The use of T‐DNAtagging to isolate mutants ofColletotrichum gloeosporioidesandColletotrichum acutatumwith reduced virulence againstHevea brasiliensis

Abstract

Agrobacterium tumefaciens-mediated transformation (ATMT) is being increasingly recognized as an effective insertional mutagenesis tool in studies of filamentous fungi for gene discovery and functional analysis. We developed and optimized ATMT for 2 Colletotrichum species, Colletotrichum gloeosporioides and Colletotrichum acutatum, the causative agents of Colletotrichum leaf disease in rubber trees in Southern China. A. tumefaciens strain AGL-1 carrying an ILV1 gene and a green fluorescent protein gene were used to transform the conidia of these 2 Colletotrichum species. The transformation efficiency was correlated with the co-cultivation duration and bacterial cell concentrations, which reached 300-400 transformants per 1 9 10 6 conidia after optimization. Southern blot analysis indicated that about 60.0% of the C. gloeosporioides transformants and 46.2% of the C. acutatum transformants had a single copy of T-DNA in their genomes. Fungal genomic DNA segments flanking the T-DNA were identified in the transformants through thermal asymmetrical interlaced polymerase chain reaction followed by sequencing. The flanking sequences from 4 C. acutatum and 7 C. gloeosporioides transformants showed moderate or weak homology to the NCBI database entries. Some sequences matching those reported virulence-related genes. The results suggest that the T-DNA inserted mutants banks constructed are useful for the discovery of new or important genes and to elucidate their function in the 2 Colletotrichum species.

The Colletotrichum gloeosporioides perilipin homologue CAP 20 regulates functional appressorial formation and fungal virulence

“…Thereafter, the pCB1532‐Cap20‐GFP plasmid was introduced into the △CAP20‐16 mutant by transformation, which resulted in CAP20‐16‐GFP. The transformants grown on sulfonylurea at 100 μg/ml were harvested (Lin et al.,).…”

“…Scientists previously focused on the taxonomy, population structure, epidemiological investigations, etc., but little is known about the molecular pathogenicity mechanism of the disease (Cai & Huang, ). To gain more insight into the molecular mechanisms of the pathogenesis of C. gloeosporioides , Agrobacterium tumefaciens ‐mediated transformation (ATMT) libraries of C. gloeosporioides were generated and some transformants with an impaired pathogenicity were screened in our research group (Cai et al., ; Lin et al., ). The T‐DNA flanking sequences of the transformants were analysed by thermal asymmetric interlaced PCR (TAIL‐PCR) and showed homology with the pathogenesis‐associated gene CAP20 (Lin et al., ).…”

“…To gain more insight into the molecular mechanisms of the pathogenesis of C. gloeosporioides , Agrobacterium tumefaciens ‐mediated transformation (ATMT) libraries of C. gloeosporioides were generated and some transformants with an impaired pathogenicity were screened in our research group (Cai et al., ; Lin et al., ). The T‐DNA flanking sequences of the transformants were analysed by thermal asymmetric interlaced PCR (TAIL‐PCR) and showed homology with the pathogenesis‐associated gene CAP20 (Lin et al., ). CAP20 is expressed during appressorium formation, and null mutants show a drastically decreased virulence in avocado and tomato fruits (Hwang, Flaishman, & Kolattukudy, ).…”