MoSNF1 regulates sporulation and pathogenicity in the rice blast fungus Magnaporthe oryzae

The protein kinase Snf1 is a major component of the glucose derepression pathway in yeast and a regulator of gene expression for the cell wall degrading enzyme (CWDE) in some plant pathogenic fungi. To address the molecular function of Snf1 in Magnaporthe oryzae, which causes the rice blast disease, MoSNF1 was cloned and functionally characterized using gene knock-out strategies. MoSNF1 functionally complemented the growth defect of the yeast snf1 mutant on a non-fermenting carbon source. However, the growth rate of the Δmosnf1 mutant on various carbon sources was reduced independent of glucose, and the expression of the CWDE genes in the mutant was induced during derepressing condition like the wild type. The pre-penetration stage including conidial germination and appressorium formation of the Δmosnf1 was largely impaired, and the pathogenicity of the Δmosnf1 was significantly reduced. Most strikingly, the Δmosnf1 mutant produced only a few conidia and had a high frequency of abnormally shaped conidia compared to the wild type. Our results suggest that MoSNF1 is a functional homolog of yeast Snf1, but its contribution to sporulation, vegetative growth and pathogenicity is critical in M. oryzae.     

Autophagy During Conidiation and Conidial Germination in Filamentous Fungi

Filamentous fungi form aerial hyphae on solid medium, and some of these differentiate into conidiophores for asexual sporulation (conidiation). In the filamentous deuteromycete, Aspergillus oryzae, aerial hyphae are formed from the foot cells and some differentiate into conidiophores, which are composed of vesicles, phialides and conidia. Recently, we isolated the yeast ATG8 gene homologue Aoatg8 from A. oryzae, and visualized autophagy by the expression of an EGFP (enhanced green fluorescent protein)–AoAtg8 fusion protein and DsRed2 protein in this fungus. Furthermore, by constructing the Aoatg8 deletion and conditional mutants, we demonstrated that autophagy functions during the process of differentiation of aerial hyphae, conidiation and conidial germination in A. oryzae. Here, we discuss the contribution of autophagy towards the differentiation and germination processes in filamentous fungi.

Addendum to:

Functional Analysis of the ATG8 Homologue Aoatg8 and Role of Autophagy in Differentiation and Germination in Aspergillus oryzae

T. Kikuma, M. Ohneda, M. Arioka and K. Kitamoto

Eukaryot Cell 2006; 5:1328-36     

Cloning and Characterization of the <Emphasis Type="Italic">BLR2</Emphasis>, the Homologue of the Blue-Light Regulator of <Emphasis Type="Italic">Neurospora crassa</Emphasis> WC-2, in the Phytopathogenic Fungus <Emphasis Type="Italic">Bipolaris oryzae</Emphasis>

Bipolaris oryzae is a filamentous ascomycetous fungus that causes brown leaf spot disease in rice. We isolated and characterized BLR2, a gene that encodes a putative blue-light regulator similar to Neurospora crassa white collar-2 (WC-2). The deduced amino acid sequence of the BLR2 showed significant homology to other fungal blue-light regulator proteins in the Per-Arnt-Sim (PAS) protein–protein interaction domain, nuclear localization signal, and GATA zinc finger DNA-binding domains. The BLR2-silenced transformants hardly produced conidia in the subsequent dark condition after near-ultraviolet (NUV) irradiation. Furthermore, the BLR2-silenced transformants suppressed the photolyase (PHR1) gene expression enhanced by NUV irradiation. These results indicate that BLR2 is necessary not only for conidial formation, but also for NUV radiation-enhanced photolyase gene expression in B. oryzae. The DDBJ accession number for the sequence reported in this paper is AB282674.     

Involvement of a Magnaporthe grisea serine/threonine kinase gene, MgATG1, in appressorium turgor and pathogenesis

We isolated an MgATG1 gene encoding a serine/threonine protein kinase from the rice blast fungus Magnaporthe grisea. In the DeltaMgatg1 mutant, in which the MgATG1 gene had been deleted, autophagy was blocked; the mutant also showed fewer lipid droplets in its conidia, lower turgor pressure of the appressorium, and such defects in morphogenesis as delayed initiation and slower germination of conidia. As a result of lower turgor pressure of the appressorium, the DeltaMgatg1 mutant lost its ability to penetrate and infect the two host plants, namely, rice and barley. However, normal values of the parameters and infective abilities were restored on reintroducing an intact copy of the MgATG1 gene into the mutant. Autophagy is thus necessary for turnover of organic matter during the formation of conidia and appressoria and for normal development and pathogenicity in M. grisea.     

Effects of Conidia of Various Aspergillus Species on Apoptosis of Human Pneumocytes and Bronchial Epithelial Cells

Aspergillus species can cause mycoses in human and animals. Previously, we demonstrated that A. fumigatus conidia from a human isolate inhibited apoptosis in human pneumocytes and bronchial epithelial cells. In the current study, we studied the effects of A. fumigatus conidia non-human origin and A. flavus, A. nidulans, A. niger and A. oryzae conidia on human cells apoptosis. Human pneumocytes or bronchial epithelial cells were simultaneously exposed to apoptotic inductors and aspergilli conidia. The cell cultures were analyzed by flow cytometry, immunoblotting, and examination of nuclear morphology. Similar to A. fumigatus conidia, A. flavus conidia inhibited cellular apoptosis while A. nidulans, A. niger and A. oryzae conidia did not affect apoptosis. We further studied the species specificity of conidia: there were no differences in the inhibition of apoptosis by A. fumigatus conidia from either human or bird isolates. In order to determine whether the inhibition of apoptosis by conidia is limited to certain strains, the effect on human cell apoptosis of different A. fumigatus human clinical isolates and A. fumigatus of environmental origin was evaluated. All A. fumigatus isolates inhibited apoptosis; an anti-apoptotic factor was released by conidia. For TNF-induced apoptosis, the anti-apoptotic effect of conidia of all isolates was found to be associated with a reduction of caspase-3 in human cells. The results suggest that suppression of apoptosis may play a role in reducing the efficacy of host defense mechanisms during infection with Aspergillus species. F. Féménia and D. Huet made an equal contribution to this work.     

橡胶树暹罗炭疽菌Zn2Cys6型转录因子CsGcc1的生物学功能

【背景】暹罗炭疽菌（Colletotrichum siamense）是一种重要的病原真菌,可以引起炭疽病,给全球橡胶产业带来巨大的经济损失。Zn₂Cys₆型转录因子是真菌特有的锌指类转录因子,通常参与调控真菌的生长发育过程。【目的】在暹罗炭疽菌中鉴定了一个与稻瘟病菌Gcc1同源的Zn₂Cys₆型转录因子CsGcc1,并研究其功能。【方法】根据同源重组原理构建CsGCC1的基因敲除突变体,并通过营养生长、H₂O₂敏感性、分生孢子产生及萌发、玻璃纸试验和致病性分析,明确CsGcc1的功能。【结果】CsGcc1编码一个含有646个氨基酸的蛋白,而且含有一个GAL4结构域。CsGCC1基因在培养36 h的菌丝及分生孢子中具有较高的表达量。CsGCC1基因敲除突变株营养生长速率降低且对H₂O₂更加敏感。相较于野生型菌株,突变株的分生孢子产量、萌发率及附着胞形成率均降低。此外,CsGCC1的敲除可以明显降低分生孢子的穿透能力,突变株对橡胶叶片的致病力减弱。【结论】Zn₂Cys₆型转录因子CsGcc1参与调控暹罗炭疽菌的营养生长、氧化应激、分生孢子发育及致病性等过程。     

The Sch9 Kinase Regulates Conidium Size,Stress Responses,and Pathogenesis in Fusarium graminearum

Fusarium head blight caused by Fusarium graminearum is an important disease of wheat and barley worldwide. In a previous study on functional characterization of the F. graminearum kinome, one protein kinase gene important for virulence is orthologous to SCH9 that is functionally related to the cAMP-PKA and TOR pathways in the budding yeast. In this study, we further characterized the functions of FgSCH9 in F. graminearum and its ortholog in Magnaporthe oryzae. The ΔFgsch9 mutant was slightly reduced in growth rate but significantly reduced in conidiation, DON production, and virulence on wheat heads and corn silks. It had increased tolerance to elevated temperatures but became hypersensitive to oxidative, hyperosmotic, cell wall, and membrane stresses. The ΔFgsch9 deletion also had conidium morphology defects and produced smaller conidia. These results suggest that FgSCH9 is important for stress responses, DON production, conidiogenesis, and pathogenesis in F. graminearum. In the rice blast fungus Magnaporthe oryzae, the ΔMosch9 mutant also was defective in conidiogenesis and pathogenesis. Interestingly, it also produced smaller conidia and appressoria. Taken together, our data indicate that the SCH9 kinase gene may have a conserved role in regulating conidium size and plant infection in phytopathogenic ascomycetes.     

A conidial protein (CP15) of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> contributes to the conidial tolerance of the entomopathogenic fungus to thermal and oxidative stresses

Aerial conidia are central dispersing structures for most fungi and represent the infectious propagule for entomopathogenic fungus Beauveria bassiana, thus the active ingredients of commercial mycoinsecticides. Although a number of formic-acid-extractable (FAE) cell wall proteins from conidia have been characterized, the functions of many such proteins remain obscure. We report that a conidial FAE protein, termed CP15, isolated from B. bassiana is related to fungal tolerance to thermal and oxidative stresses. The full-length genomic sequence of CP15 was shown to lack introns, encoding for a 131 amino acid protein (15.0 kDa) with no sequence identity to any known proteins in the NCBI database. The function of this new gene with two genomic copies was examined using the antisense-RNA method. Five transgenic strains displayed various degrees of silenced CP15 expression, resulting in significantly reduced conidial FAE protein profiles. The FAE protein contents of the strains were linearly correlated to the survival indices of their conidia when exposed to 30-min wet stress at 48°C (r ² = 0.93). Under prolonged 75-min heat stress, the median lethal times (LT₅₀s) of their conidia were significantly reduced by 13.6–29.5%. The CP15 silenced strains were also 20–50% less resistant to oxidative stress but were not affected with respect to UV-B or hyperosmotic stress. Our data indicate that discrete conidial proteins may mediate resistance to some abiotic stresses, and that manipulation of such proteins may be a viable approach to enhancing the environmental fitness of B. bassiana for more persisting control of insect pests in warmer climates.     

<Emphasis Type="Italic">Trichophyton rubrum</Emphasis> manipulates the innate immune functions of human keratinocytes

Evasion or subversion of host immune responses have been shown for a variety of microorganisms, and this might be the case for Trichophyton rubrum, the most common pathogenic fungus causing chronic dermatophytosis in humans. Keratinocytes, the main epidermal cells, have important roles as a first defense against microbial challenges in local immune reactions. Epidermal keratinocytes express several Toll-like receptors and produce host defense peptides, cytokines and chemokines in response to various stimuli. We analyzed the expression of Toll-Like receptor TLR2, TLR4, TLR6, and Human Beta Defensin (HBD)-1, HBD-2, Interleukin IL-1b and IL-8 production, when exposing primary keratinocyte cultures to T. rubrum. We observed changes in size and granularity of keratinocytes stimulated with either whole conidia or conidial homogenates compared to other treatments. Intact conidia decreased keratinocytes’ TLR2 and TLR6 expression without affecting that of TLR4, while conidial homogenates increased the expression of these three receptors. Interestingly, whole conidia decreased HBD-1 and HBD-2 production, whereas conidial homogenate increased it. No changes were observed in IL-1b and IL-8 production after stimulation with conidia or conidial homogenate. CONCLUSIONS. Our results suggest that: 1) Keratinocytes can recognize and respond to cell wall components of T. rubrum; 2) Viable intact conidia inhibit TLR-2 and TLR6 expression and decrease HBD-1 and HBD-2 production; 3) Conidial homogenate from T. rubrum increases the expression of TLR2, TLR4 and TLR6 and induces HBD-1 and HBD-2 production; 4) Therefore, innate immune functions of keratinocytes as the first level of local skin immunity are apparently manipulated by T. rubrum, likely to ensure its establishment, persistence and survival.     

Endogenous purine metabolism in the conidia of wild type and certain adenine mutants of neurospora crassa I. The nature of the reserve pools and pool utilization during adenine starvation

Conidia of four adenine auxotrophs (ad 9, ad 3B, ad 8 and ad 4 of Neurospora crassa differ in their ability to germinate on adenine-deficient medium. A large percentage of the ad 9 and ad 3B mutant conidia germinate while those of ad 8 and ad 4 mutant do not. No correlation was found between the size of the conidial purine reserves and the conidial ability to germinate. In all the strains the major fraction of the conidial purine reserved pools was inosine. The ad 8 and ad 4 mutants are blocked after IMP formation in the adenine biosynthetic pathway and therefore cannot use the stored inosine for germination. Pool-utilization studies indicated that in all strains investigated some of the purine reserved were lost from the conidia during incubation. In the most readily germinating strain, ad 9, only small amounts of the purine pool were lost from the conidia and a large portion of the reserve pool was used for nucleic acid synthesis. The nature of the purine reserves present in the conidia, and the ability of the strains to prevent loss of the stored purines from the conidia appear to be among the factors influencing the conidial germination of the adenine mutants of N. crassa.     

Laboratory studies on the development of a conidial formulation of Esteya vermicola

The endoparasitic nematophagous fungus, Esteya vermicola, has potential as a biocontrol agent against pinewood nematode, Bursaphelenchus xylophilus. An E. vermicola conidial formulation was developed to improve conidial resistance to ultraviolet (UV), drought and heat stress. The effective concentration of each protective additive [UV protectant [fulvic acid (FA) and skim milk (SM)]; drought protectant (sorbitol) and heat protectant (calcium chloride)] was determined based on the germination rate of E. vermicola conidia after exposure to the different stressors. A combination of 0.2% FA and 4% SM, 5% sorbitol and 0.05% calcium chloride provided the most effective protection. In addition, the concentrations of spreader–sticker and antibiotic were also decided. The final formulation could be used to improve the resistance of E. vermicola conidia to multiple stressors and to increase nematode mortality compared with unformulated conidia.     

MoCDC14 is important for septation during conidiation and appressorium formation in Magnaporthe oryzae

As a typical foliar pathogen, appressorium formation and penetration are critical steps in the infection cycle of Magnaporthe oryzae. Because appressorium formation and penetration are closely co‐regulated with the cell cycle, and Cdc14 phosphatases have an antagonistic relationship with cyclin‐dependent kinases (CDKs) on proteins related to mitotic exit and cytokinesis, in this study, we functionally characterized the MoCDC14 gene in M. oryzae. The Mocdc14 deletion mutant showed significantly reduced growth rate and conidiation. It was also defective in septum formation and nuclear distribution. Septation was irregular in Mocdc14 hyphae and hyphal compartments became multi‐nucleate. Mutant conidia often showed incomplete septa or lacked any septum. During appressorium formation, the septum delimiting appressoria from the rest of the germ tubes was often formed far away from the neck of the appressoria or not formed at all. Unlike the wild‐type, some mutant appressoria had more than one nucleus at 24 h. In addition to appressoria, melanization occurred on parts of the germ tubes and conidia, depending on the irregular position of the appressorium‐delimiting septum. The Mocdc14 mutant was also defective in glycogen degradation during appressorium formation and appressorial penetration of intact plant cells. Similar defects in septum formation, melanization and penetration were observed with appressorium‐like structures formed at hyphal tips in the Mocdc14 mutant. Often a long fragment of mutant hyphae was melanized, together with the apical appressorium‐like structures. These results indicate that MoCDC14 plays a critical role in septation, nuclear distribution and pathogenesis in M. oryzae, and correct septum formation during conidiogenesis and appressorium formation requires the MoCdc14 phosphatase.     

Metal ion binding byPithomyces chartarum conidia

Summary The binding of metals (Cu, Fe, Mn and Zn) commonly found in soil and decomposing plant material was studied in the saprophytic fungus,Pithomyces chartarum. Binding of metallic divalent cations was pH-dependent and temperature-independent; equilibrium occurred within 10 min in stirred suspensions of conidia, but mycelia had no detectable affinity for the metals. Germ tube emergence and elongation were stimulated by high concentrations of Mn⁺⁺ and Zn⁺⁺, byt not by Cu⁺⁺ or Fe⁺⁺. Metal binding did not obey a simple adsorption isotherm; Scatchard plot analysis indicated two classes of binding sites on the conidial surfaces, one class having association constants about 35-fold greater than those of the other. Calculations based on the conidial surface area as a smooth ellipsoid and the radii of the divalent cations indicated a multilayered coverage of the conidia by the metals at saturation concentrations. Binding sites were stable to boiling, dilute acid and base and lipid solvent extraction. The metals competed with the fungicide, thiabendazole, for binding sites on conidial surfaces.     

Perilipin LDP1 coordinates lipid droplets formation and utilization for appressorium-mediated infection in Magnaporthe oryzae

Lipid droplets (LDs) serve as one of the major reservoirs in conidia of Magnaporthe oryzae and are quickly utilized during appressorium formation. Here, we identified a gene, LDP1, encoding a perilipin that is important for LD formation and utilization during appressorium maturation. LDP1 is highly expressed in conidium and immature appressorium. Disruption mutants of LDP1 were significantly reduced in virulence, due to appressorial turgor reduction and difficulty in penetration. LDs were significantly reduced in the Δldp1 mutant, indicating LDP1 was required for LDs formation. LDP1 was colocalized with the LDs in conidium and immature appressorium but was gradually separated during appressorium maturation. A typical intracellular triacylglycerol lipase, TGL1-2, was clearly separated with LDs in conidium and immature appressorium but was well colocalized with LDs during appressorium maturation. The subcellular localization of TGL1-2 was affected by LDP1. These data suggested that LDP1 was bound to LDs for protecting from utilization in conidia and at the early appressorium stage but was separated from LDs for lipase entering and degradation. LDP1 was phosphorylated by CPKA at Thr96, which was essential for its localization and functions. These data indicate perilipin LDP1 can coordinate LD formation and utilization for appressorium-mediated infection of M. oryzae.     

The effects of culture media, solid substrates, and relative humidity on growth, sporulation and conidial discharge of Valdensinia heterodoxa

Valdensinia heterodoxa (Sclerotiniacae) is a potential fungal bioherbicide for control of salal (Gaultheria shallon). The effect of culture media, substrates and relative humidity (RH) on growth, sporulation and conidial discharge of V. heterodoxa was determined for two isolates PFC2761 and PFC3027 in vitro. Culture media significantly affected the growth, sporulation, and conidial discharge of V. heterodoxa. Of eight agar media used, colony radial growth was optimal on salal oatmeal agar and salal potato dextrose agar for isolates PFC2761 and PFC3027, respectively; whereas sporulation was at an optimum on salal oatmeal agar for both isolates. Of the eight liquid media tested, mycelial production was highest on wheat bran–salal–potato dextrose broth. Growth on solid substrates greatly stimulated sporulation and conidial discharge of V. heterodoxa. Of the 12 solid substrates used, the greatest numbers of discharged conidia were observed from wheat bran and wheat bran–salal within 14 d of sporulation. Sporulation on solid substrates continued for 42 d. RH significantly affected the sporulation and conidial discharge for both isolates across all solid substrates tested. No conidia were produced or discharged below 93 % RH on wheat bran–salal and millet. With an increase of the RH from 93 to 97 %, sporulation and the number of discharged conidia increased significantly for both isolates on wheat bran–salal, but not on millet.     

Identification and molecular cloning Moplaa gene,a homologue of Homo sapiens PLAA,in Magnaporthe oryzae

Magnaporthe oryzae has been used as a model fungal pathogen to study the molecular basis of plant–fungus interactions due to its economic and genetic importance. In this study, we identified a novel gene, Moplaa, which is the homologue of Homo sapiens PLAA encoding a phospholipase A₂-activating protein. Moplaa is conserved in some eukaryotic organisms by multiple alignment analysis. The function of the Moplaa gene was studied using the gene target replacement method. The Moplaa deletion mutant exhibited retarded growth and conidial germination, reduced conidiation, appressorial turgor pressure and pathogenicity to rice CO-39. Reintroduction of the gene restored defects of the Moplaa deletion mutant.