John W. Pitkin

A Partial Chromosomal Deletion Caused by Random Plasmid Integration Resulted in a Reduced Virulence Phenotype in Fusarium graminearum

Fusarium graminearum (teleomorph: Gibberella zeae) is an Ascomycete fungal plant pathogen which infects a range of agriculturally important crops, including wheat, barley, and maize. A random plasmid insertion mutagenesis approach was used to analyze the pathogenicity of the PH-1 strain, for which full genomic information is available. Fungal transformants were initially screened for their ability to infect wheat ears. From a total of 1,170 transformants screened, eight were confirmed to be highly reduced in pathogenicity toward wheat ears and roots. These were designated disease-attenuated F. graminearum (daf) mutants. The in vitro growth rate and appearance of each daf mutant was equivalent to the parental strain. Deoxynivalenol (DON) was not detected in threshed grain recovered from ears inoculated with the daf10 mutant. Plasmid rescue and sequencing of the mutant daf10 revealed a deletion of approximately 350 kb from one end of chromosome 1. This chromosome segment is predicted to contain 146 genes. Microarray analysis of daf10 gene expression during growth in DON-inducing conditions confirmed the large deletion. The identities of the genes deleted and their potential role in DON production, pathogenesis, and other life processes are discussed.

Enzymology, Molecular Genetics, and Regulation of Biosynthesis of the Host-Selective Toxin HC-Toxin

HC-toxin is the critical determinant of virulence and specificity in the interaction between race 1 of Cochliobolus carbonum and maize of genotype hm/hm [21]. HC-toxin is a cyclic peptide of structure cyclo(D-Pro-L-Ala-D-Ala-L-Aeo), where Aeo stands for 2-amino-9,10-epoxi-8-oxodecanoic acid. Earlier genetics studies had indicated that a single locus, TOX2, controls HC-toxin production. The overall aim of our research has been to analyze the TOX2 locus at the molecular level. We began our approach to this problem by identifying enzyme activities characteristic of the putative cyclic peptide synthetase (CPS) necessary for HC-toxin synthesis (named HC-toxin synthetase) in extracts of C. carbonum. We discovered and characterized activities that could catalyze D-Ala, L-Ala, and L-Pro-dependent ATP/PPj exchange [20, 23]. The enzyme activities are present only in race 1 (toxin-producing or Tox2+) isolates of C. carbonum. We purified the polypeptide that catalyzes L-Pro-dependent exchange and used antibodies and synthetic oligonucleotides based on peptide sequences to isolate a partial cDNA encoding the corresponding gene, HTS1. By sequencing the surrounding genomic DNA, we found that HTS1 contains an intronless 16-kb open reading frame encoding a CPS with four characteristic 600-amino acid CPS domains, one for activation of each of the four amino acids. The predicted molecular weight of the product of HTSl, HTS, is 570 kDa [17]. HTS1 is present only in isolates of C. carbonum that make HC-toxin, and in all natural Tox2+ isolates it is present in two, linked copies [13]. Gene disruption experiments proved that HTS1 is necessary for HC-toxin production, HTS activity, and pathogenicity [13].