthy or wounded leaves of Arabidopsis thaliana after inoculated with mutant Pch-1B30, and only normal appressorium, very few primary hypha but no secondary hypha were produced on leaves at4DPI. Southern blot analysis showed that mutant Pch-1B30was double-copied. The tagged genes were isolated from this mutant by obtaining the DNA sequence flanking the T-DNA borders with inverse PCR. Based on the flanking DNA, the sequences of two genes were obtained. Sequence analysis revealed that one T-DNA insertion damaged a possible gene, of which sequence contains a4612bp open reading frame (ORF) with three introns and four exons. The ORF encoded a putative protein containing991amino acids. The deduced amino acid sequence showed98%similarity with the Metarhizium anisopliae DUF221, and the function of this protein is unknown. The other gene contains a694bp open reading frame (ORF) with one intron and two exons. The ORF encoded a putative protein with208amino acids. The deduced protein amino acid sequence showed that the sequence had high homology with the EXOSC1/CSL4(exosome component) from Glomerella graminicola, with a similarity of86%. The protein was a complex exosome subunit, which was used for eliminating wrong RNA and maintaining the normal level of gene. By gene expression test, expression of DUF221gene was not detected in mycelia from mutant Pch-1B30, and it indicated that T-DNA insertion of DUF221gene might cause pathogenicity losing in mutant Pch-1B30.After inoculated with mutant Pch-1E240, no lesion was seen on detached and attached leaves of Arabidopsis thaliana. However, normal symptoms were caused on wounded leaves. Test showed that normal appressorium and primary hypha were formed at4DPI while secondary hypha were not observed. Southern blot analysis showed that T-DNA of mutant Pch-1E240was single-copied. The tagged genes were isolated from this mutant by obtaining the DNA sequence flanking the T-DNA borders with inverse PCR. Based on the flanking DNA, the sequence of a gene was obtained. Sequence analysis revealed that among the genes damaged by the T-DNA insertion, one contained a2646bp open reading frame (ORF) with five introns and six exons. The ORF encoded a putative protein with669amino acids. Homology comparison was carried out for the deduced protein amino acid sequence (BLASTP), and the results showed the sequence had high homology with the acetate-Coa ligase of Glomerella graminicola, with a similarity of98%, and the acetate-Coa ligase was an important intermediate product for energy material metabolism, and a pivot substance of in vivo energy material metabolism.After inoculated with mutant Pch-1E590, no lesion was observed on detached and attached leaves of Arabidopsis thaliana while normal symptoms were seen on wounded or young leaves. All structures including appressorium, primary hypha and secondary hypha were all produced on leaves. Southern blot analysis showed the T-DNA insertions of the stain were double-copied. The tagged genes were isolated from this mutant by obtaining the DNA sequence flanking the T-DNA insertions with inverse PCR. Based on the flanking DNA, only part of sequence was obtained. Sequence analysis revealed that T-DNA insertion failed to insert into open reading frame of this gene.T-DNA insertion of transformant Pch-1E409with slow growth was single-copied. The tagged genes were isolated from this mutant by obtaining the DNA sequence flanking the T-DNA insertions with inverse PCR. Sequence analysis revealed that among the genes damaged by the T-DNA insertion, one contains a1947bp open reading frame (ORF) with three introns and four exons. The ORF encoded a putative protein with576amino acids. Homology comparison was carried out for the deduced protein amino acid sequence (BLASTP), and the results showed the sequence had high homology with the Septin Gene Family of Glomerella graminicola, with a similarity of100%. The protein has been involved in the processes of cell division, cell polarization, vesicular transport and cell membrane reconstruction, etc.