Genetic transformation of Ascochyta rabiei using Agrobacterium-mediated transformation

Abstract

In order to study pathogenic mechanisms of the plant pathogen Ascochyta rabiei, conditions for efficient transformation using Agrobacterium-mediated transformation were investigated. Hygromycin B resistance (hph) was superior to geneticin resistance (nptII) for selecting transformants, and the hph gene was more efficiently expressed by the Aspergillus nidulans trpC promoter than by the Cauliflower mosaic virus 35S promoter CaMV35S. Co-cultivation on solid media for 72 h was optimal for generating transformants, but increasing the ratio of bacterial cells to conidia did not affect transformation efficiency. All hygromycin B-resistant transformants carried transfer-DNA (T-DNA) as determined by polymerase chain reaction (PCR) and the T-DNA integrations appeared to be random and in single copy as detected by Southern hybridization. Transformants remained resistant to hygromycin B in the absence of selection. Variations in colony morphology were observed in the presence of hygromycin B under different culture conditions, and a variety of altered phenotypes including reduced virulence were observed among 550 transformants. Inverse PCR was more efficient than TAIL-PCR in identifying flanking genomic sequences from T-DNA borders, and the possible causes are discussed. This transformation technique and recovery of flanking DNA using inverse PCR will provide a useful tool for genetic studies of A. rabiei.

Identification of Pathogenicity-Related Genes in the Vascular Wilt Fungus Verticillium dahliae by Agrobacterium tumefaciens-Mediated T-DNA Insertional Mutagenesis

“…iPCR is a rapid and an efficient tool for isolating sequences adjacent to inserted T-DNA in the transformants of fungal species [32, 33]. For iPCR amplification of genomic DNA flanking the T-DNA insertion site, we used two primers, RB3 and RBn1, which are designed to amplify the right T-DNA border sequence and adjacent sequence from V. dahliae .…”

Towards identifying pathogenic determinants of the chickpea pathogen Ascochyta rabiei

“…Genomic DNA from transformants and wild-type AR628 were digested with XhoI (New England Biolabs, Ipswich, MA, USA), separated on an agarose gel, and transferred to a nylon membrane. A DIGlabelled DNA probe was synthesized from an internal region of the hygromycin B gene using PCR (White and Chen 2006). Probed membranes were processed according to the manufacturer's instructions, and detected using the anti-DIGalkaline phosphatase conjugate antibody and the chemiluminescent substrate CSPD (Roche) by exposure to autoradiograph film to visualize hybridized fragments.…”

“…Genomic DNA of selected transformants was digested with XhoI to isolate DNA flanking the right border, and digested with either SacI, SalI or KpnI to isolate DNA flanking the left border. Digested DNA was ligated to itself and used as template for the inverse PCR using primers LB5IP and RB5IP (White and Chen 2006). Products were isolated from agarose gels and ligated to the pGEM-T Easy vector (Promega, Madison, WI, USA) for further analysis.…”

“…The transformation was carried out as previously described (White and Chen 2006). Briefly, conidia of strain AR628 were co-cultured with cells of Agrobacterium tumefaciens carrying T-DNA.…”

“…The hydrolytic enzymes are considered necessary for fungal nutrition and to facilitate spatial spread of fungi (Walton 1994). Ascochyta rabiei was first transformed with the protoplast/PEG protocol with a GUS reporter gene for observing the infection process (Kohler et al 1995), and later transformed with Agrobacterium-mediated transformation (AMT) for studying pathogenicity factors (White and Chen 2006;Morgensen et al 2006). However, little information is currently available about the genetic determinants of pathogenicity of the ascochyta pathogens.…”