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The homothallic ascomycete fungus Gibberella zeae (anamorph: Fusarium graminearum) is a major toxigenic plant pathogen that causes head blight disease on small-grain cereals. The fungus produces the mycotoxins deoxynivalenol (DON) and zearalenone (ZEA) in infected hosts, posing a threat to human and animal health. Despite its agricultural and toxicological importance, the molecular mechanisms underlying its growth, development and virulence remain largely unknown. To better understand such mechanisms, we studied the heterotrimeric G proteins of G. zeae, which are known to control crucial signalling pathways that regulate various cellular and developmental responses in fungi. Three putative G[alpha] subunits, GzGPA 1, GzGPA2 and GzGPA3, and one G[beta] subunit, GzGPB1, were identified in the F. graminearum genome. Deletion of GzGPA 1, a homologue of the Aspergillus nidulans G[alpha] gene fadA, resulted in female sterility and enhanced DON and ZEA production, suggesting that GzGPA 1 is required for normal sexual reproduction and repression of toxin biosynthesis. The production of DON and ZEA was also enhanced in the GzGPB1 mutant, suggesting that both G[alpha] GzGPA 1 and G[beta] GzGPB1 negatively control mycotoxin production. Deletion of GzGPA2, which encodes a G[alpha] protein similar to A. nidulans GanB, caused reduced pathogenicity and increased chitin accumulation in the cell wall, implying that GzGPA2 has multiple functions. Our study shows that G. zeae heterotrimeric G protein subunits can regulate vegetative growth, sexual development, toxin production and pathogenicity.

The asexual spore is one of the most crucial factors contributing to the fecundity and fitness of filamentous fungi. Although the developmental activator FluG was shown to be necessary for activation of asexual sporulation (conidiation) and production of the carcinogenic mycotoxin sterigmatocystin (ST) in the model filamentous fungus Aspergillus nidulans, the molecular mechanisms underlying the developmental switch have remained elusive. In this study, we report that the FluG-mediated conidiation in A. nidulans occurs via derepression. Suppressor analyses of fluG led to the identification of the sfgA gene encoding a novel protein with the Gal4-type Zn[(II).sub.2][Cys.sub.6] binuclear cluster DNA-binding motif at the N terminus. Deletion ([DELTA]) and 31 other loss-of-function sfgA mutations bypassed the need for fluG in conidiation and production of ST. Moreover, both [DELTA]sfgA and [DELTA]sfgA [DELTA]fluG mutations resulted in identical phenotypes in growth, conidiation, and ST production, indicating that the primary role of FluG is to remove repressive effects imposed by SfgA. In accordance with the proposed regulatory role of SfgA, overexpression of sfgA inhibited conidiation and delayed/reduced expression of conidiation- and ST-specific genes. Genetic analyses demonstrated that SfgA functions downstream of FluG but upstream of transcriptional activators (F1bD, F1bC, FlbB, and Br1A) necessary for normal conidiation.

Vegetative growth signaling in the filamentous fungus Aspergillus nidulans is primarily mediated by the heterotrimeric G-protein composed of FadA (G[alpha]), SfaD (G[beta]), and a presumed G[gamma]. Analysis of the A. nidulans genome identified a single gene named gpgA encoding a putative G[gamma]-subunit. The predicted GpgA protein consists of 90 amino acids showing 72% similarity with yeast Ste18p. Deletion ([DELTA]) of gpgA resulted in restricted vegetative growth and lowered asexual sporulation. Moreover, similar to the [DELTA]sfaD mutant, the [DELTA]gpgA mutant was unable to produce sexual fruiting bodies (cleistothecia) in self-fertilization and was severely impaired with cleistothecial development in outcross, indicating that both SfaD and GpgA are required for fruiting body formation. Developmental and morphological defects caused by deletion of flbA encoding an RGS protein negatively con trolling FadA-mediated vegetative growth signaling were suppressed by [DELTA]gpgA, indicating that GpgA functions in FadA-SfaD-mediated vegetative growth signaling. However, deletion of gpgA could not bypass the need for the early developmental activator FluG in asexual sporulation, suggesting that GpgA functions in a separate signaling pathway. We propose that GpgA is the only A. nidulans G[gamma]-subunit and is required for normal vegetative growth as well as proper asexual and sexual developmental progression.

The role of heterotrimeric G-proteins in cAMP-dependent germination of conidia was investigated in the filamentous ascomycete Aspergillus nidulans. We demonstrate that the Get-subunit GanB mediates a rapid and transient activation of cAMP synthesis in response to glucose during the early period of germination. Moreover, deletion of individual G-protein subunits resulted in defective trehalose mobilization and altered germination kinetics, indicating that GanB([alpha])-SfaD([beta])-GpgA([gamma]) constitutes a functional heterotrimer and controls cAMP/PKA signaling in response to glucose as well as conidial germination. Further genetic analyses suggest that GanB plays a primary role in cAMP/PKA signaling, whereas the SfaD-GpgA (G[beta][gamma]) heterodimer is crucial for proper activation of GanB signaling sensitized by glucose. In addition, the RGS protein RgsA is also involved in regulation of the cAMP/PKA pathway and germination via attenuation of GanB signaling. Genetic epistatic analyses led us to conclude that all controls exerted by GanB([alpha])-SfaD([beta])-GpgA([gamma]) on conidial germination are mediated through the cAMP/PKA pathway. Furthermore, GanB may function in sensing various carbon sources and subsequent activation of downstream signaling for germination.

Asexual sporulation (conidiation) in the filamentous fungus Aspergillus nidulans requires the early developmental activator fluG. Loss of fluG results in the blockage of both conidiation and production of the mycotoxin sterigmatocystin (ST). To investigate molecular mechanisms of fluG-dependent developmental activation, 40 suppressors of fluG (SFGs) that conidiate without fluG have been isolated and characterized. Genetic analyses showed that an individual suppression is caused by a single second-site mutation, and that all sfg mutations but one are recessive. Pairwise meiotic crosses grouped mutations to four loci, 31 of them to sfgA, 6 of them to sfgB, and 1 each to sfgC and sfgD, respectively. The only dominant mutation, sfgA38, also mapped to the sfgA locus, suggesting a dominant negative mutation. Thirteen sfgA and 1 sfgC mutants elaborated conidiophores in liquid submerged culture, indicating that loss of either of these gene functions not only bypasses fluG function but also results in hyperactive conidiation. While sfg mutants show varying levels of restored conidiation, all recovered the ability to produce ST at near wild-type levels. The fact that at least four loci are defined by recessive sfg mutations indicates that multiple genes negatively regulate conidiation downstream of fluG and that the activity of fluG is required to remove such repressive effects.

High-performance liquid chromatography was used in analyzing 44 moldy corn samples for the presence of the B and C series of fumonisins. The latter are mycotoxins found in corn and corn-based products. Fumonisin-contaminated corn causes leukoencephalomalacia in horses, pulmonary edema in swine and human esophageal cancer in South Africa and China. Thirty-two samples were found to be contaminated with the B and C fumonisins while six were infected with the B series only.

The role of apicidins on the occurrence of hemorrhagic disease syndrome on laboratory rats has been investigated. Apicidins are antiprotozoal agents found in Fusarium species that inhibit parasite histone deacetylase activity. The apicidins used in the study were isolated from wheat cultures of Fusarium sp. strain KCTC 16677. The Fusarium species were originally obtained from soybean seeds from South Korea. Results reveal that rats fed with a diet containing apicidins suffered from body weight loss, hemorrhage in the stomach, intestines and bladder and then died ultimately.