Shaojie Li

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.

FvVE1 regulates biosynthesis of the mycotoxins fumonisins and fusarins in Fusarium verticillioides.

The veA gene positively regulates sterigmatocystin production in Aspergillus nidulans and aflatoxin production in Aspergillus parasiticus and Aspergillus flavus . Whether veA homologues have a role in regulating secondary metabolism in other fungal genera is unknown. In this study, we examined the role of the veA homologue, FvVE1, on the production of two mycotoxin families, fumonisins and fusarins, in the important corn pathogen Fusarium verticillioides . We found that FvVE1 deletion completely suppressed fumonisin production on two natural substrates, corn and rice. Furthermore, our results revealed that FvVE1 is necessary for the expression of the pathway-specific regulatory gene FUM21 and structural genes in the fumonisin biosynthetic gene (FUM) cluster. FvVE1 deletion also blocked production of fusarins. The effects of FvVE1 deletion on the production of these toxins were found to be the same in two separate mating types. Our results strongly suggest that FvVE1 plays an important role in regulating mycotoxin production in F. verticillioides .

Setting the pace of the Neurospora circadian clock by multiple independent FRQ phosphorylation events

Protein phosphorylation plays essential roles in eukaryotic circadian clocks. Like PERIOD in animals, the Neurospora core circadian protein FRQ is progressively phosphorylated and becomes extensively phosphorylated before its degradation. In this study, by using purified FRQ protein from Neurospora, we identified 43 in vivo FRQ phosphorylation sites by mass spectrometry analysis. In addition, we show that CK-1a and CKII are responsible for most FRQ phosphorylation events and identify an additional 33 phosphorylation sites by in vitro kinase assays. Whole-cell metabolic isotope labeling and quantitative MS analyses suggest that circadian oscillation of the FRQ phosphorylation profile is primarily due to progressive phosphorylation at the majority of these newly discovered phosphorylation sites. Furthermore, systematic mutations of the identified FRQ phosphorylation sites led to either long or short period phenotypes. These changes in circadian period are attributed to increases or decreases in FRQ stability, respectively. Together, this comprehensive study of FRQ phosphorylation reveals that regulation of FRQ stability by multiple independent phosphorylation events is a major factor that determines the period length of the clock. A model is proposed to explain how FRQ stability is regulated by multiple phosphorylation events.

Specific adaptation of Ustilaginoidea virens in occupying host florets revealed by comparative and functional genomics

Ustilaginoidea virens (Cooke) Takah is an ascomycetous fungus that causes rice false smut, a devastating emerging disease worldwide. Here we report a 39.4 Mb draft genome sequence of U. virens that encodes 8,426 predicted genes. The genome has ~25% repetitive sequences that have been affected by repeat-induced point mutations. Evolutionarily, U. virens is close to the entomopathogenic Metarhizium spp., suggesting potential host jumping across kingdoms. U. virens possesses reduced gene inventories for polysaccharide degradation, nutrient uptake and secondary metabolism, which may result from adaptations to the specific floret infection and biotrophic lifestyles. Consistent with their potential roles in pathogenicity, genes for secreted proteins and secondary metabolism and the pathogen-host interaction database genes are highly enriched in the transcriptome during early infection. We further show that 18 candidate effectors can suppress plant hypersensitive responses. Together, our analyses offer new insights into molecular mechanisms of evolution, biotrophy and pathogenesis of U. virens.

Genetic diversity and population structure of rice pathogen Ustilaginoidea virens in China.

Rice false smut caused by the fungal pathogen Ustilaginoidea virens is becoming a destructive disease throughout major rice-growing countries. Information about its genetic diversity and population structure is essential for rice breeding and efficient control of the disease. This study compared the genome sequences of two U . virens isolates. Three SNP-rich genomic regions were identified as molecular markers that could be used to analyze the genetic diversity and population structure of U . virens in China. A total of 56 multilocus sequence types (haplotypes) were identified out of 162 representative isolates from 15 provinces covering five major rice-growing areas in China. However, the phylogeny, based on sequences at individual SNP-rich regions, strongly conflicted with each other and there were significant genetic differences between different geographical populations. Gene flow between the different geographical populations and genetic differentiation within each geographical population were also detected. In addition, genetic recombination and genetic isolation resulting from geographic separation was also found.

Homologous recombination as a mechanism to recognize repetitive DNA sequences in an RNAi pathway

Quelling is an RNAi-related phenomenon that post-transcriptionally silences repetitive DNA and transposons in Neurospora. We previously identified a type of DNA damage-induced small RNA called qiRNA that originates from ribosomal DNA. To understand how small RNAs are generated from repetitive DNA, we carried out a genetic screen to identify genes required for qiRNA biogenesis. Factors directly involved in homologous recombination (HR) and chromatin remodeling factors required for HR are essential for qiRNA production. HR is also required for quelling, and quelling is also the result of DNA damage, indicating that quelling and qiRNA production share a common mechanism. Together, our results suggest that DNA damage-triggered HR-based recombination allows the RNAi pathway to recognize repetitive DNA to produce small RNA.

basA Regulates Cell Wall Organization and Asexual/Sexual Sporulation Ratio in Aspergillus nidulans

Sphingolipid C4 hydroxylase catalyzes the conversion of dihydrosphingosine to phytosphingosine. In Saccharomyces cerevisiae, Sur2 is essential for sphingolipid C4 hydroxylation activity but not essential for normal growth. Here we demonstrate that the Aspergillus nidulans Sur2 homolog BasA is also required for phytosphingosine biosynthesis but is also essential for viability. We previously reported that a point missense mutation in basA resulted in aberrant cell wall thickening. Here our data suggest that accumulation of dihydrosphingosine is responsible for this phenotype. In addition, two different mutations in basA consistently accelerated the transition from asexual development to sexual development compared to the wild-type strain. The phenotype could be suppressed by exogenous addition of phytosphingosine. Northern analysis suggests that faster sexual development in the basA mutant might be due to a higher transcription level of ppoA and steA, genes demonstrated to coordinate a balance between asexual and sexual development in A. nidulans. Consistent with these findings, mutations in the ceramide-synthase-encoding genes barA and lagA also caused faster transition from asexual to sexual development, supporting the involvement of sphingolipid metabolism in fungal morphogenesis.

Role of Individual Subunits of the Neurospora crassa CSN Complex in Regulation of Deneddylation and Stability of Cullin Proteins

The Cop9 signalosome (CSN) is an evolutionarily conserved multifunctional complex that controls ubiquitin-dependent protein degradation in eukaryotes. We found seven CSN subunits in Neurospora crassa in a previous study, but only one subunit, CSN-2, was functionally characterized. In this study, we created knockout mutants for the remaining individual CSN subunits in N. crassa. By phenotypic observation, we found that loss of CSN-1, CSN-2, CSN-4, CSN-5, CSN-6, or CSN-7 resulted in severe defects in growth, conidiation, and circadian rhythm; the defect severity was gene-dependent. Unexpectedly, CSN-3 knockout mutants displayed the same phenotype as wild-type N. crassa. Consistent with these phenotypic observations, deneddylation of cullin proteins in csn-1, csn-2, csn-4, csn-5, csn-6, or csn-7 mutants was dramatically impaired, while deletion of csn-3 did not cause any alteration in the neddylation/deneddylation state of cullins. We further demonstrated that CSN-1, CSN-2, CSN-4, CSN-5, CSN-6, and CSN-7, but not CSN-3, were essential for maintaining the stability of Cul1 in SCF complexes and Cul3 and BTB proteins in Cul3-BTB E3s, while five of the CSN subunits, but not CSN-3 and CSN-5, were also required for maintaining the stability of SKP-1 in SCF complexes. All seven CSN subunits were necessary for maintaining the stability of Cul4-DDB1 complexes. In addition, CSN-3 was also required for maintaining the stability of the CSN-2 subunit and FWD-1 in the SCFFWD-1 complex. Together, these results not only provide functional insights into the different roles of individual subunits in the CSN complex, but also establish a functional framework for understanding the multiple functions of the CSN complex in biological processes.

Correction: Composition and Expression of Genes Encoding Carbohydrate-Active Enzymes in the Straw-Degrading Mushroom Volvariella volvacea

Volvariella volvacea is one of a few commercial cultivated mushrooms mainly using straw as carbon source. In this study, the genome of V. volcacea was sequenced and assembled. A total of 285 genes encoding carbohydrate-active enzymes (CAZymes) in V. volvacea were identified and annotated. Among 15 fungi with sequenced genomes, V. volvacea ranks seventh in the number of genes encoding CAZymes. In addition, the composition of glycoside hydrolases in V. volcacea is dramatically different from other basidiomycetes: it is particularly rich in members of the glycoside hydrolase families GH10 (hemicellulose degradation) and GH43 (hemicellulose and pectin degradation), and the lyase families PL1, PL3 and PL4 (pectin degradation) but lacks families GH5b, GH11, GH26, GH62, GH93, GH115, GH105, GH9, GH53, GH32, GH74 and CE12. Analysis of genome-wide gene expression profiles of 3 strains using 3′-tag digital gene expression (DGE) reveals that 239 CAZyme genes were expressed even in potato destrose broth medium. Our data also showed that the formation of a heterokaryotic strain could dramatically increase the expression of a number of genes which were poorly expressed in its parental homokaryotic strains.

Transcription of the major neurospora crassa microRNA-like small RNAs relies on RNA polymerase III.

Most plant and animal microRNAs (miRNAs) are transcribed by RNA polymerase II. We previously discovered miRNA–like small RNAs (milRNAs) in the filamentous fungus Neurospora crassa and uncovered at least four different pathways for milRNA production. To understand the evolutionary origin of milRNAs, we determined the roles of polymerases II and III (Pol II and Pol III) in milRNA transcription. Our results show that Pol III is responsible for the transcription of the major milRNAs produced in this organism. The inhibition of Pol III activity by an inhibitor or by gene silencing abolishes the production of most abundant milRNAs and pri–milRNAs. In addition, Pol III associates with these milRNA producing loci. Even though silencing of Pol II does not affect the synthesis of the most abundant milRNAs, Pol II or both Pol II and Pol III are associated with some milRNA–producing loci, suggesting a regulatory interaction between the two polymerases for some milRNA transcription. Furthermore, we show that one of the Pol III–transcribed milRNAs is derived from a tRNA precursor, and its biogenesis requires RNase Z, which cleaves the tRNA moiety to generate pre–milRNA. Our study identifies the transcriptional machinery responsible for the synthesis of fungal milRNAs and sheds light on the evolutionary origin of eukaryotic small RNAs.