Transformation ofConiothyrium minitans, a parasite ofSclerotinia sclerotiorum, withAgrobacterium tumefaciens

Abstract

Coniothyrium minitans is a potential biological control agent of the plant pathogenic fungus Sclerotinia sclerotiorum. In this research, T-DNA insertional transformation of strain ZS-1 of C. minitans mediated by Agrobacterium tumefaciens was obtained, with optimization of spore maturity for transformation. After confirmation by PCR, transformants were subjected to Southern blot analysis, and results showed that more than 82.7% of transformants had single T-DNA insertions, and 12.1% of transformants had two copies T-DNA insertions. The genomic DNA segments of transformants flanking the T-DNA could be amplified from both borders with TAIL-PCR. Four types of mutants were screened and identified from the T-DNA insertional library, which comprised sporulation deficient mutants, pathogenicity deficient mutants, pigment change mutants and antibiotic deficient mutant, and some of the mutants were described; the number and frequency of each type of mutant from the library were calculated, and the frequency of each type is 3.27 x 10(-3), 1.0 x 10(-4), 1.4 x 10(-4), 2.5 x 10(-4), respectively. The successful creation of the T-DNA insertional transformation library may help us to unravel the interaction between a parasite and its host at a molecular level, to clarify the differentiation and development of this fungus, and to analyze and clone functional genes from the biocontrol microorganism in tripartite associations.

High-throughput FACS-based mutant screen identifies a gain-of-function allele of the Fusarium graminearum adenylyl cyclase causing deoxynivalenol over-production

“…Huang et al(2015) also used a different mutagen, which may have been more effective than EMS. Other effective mutagens, which have previously been used with considerable success in fungi are gene tagging by direct DNA transfer (Seong et al, 2005), restriction enzyme mediated integration (REMI) (Brown et al, 1998), Agrobacterium mediated T-DNA (Li et al, 2005) and transposon insertions (Firon et al, 2003). However, in some cases, these mutagens can induce chromosomal rearrangements and/or multiple insertions (Brown et al, 1998;Chung et al, 2003), causing difficulties in determining the causative mutation behind an observed phenotype (reviewed in Weld et al, 2006).…”

A geminivirus-related DNA mycovirus that confers hypovirulence to a plant pathogenic fungus

“…RNA samples were treated with DNase I (RNase Free) before use. Southern and Northern blot analyses were conducted as described previously (19,42).…”

“…To test the possible transmission of hypovirulence and SsHADV-1 from strain DT-8 to a vegetatively incompatible virulent strain (Ep-1PNA367), strain Ep-1PNA367 was labeled with the hygromycin B resistance gene (hph) using Agrobacterium-mediated transformation as described by Li et al (42). The transmission test between DT-8 and labeled Ep-1PNA367 (named strain Ep-1PNA367 R ) was conducted using the method described by Zhang et al (40).…”

Approaches to functional genomics in filamentous fungi

“…Fungi that have been recalcitrant to transformation by other systems have been successfully transformed by co-cultivation with Agrobacterium [27]. AMT is a relatively simple system to work with, does not require the production of protoplasts, and is suitable for both gene-replacement by homologous recombination [1,28,29] and insertional mutagenesis by random integration [30,31].…”

“…T-DNA tagging has been used successfully to find new genes [30,70] and T-DNA tagging projects on fungi have recently been initiated in many laboratories around the world [31,68]. AMT is very suitable for insertional mutagenesis as it can cause a relatively high frequency of transformation [71][72][73] and often creates single-copy integrations [27,31].…”