Identification of virulence genes in the corn pathogenColletotrichum graminicolabyAgrobacterium tumefaciens‐mediated transformation

Abstract

A previously developed Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for the plant pathogenic fungus Colletotrichum graminicola led to high rates of tandem integration of the whole Ti-plasmid, and was therefore considered to be unsuitable for the identification of pathogenicity and virulence genes by insertional mutagenesis in this pathogen. We used a modified ATMT protocol with acetosyringone present only during the co-cultivation of C. graminicola and A. tumefaciens. Analysis of 105 single-spore isolates randomly chosen from a collection of approximately 2000 transformants, indicated that almost 70% of the transformants had single T-DNA integrations. Of 500 independent transformants tested, 10 exhibited attenuated virulence in infection assays on whole plants. Microscopic analyses primarily revealed defects at different pre-penetration stages of infection-related morphogenesis. Three transformants were characterized in detail. The identification of the T-DNA integration sites was performed by amplification of genomic DNA ends after endonuclease digestion and polynucleotide tailing. In one transformant, the T-DNA had integrated into the 5'-flank of a gene with similarity to allantoicase genes of other Ascomycota. In the second and third transformants, the T-DNA had integrated into an open reading frame (ORF) and into the 5'-flank of an ORF. In both cases, the ORFs have unknown function.

Agrobacterium tumefaciens-Mediated Transformation for Investigating Pathogenicity Genes of the Phytopathogenic Fungus Colletotrichum sansevieriae

“…The procedures among those reports are almost the same, but the major difference is strains of A. tumefaciens used and Ti plasmids they carried. In fact, we used the different strain (GV2260) of A. tumefaciens and it is notable that Münch et al [21] has successfully established the AtMT method for C. graminicola using the strain LBA1100 that Flowers and Vaillancourt [6] did not use, suggesting that combination of a strain of A. tumefaciens a M850 is not affected in its pathogenicity and shown as a reference Fig. 6 Sequences of the T-DNA end and flanking genomic DNA of pathogenicity-deficient transformants.…”

“…Good results were also obtained with the AtMT of Colletotrichum spp. such as C. gloeosporioides, C. acutatum, C. trifolii, C. orbiculare and so on [4,19,21,32,33]. Major advantages of AtMT are that intact cells, such as spores and mycelial fragments, can be used as host cells, and it is unnecessary to conduct the time-consuming work of protoplast production.…”

Draft Genome Sequence ofUmbilicaria muehlenbergiiKoLRILF000956, a Lichen-Forming Fungus Amenable to Genetic Manipulation

“…The lichens produce useful metabolites with properties of pharmaceutical interest such as anti-HIV, antitumor, antimicrobial, anti-inflammatory, and antioxidant activities. However, unlike other well-studied ascomycetous fungi, including model fungi (1–3) and phytopathogenic fungi (4–7), lichen-forming fungi had no well-established transformation system. Recently, the first successful transformation of a lichen fungus, Umbilicaria muehlenbergii , via Agrobacterium tumefaciens has been reported (8).…”

Hexose Transporters of a Hemibiotrophic Plant Pathogen

“…The C. graminicola strain M2 was used in this study. Plant infection assays with C. graminicola were performed as described (15). Briefly, 2-week-old maize plants were inoculated with fungal spore suspensions (10 6 spores per plant) and incubated at appropriate conditions in the climate chamber.…”