Fungal biology and agriculture: revisiting the field.

Plant pathology has made significant progress over the years, a process that involved overcoming a variety of conceptual and technological hurdles. Descriptive mycology and the advent of chemical plant-disease management have been followed by biochemical and physiological studies of fungi and their hosts. The later establishment of biochemical genetics along with the introduction of DNA-mediated transformation have set the stage for dissection of gene function and advances in our understanding of fungal cell biology and plant-fungus interactions. Currently, with the advent of high-throughput technologies, we have the capacity to acquire vast data sets that have direct relevance to the numerous subdisciplines within fungal biology and pathology. These data provide unique opportunities for basic research and for engineering solutions to important agricultural problems. However, we also are faced with the challenge of data organization and mining to analyze the relationships between fungal and plant genomes and to elucidate the physiological function of pertinent DNA sequences. We present our perspective of fungal biology and agriculture, including administrative and political challenges to plant protection research.

Overexpression of White Collar-1 (WC-1) activates circadian clock-associated genes, but is not sufficient to induce most light-regulated gene expression in Neurospora crassa.

Many processes in fungi are regulated by light, but the molecular mechanisms are not well understood. The White Collar-1 (WC-1) protein is required for all known blue-light responses in Neurospora crassa. In response to light, WC-1 levels increase, and the protein is transiently phosphorylated. To test the hypothesis that the increase in WC-1 levels after light treatment is sufficient to activate light-regulated gene expression, we used microarrays to identify genes that respond to light treatment. We then overexpressed WC-1 in dark-grown tissue and used the microarrays to identify genes regulated by an increase in WC-1 levels. We found that 3% of the genes were responsive to light, whereas 7% of the genes were responsive to WC-1 overexpression in the dark. However, only four out of 22 light-induced genes were also induced by WC-1 overexpression, demonstrating that changes in the levels of WC-1 are not sufficient to activate all light-responsive genes. The WC proteins are also required for circadian rhythms in dark-grown cultures and for light entrainment of the circadian clock, and WC-1 protein levels show a circadian rhythm in the dark. We found that representative samples of the mRNAs induced by over-expression of WC-1 show circadian fluctuations in their levels. These data suggest that WC-1 can mediate both light and circadian responses, with an increase in WC-1 levels affecting circadian clock-responsive gene regulation and other features of WC-1, possibly its phosphorylation, affecting light-responsive gene regulation.

Independent and Synergistic Activity of Synthetic Peptides Against Thiabendazole-Resistant Fusarium sambucinum.

ABSTRACT Two heptapeptides with broad antifungal activity were identified and assessed for their ability to act synergistically with thiabendazole. The hexapeptide 66-10 was the progenitor of the heptapeptides and exhibited minimal inhibitory concentrations (MICs) of 9.3 to 9.8 mug/ml for thiabendazole (TBZ) resistant Fusarium sambucinum strains (MIC of 186 to 312 mug/ml). Heptapeptide derivatives 77-3 and 77-12 exhibited MICs between 3.8 and 7.5 mug/ml against the same strains. Incubation of conidia or mycelia with the peptide 77-3 showed that treated fungal structures were stained by the membrane impermeant dye SYTOX Green indicating disruption of membranes. Conidia incubated with peptide 77-3 at 10 mug/ml showed a 91 +/- 3.6% reduction in viability in 15 min. A checkerboard method was used to test the peptides and TBZ individually and in combination to determine potential synergistic activity. The results indicate that small peptides can act synergistically with TBZ against TBZ-resistant F. sambucinum.

Functional analysis of pathogenicity genes in a genomics world.

Genome-wide mutational and expression analyses have been performed in yeast and provide a model for large-scale analysis of gene function in filamentous fungi. The recent completion of the Neurospora crassa genome offers a resource for comparative analysis with plant pathogenic filamentous fungi. These advances have important implications for molecular genetic studies of pathogenicity genes.

vvd is required for light adaptation of conidiation-specific genes of Neurospora crassa, but not circadian conidiation.

con-10 and con-6 are two of the conidiation (con) genes of Neurospora crassa that were identified based on their preferential expression during macroconidiophore development. They are also regulated by several other environmental stimuli independent of development, including a transient induction by light. We identified an allele of vivid (vvd) in a mutant screen designed to obtain strains with altered expression of con-10. vvd mutants display enhanced carotenoid pigmentation in response to light. In addition, con-10 and con-6 show a heightened response to photoinduction. We tested the function of the light-responsive circadian clock in the vvd mutant and found no major defect in the circadian rhythm of conidiation or light regulation of a key clock component, frequency (frq). We conclude that vvd is primarily involved in a process of light-dependent gene repression, called light adaptation. Although a number of gene products are known to control light induction in fungi, vvd is the first gene shown to have a role in adaptation to constant light.

Isolation of pheromone precursor genes of Magnaporthe grisea.

In heterothallic ascomycetes one mating partner serves as the source of female tissue and is fertilized with spermatia from a partner of the opposite mating type. The role of pheromone signaling in mating is thought to involve recognition of cells of the opposite mating type. We have isolated two putative pheromone precursor genes of Magnaporthe grisea. The genes are present in both mating types of the fungus but they are expressed in a mating type-specific manner. The MF1-1 gene, expressed in Mat1-1 strains, is predicted to encode a 26-amino-acid polypeptide that is processed to produce a lipopeptide pheromone. The MF2-1 gene, expressed in Mat1-2 strains, is predicted to encode a precursor polypeptide that is processed by a Kex2-like protease to yield a pheromone with striking similarity to the predicted pheromone sequence of a close relative, Cryphonectria parasitica. Expression of the M. grisea putative pheromone precursor genes was observed under defined nutritional conditions and in field isolates. This suggests that the requirement for complex media for mating and the poor fertility of field isolates may not be due to limitation of pheromone precursor gene expression. Detection of putative pheromone precursor gene mRNA in conidia suggests that pheromones may be important for the fertility of conidia acting as spermatia.

Analysis of two transcription activation elements in the promoter of the developmentally regulated con-10 gene of Neurospora crassa.

The con-10 gene of Neurospora crassa is activated during conidiation. CRS-B (conidiation response sequence-B) and CGE (con-10 general enhancer) elements have been proposed to function as sites of transcriptional activation and as possible elements that confer developmental regulation to con-10. In a specific analysis of the roles of these elements we found that two CRS-B elements are necessary for full activation of con-10 during macroconidiation, whereas two CGE elements are functionally redundant, such that a single CGE is sufficient for maximal expression. However, CRS-B and CGE elements are not sufficient for developmental activation of a reporter gene. The CRS-B element was further dissected and one of the CRS-B elements appears to function in repression as well as activation. con-10 is also highly expressed during microconidiation, a different form of asexual sporulation, but we show here that CRS-B elements do not play a significant role in con-10 expression during microconidiation. Both CRS-B elements contribute to basal con-10 expression during mycelial growth. con-10 is also regulated by light, and CRS-B and CGE elements may play minor roles in controlling con-10 expression in response to light.

Tissue-specific repression of starvation and stress responses of the Neurospora crassa con-10 gene is mediated by RCO1.

The Neurospora crassa con-10 gene is weakly expressed in mycelia but is induced approximately 1000-fold during macroconidiation. Studies of the promoter elements and trans-acting factors that regulate con-10 expression are needed to gain a detailed understanding of developmental regulation. The rco-1 mutant displays a 10-fold elevated basal level of expression of con-10. In contrast to the wild type, con-10 expression in mycelia of the rco-1 mutant was rapidly induced to high levels by starvation for carbon or nitrogen and by heat shock. Although con-10 is developmentally induced late in conidiation, con-10 was inducible by heat shock shortly after exposure of the wild-type mycelium to air. These findings support the view that RCO1 is a cell type-specific repressor of con-10. We propose that inactivation of RCO1 allows developing conidiophores to adjust the timing of con-10 induction in response to stress.

The fluffy gene of Neurospora crassa encodes a Gal4p-type C6 zinc cluster protein required for conidial development.

Neurospora crassa fluffy (fl) mutants are unable to produce macroconidia. We cloned the fl gene to determine its role in regulating conidiation. A cosmid clone containing fl was identified by complementation. The sequence of fl revealed that it encodes a Gal4p-type C6 zinc cluster protein with greatest similarity to the N. crassa NIT4 protein that regulates genes required for nitrate utilization. Analysis of several fl mutant alleles demonstrated that null mutants are blocked in the budding phase of development required to produce conidiophores. fl mRNA is transiently induced just prior to the developmental commitment to budding growth. This timing of fl expression is consistent with a role for FL protein in activation of the previously characterized conidiation-specific (con) genes, con-6 and con-10. These data suggest that FL acts as a developmentally regulated transcription factor required for conidiophore morphogenesis.

The Neurospora rca-1 gene complements an Aspergillus flbD sporulation mutant but has no identifiable role in Neurospora sporulation.

The Aspergillus nidulans flbD gene encodes a protein with a Myb-like DNA-binding domain that is proposed to act in concert with other developmental regulators to control initiation of conidiophore development. We have identified a Neurospora crassa gene called rca-1 (regulator of conidiation in Aspergillus) based on its sequence similarity to flbD. We found that N. crassa rca-1 can complement the conidiation defect of an A. nidulans flbD mutant and that induced expression of rca-1 caused conidiation in submerged A. nidulans cultures just as was previously observed for overexpression of flbD. Thus, the N. crassa gene appears to be a functional homologue of A. nidulans flbD and this is the first demonstration of functional complementation of an A. nidulans sporulation defect using a gene from an evolutionarily distant fungus. However, deletion of the rca-1 gene in N. crassa had no major effect on growth rate, macroconidiation, microconidiation, or ascospore formation. The only phenotype displayed by the rca-1 mutant was straight or counterclockwise hyphal growth rather than the clockwise spiral growth observed for wild type. Thus, if rca-1 is involved in N. crassa development, its role is subtle or redundant.

Hydrophobins and fungal infection of plants and animals.

Hydrophobins are among the most important structural proteins produced by fungi. Their least-understood function is how they act to promote infection-related morphogenesis. Although the hydrophobin of at least one plant pathogen appears to be involved as a signal molecule in pathogenesis, a role for hydrophobins in animal pathogenesis has not been convincingly documented.

rco-3, a gene involved in glucose transport and conidiation in Neurospora crassa.

Macroconidiation in Neurospora crassa is influenced by a number of environmental cues, including the nutritional status of the growing organism. Conidia formation is normally observed when the fungus is exposed to air. However, carbon limitation can induce conidiation in mycclia submerged in an aerated liquid medium. A mutant was previously isolated that could conidiate in submerged culture without imposing nutrient limitation and the gene responsible for this phenotype (rco-3) has now been cloned. RCO3 exhibits sequence similarity to members of the sugar transporter gene superfamily, with greatest similarity to glucose transporters of yeast. Consistent with this structural similarity, we find that glucose transport activity is altered in the mutant. However, growth of the mutant in media containing alternate carbon sources does not suppress conidiation in submerged culture. The properties of the mutant suggest that RCO3 is required for expression of glucose transport activity, glucose regulation of gene expression, and general carbon repression of development.

Inhibition of pathogenicity of the rice blast fungus by Saccharomyces cerevisiae alpha-factor.

Magnaporthe grisea is a fungal pathogen with two mating types, MAT1-1 and MAT1-2, that forms a specialized cell necessary for pathogenesis, the appressorium. Saccharomyces cerevisiae alpha-factor pheromone blocked appressorium formation in a mating type-specific manner and protected plants from infection by MAT1-2 strains. Experiments with alpha-factor analogs suggest that the observed activity is due to a specific interaction of alpha-factor with an M. grisea receptor. Culture filtrates of a MAT1-1 strain contained an activity that inhibited appressorium formation of mating type MAT1-2 strains. These findings provide evidence that a pheromone response pathway exists in M. grisea that can be exploited for plant protection.

Cross-pathway and pathway-specific control of amino acid biosynthesis in Magnaporthe grisea.

The gene encoding the small subunit of the arginine-specific carbamoyl phosphate synthetase, ARG2, of Magnaporthe grisea was characterized to examine the basic regulation of biosynthetic genes in this plant pathogen. The transcript of the ARG2 gene contains an upstream open reading frame (uORF) that is similar to uORFs found in the homologous genes of Neurospora crassa (arg-2) and Saccharomyces cerevisiae (CPA1), suggesting that the M. grisea gene is translationally regulated by a mechanism that is conserved in these fungi. Amino acid imbalance leads to elevated levels of ARG2 mRNA, indicating that in addition to translational control, ARG2 is subject to cross-pathway transcriptional control. A DNA-binding activity that has properties similar to those of the global transcriptional regulator mediating cross-pathway control in N. crassa was detected in M. grisea cell extracts. Thus, it appears that both specific regulation of ARG2 by arginine and global regulation of amino acid biosynthesis are present in M. grisea and highly conserved among M. grisea, N. crassa, and S. cerevisiae.

Characterization of rco-1 of Neurospora crassa, a pleiotropic gene affecting growth and development that encodes a homolog of Tup1 of Saccharomyces cerevisiae.

The filamentous fungus Neurospora crassa undergoes a well-defined developmental program, conidiation, that culminates in the production of numerous asexual spores, conidia. Several cloned genes, including con-10, are expressed during conidiation but not during mycelial growth. Using a previously described selection strategy, we isolated mutants that express con-10 during mycelial growth. Selection was based on expression of an integrated DNA fragment containing the con-10 promoter-regulatory region followed by the initial segment of the con-10 open reading frame fused in frame with the bacterial hygromycin B phosphotransferase structural gene (con10'-'hph). Resistance to hygromycin results from mutational alterations that allow mycelial expression of the con-10'-'hph gene fusion. A set of drug-resistant mutants were isolated; several of these had abnormal conidiation phenotypes and were trans-acting, i.e., they allowed mycelial expression of the endogenous con-10 gene. Four of these had alterations at a single locus, designated rco-1 (regulation of conidiation). Strains with the rco-1 mutant alleles were aconidial, female sterile, had reduced growth rates, and formed hyphae that coiled in a counterclockwise direction, opposite that of the wild type. The four rco-1 mutants had distinct conidiation morphologies, suggesting that conidiation was blocked at different stages. Wild-type rco-1 was cloned by a novel procedure employing heterokaryon-assisted transformation and ligation-mediated PCR. The predicted RCO1 polypeptide is a homolog of Tup1 of Saccharomyces cerevisiae, a multidomain protein that mediates transcriptional repression of genes concerned with a variety of processes. Like tup1 mutants, null mutants of rco-1 are viable and pleiotropic. A promoter element was identified that could be responsible for RCO1-mediated vegetative repression of con-10 and other conidiation genes.

MPG1, a gene encoding a fungal hydrophobin of Magnaporthe grisea, is involved in surface recognition.

Upon encountering a leaf surface, emergent germ tubes from conidia of the rice blast fungus, Magnaporthe grisea, form infection structures called appressoria that allow direct penetration of plant cells. The MPG1 gene encodes a fungal hydrophobin of M. grisea that is expressed during development of aerial hyphae, conidia, and appressoria. Deletion of MPG1 reduces the efficiency of appressorium formation. We found that yeast extract repressed MPG1 expression in vitro and inhibited appressorium development of the rice pathogen, strain Guy11. Appressorium formation of mpg1 mutants is rescued in trans by coinoculation with wild-type cells. MPG1 is required for efficient induction of appressoria in response to a host surface or highly hydrophobic artificial substrates. However, we identified several artificial substrates that can support efficient appressorium formation of mpg1 strains. This finding suggests that Mpg1p is not specifically required for appressorium formation, but is involved in the interaction with, and recognition of, the host surface. Additionally, a time window of competence to form appressoria was identified; the decision to form appressoria occurs approximately 6 to 8 h following conidial germination. After this critical time, cells are no longer able to form appressoria in response to inductive cues. These studies indicate that MPG1 hydrophobin is required for host recognition and that it acts as a morphogenetic signal for cellular differentiation.

Light and developmental regulation of the gene con-10 of Neurospora crassa.

The gene con-10 of Neurospora crassa is expressed preferentially during conidiation and following illumination of vegetative mycelia with blue light. In this study we have examined the segmental locations of the genetic elements associated with con-10 that are responsible for light and developmental expression. A translational fusion was prepared between the initial segment of con-10 and Escherichia coli lacZ. Deletions were then introduced into the con-10 upstream region associated with this translational fusion. Each construct was integrated at the his-3 locus of N. crassa by transformation and homologous recombination. Photoinduction of mycelia containing the translational fusion with the intact upstream region revealed a two phase stimulus-response curve. Exposure to light for as little as 5 sec induced a transcriptional response. Following this initial induction, a period of 15 min in the dark or light was required for appearance of a second phase response. Only a brief light treatment was necessary for induction of the second phase response. Deletions within the upstream region altered normal light and developmental expression of constructs containing the con-10-lacZ translational fusion. The deleted segments appear to contain a mycelial repression site, two conidiation activation sites, and two dark repression sites. A repeated 17-bp sequence acted as a transcriptional enhancer. One copy of this enhancer is in the upstream region. The second copy, with the opposite orientation, is located in the first con-10 intron. The enhancer was required for proper mycelial and conidial expression of the con-10-lacZ fusion. The initial 10 bp of this enhancer sequence were sufficient to restore conidial expression to a deletion construct lacking both copies of the 17-bp repeat. Proteins were detected in extracts of mycelia and conidia that specifically bound to the enhancer sequence in vitro. Our findings suggest that conidiation-specific and mycelial-specific expression of con-10 requires the action of several factors acting independently and/or in concert at distinct sites located in the regulatory regions for con-10.

Mutants of Neurospora crassa that alter gene expression and conidia development.

Several genes have been identified that are highly expressed during conidiation. Inactivation of these genes has no observable phenotypic effect. Transcripts of two such genes, con-6 and con-10, are normally absent from vegetative mycelia. To identify regulatory genes that affect con-6 and/or con-10 expression, strains were prepared in which the regulatory regions for these genes were fused to a gene conferring hygromycin resistance. Mutants were then selected that were resistant to the drug during mycelial growth. Mutations in several of the isolates had trans effects; they activated transcription of the corresponding intact gene and, in most isolates, one or more of the other con genes. Most interestingly, resistant mutants were obtained that were defective at different stages of conidiation. One mutant conidiated under conditions that do not permit conidiation in wild type.

Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea.

Differential cDNA cloning was used to identify genes expressed during infectious growth of the fungal pathogen Magnaporthe grisea in its host, the rice plant. We characterized one of these genes, MPG1, in detail. Using a novel assay to determine the proportion of fungal biomass present in the plant, we determined that the MPG1 transcript was 60-fold more abundant during growth in the plant than in culture. Mpg1 mutants have a reduced ability to cause disease symptoms that appears to result from an impaired ability to undergo appressorium formation. MPG1 mRNA was highly abundant very early in plant infection concomitant with appressorium formation and was also abundant at the time of symptom development. The MPG1 mRNA was also expressed during conidiation and in mycelial cultures starved for nitrogen or carbon. MPG1 potentially encodes a small, secreted, cysteine-rich, moderately hydrophobic protein with the characteristics of a fungal hydrophobin. Consistent with the role of the MPG1 gene product as a hydrophobin, Mpg1 mutants show an "easily wettable" phenotype. Our results suggest that hydrophobins may have a role in the elaboration of infective structures by fungi and may fulfill other functions in fungal phytopathogenesis.