Virulence gene expression during conidial germination in Cochliobolus carbonum

The fungal pathogen Cochliobolus carbonum race 1 produces a host-selective toxin (HC-toxin) that is responsible for increased virulence on susceptible genotypes of maize. The toxin is synthesized by a peptide synthetase, which is a product of the HTS1 gene. Because the toxin is not stored in dormant conidia, early expression of HTS1 is crucial for extensive colonization of susceptible leaf tissue. To detect the HTS1 transcript and determine the onset of HTS1 gene expression, we analyzed RNA preparations from ungerminated and germinated conidia by reverse transcription-polymerase chain reaction using oligonucleotide primers within the 15.7-kb open reading frame of HTS1. With primer pairs near both the 3'- and the 5'-termini, amplified products of the HTS1 transcript were detected in RNA prepared from dormant conidia. With all primer pairs used, the quantities of transcript increased substantially during germ tube emergence and elongation, indicating that expression of HTS1 is up regulated during spore germination. Digestion with restriction endonucleases confirmed the identity of the amplified products. Amplification of the constitutively expressed beta-tubulin transcript, which is processed to remove introns, as well as the absence of amplification products with primers spanning the HTS1 coding sequence established that cDNA was amplified and not contaminating genomic DNA.