The Colletotrichum lagenarium MAP kinase gene CMK1 regulates diverse aspects of fungal pathogenesis

The infection process of Colletotrichum lagenarium, the causal agent of cucumber anthracnose disease, involves several key steps: germination; formation of melanized appressoria; appressorial penetration; and subsequent invasive growth in host plants. Here we report that the C. lagenarium CMK1 gene encoding a mitogen-activated protein (MAP) kinase plays a central role in these infection steps. CMK1 can complement appressorium formation of the Pmk1 MAP kinase mutant of Magnaporthe grisea. Deletion of CMK1 causes reduction of conidiation and complete lack of pathogenicity to the host plant. Surprisingly, in contrast to M. grisea pmk1 mutants, conidia of cmk1 mutants fail to germinate on both host plant and glass surfaces, demonstrating that the CMK1 MAP kinase regulates conidial germination. However, addition of yeast extract rescues germination, indicating the presence of a CMK1-independent pathway for regulation of conidial germination. Germinating conidia of cmk1 mutants fail to form appressoria and the mutants are unable to grow invasively in the host plant. This strongly suggests that MAP kinase signaling pathways have general significance for infection structure formation and pathogenic growth in phytopathogenic fungi. Furthermore, three melanin genes show no or slight expression in the cmk1 mutant when conidia fail to germinate, suggesting that CMK1 plays a role in gene expression required for appressorial melanization.     

A Large Cluster of Highly Expressed Genes Is Dispensable for Growth and Development in Aspergillus Nidulans

We investigated the functions of the highly expressed, sporulation-specific SpoC1 genes of Aspergillus nidulans by deleting the entire 38-kb SpoC1 gene cluster. The resultant mutant strain did not differ from the wild type in (1) growth rate, (2) morphology of specialized reproductive structures formed during completion of the asexual or sexual life cycles, (3) sporulation efficiency, (4) spore viability or (5) spore resistance to environmental stress. Thus, deletion of the SpoC1 gene cluster, representing 0.15% of the A. nidulans genome, had no readily detectable phenotypic effects. Implications of this result are discussed in the context of major alterations in gene expression that occur during A. nidulans development.     

A single regulatory module of the carbamoylphosphate synthetase I gene executes its hepatic program of expression

A 469-base pair (bp) upstream regulatory fragment (URF) and the proximal promoter of the carbamoylphosphate synthetase I (CPS) gene were analyzed for their role in the regulation of spatial, developmental, and hormone-induced expression in vivo. The URF is essential and sufficient for hepatocyte-specific expression, periportal localization, perinatal activation and induction by glucocorticoids, and cAMP in transgenic mice. Before birth, the transgene is silent but can be induced by cAMP and glucocorticoids, indicating that these compounds are responsible for the activation of expression at birth. A 102-bp glucocorticoid response unit within the URF, containing binding sites for HNF3, C/EBP, and the glucocorticoid receptor, is the main determinant of the hepatocyte-specific and hormone-controlled activity. Additional sequences are required for a productive interaction between this minimal response unit and the core CPS promoter. These results show that the 469-bp URF, and probably only the 102-bp glucocorticoid response unit, functions as a regulatory module, in that it autonomously executes a correct spatial, developmental and hormonal program of CPS expression in the liver.     

Germination of penicillium paneum Conidia is regulated by 1-octen-3-ol, a volatile self-inhibitor

Penicillium paneum is an important contaminant of cereal grains which is able to grow at low temperature, low pH, high levels of carbon dioxide, and under acid conditions. P. paneum produces mycotoxins, which may be harmful to animals and humans. We found that conidia in dense suspensions showed poor germination, suggesting the presence of a self-inhibitor. A volatile compound(s) produced by these high-density conditions also inhibited mycelial growth of different species of fungi belonging to a variety of genera, suggesting a broad action range. The heat-stable compound was isolated by successive centrifugation of the supernatant obtained from spore suspensions with a density of 10(9) conidia ml(-1). By using static headspace analyses, two major peaks were distinguished, with the highest production of these metabolites after 22 h of incubation at 25 degrees C and shaking at 140 rpm. Gas chromatography coupled with mass spectra analysis revealed the compounds to be 3-octanone and 1-octen-3-ol. Notably, only the latter compound appeared to block the germination process at different developmental stages of the conidia (swelling and germ tube formation). In this study, 1-octen-3-ol influenced different developmental processes during the P. paneum life cycle, including induction of microcycle conidiation and inhibition of spore germination. Therefore, the compound can be considered a fungal hormone during fungal development.     

Conidial germination in the filamentous fungus Fusarium graminearum

The ascomycetous fungus Fusarium graminearum is an important plant pathogen causing Fusarium head blight disease of wheat and barley. To understand early developmental stages of this organism, we followed the germination of macroconidia microscopically to understand the timing of key events. These events, recorded after suspension of spores in liquid germination medium, included spore swelling at 2h, germination tube emergence and elongation from conidia at 8h and hyphal branching at 24h. To understand changes in gene expression during these developmental changes, RNA was isolated from spores and used to interrogate the F. graminearum Affymetrix GeneChip. RNAs corresponding to 5813 genes were detected in fresh spores and 5146, 5249 and 5993, respectively, in spores incubated in germination medium after 2, 8 or 24h (P<0.001). Gene expression data were used to predict the cellular and physiological state of each developmental stage for known processes. Predictions were confirmed microscopically for several previously unreported developmental events such as manifestation of peroxisomes in fresh spores and nuclear division resulting in binuclear cells within macroconidia prior to spore germination. Knowledge of stage-specific gene expression and changes in gene expression levels between developmental stages are an important first step to understanding the molecular mechanisms responsible for spore germination and development.     

Conidial germination in the filamentous fungus Fusarium graminearum.

The ascomycetous fungus Fusarium graminearum is an important plant pathogen causing Fusarium head blight disease of wheat and barley. To understand early developmental stages of this organism, we followed the germination of macroconidia microscopically to understand the timing of key events. These events, recorded after suspension of spores in liquid germination medium, included spore swelling at 2h, germination tube emergence and elongation from conidia at 8h and hyphal branching at 24h. To understand changes in gene expression during these developmental changes, RNA was isolated from spores and used to interrogate the F. graminearum Affymetrix GeneChip. RNAs corresponding to 5813 genes were detected in fresh spores and 5146, 5249 and 5993, respectively, in spores incubated in germination medium after 2, 8 or 24h (P<0.001). Gene expression data were used to predict the cellular and physiological state of each developmental stage for known processes. Predictions were confirmed microscopically for several previously unreported developmental events such as manifestation of peroxisomes in fresh spores and nuclear division resulting in binuclear cells within macroconidia prior to spore germination. Knowledge of stage-specific gene expression and changes in gene expression levels between developmental stages are an important first step to understanding the molecular mechanisms responsible for spore germination and development.     

Impaired ribosome biogenesis disrupts the integration between morphogenesis and nuclear duplication during the germination of Aspergillus fumigatus

Aspergillus fumigatus is an important opportunistic fungal pathogen that is responsible for high mortality rates in the immunosuppressed population. CgrA, the A. fumigatus ortholog of a Saccharomyces cerevisiae nucleolar protein involved in ribosome biogenesis, contributes to the virulence of this fungus by supporting rapid growth at 37 degrees C. To determine how CgrA affects ribosome biogenesis in A. fumigatus, polysome profile and ribosomal subunit analyses were performed on both wild-type A. fumigatus and a DeltacgrA mutant. The loss of CgrA was associated with a reduction in the level of 80S monosomes as well as an imbalance in the 60S:40S subunit ratio and the appearance of half-mer ribosomes. The gene expression profile in the DeltacgrA mutant revealed increased abundance of a subset of translational machinery mRNAs relative to the wild type, suggesting a potential compensatory response to CgrA deficiency. Although DeltacgrA conidia germinated normally at 22 degrees C, they swelled excessively when incubated at 37 degrees C and accumulated abnormally high numbers of nuclei. This hypernucleated phenotype could be replicated pharmacologically by germinating wild-type conidia under conditions of reductive stress. These findings indicate that the germination process is particularly vulnerable to global disruption of protein synthesis and suggest that CgrA is involved in both ribosome biogenesis and polarized cell growth in A. fumigatus.     

A 368-base-pair cis-acting HWP1 promoter region, HCR, of Candida albicans confers hypha-specific gene regulation and binds architectural transcription factors Nhp6 and Gcf1p

To elucidate the molecular mechanisms controlling the expression of the hypha-specific adhesin gene HWP1 of Candida albicans, its promoter was dissected and analyzed using a green fluorescent protein reporter gene. A 368-bp region, the HWP1 control region (HCR), was critical for activation under hypha-inducing conditions and conferred developmental regulation to a heterologous ENO1 promoter. A more distal region of the promoter served to amplify the level of promoter activation. Using gel mobility shift assays, a 249-bp subregion of HCR, HCRa, was found to bind at least four proteins from crude extracts of yeasts and hyphae with differing binding patterns dependent on cell morphology. Four proteins with DNA binding activities were identified by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis after separation by anion-exchange and heparin-Sepharose chromatography. One protein with high similarity to Nhp6, an HMG1 family member in Saccharomyces cerevisiae, and another with weak similarity to an HMG-like condensation factor from Physarum polycephalum implicated changes in chromatin structure as a critical process in hypha-specific gene regulation. Proteins with strong homology to histones were also found. These studies are the first to identify proteins that bind to a DNA segment that confers developmental gene regulation in C. albicans and suggest a new model for hypha-specific gene regulation.     

Conidium differentiation in Aspergillus nidulans wild-type and wet-white (wetA) mutant strains

Conidium (asexual spore) differentiation in wild-type and the wet-white (wetA) mutant of Aspergillus nidulans was compared in intact chains of successively older conidia. Carbohydrate cytochemistry helped define three stages (Stages I, II, and III) of wild-type conidium maturation on the basis of changes in the ultrastructure and composition of the conidium wall. Conidia of the wetA6 mutant strain formed normally but failed to mature during Stages II and III. Specifically, the inner wall layer of wetA6 conidia did not condense during Stage II and two wall layers that stained for carbohydrates did not form during the transition to Stage III. Concomitantly, wetA6 conidia formed large cytoplasmic vacuoles and underwent lysis. The wetA gene appears to have a conidium-specific function for the modification of the conidium wall during Stages II and III. These modifications of the conidium wall are essential for the stability of mature, dormant conidia.     

The FUS3/KSS1-type MAP kinase gene FPK1 is involved in hyphal growth, conidiation and plant infection of Fusarium proliferatum

Fusarium proliferatum is an important pathogen of maize that is responsible for ear rots, stalk rots and seeding blight worldwide. During the past decade, F. proliferatum has caused several severe epidemics of maize seedling blight in many areas of China, which led to significant losses in maize. To understand the molecular mechanisms in the fungal developmental regulation and pathogenicity, we isolated and characterized the FPK1 gene (GenBank accession No. HQ844224) encoding a MAP kinase homolog of FUS3/KSS1 in yeast. The gene includes a 1,242-bp DNA sequence from ATG to TAA, with a coding region of 1,068 bp, 3 introns (58 bp, 56 bp and 60 bp) and a predicted protein of 355 aa.The mutant ΔFPK1, which has a disruption of the FPK1 gene, showed reduced vegetative growth, fewer and shorter aerial mycelia, strongly impaired conidiation and spore germination, as well as deviant germ tube outgrowth. When the strain was inoculated in susceptible maize varieties, the infection of the mutant ΔFPK1 was delayed, and the infection efficiency was reduced compared to the wild-type strain. Complementation of the disruptions within the FPK1 open reading frame restored wild-type levels of conidiation, growth rate and virulence to maize seedlings. Our results indicated that the FPK1 gene functioned in hyphal growth, conidiation, spore germination and virulence in F. proliferatum.