A Novel Gene,ROA, Is Required for Normal Morphogenesis and Discharge of Ascospores in Gibberella zeae

Head blight, caused by Gibberella zeae, is a significant disease among cereal crops, including wheat, barley, and rice, due to contamination of grain with mycotoxins. G. zeae is spread by ascospores forcibly discharged from sexual fruiting bodies forming on crop residues. In this study, we characterized a novel gene, ROA, which is required for normal sexual development. Deletion of ROA (Δroa) resulted in an abnormal size and shape of asci and ascospores but did not affect vegetative growth. The Δroa mutation triggered round ascospores and insufficient cell division after spore delimitation. The asci of the Δroa strain discharged fewer ascospores from the perithecia but achieved a greater dispersal distance than those of the wild-type strain. Turgor pressure within the asci was calculated through the analysis of osmolytes in the epiplasmic fluid. Deletion of the ROA gene appeared to increase turgor pressure in the mutant asci. The higher turgor pressure of the Δroa mutant asci and the mutant spore shape contributed to the longer distance dispersal. When the Δroa mutant was outcrossed with a Δmat1-2 mutant, a strain that contains a green fluorescence protein (GFP) marker in place of the MAT1-2 gene, unusual phenotypic segregation occurred. The ratio of GFP to non-GFP segregation was 1:1; however, all eight spores had the same shape. Taken together, the results of this study suggest that ROA plays multiple roles in maintaining the proper morphology and discharge of ascospores in G. zeae.Gibberella zeae (anamorph: Fusarium graminearum) causes Fusarium head blight in wheat, barley, and rice, as well as ear rot and stalk rot in maize (20, 23). The infected grains are frequently contaminated by mycotoxins, such as trichothecenes and zearalenone, which are harmful to humans and animals (6). The fungus overwinters in crop debris in the form of storage hyphae and develops ephemeral fruiting bodies (perithecia) at warmer temperatures. Ascospores formed within the perithecia are forcibly discharged into the air and are believed to serve as the primary inoculum of the disease (7, 27, 37, 39,–42). Therefore, sexual development and ascospore discharge are important factors in fungal survival and disease initiation.In fungi of the phylum Ascomycota, the sexual cycle is initiated when two genetically distinct nuclei combine to form a binucleate cell (31). As a homothallic fungus, G. zeae possesses the two mating type genes MAT1-1 and MAT1-2 in the haploid genome and therefore does not require a mating partner for sexual development (22, 46). Perithecium initials give rise to small, coiled initials that develop into perithecia filled with asci, tubular sacs of ascospores, which are the products of meiosis. Mature asci extend through the ostiole of perithecia and discharge their ascospores (40).Unique features of cell differentiation are involved in ascus and ascospore morphogenesis. Ascospore delimitation within the ascus and the development of a cell wall between the ascus and ascospore membranes are unique features of the process (31). Most studies of morphogenesis have described these changes in detail; however, much of these data have been limited to microscopic observations. Several genes involved in ascospore morphogenesis have been identified in Neurospora crassa (30), but the detailed mechanisms and genes involved in ascus and ascospore morphogenesis remain to be elucidated. The Round spore (R) mutant of N. crassa was shown to have round ascospores (24), and the gene responsible for this phenotype, rsp, was subsequently cloned (28). However, in G. zeae, no genes have been identified that are involved in ascus and ascospore morphogenesis.Although recent research has shed light on the physiological basis of ascospore discharge, the genetic basis remains largely unknown (38). The main force responsible for the observed shooting is turgor pressure within the extended asci. In G. zeae, a buildup of K⁺ and Cl⁻ ions drives the influx of water and causes turgor pressure that stretches the asci (41). Asci can accumulate polyols as well as ions. In a previous study, it was shown that the polyols are comprised mainly of mannitol and glucose; however, the concentration of these polyols is too low to make a significant contribution to turgor pressure (42). When the turgor pressure exceeds the threshold of the asci, apical pores rupture and ascospores are forcibly discharged (38). Trail et al. (41) estimated that the acceleration of ascospores in G. zeae is 8,500,000 m s⁻² using an iterative model to predict initial velocity. Recently, Yafetto et al. (44) used high-speed video photography to examine several large-spore fungi, including Ascobolus immerses, and to predict acceleration during dispersal. The asci of A. immerses are more than 12-fold larger in diameter than the asci of G. zeae (38). The size difference between these fungi greatly affects the behavior of their projectiles and results in an initial speed for G. zeae that is too great for application of the video photography method (for further discussion, see the supplemental material).To date, only one gene from G. zeae, the calcium ion channel gene cch1, has been shown to be involved in ascospore discharge (12). Deletion of this gene was shown to arrest ascospore discharge without affecting spore and ascus morphology. Since the genomic sequence of G. zeae is now available, the functional analysis of genes involved in sexual development has been accelerated. Random insertional mutagenesis is one strategy that has been used to identify novel genes associated with sexual development (13, 34). Previously, we produced a collection of more than 20,000 mutants from G. zeae by using the restriction enzyme-mediated integration (REMI) transformation procedure (13). In this study, the G. zeae mutant Z43R9901, which was isolated from a screening of REMI transformants, showed an unusual phenotype during sexual development. Further analysis demonstrated that the novel gene ROA is involved in ascospore morphogenesis and discharge in G. zeae. The results of this study increase our understanding of sexual development in the fungus.     

The Kinesin-related Proteins, Kip2p and Kip3p, Function Differently in Nuclear Migration in Yeast

The roles of two kinesin-related proteins, Kip2p and Kip3p, in microtubule function and nuclear migration were investigated. Deletion of either gene resulted in nuclear migration defects similar to those described for dynein and kar9 mutants. By indirect immunofluorescence, the cytoplasmic microtubules in kip2Δwere consistently short or absent throughout the cell cycle. In contrast, in kip3Δ strains, the cytoplasmic microtubules were significantly longer than wild type at telophase. Furthermore, in the kip3Δ cells with nuclear positioning defects, the cytoplasmic microtubules were misoriented and failed to extend into the bud. Localization studies found Kip2p exclusively on cytoplasmic microtubules throughout the cell cycle, whereas GFP-Kip3p localized to both spindle and cytoplasmic microtubules. Genetic analysis demonstrated that the kip2Δ kar9Δ double mutants were synthetically lethal, whereas kip3Δ kar9Δ double mutants were viable. Conversely, kip3Δ dhc1Δ double mutants were synthetically lethal, whereas kip2Δ dhc1Δ double mutants were viable. We suggest that the kinesin-related proteins, Kip2p and Kip3p, function in nuclear migration and that they do so by different mechanisms. We propose that Kip2p stabilizes microtubules and is required as part of the dynein-mediated pathway in nuclear migration. Furthermore, we propose that Kip3p functions, in part, by depolymerizing microtubules and is required for the Kar9p-dependent orientation of the cytoplasmic microtubules.     

fbfB, a Gene Encoding a Putative Galactose Oxidase, Is Involved in Stigmatella aurantiaca Fruiting Body Formation

Stigmatella aurantiaca is a gram-negative bacterium which forms, under conditions of starvation in a multicellular process, characteristic three-dimensional structures: the fruiting bodies. For studying this complex process, mutants impaired in fruiting body formation have been induced by transposon insertion with a Tn5-derived transposon. The gene affected (fbfB) in one of the mutants (AP182) was studied further. Inactivation of fbfB results in mutants which form only clumps during starvation instead of wild-type fruiting bodies. This mutant phenotype can be partially rescued, if cells of mutants impaired in fbfB function are mixed with those of some independent mutants defective in fruiting before starvation. The fbfB gene is expressed about 14 h after induction of fruiting body formation as determined by measuring β-galactosidase activity in a merodiploid strain harboring the wild-type gene and an fbfB-Δtrp-lacZ fusion gene or by Northern (RNA) analysis with the Rhodobacter capsulatus pufBA fragment fused to fbfB as an indicator. The predicted polypeptide FbfB has a molecular mass of 57.8 kDa and shows a significant homology to the galactose oxidase (GaoA) of the fungus Dactylium dendroides. Galactose oxidase catalyzes the oxidation of galactose and primary alcohols to the corresponding aldehydes.     

The MET13 Methylenetetrahydrofolate Reductase Gene Is Essential for Infection-Related Morphogenesis in the Rice Blast Fungus Magnaporthe oryzae

Methylenetetrahydrofolate reductases (MTHFRs) play a key role in the biosynthesis of methionine in both prokaryotic and eukaryotic organisms. In this study, we report the identification of a novel T-DNA-tagged mutant WH672 in the rice blast fungus Magnaporthe oryzae, which was defective in vegetative growth, conidiation and pathogenicity. Analysis of the mutation confirmed a single T-DNA insertion upstream of MET13, which encodes a 626-amino-acid protein encoding a MTHFR. Targeted gene deletion of MET13 resulted in mutants that were non-pathogenic and significantly impaired in aerial growth and melanin pigmentation. All phenotypes associated with Δmet13 mutants could be overcome by addition of exogenous methionine. The M. oryzae genome contains a second predicted MTHFR-encoding gene, MET12. The deduced amino acid sequences of Met13 and Met12 share 32% identity. Interestingly, Δmet12 mutants produced significantly less conidia compared with the isogenic wild-type strain and grew very poorly in the absence of methionine, but were fully pathogenic. Deletion of both genes resulted in Δmet13Δmet12 mutants that showed similar phenotypes to single Δmet13 mutants. Taken together, we conclude that the MTHFR gene, MET13, is essential for infection-related morphogenesis by the rice blast fungus M. oryzae.     

Innate immunity to yeast prions: Btn2p and Cur1p curing of the [URE3] prion is prevented by 60S ribosomal protein deficiency or ubiquitin/proteasome system overactivity

[URE3] is an amyloid-based prion of Ure2p, a negative regulator of poor nitrogen source catabolism in Saccharomyces cerevisiae. Overproduced Btn2p or its paralog Cur1p, in processes requiring Hsp42, cure the [URE3] prion. Btn2p cures by collecting Ure2p amyloid filaments at one place in the cell. We find that rpl4aΔ, rpl21aΔ, rpl21bΔ, rpl11bΔ, and rpl16bΔ (large ribosomal subunit proteins) or ubr2Δ (ubiquitin ligase targeting Rpn4p, an activator of proteasome genes) reduce curing by overproduced Btn2p or Cur1p. Impaired curing in ubr2Δ or rpl21bΔ is restored by an rpn4Δ mutation. No effect of rps14aΔ or rps30bΔ on curing was observed, indicating that 60S subunit deficiency specifically impairs curing. Levels of Hsp42p, Sis1p, or Btn3p are unchanged in rpl4aΔ, rpl21bΔ, or ubr2Δ mutants. Overproduction of Cur1p or Btn2p was enhanced in rpn4Δ and hsp42Δ mutants, lower in ubr2Δ strains, and restored to above wild-type levels in rpn4Δ ubr2Δ strains. As in the wild-type, Ure2N-GFP colocalizes with Btn2-RFP in rpl4aΔ, rpl21bΔ, or ubr2Δ strains, but not in hsp42Δ. Btn2p/Cur1p overproduction cures [URE3] variants with low seed number, but seed number is not increased in rpl4aΔ, rpl21bΔ or ubr2Δ mutants. Knockouts of genes required for the protein sorting function of Btn2p did not affect curing of [URE3], nor did inactivation of the Hsp104 prion-curing activity. Overactivity of the ubiquitin/proteasome system, resulting from 60S subunit deficiency or ubr2Δ, may impair Cur1p and Btn2p curing of [URE3] by degrading Cur1p, Btn2p or another component of these curing systems.     

The White Collar Complex Is Involved in Sexual Development of Fusarium graminearum

Sexual spores (ascospores) of Fusarium graminearum, a homothallic ascomycetous fungus, are believed to be the primary inocula for epidemics of the diseases caused by this species in cereal crops. Based on the light requirement for the formation of fruiting bodies (perithecia) of F. graminearum under laboratory conditions, we explored whether photoreceptors play an important role in sexual development. Here, we evaluated the roles of three genes encoding putative photoreceptors [a phytochrome gene (FgFph) and two white collar genes (FgWc-1 and FgWc-2)] during sexual development in F. graminearum. For functional analyses, we generated transgenic strains lacking one or two genes from the self-fertile Z3643 strain. Unlike the wild-type (WT) and add-back strains, the single deletion strains (ΔFgWc-1 and ΔFgWc-2) produced fertile perithecia under constant light on complete medium (CM, an unfavorable medium for sexual development) as well as on carrot agar (a perithecial induction condition). The expression of mating-type (MAT) genes increased significantly in the gene deletion strains compared to the WT under both conditions. Deletion of FgFph had no significant effect on sexual development or MAT gene expression. In contrast, all of the deletion strains examined did not show significant changes in other traits such as hyphal growth, mycotoxin production, and virulence. A split luciferase assay confirmed the in vivo protein-protein interactions among three photoreceptors along with FgLaeA, a global regulator of secondary metabolism and fungal development. Introduction of an intact copy of the A. nidulans LreA and LreB genes, which are homologs of FgWc-1 and FgWc-2, into the ΔFgWc-1 and ΔFgWc-2 strains, respectively, failed to repress perithecia formation on CM in the gene deletion strains. Taken together, these results demonstrate that FgWc-1 and FgWc-2, two central components of the blue-light sensing system, negatively regulate sexual development in F. graminearum, which differs from the regulation pattern in A. nidulans.     

HST3/HST4-dependent deacetylation of lysine 56 of histone H3 in silent chromatin

The composition of posttranslational modifications on newly synthesized histones must be altered upon their incorporation into chromatin. These changes are necessary to maintain the same gene expression state at individual chromosomal loci before and after DNA replication. We have examined how one modification that occurs on newly synthesized histone H3, acetylation of K56, influences gene expression at epigenetically regulated loci in Saccharomyces cerevisiae. H3 K56 is acetylated by Rtt109p before its incorporation into chromatin during S phase, and this modification is then removed by the NAD⁺-dependent deacetylases Hst3p and Hst4p during G2/M phase. We found silenced loci maintain H3 K56 in a hypoacetylated state, and the absence of this modification in rtt109 mutants was compatible with HM and telomeric silencing. In contrast, loss of HST3 and HST4 resulted in hyperacetylation of H3 K56 within silent loci and telomeric silencing defects, despite the continued presence of Sir2p throughout these loci. These silencing defects in hst3Δ hst4Δ mutants could be suppressed by deletion of RTT109. In contrast, overexpression of Sir2p could not restore silencing in hst3Δ hst4Δ mutants. Together, our findings argue that HST3 HST4 play critical roles in maintaining the hypoacetylated state of K56 on histone H3 within silent chromatin.     

Identification of Highly Attenuated Mutants of Simian Immunodeficiency Virus

Deletion mutants of the pathogenic clone of simian immunodeficiency virus isolate 239 (SIVmac239) were derived that are missing nef, vpr, and upstream sequences (US) in the U3 region of the LTR (SIVmac239Δ3), nef, vpx, and US (SIVmac239Δ3x), and nef, vpr, vpx, and US (SIVmac239Δ4). These multiply deleted derivatives replicated well in the continuously growing CEMx174 cell line and were infectious for rhesus monkeys. However, on the basis of virus load measurements, strength of antibody responses, and lack of disease progression, these mutants were highly attenuated. Measurements of cell-associated viral load agreed well with assays of plasma viral RNA load and with the strengths of the antibody responses; thus, these measurements likely reflected the extent of viral replication in vivo. A derivative of SIVmac239 lacking vif sequences (SIVmac239Δvif) could be consistently grown only in a vif-complementing cell line. This Δvif virus appeared to be very weakly infectious for rhesus monkeys on the basis of sensitive antibody tests only. The weak antibody responses elicited by SIVmac239Δvif were apparently in response to low levels of replicating virus since they were not elicited by heat-inactivated virus and the anti-SIV antibody responses persisted for greater than 1 year. These results, and the results of previous studies, allow a rank ordering of the relative virulence of nine mutant strains of SIVmac according to the following order: Δvpr > Δvpx > ΔvprΔvpx Δnef > Δ3 > Δ3x ≥ Δ4 > Δvif > Δ5. The results also demonstrate that almost any desired level of attenuation can be achieved, ranging from still pathogenic in a significant proportion of animals (Δvpr and Δvpx) to not detectably infectious (Δ5), simply by varying the number and location of deletions in these five loci.     

Integration of Metabolic Modeling and Phenotypic Data in Evaluation and Improvement of Ethanol Production Using Respiration-Deficient Mutants of Saccharomyces cerevisiae

Flux balance analysis and phenotypic data were used to provide clues to the relationships between the activities of gene products and the phenotypes resulting from the deletion of genes involved in respiratory function in Saccharomyces cerevisiae. The effect of partial or complete respiratory deficiency on the ethanol production and growth characteristics of hap4Δ/hap4Δ, mig1Δ/mig1Δ, qdr3Δ/qdr3Δ, pdr3Δ/pdr3Δ, qcr7Δ/qcr7Δ, cyt1Δ/cyt1Δ, and rip1Δ/rip1Δ mutants grown in microaerated chemostats was investigated. The study provided additional evidence for the importance of the selection of a physiologically relevant objective function, and it may improve quantitative predictions of exchange fluxes, as well as qualitative estimations of changes in intracellular fluxes. Ethanol production was successfully predicted by flux balance analysis in the case of the qdr3Δ/qdr3Δ mutant, with maximization of ethanol production as the objective function, suggesting an additional role for Qdr3p in respiration. The absence of similar changes in estimated intracellular fluxes in the qcr7Δ/qcr7Δ mutant compared to the rip1Δ/rip1Δ and cyt1Δ/cyt1Δ mutants indicated that the effect of the deletion of this subunit of complex III was somehow compensated for. Analysis of predicted flux distributions indicated self-organization of intracellular fluxes to avoid NAD⁺/NADH imbalance in rip1Δ/rip1Δ and cyt1Δ/cyt1Δ mutants, but not the qcr7Δ/qcr7Δ mutant. The flux through the glycerol efflux channel, Fps1p, was estimated to be zero in all strains under the investigated conditions. This indicates that previous strategies for improving ethanol production, such as the overexpression of the glutamate synthase gene GLT1 in a GDH1 deletion background or deletion of the glycerol efflux channel gene FPS1 and overexpression of GLT1, are unnecessary in a respiration-deficient background.     

Fission Yeast Rgf2p Is a Rho1p Guanine Nucleotide Exchange Factor Required for Spore Wall Maturation and for the Maintenance of Cell Integrity in the Absence of Rgf1p

Schizosaccharomyces pombe Rho1p is essential, directly activates β-1,3-glucan synthase, and participates in the regulation of morphogenesis. In S. pombe, Rho1p is activated by at least three guanine nucleotide exchange factors (GEFs): Rgf1p, Rgf2p, and Rgf3p. In this study we show that Rgf2p is a Rho1p GEF required for sporulation. The rgf2⁺ deletion did not affect forespore membrane formation and the nuclei were encapsulated properly. However, the mutant ascospores appeared dark and immature. The rgf2Δ zygotes were not able to release the ascospores spontaneously, and the germination efficiency was greatly reduced compared to wild-type (wt) spores. This phenotype resembles that of the mutants in bgs2⁺, which encodes a sporulation-specific glucan synthase subunit. In fact, glucan synthase activity was diminished in sporulating rgf2Δ diploids. Rgf2p also plays a role in β-glucan biosynthesis during vegetative growth. Overexpression of rgf2⁺ specifically increased GTP-bound Rho1p, caused changes in cell morphology, and elicited an increase in β-1,3-glucan synthase activity. Moreover, the simultaneous disruption of rgf1⁺ and rgf2⁺ was lethal and both Rgf1p and Rgf2p were able to partially substitute for each other. Our results suggest that Rgf1p and Rgf2p are alternative GEFs with an essential overlapping function in Rho1p activation during vegetative growth.     

The Ras/cAMP Pathway and the CDK-Like Kinase Ime2 Regulate the MAPK Smk1 and Spore Morphogenesis in Saccharomyces cerevisiae

Meiotic development (sporulation) in the yeast Saccharomyces cerevisiae is induced by nutritional deprivation. Smk1 is a meiosis-specific MAP kinase homolog that controls spore morphogenesis after the meiotic divisions have taken place. In this study, recessive mutants that suppress the sporulation defect of a smk1-2 temperature-sensitive hypomorph were isolated. The suppressors are partial function alleles of CDC25 and CYR1, which encode the Ras GDP/GTP exchange factor and adenyl cyclase, respectively, and MDS3, which encodes a kelch-domain protein previously implicated in Ras/cAMP signaling. Deletion of PMD1, which encodes a Mds3 paralog, also suppressed the smk1-2 phenotype, and a mds3-Δ pmd1-Δ double mutant was a more potent suppressor than either single mutant. The mds3-Δ, pmd1-Δ, and mds3-Δ pmd1-Δ mutants also exhibited mitotic Ras/cAMP phenotypes in the same rank order. The effect of Ras/cAMP pathway mutations on the smk1-2 phenotype required the presence of low levels of glucose. Ime2 is a meiosis-specific CDK-like kinase that is inhibited by low levels of glucose via its carboxy-terminal regulatory domain. IME2-ΔC241, which removes the carboxy-terminal domain of Ime2, exacerbated the smk1-2 spore formation phenotype and prevented cyr1 mutations from suppressing smk1-2. Inhibition of Ime2 in meiotic cells shortly after Smk1 is expressed revealed that Ime2 promotes phosphorylation of Smk1's activation loop. These findings demonstrate that nutrients can negatively regulate Smk1 through the Ras/cAMP pathway and that Ime2 is a key activator of Smk1 signaling.