Cgl-SLT2 is required for appressorium formation, sporulation and pathogenicity in Colletotrichum gloeosporioide

The mitogen-activated protein (MAP) kinase pathways has been implicated in the pathogenicity of various pathogenic fungi and plays important roles in regulating pathogenicity-related morphogenesis. This work describes the isolation and characterization of MAP kinase gene, Cgl-SLT2, from Colletotrichum gloeosporioides. A DNA sequence, including 1,633 bp of Cgl-SLT2 open-reading frame and its promoter and terminator regions, was isolated via DNA walking and cloned. To analyze gene function, a gene disruption cassette containing hygromycin-resistant gene was constructed, and Cgl-SLT2 was inactivated via gene deletion. Analysis on Cgl-slt2 mutant revealed a defect in vegetative growth and sporulation as compared to the wild-type strain. When grown under nutrient-limiting conditions, hyperbranched hyphal morphology was observed in the mutant. Conidia induction for germination on rubber wax-coated hard surfaces revealed no differences in the percentage of conidial germination between the wild-type and Cgl-slt2 mutant. However, the percentage of appressorium formation in the mutant was greatly reduced. Bipolar germination in the mutant was higher than in the wild-type at 8-h post-induction. A pathogenicity assay revealed that the mutant was unable to infect either wounded or unwounded mangoes. These results suggest that the Cgl-SLT2 MAP kinase is required for C. gloeosporioides conidiation, polarized growth, appressorium formation and pathogenicity.

Cgl-SLT2 is required for appressorium formation, sporulation and pathogenicity in Colletotrichum gloeosporioides.

The mitogen-activated protein (MAP) kinase pathways has been implicated in the pathogenicity of various pathogenic fungi and plays important roles in regulating pathogenicity-related morphogenesis. This work describes the isolation and characterization of MAP kinase gene, Cgl-SLT2, from Colletotrichum gloeosporioides. A DNA sequence, including 1,633 bp of Cgl-SLT2 open-reading frame and its promoter and terminator regions, was isolated via DNA walking and cloned. To analyze gene function, a gene disruption cassette containing hygromycin-resistant gene was constructed, and Cgl-SLT2 was inactivated via gene deletion. Analysis on Cgl-slt2 mutant revealed a defect in vegetative growth and sporulation as compared to the wild-type strain. When grown under nutrient-limiting conditions, hyperbranched hyphal morphology was observed in the mutant. Conidia induction for germination on rubber wax-coated hard surfaces revealed no differences in the percentage of conidial germination between the wild-type and Cgl-slt2 mutant. However, the percentage of appressorium formation in the mutant was greatly reduced. Bipolar germination in the mutant was higher than in the wild-type at 8-h post-induction. A pathogenicity assay revealed that the mutant was unable to infect either wounded or unwounded mangoes. These results suggest that the Cgl-SLT2 MAP kinase is required for C. gloeosporioides conidiation, polarized growth, appressorium formation and pathogenicity.

L-Mandelate dehydrogenase from Rhodotorula graminis: cloning, sequencing and kinetic characterization of the recombinant enzyme and its independently expressed flavin domain.

The l-mandelate dehydrogenase (L-MDH) from the yeast Rhodotorula graminis is a mitochondrial flavocytochrome b2 which catalyses the oxidation of mandelate to phenylglyoxylate coupled with the reduction of cytochrome c. We have used the N-terminal sequence of the enzyme to isolate the gene encoding this enzyme using the PCR. Comparison of the genomic sequence with the sequence of cDNA prepared by reverse transcription PCR revealed the presence of 11 introns in the coding region. The predicted amino acid sequence indicates a close relationship with the flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala, with about 40% identity to each. The sequence shows that a key residue for substrate specificity in S. cerevisiae flavocytochrome b2, Leu-230, is replaced by Gly in L-MDH. This substitution is likely to play an important part in determining the different substrate specificities of the two enzymes. We have developed an expression system and purification protocol for recombinant L-MDH. In addition, we have expressed and purified the flavin-containing domain of L-MDH independently of its cytochrome domain. Detailed steady-state and pre-steady-state kinetic investigations of both L-MDH and its independently expressed flavin domain have been carried out. These indicate that L-MDH is efficient with both physiological (cytochrome c, kcat=225 s-1 at 25 degrees C) and artificial (ferricyanide, kcat=550 s-1 at 25 degrees C) electron acceptors. Kinetic isotope effects with [2-2H]mandelate indicate that H-C-2 bond cleavage contributes somewhat to rate-limitation. However, the value of the isotope effect erodes significantly as the catalytic cycle proceeds. Reduction potentials at 25 degrees C were measured as -120 mV for the 2-electron reduction of the flavin and -10 mV for the 1-electron reduction of the haem. The general trends seen in the kinetic studies show marked similarities to those observed previously with the flavocytochrome b2 (L-lactate dehydrogenase) from S. cerevisiae.