Identification of virulence genes in the corn pathogen Colletotrichum graminicola by Agrobacterium tumefaciens-mediated transformation.
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.
Martin-Luther-University Halle-Wittenberg, Faculty of Agricultural and Nutritional Sciences, Phytopathology and Plant Protection, Betty-Heimann-Str. 3, 06120 Halle (Saale), Germany.
Steffen
S
Münch
Nancy
N
Ludwig
Daniela S
DS
Floss
Janyce A
JA
Sugui
Anna M
AM
Koszucka
Lars M
LM
Voll
Uwe
U
Sonnewald
Holger B
HB
Deising
D016960:Agrobacterium tumefaciens / Q000235:genetics; D020231:Colletotrichum / Q000235:genetics* / Q000472:pathogenicity*; D004271:DNA, Fungal / Q000235:genetics; D005800:Genes, Fungal; D015698:Genomic Library; D054884:Host-Pathogen Interactions / Q000235:genetics; D016254:Mutagenesis, Insertional; D010788:Photosynthesis; D010935:Plant Diseases / Q000382:microbiology*; D036741:Plant Tumor-Inducing Plasmids / Q000235:genetics; D014170:Transformation, Genetic; D014774:Virulence / Q000235:genetics; D003313:Zea mays / Q000378:metabolism / Q000382:microbiology*
D004271:DNA, Fungal