Genotyping of benzimidazole-resistant and dicarboximide-resistant mutations in Botrytis cinerea using real-time polymerase chain reaction assays.

Botrytis cinerea, an economically important gray mold pathogen, frequently exhibits multiple fungicide resistance. A fluorescence resonance energy transfer-based real-time polymerase chain reaction assay has been developed to detect benzimidazole- and dicarboximide-resistant mutations. Three benzimidazole-resistant mutations-(198)Glu to Ala (E198A), F200Y, and E198K-in beta-tubulin BenA were detected using a single set of fluorescence-labeled sensor and anchor probes by melting curve analysis.

A Point Mutation in the Two-Component Histidine Kinase BcOS-1 Gene Confers Dicarboximide Resistance in Field Isolates of Botrytis cinerea.

ABSTRACT Partial DNA fragments of Botrytis cinerea field isolates encoding the putative osmosensor histidine kinase gene (BcOS1) were cloned by polymerase chain reaction amplification and the predicted amino acid sequences were compared between dicarboximide-sensitive and resistant field isolates. The predicted BcOS1p is highly homologous to osmosensor histidine kinase OS1p from Neurospora crassa including the N-terminal six tandem repeats of approximately 90 amino acids. Four dicarboximide-resistant isolates of B.

Involvement of OS-2 MAP kinase in regulation of the large-subunit catalases CAT-1 and CAT-3 in Neurospora crassa.

Neurospora crassa has four catalase genes--cat-1, cat-2, cat-3, and ctt-1/cat-4. cat-1 and cat-3 encode two fungal-specific large-subunit catalases CAT-1 and CAT-3 normally produced in conidia and growing hyphae, respectively. cat-2 encodes CAT-2 catalase-peroxidase normally produced in conidia.

Roles of putative His-to-Asp signaling modules HPT-1 and RRG-2, on viability and sensitivity to osmotic and oxidative stresses in Neurospora crassa.

Neurospora crassa has a putative histidine phosphotransfer protein (HPT-1) that transfers signals from 11 histidine kinases to two putative response regulators (RRG-1 and RRG-2) in its histidine-to-aspartate phosphorelay system. The hpt-1 gene was successfully disrupted in the os-2 (MAP kinase gene) mutant, but not in the wild-type strain in this study. Crossing the resultant hpt-1; os-2 mutants with the wild-type or os-1 (histidine kinase gene) mutant strains produced no progeny with hpt-1 or os-1; hpt-1 mutation, strongly suggesting that hpt-1 is essential for growth unless downstream OS-2 is inactivated.

Identification of OS-2 MAP kinase-dependent genes induced in response to osmotic stress, antifungal agent fludioxonil, and heat shock in Neurospora crassa.

Two-component signal transduction comprising of OS-1 (histidine kinase), OS-4 (MAPKK kinase), OS-5 (MAPK kinase), and OS-2 (MAP kinase) plays an important role in osmotic regulation in Neurospora crassa. To identify the genes regulated downstream of OS-2 MAP kinase, quantitative real-time RT-PCR analysis was conducted in selected genes based on Hog1 MAP kinase regulated genes in yeast. In response to osmotic stress and fludioxonil, expression of six genes that for glycerol synthesis (gcy-1, gcy-3, and dak-1), gluconeogenesis (fbp-1 and pck-1), and catalase (ctt-1) was activated in the wild-type strain, but not in the os-2 mutant.

A catalytic subunit of cyclic AMP-dependent protein kinase, PKAC-1, regulates asexual differentiation in Neurospora crassa.

A cyclic AMP (cAMP)-dependent protein kinase pathway has been shown to regulate growth, morphogenesis and virulence in filamentous fungi. However, the precise mechanisms of regulation through the pathway remain poorly understood. In Neurospora crassa, the cr-1 adenylate cyclase mutant exhibits colonial growth with short aerial hyphae bearing conidia, and the mcb mutant, a mutant of the regulatory subunit of cAMP-dependent protein kinase (PKA), shows the loss of growth polarity at the restrictive temperature.

Cloning and characterization of genes specifically expressed during infection stages in the rice blast fungus.

We isolated promoters of 12 genes from the rice blast fungus based on the sequences of randomly selected expressed sequence tags (ESTs) (appressorium formation stage cDNA library of Magnaporthe available from GenBank). These promoters (and the 5' coding regions if any) were fused in frame with egfp, and their expression patterns were examined under the epifluorescence microscope. Among them, two turned out to be specifically active in structures necessary for infection, viz.