Three proline rotamases involved in calcium homeostasis play differential roles in stress tolerance,virulence and calcineurin regulation of Beauveria bassiana

FK506‐sensitive proline rotamases (FPRs), also known as FK506‐binding proteins (FKBPs), can mediate immunosuppressive drug resistance in budding yeast but their physiological roles in filamentous fungi remain opaque. Here, we report that three FPRs (cytosolic/nuclear 12.15‐kD Fpr1, membrane‐associated 14.78‐kD Fpr2 and nuclear 50.43‐kD Fpr3) are all equally essential for cellular Ca²⁺ homeostasis and contribute significantly to calcineurin activity at different levels in the insect‐pathogenic fungus Beauveria bassiana although the deletion of fpr1 alone conferred resistance to FK506. Radial growth, conidiation, conidial viability and virulence were less compromised in the absence of fpr1 or fpr2 than in the absence of fpr3, which abolished almost all growth on scant media and reduced growth moderately on rich media. The Δfpr3 mutant was more sensitive to Na⁺, K⁺, Mn²⁺, Ca²⁺, Cu²⁺, metal chelate, heat shock and UVB irradiation than was Δfpr2 while both mutants were equally sensitive to Zn²⁺, Mg²⁺, Fe²⁺, H₂O₂ and cell wall‐perturbing agents. In contrast, the Δfpr1 mutant was less sensitive to fewer stress cues. Most of 32 examined genes involved in DNA damage repair, Na⁺/K⁺ detoxification or osmotolerance and Ca²⁺ homeostasis were downregulated sharply in Δfpr2 and Δfpr3 but rarely so affected in Δfpr1, coinciding well with their phenotypic changes. These findings uncover important, but differential, roles of three FPRs in the fungal adaptation to insect host and environment and provide novel insight into their essential roles in calcium signalling pathway.     

The transmembrane protein Sho1 cooperates with the mucin Msb2 to regulate invasive growth and plant infection in Fusarium oxysporum

In the vascular wilt pathogen Fusarium oxysporum, the mitogen‐activated protein kinase (MAPK) Fmk1 is essential for plant infection. The mucin‐like membrane protein Msb2 regulates a subset of Fmk1‐dependent functions. Here, we examined the role of the tetraspan transmembrane protein Sho1 as an additional regulator of the Fmk1 pathway and determined its genetic interaction with Msb2. Targeted Δsho1 mutants were generated in wild‐type and Δmsb2 backgrounds to test possible interactions between the two genes. The mutants were examined for hyphal growth under different stress conditions, phosphorylation of the MAPK Fmk1 and an array of Fmk1‐dependent virulence functions. Similar to Msb2, Sho1 was required for the activation of Fmk1 phosphorylation, as well as Fmk1‐dependent gene expression and invasive growth functions, including extracellular pectinolytic activity, cellophane penetration, plant tissue colonization and virulence on tomato plants. Δsho1 mutants were hypersensitive to the cell wall‐perturbing compound Calcofluor White, and this phenotype was exacerbated in the Δmsb2 Δsho1 double mutant. These results highlight that Sho1 and Msb2 have partially overlapping functions upstream of the Fmk1 MAPK cascade, to promote invasive growth and plant infection, as well as cell wall integrity, in F. oxysporum.     

FraC/FraD-dependent intercellular molecular exchange in the filaments of a heterocyst-forming cyanobacterium, Anabaena sp

The filamentous, heterocyst‐forming cyanobacteria are multicellular organisms in which two different cell types, the CO₂‐fixing vegetative cells and the N₂‐fixing heterocysts, exchange nutrients and regulators. In Anabaena sp. strain PCC 7120, inactivation of sepJ or genes in the fraC operon (fraC, fraD and fraE) produce filament fragmentation. SepJ, FraC and FraD are cytoplasmic membrane proteins located in the filament's intercellular septa that are needed for intercellular exchange of the fluorescent tracer calcein (622 Da). Transmission electron microscopy showed an alteration in the heterocyst cytoplasmic membrane at the vegetative cell‐heterocyst septa in ΔfraC and ΔfraD mutants. Immunogold labelling of FraD confirmed its localization in the intercellular septa and clearly showed the presence of part of the protein between the cytoplasmic membranes of the adjacent cells. This localization seemed to be affected in the ΔfraC mutant but was not impaired in a ΔsepJ mutant. Intercellular transfer of a smaller fluorescent tracer, 5‐carboxyfluorescein (374 Da), was largely impaired in ΔfraC, ΔfraD and double ΔfraC‐ΔfraD mutants, but much less in the ΔsepJ mutant. These results show the existence in the Anabaena filaments of a FraC/FraD‐dependent intercellular molecular exchange that does not require SepJ.     

Proteomic identification of potential target proteins regulated by the SCFFbp1‐mediated proteolysis pathway in Fusarium oxysporum

F‐box proteins function in the recruitment of proteins for SCF ubiquitination and proteasome degradation. Here, we studied the role of Fbp1, a nonessential F‐box protein of the tomato pathogen Fusarium oxysporum f. sp. lycopersici. The Δfbp1 mutant showed a significant delay in the production of wilt symptoms on tomato plants and was impaired in invasive growth on cellophane membranes and on living plant tissue. To search for target proteins recruited by Fbp1, a combination of sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and matrix‐assisted laser desorption/ionization time‐of‐flight/time‐of‐flight (MALDI‐TOF/TOF) was used to compare proteins in mycelia of the wild‐type and Δfbp1 mutant. The proteomic approach identified 41 proteins differing significantly in abundance between the two strains, 17 of which were more abundant in the Δfbp1 mutant, suggesting a possible regulation by proteasome degradation. Interestingly, several of the identified proteins were related to vesicle trafficking. Microscopic analysis revealed an impairment of the Δfbp1 strain in directional growth and in the structure of the Spitzenkörper, suggesting a role of Fbp1 in hyphal orientation. Our results indicate that Fbp1 regulates protein turnover and pathogenicity in F. oxysporum.     

The F‐box protein Fbp1 functions in the invasive growth and cell wall integrity mitogen‐activated protein kinase (MAPK) pathways in Fusarium oxysporum

F‐box proteins determine substrate specificity of the ubiquitin–proteasome system. Previous work has demonstrated that the F‐box protein Fbp1, a component of the SCF^Fbp1 E3 ligase complex, is essential for invasive growth and virulence of the fungal plant pathogen Fusarium oxysporum. Here, we show that, in addition to invasive growth, Fbp1 also contributes to vegetative hyphal fusion and fungal adhesion to tomato roots. All of these functions have been shown previously to require the mitogen‐activated protein kinase (MAPK) Fmk1. We found that Fbp1 is required for full phosphorylation of Fmk1, indicating that Fbp1 regulates virulence and invasive growth via the Fmk1 pathway. Moreover, the Δfbp1 mutant is hypersensitive to sodium dodecylsulfate (SDS) and calcofluor white (CFW) and shows reduced phosphorylation levels of the cell wall integrity MAPK Mpk1 after SDS treatment. Collectively, these results suggest that Fbp1 contributes to both the invasive growth and cell wall integrity MAPK pathways of F. oxysporum.     

A Trichoderma atroviride stress‐activated MAPK pathway integrates stress and light signals

Cells possess stress‐activated protein kinase (SAPK) signalling pathways, which are activated practically in response to any cellular insult, regulating responses for survival and adaptation to harmful environmental changes. To understand the function of SAPK pathways in T. atroviride, mutants lacking the MAPKK Pbs2 and the MAPK Tmk3 were analysed under several cellular stresses, and in their response to light. All mutants were highly sensitive to cellular insults such as osmotic and oxidative stress, cell wall damage, high temperature, cadmium, and UV irradiation. Under oxidative stress, the Tmk3 pathway showed specific roles during development, which in conidia are essential for tolerance to oxidant agents and appear to play a minor role in mycelia. The function of this pathway was more evident in Δpbs2 and Δtmk3 mutant strains when combining oxidative stress or cell wall damage with light. Light stimulates tolerance to osmotic stress through Tmk3 independently of the photoreceptor Blr1. Strikingly, photoconidiation and expression of blue light regulated genes was severally affected in Δtmk3 and Δpbs2 strains, indicating that this pathway regulates light responses. Furthermore, Tmk3 was rapidly phosphorylated upon light exposure. Thus, our data indicate that Tmk3 signalling cooperates with the Blr photoreceptor complex in the activation of gene expression.     

Deciphering the role of the chitin synthase families 1 and 2 in the in vivo and in vitro growth of Aspergillus fumigatus by multiple gene targeting deletion

Although chitin is an essential component of the fungal cell wall (CW), its biosynthesis and role in virulence is poorly understood. In Aspergillus fumigatus, there are eight chitin synthase (CHS) genes belonging to two families CHSA‐C, CHSG in family 1 and CHSF, CHSD, CSMA, CSMB in family 2). To understand the function of these CHS genes, their single and multiple deletions were performed using β‐rec/six system to be able to delete all genes within each family (up to a quadruple ΔchsA/C/B/G mutant in family 1 and a quadruple ΔcsmA/csmB/F/D mutant in family 2). Radial growth, conidiation, mycelial/conidial morphology, CW polysaccharide content, Chs‐activity, susceptibility to antifungal molecules and pathogenicity in experimental animal aspergillosis were analysed for all the mutants. Among the family 1 CHS, ΔchsA, ΔchsB and ΔchsC mutants showed limited impact on chitin synthesis. In contrast, there was reduced conidiation, altered mycelial morphotype and reduced growth and Chs‐activity in the ΔchsG and ΔchsA/C/B/G mutants. In spite of this altered phenotype, these two mutants were as virulent as the parental strain in the experimental aspergillosis models. Among family 2 CHS, phenotypic defects mainly resulted from the CSMA deletion. Despite significant morphological mycelial and conidial growth phenotypes in the quadruple ΔcsmA/csmB/F/D mutant, the chitin content was poorly affected by gene deletions in this family. However, the entire mycelial cell wall structure was disorganized in the family 2 mutants that may be related to the reduced pathogenicity of the quadruple ΔcsmA/csmB/F/D mutant strain compared to the parental strain, in vivo. Deletion of the genes encompassing the two families (ΔcsmA/csmB/F/G) showed that in spite of being originated from an ancient divergence of fungi, these two families work cooperatively to synthesize chitin in A. fumigatus and demonstrate the essentiality of chitin biosynthesis for vegetative growth, resistance to antifungal drugs, and virulence of this filamentous fungus.     

Reduced susceptibility to Fusarium head blight in Brachypodium distachyon through priming with the Fusarium mycotoxin deoxynivalenol

The fungal cereal pathogen Fusarium graminearum produces deoxynivalenol (DON) during infection. The mycotoxin DON is associated with Fusarium head blight (FHB), a disease that can cause vast grain losses. Whilst investigating the suitability of Brachypodium distachyon as a model for spreading resistance to F. graminearum, we unexpectedly discovered that DON pretreatment of spikelets could reduce susceptibility to FHB in this model grass. We started to analyse the cell wall changes in spikelets after infection with F. graminearum wild‐type and defined mutants: the DON‐deficient Δtri5 mutant and the DON‐producing lipase disruption mutant Δfgl1, both infecting only directly inoculated florets, and the mitogen‐activated protein (MAP) kinase disruption mutant Δgpmk1, with strongly decreased virulence but intact DON production. At 14 days post‐inoculation, the glucose amounts in the non‐cellulosic cell wall fraction were only increased in spikelets infected with the DON‐producing strains wild‐type, Δfgl1 and Δgpmk1. Hence, we tested for DON‐induced cell wall changes in B. distachyon, which were most prominent at DON concentrations ranging from 1 to 100 ppb. To test the involvement of DON in defence priming, we pretreated spikelets with DON at a concentration of 1 ppm prior to F. graminearum wild‐type infection, which significantly reduced FHB disease symptoms. The analysis of cell wall composition and plant defence‐related gene expression after DON pretreatment and fungal infection suggested that DON‐induced priming of the spikelet tissue contributed to the reduced susceptibility to FHB.     

胶孢炭疽菌C2H2型转录因子CgGcp1的生物学功能

【背景】胶孢炭疽菌(Colletotrichum gloeosporioides)可以寄生于多种植物,侵染方式多样,能够引起严重的农业危害。在胶孢炭疽菌中,CgGcp1是一个C2H2型的转录因子,关于其生物学功能的研究未见报道。【目的】明确CgGcp1的生物学功能,为深入解析该病菌的致病机制奠定一定的理论依据。【方法】构建CgGCP1基因的敲除载体,利用同源重组得到敲除突变体。通过表型分析,包括营养生长、胁迫响应、孢子产生、附着胞形成及致病性分析等,明确该基因的生物学功能。【结果】CgGCP1基因敲除突变体生长速率较野生型减慢,对SDS、刚果红、NaCl和甘油更加敏感,孢子产量显著降低,附着胞的形成率降低且侵入能力减弱,在橡胶叶片上的致病力明显下降。【结论】CgGcp1参与调控胶孢炭疽菌营养生长、细胞壁完整性、分生孢子产生、附着胞形成与侵入和致病性。     

Involvement of FgERG4 in ergosterol biosynthesis,vegetative differentiation and virulence in Fusarium graminearum

The ergosterol biosynthesis pathway is well understood in Saccharomyces cerevisiae, but currently little is known about the pathway in plant‐pathogenic fungi. In this study, we characterized the Fusarium graminearum FgERG4 gene encoding sterol C‐24 reductase, which catalyses the conversion of ergosta‐5,7,22,24‐tetraenol to ergosterol in the final step of ergosterol biosynthesis. The FgERG4 deletion mutant ΔFgErg4‐2 failed to synthesize ergosterol. The mutant exhibited a significant decrease in mycelial growth and conidiation, and produced abnormal conidia. In addition, the mutant showed increased sensitivity to metal cations and to various cell stresses. Surprisingly, mycelia of ΔFgErg4‐2 revealed increased resistance to cell wall‐degrading enzymes. Fungicide sensitivity tests revealed that ΔFgErg4‐2 showed increased resistance to various sterol biosynthesis inhibitors (SBIs), which is consistent with the over‐expression of SBI target genes in the mutant. ΔFgErg4‐2 was impaired dramatically in virulence, although it was able to successfully colonize flowering wheat head and tomato, which is in agreement with the observation that the mutant produces a significantly lower level of trichothecene mycotoxins than does the wild‐type progenitor. All of these phenotypic defects of ΔFgErg4‐2 were complemented by the reintroduction of a full‐length FgERG4 gene. In addition, FgERG4 partially rescued the defect of ergosterol biosynthesis in the Saccharomyces cerevisiae ERG4 deletion mutant. Taken together, the results of this study indicate that FgERG4 plays a crucial role in ergosterol biosynthesis, vegetative differentiation and virulence in the filamentous fungus F. graminearum.     

Studies of the unfolding of an unstable mutant of staphylococcal nuclease: Evidence for low temperature unfolding and compactness of the high temperature unfolded state

Fluorescence and circular dichroism data as a function of temperature were obtained to characterize the unfolding of nuclease A and two of its less stable mutants. These spectroscopic data were obtained with a modified instrument that enables the nearly simultaneous detection of both fluorescence and CD data on the same sample. A global analysis of these multiple datasets yielded an excellent fit of a model that includes a change in the heat capacity change, ΔC_p, between the unfolded and native states. This analysis gives a ΔC_p of 2.2 kcal/mol/·K for thermal unfolding of the WT protein and 1.3 and 1.8 kcal/mol/K for the two mutants. These ΔC_p values are consistent with significant population of the cold unfolded state at ∼0°C. Independent evidence for the existence of a cold unfolded state is the observation of a separately migrating peak in size exclusion chromatography. The new chromatographic peak is seen near 0°C, has a partition coefficient corresponding to a larger hydrodynamic radius, and shows a red-shifted fluorescence spectrum, as compared to the native protein. Data also indicate that the high-temperature unfolded form of mutant nuclease is relatively compact. Size exclusion chromatography shows the high temperature unfolded form to have a hydrodynamic radius that is larger than that for the native form, but smaller than that for the urea or pH-induced unfolded forms. Addition of chemical denaturants to the high-temperature unfolded form causes a further unfolding of the protein, as indicated by an increase in the apparent hydrodynamic radius and a decrease in the rotational correlation time for Trp140 (as determined by fluorescence anisotropy decay measurements). Proteins 28:227–240, 1997 © 1997 Wiley-Liss Inc.