Establishment of a new method to quantitatively evaluate hyphal fusion ability in Aspergillus oryzae

Hyphal fusion is involved in the formation of an interconnected colony in filamentous fungi, and it is the first process in sexual/parasexual reproduction. However, it was difficult to evaluate hyphal fusion efficiency due to the low frequency in Aspergillus oryzae in spite of its industrial significance. Here, we established a method to quantitatively evaluate the hyphal fusion ability of A. oryzae with mixed culture of two different auxotrophic strains, where the ratio of heterokaryotic conidia growing without the auxotrophic requirements reflects the hyphal fusion efficiency. By employing this method, it was demonstrated that AoSO and AoFus3 are required for hyphal fusion, and that hyphal fusion efficiency of A. oryzae was increased by depleting nitrogen source, including large amounts of carbon source, and adjusting pH to 7.0.     

Heterokaryon incompatibility is suppressed following conidial anastomosis tube fusion in a fungal plant pathogen

It has been hypothesized that horizontal gene/chromosome transfer and parasexual recombination following hyphal fusion between different strains may contribute to the emergence of wide genetic variability in plant pathogenic and other fungi. However, the significance of vegetative (heterokaryon) incompatibility responses, which commonly result in cell death, in preventing these processes is not known. In this study, we have assessed this issue following different types of hyphal fusion during colony initiation and in the mature colony. We used vegetatively compatible and incompatible strains of the common bean pathogen Colletotrichum lindemuthianum in which nuclei were labelled with either a green or red fluorescent protein in order to microscopically monitor the fates of nuclei and heterokaryotic cells following hyphal fusion. As opposed to fusion of hyphae in mature colonies that resulted in cell death within 3 h, fusions by conidial anastomosis tubes (CAT) between two incompatible strains during colony initiation did not induce the vegetative incompatibility response. Instead, fused conidia and germlings survived and formed heterokaryotic colonies that in turn produced uninucleate conidia that germinated to form colonies with phenotypic features different to those of either parental strain. Our results demonstrate that the vegetative incompatibility response is suppressed during colony initiation in C. lindemuthianum. Thus, CAT fusion may allow asexual fungi to increase their genetic diversity, and to acquire new pathogenic traits.     

Ultraviolet Light-Induced Heterokaryon Formation and Parasexuality in Cryphonectria parasitica

Rizwana, R., and Powell, W. A. 1995. Ultraviolet light-induced heterokaryon formation and parasexuality in Cryphonectria parasitica. Experimental Mycology 19, 48-60. The effect of ultraviolet-light on heterokaryon formation, vegetative compatibility, and parasexuality in Cryphonectria parasitica was examined. Heterokaryons of complementary auxotrophic strains could not be made by hyphal anastomosis if the strains belonged to different vegetative compatibility groups. Protoplast fusions overcame incompatibility of strains differing in the alleles of a single but not multiple vegetative incompatibility loci. Fusion of protoplasts from ultraviolet light-treated complementary auxotrophs increased heterokaryon formation by 10⁴ to 10⁵ using the strains differing in alleles of a single vegetative incompatibility gene but had no detectable effect on strains differing in multiple vegetative incompatibility genes. Vegetative compatibility tests of single conidial isolates resolved from these heterokaryons suggest that diploids had formed followed by the loss of one of the VIC alleles. Presence of both auxotrophic markers in some of these single conidial isolates confirms the occurrence of a parasexual cycle. These experiments demonstrate that ultraviolet-light can enhance heterokaryon formation and parasexuality in C. parasitica .     

Auxotrophic Mutations Reduce Tolerance of Saccharomyces cerevisiae to Very High Levels of Ethanol Stress

Very high ethanol tolerance is a distinctive trait of the yeast Saccharomyces cerevisiae with notable ecological and industrial importance. Although many genes have been shown to be required for moderate ethanol tolerance (i.e., 6 to 12%) in laboratory strains, little is known of the much higher ethanol tolerance (i.e., 16 to 20%) in natural and industrial strains. We have analyzed the genetic basis of very high ethanol tolerance in a Brazilian bioethanol production strain by genetic mapping with laboratory strains containing artificially inserted oligonucleotide markers. The first locus contained the ura3Δ0 mutation of the laboratory strain as the causative mutation. Analysis of other auxotrophies also revealed significant linkage for LYS2, LEU2, HIS3, and MET15. Tolerance to only very high ethanol concentrations was reduced by auxotrophies, while the effect was reversed at lower concentrations. Evaluation of other stress conditions showed that the link with auxotrophy is dependent on the type of stress and the type of auxotrophy. When the concentration of the auxotrophic nutrient is close to that limiting growth, more stress factors can inhibit growth of an auxotrophic strain. We show that very high ethanol concentrations inhibit the uptake of leucine more than that of uracil, but the 500-fold-lower uracil uptake activity may explain the strong linkage between uracil auxotrophy and ethanol sensitivity compared to leucine auxotrophy. Since very high concentrations of ethanol inhibit the uptake of auxotrophic nutrients, the active uptake of scarce nutrients may be a major limiting factor for growth under conditions of ethanol stress.     

Heterokaryosis and Alkaloid Production in Claviceps purpurea

Strain 275 FI of Claviceps purpurea, which produces large amounts of peptide alkaloids in submerged culture, is a heterokaryon; after several generations on agar media, it segregates the single components. These components (by us labeled V, C, and W) are stable and produce almost no alkaloids under described conditions of submerged culture. The mycelium of strain 275 FI consists of hyphae with multinucleate cells and does not produce conidia. Strains V, C, and W form numerous anastomoses when grown together on agar. By combining strains V and C, a heterokaryon similar to 275 FI in appearance has been obtained. This new strain produces amounts of alkaloids much larger than those produced by V and C separately or in associated submerged culture. We conclude, therefore, that in strain 275 FI the heterokaryotic condition is favorable to the production of alkaloids. Several conidiaproducing cultures of C. purpurea of various origins, as well as sclerotia of the same species, have been examined. The results demonstrated that the heterokaryotic condition is rare in the cultures, but it is frequent in the mycelium from sclerotia. Since it is known that the production of alkaloids is typical of the sclerotial phase in C. purpurea, it is suggested that this capacity is related to the heterokaryosis of the producing strains.     

Trikaryon formation and nuclear selection in pairings between heterokaryons and homokaryons of the root rot pathogen Heterobasidion parviporum

Pairings between heterokaryons and homokaryons of Agaricomycete fungi (he-ho pairings) can lead to either heterokaryotization of the homokaryon or displacement of the homokaryotic nucleus through migration of nuclei from the heterokaryon into the homokaryon. In species of Agaricomycetes with multinucleate cells (>2 nuclei per cell), he-ho pairings could result in the stable or transient formation of a hypha with three genetically different nuclei (trikaryons). In this study, he-ho pairings were conducted using the multinucleate Agaricomycete Heterobasidion parviporum to determine whether trikaryons can be formed in the laboratory and whether nuclear genotype affects migration and heterokaryon formation. Nuclei were tracked by genotyping the heterokaryotic mycelium using nucleus-specific microsatellite markers. The data indicated that certain nuclear combinations were favored, and that nuclei from some strains had a higher rate of migration. A high percentage of trikaryons (19 %) displaying three microsatellite alleles per locus were identified among subcultures of the he-ho pairings. Using hyphal tip and conidial isolation, we verified that nuclei of three different mating types can inhabit the same mycelium, and one of the trikaryotic strains was judged to be semi-stable over multiple sub-culturing steps, with some hyphal tips that retained three alleles and others that reduced to two alleles per locus. These results demonstrate that nuclear competition and selection are possible outcomes of heterokaryon-homokaryon interactions in H. parviporum and confirm that ratios of component nuclei in heterokaryons are not strictly 1:1. The high rate of trikaryon formation in this study suggests that fungi with multinucleate cells may have the potential for greater genetic diversity and recombination relative to dikaryotic fungi.     

Intergeneric hybridization betweenMonascus anka andAspergillus oryzae by protoplast fusion

Summary To breed industrially useful strains of a slow-growing, red-pigment-producing strain ofMonascus anka, protoplasts ofM. anka MAK1 (arg) andAspergillus oryzae AOK1 (met, thr) were fused. A mixture of protoplasts prepared from mycelia ofM. anka MAK1 treated with 2% Usukizyme and ofA. oryzae AOK1 treated with 2% Usukizyme and 0.2% NovoZym 234 was incubated with 30% (w/v) polyethylene glycol no. 6000. Heterokaryon fusants complementing the auxotrophies of both mutants were isolated on minimal medium, but segregated into red (MAK1) and white (AOK1) sectors after being cultured on a complete medium. After irradiation with UV light, the fusants gave stable heterozygous diploids that formed long white hyphae. These diploids, which had twice as much DNA in the nucleus as their parents, grew more rapidly than the parent strain YZT1, and produced ethanol earlier than the parents. Production of amylase, protease, and kojic acid by the fusants was intermediate in amount between that of the two parents.     

Mating-type heterokaryosis and selfing in Cryphonectria parasitica

Selfing in the chestnut blight fungus, Cryphonectria parasitica, occurs by two different genetic mechanisms. Most self-fertile isolates of C. parasitica are heterokaryotic for mating type, and the progeny from selfing segregate for mating type. Further, we resolved mating-type (MAT) heterokaryons into homokaryons of both mating types by isolating uninucleate asexual spores (conidia). However, because ascospore progeny, with rare exceptions, are not MAT heterokaryons, C. parasitica must lack a regular mechanism to maintain heterokaryosis by selfing. We hypothesize that heterokaryon formation may occur either because of recurrent biparental inbreeding, or by mating-type switching, possibly one involving some kind of parasexual process. The second mechanism found for selfing in C. parasitica occurred less frequently. Three single-conidial isolates (MAT-1 and MAT-2) selfed and produced progeny that did not segregate for mating type. It is currently not known if meiosis occurs during ascospore formation by this mechanism.     

An atypical forkhead‐containing transcription factor SsFKH1 is involved in sclerotial formation and is essential for pathogenicity in Sclerotinia sclerotiorum

Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic plant pathogen with a worldwide distribution. The sclerotia of S. sclerotiorum are pigmented multicellular structures formed from the aggregation of vegetative hyphae. These survival structures play a central role in the life and infection cycles of this pathogen. Here, we characterized an atypical forkhead (FKH)‐box‐containing protein, SsFKH1, involved in sclerotial development and virulence. To investigate the role of SsFkh1 in S. sclerotiorum, the partial sequence of SsFkh1 was cloned and RNA interference (RNAi)‐based gene silencing was employed to alter the expression of SsFkh1. RNA‐silenced mutants with significantly reduced SsFkh1 RNA levels exhibited slow hyphal growth and sclerotial developmental defects. In addition, the expression levels of a set of putative melanin biosynthesis‐related laccase genes and a polyketide synthase‐encoding gene were significantly down‐regulated in silenced strains. Disease assays demonstrated that pathogenicity in RNAi‐silenced strains was significantly compromised with the development of a smaller infection lesion on tomato leaves. Collectively, the results suggest that SsFkh1 is involved in hyphal growth, virulence and sclerotial formation in S. sclerotiorum.     

The A mating type and blue light regulate all known differentiation processes in the basidiomycete Coprinus cinereus

Monokaryons of Coprinus cinereus constitutively form small spores (oidia) in the aerial mycelium. Some strains also produce large, inflated single cells (chlamydospores) at the agar/air interface, and hyphal aggregates (hyphal knots) that can develop into sclerotia. Monokaryons show various reactions upon transformation with heterologous A mating type genes. Production of oidia in such A-activated transformants is repressed in the dark and induced by blue light. Five of six monokaryons tested following transformation with A genes showed induced production of hyphal knots and sclerotia in the dark, and at least three strains showed enhanced chlamydospore production in the dark. Continuous incubation under blue light inhibited formation of hyphal knots, sclerotia and chlamydospores in both competent monokaryons and in A-activated transformants. On artificial medium and on a 12?h light/12?h dark regime, A-activated transformants of one distinct monokaryon (218) formed fruit-body primordia that were arrested in development before karyogamy. Our studies show that A mating type genes control all major differentiation processes in Coprinus, but whether developmental processes can proceed depends on the genetic background of the strain.     

Sclerotial Formation of Polyporus umbellatus by Low Temperature Treatment under Artificial Conditions

Background

Polyporus umbellatus sclerotia have been used as a diuretic agent in China for over two thousand years. A shortage of the natural P. umbellatus has prompted researchers to induce sclerotial formation in the laboratory.

Methodology/Principal Finding

P. umbellatus cultivation in a sawdust-based substrate was investigated to evaluate the effect of low temperature conditions on sclerotial formation. A phenol-sulfuric acid method was employed to determine the polysaccharide content of wild P. umbellatus sclerotia and mycelia and sclerotia grown in low-temperature treatments. In addition, reactive oxygen species (ROS) content, expressed as the fluorescence intensity of mycelia during sclerotial differentiation was determined. Analysis of ROS generation and sclerotial formation in mycelia after treatment with the antioxidants such as diphenyleneiodonium chloride (DPI), apocynin (Apo), or vitamin C were studied. Furthermore, macroscopic and microscopic characteristics of sclerotial differentiation were observed. Sclerotia were not induced by continuous cultivation at 25°C. The polysaccharide content of the artificial sclerotia is 78% of that of wild sclerotia. In the low-temperature treatment group, the fluorescent intensity of ROS was higher than that of the room temperature (25°C) group which did not induce sclerotial formation all through the cultivation. The antioxidants DPI and Apo reduced ROS levels and did not induce sclerotial formation. Although the concentration-dependent effects of vitamin C (5–15 mg mL⁻¹) also reduced ROS generation and inhibited sclerotial formation, using a low concentration of vitamin C (1 mg mL⁻¹) successfully induced sclerotial differentiation and increased ROS production.

Conclusions/Significance

Exposure to low temperatures induced P. umbellatus sclerotial morphogenesis during cultivation. Low temperature treatment enhanced ROS in mycelia, which may be important in triggering sclerotial differentiation in P. umbellatus. Moreover, the application of antioxidants impaired ROS generation and inhibited sclerotial formation. Our findings may help to provide new insights into the biological mechanisms underlying sclerotial morphogenesis in P. umbellatus.     

ROS and trehalose regulate sclerotial development in Rhizoctonia solani AG-1 IA

Rhizoctonia solani AG-1 IA is the causal agent of rice sheath blight (RSB) and causes severe economic losses in rice-growing regions around the world. The sclerotia play an important role in the disease cycle of RSB. In this study, we report the effects of reactive oxygen species (ROS) and trehalose on the sclerotial development of R. solani AG-1 IA. Correlation was found between the level of ROS in R. solani AG-1 IA and sclerotial development. Moreover, we have shown the change of ROS-related enzymatic activities and oxidative burst occurs at the sclerotial initial stage. Six genes related to the ROS scavenging system were quantified in different sclerotial development stages by using quantitative RT-PCR technique, thereby confirming differential gene expression. Fluorescence microscopy analysis of ROS content in mycelia revealed that ROS were predominantly produced at the hyphal branches during the sclerotial initial stage. Furthermore, exogenous trehalose had a significant inhibitory effect on the activities of ROS-related enzymes and oxidative burst and led to a reduction in sclerotial dry weight. Taken together, the findings suggest that ROS has a promoting effect on the development of sclerotia, whereas trehalose serves as an inhibiting factor to sclerotial development in R. solani AG-1 IA.     

球孢白僵菌种群野生菌株异核现象的分子验证*

球孢白僵菌28S rRNA基因D11域由于一内含子的存在与否,引起这一区域的扩增产物有两种片段类型(约520bp及120bp),将此作为分子标记分析了球孢白僵菌的117个菌株,发现17%菌株的扩增产物为双片段类型,即为异核体。异核体在不同种群中的分布频率因地理生态条件而异。单孢分离分析表明,异核体的分离频率约为35%。RAPD检测结果说明异核体在准性循环中发生了不同程度的遗传交换及遗传重组。球孢白僵菌异核体在继代培养中极不稳定。     

Protoplast fusion between Aspergillus oryzae and Aspergillus niger in relation to their multinuclear nature

Protoplasts of cycloheximide-resistant strains from Aspergillus oryzae IFO 5239 were fused with those of kabicidin-resistant strains from Aspergillus niger IFO 4407. By nuclear staining in conidia, it appeared that all of the fusant conidia had two kinds of nuclei. Small nuclei seemed to be derived from A. oryzae and large nuclei seemed to be derived from A. niger. However, three types of antibiotic resistance were shown among the conidia of fusants. Almost all were kabicidin resistant. Conidia of fusants were multinuclear and had various DNA contents and various sizes. By the comparison with the growth rates of parental strains, the growth rates of A. niger were superior to those of A. oryzae. The inclination in the distribution of antibiotic resistance of fusant conidia seemed to owe more to the differences of growth rates between parental strains than the influence of the multinucleate nature of a parental strain.     

HETEROKARYOSIS AND PARASEXUALITY IN THE FUNGUS ASCOCHYTA IMPERFECTA

The object of this investigation was to discover whether heterokaryosis and parasexuality occur in the imperfect fungus Ascochyta imperfecta. Both phenomena have been observed. The wild type of A. imperfecta grows on a minimal medium containing only salts plus a carbon source. Auxotrophic and morphological mutants have been isolated after treatment with ultraviolet light. When 2 different mutant auxotrophs are inoculated together onto minimal medium, colonies are consistently formed. These colonies might be due, a priori, to back-mutation, diploidy, syntrophism or heterokaryosis. Back-mutation and diploidy have been eliminated, since no back-mutant nuclei have been isolated from any heterokaryon, and since the frequency of diploid nuclei is very low. The combination is primarily syntrophic (only 2% heterokaryotic hyphal tips) when the nicotinamide mutant is one component. The combination is primarily heterokaryotic (over 50% heterokaryotic hyphal tips) when both components are auxotrophs for amino acids. From the heterokaryotic hyphal tips, the 2 unaltered nuclear components have been isolated. Heterozygous diploid nuclei (4.2 X 10^−-7 per haploid nucleus) can be isolated from heterokaryons by plating, onto minimal medium, the primarily uninucleate conidia from a heterokaryon of 2 auxotrophs. The resulting colonies are isolated as potential diploids. Three properties of these isolates establish their diploid nature: (1) the isolates are wild type for nutrition and morphology; (2) their conidial length is uniformly greater than that of the haploids (1.21 times); (3) the isolates produce segregants with nonparental combinations of the marker genes. The diploid isolates are much more stable than heterokaryons. The recombinants from the diploids are still diploid, since (1) their conidial length falls in the diploid range, and (2) one of the recombinants has segregated a second-order recombinant. Many of the expected classes of recombinants have not been detected.     

THE MORPHOGENESIS AND POSSIBLE EVOLUTIONARY ORIGINS OF FUNGAL SCLEROTIA

1. Fungal sclerotia are able to survive adverse conditions for long periods and they are formed by many important plant pathogens. An understanding of the factors involved in their initiation and development may lead to a method of repressing their formation in nature, thereby reducing the chances of survival of fungi that depend on them as persistent resting stages in their life-cycles. Also, data on sclerotial morphogenesis may be applicable to other multihyphal fungal structures. 2. There are three types of sclerotial development. The most primitive and least common is the loose type, which is illustrated by Rhizoctonia solani. The sclerotium forms by irregular branching of the mycelium followed by intercalary septation and hyphal swelling. When mature, it consists of loosely interwoven hyphae that are rich in food reserves and darkly pigmented. The main types of development are terminal and lateral. The former develops from the coalescence of initials that are produced by a well-defined pattern of branching at the tip of a hypha or tips of closely associated hyphae, e.g. Botrytis cinerea. Lateral sclerotia are formed by the interweaving of side branches of one or several main hyphae. When only one main hypha is involved the sclerotium is of the lateral, simple type, e.g. Sclerotinia gladioli. If several main hyphae give rise to a sclerotium, the term strand type has been used. Sclerotium rolfsii is the classical example. 3. There is a considerable literature on the effects of environmental conditions on the initiation, development and maturation of sclerotia but few attempts have been made to interpret the data. Phenolics and/or polyphenol oxidases have been found to be connected with morphogenesis of the protoperithecium of Neurospora crassa, the perithecium of Podospora anserina and of Hypomyces sp. and the basidiocarp of Schixophyllum commune. A close correlation has been shown between melanin synthesis and microsclerotial development by Verticillium but there appears to be no literature on the role of phenolics and polyphenol oxidases in the morphogenesis of sclerotia. Possibly these substances may inhibit growth of the apices of main hyphae by changing the permeability of the membrane, by inducing a thickening of the cell wall at the tip or by reducing the plasticity of the wall. Such a check in growth could trigger-off the formation of initials close to the margin of the colony or elsewhere in the culture. Sulphydryl groups and disulphide bonds are of great significance in morphogenesis of organisms and are probably involved in sclerotial initiation. The formation of a large number of hyphal branches is a prerequisite for sclerotial initiation and mycelial branching is possible only if there is plasticity of hyphal walls. The ability of the wall to be moulded is possibly related to changes in the sulphur linkages of the protein of the protein-carbohydrate complexes of the cell wall and could be influenced by sulphur availability or the activity of specific enzymes. 4. After a sclerotial primordium has been initiated, further increase in size will depend on the continued, active translocation of nutrients to the site of development. Movement of nutrients to sclerotia is through a few translocatory hyphae. Presumably, nutrients will continue to move into the young sclerotium as long as a concentration or pressure gradient is maintained. Energy and substances for the formation of new branches are supplied in this way and as the requirements for hyphal branches are reduced, excess nutrients become available for conversion to inactive or insoluble reserves and for exudation. The exudates are often complex, consisting of proteins, including enzymes, lipids and carbohydrates. Many sclerotia have a mucilaginous matrix in which the medullary hyphae are embedded. Sclerotium-forming, fungal species that are not regarded as having such a matrix appear to secrete a layer of mucilage over the surface of sclerotial hyphae. This mucilage could have a morphogenetic function and serve as an adhesive which loosely binds hyphae together. More permanent unions are by hyphal fusions or anastomoses. 5. The sclerotium matures within a few days of attaining its maximum size. The rind effectively seals off the medullary hyphae from the surroundings and the translocatory hyphae cease to function. Thus the sclerotium is isolated both physiologically and nutritionally. The endogenous reserves enable the structure to exist in the absence of exogenous nutrients and then, when conditions become suitable, to germinate. 6. The sclerotium appears to provide an example of convergent evolution whereby analogous structures, which have become adapted to resist adverse conditions, have evolved. Data are available mainly for Typhula spp. and ScZerotinia spp. Sclerotia may be degenerate sexual reproductive structures, hyphal aggregates that have developed from closely interwoven conidiophores and undifferentiated conidia or they may be modified vegetative structures.     

Protoplast fusion and genetic recombination in Metarhizium anisopliae

Using two strains of Metarhizium anisopliae var. minor designated E6 and RJ from different origins, inter-strain crosses were readily performed by orthodox methods from mutants of strain E6 through the parasexual cycle. However, in most cases crosses between mutants of strain RJ or between RJ and E6 mutant strains were not achieved. Protoplast fusion was carried out in an attempt to cross such strains. Protoplasts were obtained after treatment of mycelium with lytic enzymes using 0.7 m KCl as osmotic stabilizer. Regeneration frequency varied from 1.2 to 3.0%. Poly (tethylene glycol) was used for the fusion of protoplasts and fusion frequencies varied from 0.9 to 44.1 in 10⁵ protoplasts according to the cross. Sectors which emerge from fusion products were analysed and recombinants were obtained even from crosses between mutant strains which could not be crossed by hyphal fusion. In this way protoplast fusion proved to be a valuable tool for further studies of genetics and breeding of Metarhizium anisopliae.     

Aspergillus oryzae AoSO Is a Novel Component of Stress Granules upon Heat Stress in Filamentous Fungi

Stress granules are a type of cytoplasmic messenger ribonucleoprotein (mRNP) granule formed in response to the inhibition of translation initiation, which typically occurs when cells are exposed to stress. Stress granules are conserved in eukaryotes; however, in filamentous fungi, including Aspergillus oryzae, stress granules have not yet been defined. For this reason, here we investigated the formation and localization of stress granules in A. oryzae cells exposed to various stresses using an EGFP fusion protein of AoPab1, a homolog of Saccharomyces cerevisiae Pab1p, as a stress granule marker. Localization analysis showed that AoPab1 was evenly distributed throughout the cytoplasm under normal growth conditions, and accumulated as cytoplasmic foci mainly at the hyphal tip in response to stress. AoSO, a homolog of Neurospora crassa SO, which is necessary for hyphal fusion, colocalized with stress granules in cells exposed to heat stress. The formation of cytoplasmic foci of AoSO was blocked by treatment with cycloheximide, a known inhibitor of stress granule formation. Deletion of the Aoso gene had effects on the formation and localization of stress granules in response to heat stress. Our results suggest that AoSO is a novel component of stress granules specific to filamentous fungi.The authors would specially like to thank Hiroyuki Nakano and Kei Saeki for generously providing experimental and insightful opinions.