Chromosomal organization of TOX2, a complex locus controlling host-selective toxin biosynthesis in Cochliobolus carbonum

Race 1 isolates of the filamentous fungus Cochliobolus carbonum are exceptionally virulent on certain genotypes of maize due to production of a cyclic tetrapeptide, HC-toxin, In crosses between toxin-producing (Tox2(+)) and toxin-nonproducing (Tox2(-)) isolates, toxin production segregates in a simple 1:1 pattern, suggesting the involvement of a single genetic locus, which has been named TOX2. Earlier work had shown that in isolate SB111, TOX2 consists in part of two copies of a gene, HTS1, that encodes a 570-kD cyclic peptide synthetase and is lacking in Tox2(-) isolates. The genomic structure of TOX2 and the relationship between the two copies of HTS1 have now been clarified by using pulsed-field gel electrophoresis and physical mapping. In isolate SB111, both copies of HTS1 are on the largest chromosome (3.5 Mb), which is not present in the related Tox2(-) strain SB114, Two other genes known or thought to be important for HC-toxin biosynthesis, TOXA and TOXC, are also on the same chromosome in multiple copies, Other independent Tox2(+) isolates also have two linked copies of HTS1, but in some isolates the size of the chromosome containing HTS1 is 2.2 Mb. Evidence obtained with Tox2(+)-unique and with random probes is consistent with a reciprocal translocation as the cause of the difference in the size of the HTS1-containing chromosome among the Tox2(+) isolates studied here, Physical mapping of the 3.5-Mb chromosome of SB111 that contains HTS1 using rare-cutting restriction enzymes and engineered restriction sites was used to map the chromosomal location of the two copies of HTS1 and the three copies of TOXC. The results indicate that TOX2 is a complex locus that extends over more than 500 kb. The capacity to produce HC-toxin did not evolve by any single, simple mechanism.