| HC-toxin is the molecular determinant in the interaction between the fungal pathogen Cochliobolus carbonum race 1 and its host plant, Zea mays L. (genotype hm1/ hm1). HC-toxin exists as a small family of cyclic tetrapeptides. The major component of HC-toxin, HC-toxin I, has the structure cyclo(D-Pro-L-Ala-D-Ala-L-Aeo), where Aeo stands for 2-amino-9,10-epoxi-8-oxo-decanoic acid. The biosynthesis of HC-toxin is controlled by a genetic locus, TOX2, that is complex at the molecular level. Previous studies have identified four genes (HTS1, TOXA, TOXC, and TOXE collectively called TOX2). These known TOX2 genes each exist as two or three functional copies per genome and are present only in HC-toxin-producing isolates (Tox2+) of C. carbonum. The TOX2 genes are distributed over &sim540 kb on a special chromosome in a standard lab strain SB111 (one copy of TOXE is located on a different chromosome). When all copies of any individual gene are mutated, the fungus loses the capability to produce HC-toxin and thus pathogenicity.In this thesis research, two additional TOX2 genes, TOXF and TOXG, have been cloned by using bacterial artificial chromosomes (BACs). TOXF and TOXG are two tightly linked genes. Both have three copies in SB111, and two functional copies in isolate 164R10, both are exclusively present in Tox2+ isolates of C. carbonum, both map to the TOX2 locus, and both are regulated by TOXE. TOXF encodes a putative branched-chain amino acid aminotransferase that we hypothesize to aminate an alpha-keto acid in the pathway to make Aeo. A null mutant of TOXF failed to produce HC-toxin and lost the ability to causes severe leaf spot disease on maize. Therefore, TOXF can be regarded as a pathogenicity gene. TOXG encodes a novel alanine racemase that catalyzes the interconversion between L-alanine and D-alanine. D-alanine is a critical constituent in HC-toxin I, III and IV, but not HC-toxin II, which has glycine in place of D-alanine. A null mutant of TOXG failed to make HC-toxin I, III and IV, but still made HC-toxin II. Feeding the TOXG null mutant with D-alanine restored the normal HC-toxin production profile. Compared to wild type, the TOXG mutant caused a delayed disease phenotype that eventually resulted in full symptoms due to the presence of HC-toxin II. Therefore, TOXG can only be classified as a virulence factor. Genetic and biochemical experiments were successfully adopted to confirm that TOXG gene product functions as alanine racemase.Another TOX2 candidate gene, TOXD, was cloned based on its linkage to TOXC. TOXD shares similarities with other known TOX2 genes, including physical linkage, copy number, gene distribution, and regulation by TOXE. However, HC-toxin production and fungal pathogenicity were unchanged in a TOXD null mutant. It appears that TOXD has no apparent role in HC-toxin production or fungal pathogenicity. |
