Mid1, a mechanosensitive calcium ion channel, affects growth, development, and ascospore discharge in the filamentous fungus Gibberella zeae

The role of Mid1, a stretch-activated ion channel capable of being permeated by calcium, in ascospore development and forcible discharge from asci was examined in the pathogenic fungus Gibberella zeae (anamorph Fusarium graminearum). The Δmid1 mutants exhibited a >12-fold reduction in ascospore discharge activity and produced predominately abnormal two-celled ascospores with constricted and fragile septae. The vegetative growth rate of the mutants was ～50% of the wild-type rate, and production of macroconidia was >10-fold lower than in the wild type. To better understand the role of calcium flux, Δmid1 Δcch1 double mutants were also examined, as Cch1, an L-type calcium ion channel, is associated with Mid1 in Saccharomyces cerevisiae. The phenotype of the Δmid1 Δcch1 double mutants was similar to but more severe than the phenotype of the Δmid1 mutants for all categories. Potential and current-voltage measurements were taken in the vegetative hyphae of the Δmid1 and Δcch1 mutants and the wild type, and the measurements for all three strains were remarkably similar, indicating that neither protein contributes significantly to the overall electrical properties of the plasma membrane. Pathogenicity of the Δmid1 and Δmid1Δcch1 mutants on the host (wheat) was not affected by the mutations. Exogenous calcium supplementation partially restored the ascospore discharge and vegetative growth defects for all mutants, but abnormal ascospores were still produced. These results extend the known roles of Mid1 to ascospore development and forcible discharge. However, Neurospora crassa Δmid1 mutants were also examined and did not exhibit defects in ascospore development or in ascospore discharge. In comparison to ion channels in other ascomycetes, Mid1 shows remarkable adaptability of roles, particularly with regard to niche-specific adaptation.     

Preferential Ascus Discharge during Cross Maturation in SORDARIA BREVICOLLIS

Crosses involving spore color mutants of Sordaria brevicollis all showed a decline in the frequency of second division asymmetric asci (2:2:2:2's) as the cross matured. This decline was due to the preferential maturation and/or discharge of these asci. The proportion of spindle overlap and recombinational asci within the group did not change as shown by ascus dissection. The preferential discharge was also found to occur in two-point crosses where the asci did not contain wild-type spores.     

Meiotic silencing in the homothallic fungus Gibberella zeae

The homothallic ascomycete fungus Gibberella zeae is an important pathogen on major cereal crops. The objective of this study was to determine whether meiotic silencing occurs in G. zeae. Cytological studies demonstrated that GFP and RFP-fusion proteins were not detected during meiosis, both in heterozygous outcrosses and homozygous selfings. The deletion of rsp-1, a homologue used for studies on meiotic silencing of Neurospora crassa, triggered abnormal ascospores from selfing, but outcrosses between the mutant and wild-type strain resulted in some ascospores with mutant phenotype (low occurrence of ascus dominance). When the ectopic mutants that carried an additional copy of rsp-1 were selfed, they primarily produced ascospores with normal shape but a few ascospores (0.23 %) were abnormal, in which both endogenous and ectopically integrated genes contained numerous point mutations. The ectopic mutants showed low occurrence of ascus dominance in outcrosses with strains that carried the wild-type allele. Approximately 10 % of ascospores were abnormal but all of the single-ascospore isolates produced normal-shaped ascospores from selfing. However, no ascus dominance was observed when the mutants were outcrossed with a sad-1 deletion mutant, which lacks the putative RNA-dependent RNA polymerase essential for meiotic silencing in N. crassa. All results were consistent with those generated from an additional gene, roa, required for ascospore morphogenesis. This study demonstrated that G. zeae possesses a functional meiotic silencing mechanism which is triggered by unpaired DNA, as in N. crassa.     

Cytology of recessive sexual-phase mutants from wild strains of Neurospora crassa.

Wild-collected strains of Neurospora crassa harbor recessive mutations that are expressed in the sexual phase when homozygous. Thirty-two representative mutants that produced barren perithecia were examined cytologically. Six of these mutants failed to form asci. Of the remaining 26, chromosome pairing was disturbed in 12 and meiosis was disturbed at pachytene or diplotene in 5. Seven mutants showed normal meiosis I but then diverged from the normal sequence, and two showed perithecial beak abnormalities. In many mutants, ascus development and nuclear divisions continued after the initial defect, albeit abnormally. Nuclear divisions were often delayed, essentially uncoupling them from other ascus events such as the formation of enlarged spindle pole body plaques, ascospore wall membranes, and spore delimitation. All 32 mutants were recessive and none showed obvious morphological abnormalities during vegetative growth. This phenotype contrasts sharply with that of numerous laboratory-induced ascus mutants, which are frequently expressed pleiotropically in the vegetative phase and several are dominant in the sexual phase.     

Distinct steps in yeast spore morphogenesis require distinct SMK1 MAP kinase thresholds

The SMK1 mitogen-activated protein kinase is required for spore morphogenesis in Saccharomyces cerevisiae. In contrast to the multiple aberrant spore wall assembly patterns seen even within a single smk1 null ascus, different smk1 missense mutants block in a coordinated fashion at intermediate stages. One smk1 mutant forms asci in which the four spores are surrounded only by prospore wall-like structures, while another smk1 mutant forms asci in which the spores are surrounded by inner but not outer spore wall layers. Stepwise increases in gene dosage of a hypomorphic smk1 allele allow for the completion of progressively later morphological and biochemical events and for the acquisition of distinct spore-resistance phenotypes. Furthermore, smk1 allelic spore phenotypes can be recapitulated by reducing wild-type SMK1 expression. The data demonstrate that SMK1 is required for the execution of multiple steps in spore morphogenesis that require increasing thresholds of SMK1 activity. These results suggest that quantitative changes in mitogen-activated protein kinase signaling play a role in coordinating multiple events of a single cellular differentiation program.     

Physiological and environmental aspects of ascospore discharge in Gibberella zeae (anamorph Fusarium graminearum)

We investigated ascospore discharge in the perithecial fungus, Gibberella zeae. In a wind tunnel study that simulated constant rain and varying day and night lengths, the rate of ascospore release was approximately 8-30% greater under light than in complete darkness. Under constant light, ascospore discharge occurred at maximal rates at relative humidity levels greater than 92%. When perithecia were placed under conditions of high external osmolarity, ascospore discharge was significantly reduced. Ascospores were discharged from asci along with droplets of fluid, the epiplasm, from within the ascus. Analysis of discharged epiplasmic fluid by GC-MASS Spectrometry revealed that mannitol was the major simple sugar component of the fluid. Activity of mannitol dehydrogenase, which catalyzes the conversion of fructose to mannitol, was higher in protein extracts from mature perithecia than in extracts from vegetative tissue. Several inhibitors of K(+) and Ca(++) ion channels inhibited ascospore discharge, which suggested that ascospore discharge resulted from the buildup of turgor pressure generated by ion fluxes and mannitol accumulation.     

Intracellular siderophores are essential for ascomycete sexual development in heterothallic Cochliobolus heterostrophus and homothallic Gibberella zeae

Connections between fungal development and secondary metabolism have been reported previously, but as yet, no comprehensive analysis of a family of secondary metabolites and their possible role in fungal development has been reported. In the present study, mutant strains of the heterothallic ascomycete Cochliobolus heterostrophus, each lacking one of 12 genes (NPS1 to NPS12) encoding a nonribosomal peptide synthetase (NRPS), were examined for a role in sexual development. One type of strain (Delta nps2) was defective in ascus/ascospore development in homozygous Delta nps2 crosses. Homozygous crosses of the remaining 11 Delta nps strains showed wild-type (WT) fertility. Phylogenetic, expression, and biochemical analyses demonstrated that the NRPS encoded by NPS2 is responsible for the biosynthesis of ferricrocin, the intracellular siderophore of C. heterostrophus. Functional conservation of NPS2 in both heterothallic C. heterostrophus and the unrelated homothallic ascomycete Gibberella zeae was demonstrated. G. zeae Delta nps2 strains are concomitantly defective in intracellular siderophore (ferricrocin) biosynthesis and sexual development. Exogenous application of iron partially restored fertility to C. heterostrophus and G. zeae Delta nps2 strains, demonstrating that abnormal sexual development of Delta nps2 strains is at least partly due to their iron deficiency. Exogenous application of the natural siderophore ferricrocin to C. heterostrophus and G. zeae Delta nps2 strains restored WT fertility. NPS1, a G. zeae NPS gene that groups phylogenetically with NPS2, does not play a role in sexual development. Overall, these data demonstrate that iron and intracellular siderophores are essential for successful sexual development of the heterothallic ascomycete C. heterostrophus and the homothallic ascomycete G. zeae.     

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.     

Genetic recombination at the buff spore colour locus in Sordaria brevicollis

Summary Asci showing aberrant segregation at the buff spore colour locus in Sordaria brevicollis were selected from crosses between buff mutants and wild type in the presence of closely-linked flanking markers. The frequency of crossing-over associated with aberrant segregations was calculated and corrected to allow for crossovers between the flanking markers incidental to the aberrant segregation. The average frequency of crossing over was found to be related to the class of aberrant ascus studied. 5+:3m and 3+:5m asci showed 16% associated marker recombination while 6+:2m and 2+:6m asci showed 27% recombination. The frequency of tritype and tetratype postmeiotic segregation asci was calculated. Only 3% tetratypes were found and this is thought to indicate a low frequency of symmetric hybrid DNA formation.     

Induction of multispored asci in two-spored strains of Saccharomyces cerevisiae by amitrole.

Although growth of two yeast strains characterized by consistent production of two diploid spores per ascus was inhibited in complex presporulation media containing amitrole, a fraction of the cells produced were able to form asci with more than two spores after transfer to acetate sporulation medium. Cells grown in a defined presporulation medium containing amitrole did not acquire this ability. The increase in spore numbers per ascus is attributed either to the induction by amitrole in growth medium of cells with more than one nucleus or to the restoration of normal meioses in the multispored asci.     

Morphology,development and cytology of Taphrina maculans Butler

Morphology, development and nuclear behavior of the ascogenous stroma and asci in the infection spots have been described inTaphrina maculans Butler. The fungus forms subcuticular and intercellular mycelium in the leaf tissues and the ascogenous layers originate through division of the subcuticular hyphal cells in the infection sites. Germination of ascogenous cells starts with their elongation in the uppermost layer forming asci and ascospores without formation of stalk cells. Meiosis of the fusion (diploid) nucleus occurs in the young ascus as in otherTaphrina species devoid of stalk cells. The haploid chromosome complement in this species consists of 3 chromosomes (n=3). All the cells in the stromatic layer are potential ascogenous cells and ascus formation continues, until all of them are exhausted in the infection spot. Eight ascospores are normally formed in each ascus, but multi-plication of ascospores may occurin situ later. Three morphologically distinct types of ascus opening are encountered, which are apparently not correlated with prevalent environment. Multiplication of ascospores after their discharge from mature asci occurs by budding proceded by a mitotic division of the spore nucleus. Blastospores (budded cells) germinate into short hyphae and binucleate condition of cells originates by mitotic division of the nucleus. Occurrence of giant cells containing 2 nuclei is often observed. Possible origin of Uredinales fromTaphrina-like ancestors has been indicated due to their close resemblance.     

Mutations at the smo genetic locus affect the shape of diverse cell types in the rice blast fungus

Teflon film surfaces are highly conducive to the formation of infection structures (appressoria) in the plant pathogenic fungus, Magnaporthe grisea. We have utilized Teflon films to screen and select for mutants of M. grisea that are defective in appressorium formation. This approach and several others yielded a group of 14 mutants with a similar phenotype. All the mutant strains make abnormally shaped conidia and appressoria. When two mutant strains are crossed, abnormally shaped asci are formed. Ascus shape is normal when a mutant strain is crossed with a wild-type strain. Despite dramatic alterations in cell shape these strains otherwise grow, form conidia, undergo meiosis, and infect plants normally. This mutant phenotype, which we have termed Smo(-), for abnormal spore morphology, segregates in simple Mendelian fashion in crosses with wild-type strains. Some ascospore lethality is associated with smo mutations. In genetic crosses between mutants, smo mutations fail to recombine and do not demonstrate complementation of the abnormal ascus shape phenotype. We conclude that the smo mutations are alleles of a single genetic locus and are recessive with regard to the the ascus shape defect. Mutations at the SMO locus also permit germinating M. grisea conidia to differentiate appressoria on surfaces that are not normally conducive to infection structure formation. A number of spontaneous smo mutations have been recovered. The frequent occurrence of this mutation suggests that the SMO locus may be highly mutable.     

Variation in yeast mitochondrial activity associated with asci

An increase in mitochondrial membrane potential (DeltaPsim) and mitochondrially produced 3-hydroxy (3-OH) oxylipins was experienced in asci of the nonfermentative yeasts Galactomyces reessii and Lipomyces starkeyi and the fermentative yeasts Pichia farinosa and Schizosaccharomyces octosporus. Strikingly, asci of Zygosaccharomyces bailii showed no increase in mitochondrial activity (DeltaPsim and oxylipin production). As expected, oxygen deprivation only inhibited ascus formation in those yeasts with increased ascus mitochondrial activity. We conclude that ascus formation in yeasts is not always dependent on mitochondrial activity. In this case, fermentation may provide enough energy for ascus formation in Z. bailii.     

The mechanism of ascus firing – Merging biophysical and mycological viewpoints

The actively discharging ascus is the unique spore-bearing cell that is responsible to dispatch spores into the atmosphere. From a physical perspective, this type of ascus is a sophisticated pressure gun that reliably discharges the spores at an extremely high velocity, without breaking apart. We identify four essential steps in discharge of spores whose order and timing may vary across species. First, asci that fire are mature, so a cue must be present that prevents discharge of immature spores and signals maturity. Second, pressure within the ascus serves to propel the spores forward; therefore a mechanism should be present to pressurize the ascus. Third, in ostiolate fruiting bodies (e.g. perithecia), the ascus extends through an opening to fire spores into the air. The extension process is a relatively unique aspect of the ascus and must be structurally facilitated. Fourth, the ascus must open at its tip for spore release in a controlled rupture. Here we discuss each of these aspects in the context of understanding the process of ascus and fruiting body function. While there is great diversity among fungi, we focus on discharge in a few model species, and then discuss how this framework may vary in other fungi. Our goal is to tie the physiological and molecular studies of ascus function with concepts in engineering that dictate structure.     

Biphasic diurnal cycle of ascus development of Taphrina maculans Butler

Taphrina maculans Butler inciting the brown leaf spot disease of turmeric (Curcuma longa L.) produces cuboid ascogenous cells several layers in depth in the subcuticular interspaces of the epidermis. The ascogenous cells germinate to produce asci centrifugally maturing toward the periphery of the stroma, expelling octosporous microcolonies of asco-blastospores on the leaf surface inciting secondary infection in favorable environment. Occurrence of a rhythmic cycle of ascus development and ascospore discharge giving 2 peaks of ascospore discharge each day has been demonstrated in Taphrina maculans. The cycle is directly affected by atmospheric temperature, availability of free moisture on the leaf surface and sunlight. Free moisture on a leaf surface appropriately soaks the infection spots (ascogenous cells) and induces ascus elongation and ascospore discharge, when a suitable atmospheric temperature is reached. Sunlight may adversely affect the cycle by increasing the temperature and lowering humidity in the atmosphere.     

A constitutive, heat shock-activated neutral trehalase occurs in Schizosaccharomyces pombe in addition to the sporulation-specific acid trehalase

Trehalase was studied in Schizosaccharomyces pombe cells growing vegetatively on minimal medium and in sporulating cultures. Acid trehalase activity, measured at pH 4.2, was absent in vegetative cells and occurred only in asci, indicating that this activity represented the sporulation-specific trehalase reported previously. In contrast, neutral trehalase, measured at pH 6.0, was constitutively present in vegetative cells during the exponential and stationary growth phase as well as in asci. In vegetative cells, neutral trehalase did not sediment with cell walls, suggesting a cytoplasmic localization. Its activity increased ten-fold when growing cells were subjected to heat treatment of 2 h. Neutral trehalase from heat-treated cells had a pH optimum of 6.0 and was almost completely inhibited by 3 mM ZnCl2. Acid trehalase activity could be measured in intact asci, indicating that it is localized in the ascus cell walls, while neutral trehalase was not detectable in intact asci and appeared to be present primarily in the walls of ascospores and in the ascus epiplasm.     

A Putative Rhamnogalacturonase Required for Sexual Development of Neurospora Crassa

In previous work, the asd-1 (ascus development) gene of the filamentous fungus Neurospora crassa was identified as a gene expressed preferentially during the sexual cycle and shown to be essential for normal sexual development. The asd-1 gene has been sequenced and further characterized. It contains two introns, the first of which is in-frame and inefficiently or differentially spliced. The predicted ASD-1 protein has extensive homology with rhamnogalacturonase B of Aspergillus aculeatus, which cleaves the backbone within the ramified hairy regions of pectin. In homozygous asd-1 crosses, sexual development is initiated and large numbers of normal-sized asci are formed. Ascospore delineation does not occur, however, and no sexual progeny are produced. As most asd-1 asci contain eight nuclei, the two meiotic divisions and subsequent mitotic division typical of normal crosses seem to occur, but the haploid nuclei are not partitioned into ascospores. In wild-type crosses, the ASD-1 protein is present in large amounts in croziers and young asci, but it is only faintly detectable in more mature asci containing developing ascospores. Models to explain the possible role of a rhamnogalacturonase in sexual development are presented.     

Ascospore linearity in the four-spored ascus ofNeurospora tetrasperma

The four-spored ascus ofNeurospora tetrasperma is linearly ordered, i.e. the order of the ascospores within the linear ascus directly reflects preceding meiotic events. This conclusion is based upon the finding of only two types of arrangements of homokaryotic ascospores in asci showing second division segregation and the failure to find any of the other four theoretically possible types of homokaryotic arrangements. The data are also consistent with the regular occurrence of nuclear passing at both the second and third meiotic divisions during ascus development. This work was supported by Public Health Service Grant GM 10672. Supported in part by Public Health Service Training Grant 5-T1-GM-767-05.     

Identification of six loci in which mutations partially restore peroxisome biogenesis and/or alleviate the metabolic defect of pex2 mutants in podospora

Peroxins (PEX) are proteins required for peroxisome biogenesis. Mutations in PEX genes cause lethal diseases in humans, metabolic defects in yeasts, and developmental disfunctions in plants and filamentous fungi. Here we describe the first large-scale screening for suppressors of a pex mutation. In Podospora anserina, pex2 mutants exhibit a metabolic defect [inability to grow on medium containing oleic acid (OA medium) as sole carbon source] and a developmental defect (inability to differentiate asci in homozygous crosses). Sixty-three mutations able to restore growth of pex2 mutants on OA medium have been analyzed. They fall in six loci (suo1 to suo6) and act as dominant, allele-nonspecific suppressors. Most suo mutations have pleiotropic effects in a pex2(+) background: formation of unripe ascospores (all loci except suo5 and suo6), impaired growth on OA medium (all loci except suo4 and suo6), or sexual defects (suo4). Using immunofluorescence and GFP staining, we show that peroxisome biogenesis is partially restored along with a low level of ascus differentiation in pex2 mutant strains carrying either the suo5 or the suo6 mutations. The data are discussed with respect to beta-oxidation of fatty acids, peroxisome biogenesis, and cell differentiation.