Open AccessIdentification of virulence genes in the corn pathogen Colletotrichum graminicola by Agrobacterium tumefaciens ‐mediated transformation
Author(s) -
MÜNCH STEFFEN,
LUDWIG NANCY,
FLOSS DANIELA S.,
SUGUI JANYCE A.,
KOSZUCKA ANNA M.,
VOLL LARS M.,
SONNEWALD UWE,
DEISING HOLGER B.
Publication year - 2011
Publication title -
molecular plant pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.945
H-Index - 103
eISSN - 1364-3703
pISSN - 1464-6722
DOI - 10.1111/j.1364-3703.2010.00651.x
Subject(s) - biology , virulence , agrobacterium tumefaciens , pathogen , transformation (genetics) , identification (biology) , microbiology and biotechnology , gene , agrobacterium , botany , genetics
SUMMARY 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.