Aerial Hyphal Growth Research Articles

WdStuAp, an APSES Transcription Factor, Is a Regulator of Yeast-Hyphal Transitions in Wangiella ( Exophiala ) dermatitidis

APSES transcription factors are well-known regulators of fungal cellular development and differentiation. To study the function of an APSES protein in the fungus Wangiella dermatitidis, a conidiogenous and polymorphic agent of human phaeohyphomycosis with yeast predominance, the APSES transcription factor gene WdSTUA was cloned, sequenced, disrupted, and overexpressed. Analysis showed that its derived protein was most similar to the APSES proteins of other conidiogenous molds and had its APSES DNA-binding domain located in the amino-terminal half. Deletion of WdSTUA in W. dermatitidis induced convoluted instead of normal smooth colony surface growth on the rich yeast maintenance agar medium yeast extract-peptone-dextrose agar (YPDA) at 37 degrees C. Additionally, deletion of WdSTUA repressed aerial hyphal growth, conidiation, and invasive hyphal growth on the nitrogen-poor, hypha-inducing agar medium potato dextrose agar (PDA) at 25 degrees C. Ectopic overexpression of WdSTUA repressed the convoluted colony surface growth on YPDA at 37 degrees C, and also strongly repressed hyphal growth on PDA at 25 degrees C and 37 degrees C. These new results provide additional insights into the diverse roles played by APSES factors in fungi. They also suggest that the transcription factor encoded by WdSTUA is both a positive and negative morphotype regulator in W. dermatitidis and possibly other of the numerous human pathogenic, conidiogenous fungi capable of yeast growth.

Identification and characterization of sawC, a whiA-like gene, essential for sporulation in Streptomyces ansochromogenes

sawC, encoding a protein homologous to the WhiA sporulation regulator of Streptomyces coelicolor, was cloned from a fairly distantly related species, Streptomyces ansochromogenes. Disruption of sawC led to formation of aerial mycelium much longer than normal spore chains and with somewhat increased coiling, indicating that sawC plays an important role in the cessation of aerial hyphal growth similar to that of whiA, and that it has an effect on cell wall structure. However, complementation of sawC and whiA mutants with the same DNA fragment gave different results, which suggested that there may be some difference in regulation or function of WhiA/SawC between the two strains. S1 nuclease mapping identified one transcription start point of sawC. Using EGFP as a reporter, the spatial expression of sawC was shown to be confined to aerial hyphae. Computer-aided structural prediction analysis of SawC/WhiA proteins revealed the presence of a fold very similar to the endonuclease domain of PI-PfuI, raising the possibility that these proteins may interact with an intermediate in DNA repair or replication.

FvVE1 regulates filamentous growth, the ratio of microconidia to macroconidia and cell wall formation in Fusarium verticillioides

The velvet gene, veA, co-ordinates asexual and sexual development in the homothallic fungal species Aspergillus nidulans. Studies in Aspergillus parasiticus and Aspergillus fumigatus demonstrated that veA also regulates morphological differentiation in these species. Whether veA has the same role in morphogenesis in other fungal genera has not been investigated. In this work, we studied the role of the veA homologue, FvVE1, in the heterothallic fungus Fusarium verticillioides. Deletion of FvVE1 suppressed aerial hyphal growth and reduced colony surface hydrophobicity on solid media. In submerged cultures, FvVE1 deletion caused alterations in hyphal polarity, marked activation of conidiation and yeast-like growth. The latter was promoted by shaking to increase aeration of cultures. In addition, FvVE1 deletion markedly increased the ratio of macroconidia to microconidia. Supplementation of osmotic stabilizers restored the wild-type phenotype to deletion mutants, suggesting phenotypic alterations caused by FvVE1 deletion are related to cell wall defects. This is consistent with the hypersensitivity of FvVE1 deletion mutants to SDS and with the significant reduction in the mannoprotein content of mutants compared with the wild-type strain. However, no dramatic cell wall alterations were observed when mutants were examined by transmission electron microscopy. Our data strongly suggest that FvVE1 is important for cell wall integrity, cell surface hydrophobicity, hyphal polarity and conidiation pattern.

Two PAK Kinase Genes, CHM1 and MST20, Have Distinct Functions in Magnaporthe grisea

In the rice blast fungus Magnaporthe grisea, the Pmk1 mitogen-activated protein (MAP) kinase is essential for appressorium formation and infectious growth. PMK1 is homologous to yeast Fus3 and Kss1 MAP kinases that are known to be regulated by the Ste20 PAK kinase for activating the pheromone response and filamentation pathways. In this study, we isolated and characterized two PAK genes, CHM1 and MST20, in M. grisea. Mutants disrupted in MST20 were reduced in aerial hyphae growth and conidiation, but normal in growth rate, appressorium formation, penetration, and plant infection. In chm1 deletion mutants, growth, conidiation, and appressorium formation were reduced significantly. Even though appressoria formed by chm1 mutants were defective in penetration, chm1 mutants were able to grow invasively on rice leaves and colonize through wounds. The chm1 mutants were altered in conidiogenesis and produced conidia with abnormal morphology. Hyphae of chm1 mutants had normal septation, but the length of hyphal compartments was reduced. On nutritionally poor oatmeal agar, chm1 mutants were unstable and produced sectors that differed from original chm1 mutants in growth rate, conidiation, or colony morphology. However, none of the monoconidial cultures derived from these spontaneous sectors were normal in appressorial penetration and fungal pathogenesis. These data suggest that MST20 is dispensable for plant infection in M. grisea, but CHM1 plays a critical role in appressorium formation and penetration. Both mst20 and chm1 deletion mutants were phenotypically different from the pmk1 mutant that is defective in appressorium formation and infectious hyphae growth. It is likely that MST20 and CHM1 individually play no critical role in activating the PMK1 MAP kinase pathway during appressorium formation and infectious hyphae growth. However, CHM1 appears to be essential for appressorial penetration and CHM1 and MST20 may have redundant functions in M. grisea.

The product of a developmental gene, crgA, that coordinates reproductive growth in Streptomyces belongs to a novel family of small actinomycete-specific proteins.

On solid media, the reproductive growth of Streptomyces involves antibiotic biosynthesis coincident with the erection of filamentous aerial hyphae. Following cessation of growth of an aerial hypha, multiple septation occurs at the tip to form a chain of unigenomic spores. A gene, crgA, that coordinates several aspects of this reproductive growth is described. The gene product is representative of a well-conserved family of small actinomycete proteins with two C-terminal hydrophobic-potential membrane-spanning segments. In Streptomyces avermitilis, crgA is required for sporulation, and inactivation of the gene abolished most sporulation septation in aerial hyphae. Disruption of the orthologous gene in Streptomyces coelicolor indicates that whereas CrgA is not essential for sporulation in this species, during growth on glucose-containing media, it influences the timing of the onset of reproductive growth, with precocious erection of aerial hyphae and antibiotic production by the mutant. Moreover, CrgA subsequently acts to inhibit sporulation septation prior to growth arrest of aerial hyphae. Overexpression of CrgA in S. coelicolor, uncoupling any nutritional and growth phase-dependent regulation, results in growth of nonseptated aerial hyphae on all media tested, consistent with a role for the protein in inhibiting sporulation septation.

A central regulator of morphological differentiation in the multicellular bacterium Streptomyces coelicolor

In the multicellular bacterium Streptomyces coelicolor, functions of developmental (bald) genes are required for the biosynthesis of SapB, a hydrophobic peptidic morphogen that facilitates aerial hyphae formation. Here, we show that aerial hyphal growth and SapB biosynthesis could be activated independently from the normal developmental cascade by providing unprogrammed expression of functionally interactive genes within the ram cluster. ramC, ramS and ramR were essential for normal growth of aerial hyphae, and ramR, a response regulator gene, was a key activator of development. The ramR gene restored growth of aerial hyphae and SapB formation in all bald strains tested (albeit only weakly in the bldC mutant), many of which are characterized by physiological defects. Disruption of the ramR gene abolished SapB biosynthesis and severely delayed growth of aerial hyphae. Transcription of ramR was developmentally controlled, and RamR function in vivo depended on its putative phosphorylation site (D53). We identified and mapped RamR targets immediately upstream of the region encoding ramC and ramS, a putative operon. Overexpression of ramR in the wild-type strain increased SapB levels and caused a distinctive wrinkled surface topology. Based on these results, we propose that phenotypes of bald mutations reflect an early stage in the Streptomyces developmental programme similar to the spo0 mutations in the unicellular bacterium Bacillus subtilis, and that RamR has analogies to Spo0A, the Bacillus response regulator that integrates physiological signals before triggering endospore formation.

Parasitism and measurement of damage to Fusarium oxysporum by species of Melanospora, Sphaerodes, and Persiciospora

Five pyrenomycete isolates found in association with different form species of Fusarium oxysporum were evaluated for their in vitro ability to parasitize the watermelon wilt pathogen (F. oxysporum f. sp. niveum). Several assays were also developed that attempted to measure damage to the wilt pathogen direcdy in culture. The pyrenomycetes used for pathogenicity and parasitism tests included three isolates of Sphaerodes retispora var. retispora, and one each of Melanospora zamiae and Persiciospora moreaui. A collection of 24 different isolates of F. oxysporum was employed to define a parasitic host range for this group of pyrenomycetes within the species oxysporum. The ability to sporulate on the Fusarium hosts was the primary criterion used for establishing host ranges. Results indicated that all five isolates were capable of parasitizing the wilt pathogen, but P. moreaui was the only one that caused no detectable damage in both the aerial hyphal reduction and growth inhibition assays. Melanospora induced significant damage with the growth inhibition assay only. Significant differences were observed among and between the pyrenomycetes and the different form species in terms of ability to parasitize and reproduce.

Isolation of a fungus from shell lesions of New Zealand abalone, Haliotis iris Martyn and H. australis Gmelin

Shell irregularities characterized by deposition of conchiolin on the inner shell surface of abalone, Haliotis spp., have been reported in populations along the South Island of New Zealand for over 50 years. A previously reported, but unidentified, fungus associated with these shell lesions is isolated for the first time from Haliotis iris, the black footed paua, and H. australis, the yellow footed paua, both obtained from Jacks Bay, South East Otago. Fungal hyphae measured 1.20 – 2.38 μm (=1.83 μm) in diameter. Occasionally, wider hyphae that measured 2.38 – 3.60 μm (= 2.60 μm) in diameter with an apical swelling that measured 4.80 – 7.20 × 7.20 – 15.00 μm (= 6.20 × 11.80 μm) were observed within lesions. Visible growth of aerial hyphae was observed on plates within 12 d at 16 °C on several media. Three morphologically indistinguishable isolates were obtained: two originated from H. iris shells and one from an H. australis shell. We examined 8 fungal and/or bacterial media and determined that the fungus grew best on a dilute medium (yeast extract, glucose, shell extract (+/-peptone agar) at 15 °C. Fungal growth was observed between 7 – 20 °C but not at room temperature (24 °C). Sporulation has not been induced , therefore the fungus is provisionally identified as a member of the Class Deuteromycotina. No significant relationship between animal length and incidence of shell mycosis was observed. Abalone condition was not influenced by the shell mycosis in either species. The number of circulating haemocytes was significantly elevated in infected H. australis (1.19 × 107 cells ml−1) relative to uninfected individuals (6.72 × 106 cells ml−1) but not between infected and uninfected H. iris. No significant relationships were observed between the degree of fungal infection and protein levels of whole haemolymph, serum or sex (immature, male or female; p<0.050; N=34). We are currently attempting to determine the taxonomic placement of the fungus and further characterize its growth physiology and pathogenicity.

A novel class of small amphipathic peptides affect aerial hyphal growth and surface hydrophobicity in Ustilago maydis.

Ustilago maydis, a fungal pathogen of corn, can alternate between yeast-like and filamentous growth. This dimorphic switch is governed by the mating-type loci. We have identified an abundant class of small SDS-insoluble cell wall proteins, designated repellents, specifically present in the filamentous form. Genetic analysis revealed that these peptides are processed from a single precursor protein, Rep1. Rep1 comprises 652 amino acids with a leader sequence for secretion. A characteristic feature of Rep1 is 12 repeats of a 37 amino acid consensus sequence; 10 of these repeats are separated by Kex2 protease cleavage sites. In (delta)rep1 mutants formation of aerial hyphae and surface hydrophobicity were reduced dramatically. This and the fact that expression of rep1 is regulated by the mating-type loci indicates that repellents play a structural role in the formation of aerial hyphae.

Redox Imbalance at the Start of Each Morphogenetic Step of Neurospora crassa Conidiation

The conidiation process of Neurospora crassa is characterized by three morphogenetic steps: hyphal adhesion, aerial hyphal formation, and production of conidia. Total protein oxidation and specific enzyme oxidation coincided with an increased oxygen-dependent chemiluminescence and indicated the occurrence of a hyperoxidant state at the onset of all three morphogenetic steps. Oxidation of NAD(P)H and excretion of glutathione disulfide was detected at the start of hyphae adhesion. Here we show that NAD(P)H and glutathione redox imbalance also occurred at the beginning of aerial hyphal growth and just before formation of conidia in the isolated cell structures. An increased loss and oxidation of NAD(P)(H) and glutathione were detected with each morphogenetic transition. These results give further support to our proposal that a hyperoxidant state develops at the start of each of the three morphogenetic processes during N. crassa conidiation.

Heterozygosity at the b mating-type locus attenuates fusion in Ustilago maydis

Mating and pathogenesis of the corn smut fungus, Ustilago maydis, are controlled by two unlinked mating-type loci, a and b. Yeast-like haploids that differ at both loci are compatible and fuse to establish a pathogenic dikaryon. Mating is assayed in vitro by co-inoculation on culture medium containing activated charcoal; compatible combinations have a characteristic "fuzzy" appearance caused by the growth of aerial hyphae. In general, this test has not been useful for assaying the mating ability of strains that are already mycelial (e.g., those heterozygous at b or at both mating-type loci). Using an assay for cytoduction involving transfer of a mitochondrial marker during transient cell fusion, and engineered strains with defined genotypes, we examined the mating abilities of strains heterozygous or hemizygous at the mating-type loci. The data (which have not been available from conventional pathogenicity or plate mating tests) show that heterozygosity at b attenuates fusion in haploid and diploid strains, whereas strains heterozygous at a retain the ability to fuse with a compatible haploid partner. It appears, therefore, that subsequent fusion events are attenuated once fusion has occurred to establish the U. maydis dikaryon.

Growth of axenic cultures of Cronartium flaccidum on callus tissue from Pinus nigra var. laricio and Pinus sylvestris

SummaryThe callus‐fungal method was employed to test the response to C. flaccidum of the highly susceptible P. nigra var. laricio and the resistant P. sylvestris, and to ascertain whether results obtained with this method matched in planta observations. Calli were inoculated with axenie cultures of C. flaccidum obtained by incubating basidiospores on modified Schenk and Hildebrandt medium. Several parameters were evaluated. Colony growth was more rapid on P. nigra var. laricio. Colonies were dense on P. nigra var. laricio, but sparse on P. sylvestris. Aerial hyphae growth was abundant on P. nigra var. laricio, but less frequent on P. sylvestris. Hyphal branching began after 18 h on P. nigra var. laricio and after 45 h on P. sylvestris. Necrosis of the host cells set in after 24 h on P. nigra var. laricio, and after 70 h on P. sylvestris. The number of cells with plasmolysis was much larger in P. nigra var. laricio than in P. sylvestris. These results were consistent with the known resistance of the two species on whole plants.

Tergitol-induced colonial growth without inhibition of conidiation

The cloning of genes from Neurospora crassa usually involves transformation of a mutant with genomic library DNA and the subsequent identification of transformants showing wild-type phenotype (Vollmer and Yanofsky 1986. Proc. Natl. Acad. Sci. 83:4869- 4873). This can be problematic in the case of morphological mutants, because the sorbose- containing medium that is typically used to promote colonial growth of the transformants tends to interfere with conidiation, and sometimes inhibits aerial hyphal growth altogether. The medium described below uses the nonionic detergent tergitol (Tatum et al. 1949. Science 109:509-511) to induce colonial vegetative growth without affecting aerial hyphal growth or morphology.

Protein oxidation related to morphogenesis in Neurospora crassa

Hyphae of Neurospora crassa exposed to the air adhere to each other, form aerial hyphae, and conidiate. During these morphogenic events, the carbonyl content in purified total protein was determined as a measurement of protein oxidation. A peak of protein carbonyl content coincided in time with hyphal adhesion, with initiation of aerial hyphae growth, and with the onset of conidia formation. These results suggest increased generation of reactive oxygen species at the start of each morphogenic process and a reversion of the hyperoxidant states with the appearance of new structures.

Enzyme patterns and protein synthesis during synchronous conidiation in Neurospora crassa

Both the growth of aerial hyphae and the differentiation of conidia from existing aerials were inhibited by cycloheximide or ethionine. These results suggest a requirement for protein synthesis throughout conidiogenesis. Several results are presented which suggest NADase may be required for the processes culminating in the differentiation of macroconidia. In particular, using a new technique to isolate aerial hyphae and conidia, I discovered that the increase in NADase activity during conidiogenesis was confined to the newly formed aerial hyphae and conidia. No NADase was detected in the old mycelium. In sharp contrast, aryl-β-glucosidase and cellobiase activities increased in both the mycelium and the aerials and conidia. The increases in all three enzyme activities during conidiogenesis were inhibited by cycloheximide and ethionine, a result suggesting the de novo synthesis of these enzymes. Other results show that aryl-β-glucosidase, cellobiase, and a fourth enzyme, trehalase, were not required to form normal numbers of conidia.