EXG1p, a novel exo-beta1,3-glucanase from the fungus Cochliobolus carbonum, contains a repeated motif present in other proteins that interact with polysaccharides.

Genomic and cDNA copies of EXG1, a gene encoding an exo-beta1, 3-glucanase from the plant pathogenic fungus Cochliobolus carbonum, were isolated. The gene contains two introns of 50 and 53 bp, and the mRNA has a 5'-untranslated region of 90 nt and a 3'-untranslated region of 159 nt. The deduced protein product, EXG1p, has a predicted signal peptide of 17 amino acids, but based on the known N-terminus of the mature protein is further processed to remove an additional 25 amino acids. The sequence of EXG1p is not closely related to any other known protein, but has a low similarity (29% overall amino acid identity) to BGN13.1, an endo-beta1,3-glucanase from the mycoparasitic fungus Trichoderma harzianum. EXG1p contains two imperfect copies of a 23-amino acid motif that is found in several other proteins that interact with polysaccharides, including plant and bacterial polygalacturonases, phage neck appendage protein, phage endoneuramidase, and bacterial mannuronan epimerase.

Reduced virulence caused by meiotic instability of the TOX2 chromosome of the maize pathogen Cochliobolus carbonum.

The mechanisms by which pathogenic fungi evolve are poorly understood. Production of the host-selective cyclic peptide HC-toxin is controlled by a complex locus, TOX2, in the plant pathogen Cochliobolus carbonum. Crosses between toxin-producing (Tox2+) and toxin-nonproducing (Tox2-) isolates, as well as crosses between isolates in which the TOX2 genes were on chromosomes of different size, yielded progeny that had lost one or more copies of one or more of the TOX2 genes. Of approximately 200 progeny analyzed, eight (4%) had lost at least one TOX2 gene. All of them still had at least one functional copy of all of the known genes required for HC-toxin production (HTS1, TOXA, TOXC, and TOXE). Most deletion strains could be explained by simple chromosome breaks resulting in the loss of major contiguous portions (0.8 to 1.4 Mb) of the 3.5-Mb TOX2 chromosome, whereas others had more complicated patterns. All deletion strains had normal growth and were fertile, indicating that the 1.4 Mb of DNA contained no essential housekeeping genes. Most strains were also still virulent (Tox2+), but two had a novel phenotype of reduced virulence (RV), characterized by smaller lesions that expanded at a reduced rate and an inability to colonize plants systemically. Although the RV strains made no detectable HC-toxin in culture, the RV phenotype was dependent on the presence of a functional copy of HTS1, which encodes the central enzyme in HC-toxin biosynthesis. We propose that the RV strains still make a low level of HC-toxin, at least in planta, and that this is due to the loss of one or more genes that contribute to, but are not absolutely required for, HC-toxin synthesis.

Transposon-like sequences at the TOX2 locus of the plant-pathogenic fungus Cochliobolus carbonum.

The ascomycete fungus Cochliobolus carbonum race 1 is pathogenic on certain genotypes of maize due to the production of HC-toxin, a host-specific cyclic peptide. HC-toxin production is controlled, at least in part, by a duplicated 22-kb region of DNA that is found only in toxin-producing isolates of the fungus. This 22-kb region of DNA is flanked by a repetitive element. We have sequenced the element and found an interrupted reading frame that would encode a product similar to transposases from the fungal transposons Fot1 of Fusarium oxysporum and Pot2 of Magnaporthe grisea. The individual element cloned from C. carbonum is likely to function neither in cis nor trans, as it had a nonsense mutation in frame and several substitutions in its terminal inverted repeats. However, similar elements in the C. carbonum genome may be active, as the putative transposase-encoding region hybridized to mRNA of the size predicted by the reading frame. The element was found in varying copy number in the genomes of all Cochliobolus spp. examined, giving a distinct fingerprint in each species and race tested. The sequence similarity of the C. carbonum repetitive element to other fungal transposons, along with its presence in multiple copies per genome, strongly suggest that the C. carbonum repetitive element is a member of the Fot1 family of fungal transposons.

Cloning and expression of the S-adenosylmethionine decarboxylase gene of Neurospora crassa and processing of its product.

S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes the formation of decarboxylated AdoMetDC, a precursor of the polyamines spermidine and spermine. The enzyme is derived from a proenzyme by autocatalytic cleavage. We report the cloning and regulation of the gene for AdoMetDC in Neurospora crassa, spe-2, and the effect of putrescine on enzyme maturation and activity. The gene was cloned from a genomic library by complementation of a spe-2 mutant. Like other AdoMetDCs, that of Neurospora is derived by cleavage of a proenzyme. The deduced sequence of the Neurospora proenzyme (503 codons) is over 100 codons longer than any other AdoMetDC sequence available in genomic databases. The additional amino acids are found only in the AdoMetDC of another fungus, Aspergillus nidulans, a cDNA for which we also sequenced. Despite the conserved processing site and four acidic residues required for putrescine stimulation of human proenzyme processing, putrescine has no effect on the rate (t0.5 approximately 10 min) of processing of the Neurospora gene product. However, putrescine is absolutely required for activity of the Neurospora enzyme (K0.5 approximately 100 microM). The abundance of spe-2 mRNA and enzyme activity is regulated 2- to 4-fold by spermidine.

Targeted mutants of Cochliobolus carbonum lacking the two major extracellular polygalacturonases.

The filamentous fungus Cochliobolus carbonum produces endo-alpha 1,4-polygalacturonase (endoPG), exo-alpha 1,4-polygalacturonase (exoPG), and pectin methylesterase when grown in culture on pectin. Residual activity in a pgn1 mutant (lacking endoPG) was due to exoPG activity, and the responsible protein has now been purified. After chemical deglycosylation, the molecular mass of the purified protein decreased from greater than 60 to 45 kDa. The gene that encodes exoPG, PGX1, was isolated with PCR primers based on peptide sequences from the protein. The product of PGX1, Pgx1p, has a predicted molecular mass of 48 kDa, 12 potential N-glycosylation sites, and 61% amino acid identity to an exoPG from the saprophytic fungus Aspergillus tubingensis. Strains of C. carbonum mutated in PGX1 were constructed by targeted gene disruption and by gene replacement. Growth of pgx1 mutant strains on pectin was reduced by ca. 20%, and they were still pathogenic on maize. A double pgn1/pgx1 mutant strain was constructed by crossing. The double mutant grew as well as the pgx1 single mutant on pectin and was still pathogenic despite having less than 1% of total wild-type PG activity. Double mutants retained a small amount of PG activity with the same cation-exchange retention time as Pgn1p and also pectin methylesterase and a PG activity associated with the mycelium. Continued growth of the pgn1/pgx1 mutant on pectin could be due to one or more of these residual activities.