Agrobacterium tumefaciens -mediated transformation for random insertional mutagenesis in Colletotrichum lagenarium

Abstract

Random insertional mutagenesis using a marker DNA fragment is an effective method for identifying fungal genes relevant to morphogenesis, metabolism, and so on. Agrobacterium tumefaciens-mediated transformation (AtMT) has long been used as a tool for the genetic modification of a wide range of plant species. Recent study has indicated that A. tumefaciens could transfer T-DNA not only to plant cells but also to fungal cells. In this study, AtMT was applied to Colletotrichum lagenarium for random insertional mutagenesis. We constructed a binary vector pBIG2RHPH2 carrying a hygromycin-resistant gene cassette between the right and left borders of T-DNA. Optimal co-cultivation of C. lagenarium wild-type 104-T with pBIG2RHPH2-introduced A. tumefaciens C58C1 led to the production of 150-300 hygromycin-resistant transformants per 10 6 conidia. Southern blot analysis revealed that T-DNA was mainly integrated at a single site in the genome and at different sites in transformants. The T-DNA inserts showed small truncations of either end, but the hygromycinresistant gene cassette inside the T-DNA was generally intact. The mode of T-DNA insertion described above resulted in highly efficient gene recovery from the transformants by thermal asymmetrical interlaced-polymerase chain reaction. The fungal genomic DNA segments flanking T-DNA were identified from five of eight mutants that had defective melanin biosynthesis. The sequence from one of the segments was identical to that of the melanin biosynthesis gene PKS1 of C. lagenarium, which we previously characterized. These results strongly support our notion that AtMT is a possible tool for tagging genes relevant to pathogenicity in the plant pathogenic fungus C. lagenarium.

Colletotrichum orbiculare Regulates Cell Cycle G1/S Progression via a Two-Component GAP and a GTPase to Establish Plant Infection

“…The AtMT protocol was applied with slight modifications of a previously described method (Tsuji et al, 2003). Hygromycin-resistant transformants were selected on PDA containing 100 µg/mL hygromycin B (Wako Chemicals) and 25 µg/mL meropenem hydrate (Sumitomo Dainippon Pharma).…”

“…The T-DNA inserted gene in the PDM1 mutant was identified using a thermal asymmetric interlaced-PCR protocol (Liu et al, 1995). Fungal genomic DNA flanking the T-DNA insert was analyzed as previously described (Tsuji et al, 2003). Amplified PCR products were sequenced with the Big-Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) and an ABI PRISM 310 automated DNA sequencer (Applied Biosystems).…”

“…orbiculare was tested for pathogenicity on detached cucumber cotyledons (Cucumis sativus) as described previously (Tsuji et al, 2003). Conidia of C. orbiculare were obtained from 7-d-old PDA cultures, and six drops of 10-mL of a conidial suspension (5 3 10 5 conidia/mL) were placed on the surface of cucumber cotyledons, and the tested strains were dropped onto five cucumber cotyledons.…”

Construction of a Binary Vector for Knockout and Expression Analysis of Rice Blast Fungus Genes

“…For functional analysis of the genes, insertional mutagenesis and gene knockout using Agrobacterium-mediated transformation (AMT) have been performed. 2,3) Characterizing the expression profile of the gene of interest is also useful in understanding its function. The green fluorescent protein (GFP) gene was used to monitor promoter activity.…”

“…A PtrpC-HPT fragment (hygromycin B phosphotransferase (HPT) under the control of the Aspergillus nidulans trpC promoter) was prepared from pBIG2RHPH2 2) and cloned into pCR8/GW/TOPO (Invitrogen) to generate an entry vector, pETH. A synthetic GFP (S65T) gene 5) was introduced into pETH to construct pETHG.…”