Regulatory functions of homeobox domain transcription factors in fungi.

Homeobox domain (HD) proteins present a crucial involvement in morphological differentiation and other functions in eukaryotes. Most HD genes encode transcription factors (TFs) that orchestrate a regulatory role in cellular and developmental decisions. In fungi, multiple studies have increased our understanding of these important HD regulators in recent years. These reports have revealed their role in fungal development, both sexual and asexual, as well as their importance in governing other biological processes in these organisms, including secondary metabolism, pathogenicity, and sensitivity to environmental stresses. Here, we provide a comprehensive review of the current knowledge on the regulatory roles of HD-TFs in fungi, with a special focus on Aspergillus species.

VmSom1 is essential for growth, development, maintenance of cell wall integrity and virulence in Valsa mali.

Apple Valsa canker disease, caused by Valsa mali Miyabe et Yamada, severely endangers the healthy growth of apple trees. The Som1, located downstream of the cyclic AMP-dependent protein kinase A (cAMP-PKA) pathway, plays crucial roles in the growth, development, morphological differentiation, and virulence of filamentous fungi. In this study, we identify and functionally characterize VmSom1, a homolog of Som1, in Valsa mali. The VmSom1 gene is located on chromosome 12, encoding an 824 amino acid protein. Phylogenetic analysis reveals VmSom1 as a fungal Som1 homolog. The VmSom1 deletion mutants exhibit slower growth rates and fail to produce pycnidia. Additionally, their hyphal growth is significantly inhibited on media containing Calcofluor White, Congo Red, NaCl, and sorbitol. The growth rate of VmSom1 deletion mutants is reduced on maltose, lactose, sucrose and fructose media but increases on glucose medium. Moreover, the mycelial growth rate of the VmSom1 deletion mutant is significantly lower than that of the wild-type strain in peptone, NH4SO4, NaNO3, and no nitrogen. Notably, the distances between the septa increase, and chitin concentration shifts to the hyphal tip in the VmSom1 deletion mutant. Furthermore, compared with the wild-type strain, the VmSom1 deletion mutant exhibits fewer diseased spots on apple fruit and branches. Overall, our findings demonstrate that VmSom1 is involved in regulating the growth and development, colony surface hydrophobicity, osmotic stress, cell wall integrity maintenance, carbon and nitrogen source utilization, septa formation, and virulence of V. mali.

Heterologous pulcherrimin production in Saccharomyces cerevisiae confers inhibitory activity on Botrytis conidiation.

Pulcherrimin is an iron (III) chelate of pulcherriminic acid that plays a role in antagonistic microbial interactions, iron metabolism, and stress responses. Some bacteria and yeasts produce pulcherriminic acid, but so far, pulcherrimin could not be produced in Saccharomyces cerevisiae. Here, multiple integrations of the Metschnikowia pulcherrima PUL1 and PUL2 genes in the S. cerevisiae genome resulted in red colonies, which indicated pulcherrimin formation. The coloration correlated positively and significantly with the number of PUL1 and PUL2 genes. The presence of pulcherriminic acid was confirmed by mass spectrometry. In vitro competition assays with the plant pathogenic fungus Botrytis caroliana revealed inhibitory activity on conidiation by an engineered, strong pulcherrimin-producing S. cerevisiae strain. We demonstrate that the PUL1 and PUL2 genes from M. pulcherrima, in multiple copies, are sufficient to transfer pulcherrimin production to S. cerevisiae and represent the starting point for engineering and optimizing this biosynthetic pathway in the future.

C-terminus of serine-arginine protein kinase-like protein, SrpkF, is involved in conidiophore formation and hyphal growth under salt stress in Aspergillus aculeatus.

The serine-arginine protein kinase-like protein, SrpkF, was identified as a regulator for the cellulose-responsive induction of cellulase genes in Aspergillus aculeatus. To analyze various aspects of SrpkF function, we examined the growth of the control strain (MR12); C-terminus deletion mutant, which produced SrpkF1-327 (ΔCsrpkF); whole gene-deletion mutant of srpkF (ΔsrpkF), srpkF overexpressing strain (OEsprkF); and the complemented strain (srpkF+) under various stress conditions. All test strains grew normally on minimal medium under control, high salt (1.5 M KCl), and high osmolality (2.0 M sorbitol and 1.0 M sucrose). However, only ΔCsrpkF showed reduced conidiation on 1.0 M NaCl media. Conidiation of ΔCsrpkF on 1.0 M NaCl media was reduced to 12% compared with that of srpkF+. Further, when OEsprkF and ΔCsrpkF were pre-cultured under salt stress conditions, germination under salt stress conditions was enhanced in both strains. By contrast, deletion of srpkF did not affect hyphal growth and conidiation under the same conditions. We then quantified the transcripts of the regulators involved in the central asexual conidiation pathway in A. aculeatus. The findings revealed that the expression of brlA, abaA, wetA, and vosA was reduced in ΔCsrpkF under salt stress. These data suggest that in A. aculeatus, SrpkF regulates conidiophore development. The C-terminus of SrpkF seems to be important for regulating SrpkF function in response to culture conditions such as salt stress.

Quantitative Proteomic Analysis Reveals Important Roles of the Acetylation of ER-Resident Molecular Chaperones for Conidiation in Fusarium oxysporum.

Fusarium oxysporum is one of the most abundant and diverse fungal species found in soils and includes nonpathogenic, endophytic, and pathogenic strains affecting a broad range of plant and animal hosts. Conidiation is the major mode of reproduction in many filamentous fungi, but the regulation of this process is largely unknown. Lysine acetylation (Kac) is an evolutionarily conserved and widespread posttranslational modification implicated in regulation of multiple metabolic processes. A total of 62 upregulated and 49 downregulated Kac proteins were identified in sporulating mycelia versus nonsporulating mycelia of F. oxysporum. Diverse cellular proteins, including glycolytic enzymes, ribosomal proteins, and endoplasmic reticulum-resident molecular chaperones, were differentially acetylated in the sporulation process. Altered Kac levels of three endoplasmic reticulum-resident molecular chaperones, PDIK70, HSP70K604, and HSP40K32 were identified that with important roles in F. oxysporum conidiation. Specifically, K70 acetylation (K70ac) was found to be crucial for maintaining stability and activity of protein disulphide isomerase and the K604ac of HSP70 and K32ac of HSP40 suppressed the detoxification ability of these heat shock proteins, resulting in higher levels of protein aggregation. During conidial formation, an increased level of PDIK70ac and decreased levels of HSP70K604ac and HSP40K32ac contributed to the proper processing of unfolded proteins and eliminated protein aggregation, which is beneficial for dramatic cell biological remodeling during conidiation in F. oxysporum.

Bbhox2 is a key regulator for conidiation and virulence in Beauveria bassiana.

Beauveria bassiana, a well-known filamentous biocontrol fungus, is the main pathogen of numerous field and forest pests. To explore the potential factors involved in the fungal pathogenicity, Bbhox2, an important and conserved functional transcription factor containing homeodomain was carried out by functional analysis. Homologous recombination was used to disrupt the Bbhox2 gene in B.bassiana. The conidia yield of the deletant fungal strain was significantly reduced. The conidial germination was faster, and stress tolerance to Congo red and high osmotic agents were decreased compared with that in the wildtype. Additionally, ΔBbhox2 showed a dramatic reduction in virulence no matter in topical inoculations or in intra-hemolymph injections against Galleria mellonella larvae, which is likely due to the failure of appressorium formation and the defect in producing hyphal body. These results indicate that the Bbhox2 gene markedly contributes to conidiation and pathogenicity in B. bassiana.

Crucial Involvement of Heme Biosynthesis in Vegetative Growth, Development, Stress Response, and Fungicide Sensitivity of Fusarium graminearum.

Heme biosynthesis is a highly conserved pathway from bacteria to higher animals. Heme, which serves as a prosthetic group for various enzymes involved in multiple biochemical processes, is essential in almost all species, making heme homeostasis vital for life. However, studies on the biological functions of heme in filamentous fungi are scarce. In this study, we investigated the role of heme in Fusarium graminearum. A mutant lacking the rate-limiting enzymes in heme synthesis, coproporphyrinogen III oxidase (Cpo) or ferrochelatase (Fc), was constructed using a homologous recombination strategy. The results showed that the absence of these enzymes was lethal to F. graminearum, but the growth defect could be rescued by the addition of hemin, so we carried out further studies with the help of hemin. The results demonstrated that heme was required for the activity of FgCyp51, and its absence increased the sensitivity to tebuconazole and led to the upregulation of FgCYP51 in F. graminearum. Additionally, heme plays an indispensable role in the life cycle of F. graminearum, which is essential for vegetative growth, conidiation, external stress response (especially oxidative stress), lipid accumulation, fatty acid ß-oxidation, autophagy, and virulence.

MoNOT3 Subunit Has Important Roles in Infection-Related Development and Stress Responses in Magnaporthe oryzae.

The multifunctional carbon catabolite repression negative on TATA-box-less complex (CCR4-NOT) is a multi-subunit complex present in all eukaryotes, including fungi. This complex plays an essential role in gene expression; however, a functional study of the CCR4-NOT complex in the rice blast fungus Magnaporthe oryzae has not been conducted. Seven genes encoding the putative CCR4-NOT complex were identified in the M. oryzae genome. Among these, a homologous gene, MoNOT3, was overexpressed during appressorium development in a previous study. Deletion of MoNOT3 in M. oryzae resulted in a significant reduction in hyphal growth, conidiation, abnormal septation in conidia, conidial germination, and appressorium formation compared to the wild-type. Transcriptional analyses suggest that the MoNOT3 gene affects conidiation and conidial morphology by regulating COS1 and COM1 in M. oryzae. Furthermore, Δmonot3 exhibited a lack of pathogenicity, both with and without wounding, which is attributable to deficiencies in the development of invasive growth in planta. This result was also observed in onion epidermal cells, which are non-host plants. In addition, the MoNOT3 gene was involved in cell wall stress responses and heat shock. Taken together, these observations suggest that the MoNOT3 gene is required for fungal infection-related cell development and stress responses in M. oryzae.

Roles of CgEde1 and CgMca in Development and Virulence of Colletotrichum gloeosporioides.

Anthracnose, induced by Colletotrichum gloeosporioides, poses a substantial economic threat to rubber tree yields and various other tropical crops. Ede1, an endocytic scaffolding protein, plays a crucial role in endocytic site initiation and maturation in yeast. Metacaspases, sharing structural similarities with caspase family proteases, are essential for maintaining cell fitness. To enhance our understanding of the growth and virulence of C. gloeosporioides, we identified a homologue of Ede1 (CgEde1) in C. gloeosporioides. The knockout of CgEde1 led to impairments in vegetative growth, conidiation, and pathogenicity. Furthermore, we characterized a weakly interacted partner of CgEde1 and CgMca (orthologue of metacaspase). Notably, both the single mutant ΔCgMca and the double mutant ΔCgEde1/ΔCgMca exhibited severe defects in conidiation and germination. Polarity establishment and pathogenicity were also disrupted in these mutants. Moreover, a significantly insoluble protein accumulation was observed in ΔCgMca and ΔCgEde1/ΔCgMca strains. These findings elucidate the mechanism by which CgEde1 and CgMca regulates the growth and pathogenicity of C. gloeosporioides. Their regulation involves influencing conidiation, polarity establishment, and maintaining cell fitness, providing valuable insights into the intricate interplay between CgEde1 and CgMca in C. gloeosporioides.

DhFIG_2, a gene of nematode-trapping fungus Dactylellina haptotyla that encodes a component of the low-affinity calcium uptake system, is required for conidiation and knob-trap formation.

Calcium ion (Ca2+) is a universal second messenger involved in regulating diverse processes in animals, plants, and fungi. The low-affinity calcium uptake system (LACS) participates in acquiring Ca2+ from extracellular environments under high extracellular Ca2+ concentration. Unlike most fungi, which encode only one protein (FIG1) for LACS, nematode-trapping fungi (NTF) encode two related proteins. AoFIG_2, the NTF-specific LACS component encoded by adhesive network-trap forming Arthrobotrys oligospora, was shown to be required for conidiation and trap formation. We characterized the role of DhFIG_2, an AoFIG_2 ortholog encoded by knob-trap forming Dactylellina haptotyla, in growth and development to expand our understanding of the role of LACS in NTF. Because repeated attempts to disrupt DhFIG_2 failed, knocking down the expression of DhFIG_2 via RNA interference (RNAi) was used to study its function. RNAi of DhFIG_2 significantly decreased its expression, severely reduced conidiation and trap formation, and affected vegetative growth and stress responses, suggesting that this component of LACS is crucial for trap formation and conidiation in NTF. Our study demonstrated the utility of RNAi assisted by ATMT for studying gene function in D. haptotyla.

Construction of RNA silencing system of Penicillium brevicompactum and genetic manipulation of the regulator pbpcz in mycophenolic acid production.

Penicillium brevicompactum is a critical industrial strain for the production of mycophenolic acid (MPA). However, the genetic background of Penicillium brevicompactum is unclear, and there are few tools available for genetic manipulation. To investigate its gene function, we first verified the feasibility of a pair of citrate synthase promoter (Pcit) and terminator (Tcit) from P. brevicompactum by constructing a fluorescent expression cassette. Based on this, an RNAi vector was designed and constructed with reverse promoters. This study focused on the functional investigation of the pbpcz gene in P. brevicompactum, a regulator belonging to the Zn(II)2Cys6 family. RNAi was used to silence the pbpcz gene, providing a valuable tool for genetic studies in P. brevicompactum. After seven days, we observed differences in the number of spores between different phenotypes strains of pbpcz gene. Compared to the wild-type strain (WT), the spore yield of the pbpcz gene silencing mutant (M2) was only 51.4 %, while that of the pbpcz gene overexpressed mutant (SE4) was increased by 50 %. Expression levels of the three genes (brlA, abaA, and wetA) comprising conidia's central regulatory pathway were significantly reduced in the pbpcz gene silencing mutant, while fluorescence localization showed that PbPCZ protein was mainly distributed in spores. The results indicated that the pbpcz gene is critical for conidia and asexual development of P. brevicompactum. In addition, overexpressing the pbpcz gene resulted in a 30.3 % increase in MPA production compared to the wild type, with a final yield of 3.57 g/L. These results provide evidence that PbPCZ acts as a positive regulator in P. brevicompactum, controlling MPA production and regulating conidia and asexual development.

AoMedA has a complex regulatory relationship with AoBrlA, AoAbaA, and AoWetA in conidiation, trap formation, and secondary metabolism in the nematode-trapping fungus Arthrobotrys oligospora.

The asexual sporulation of filamentous fungi is an important mechanism for their reproduction, survival, and pathogenicity. In Aspergillus and several filamentous fungi, BrlA, AbaA, and WetA are the key elements of a central regulatory pathway controlling conidiation, and MedA is a developmental modifier that regulates temporal expression of central regulatory genes; however, their roles are largely unknown in nematode-trapping (NT) fungi. Arthrobotrys oligospora is a representative NT fungus, which can capture nematodes by producing adhesive networks (traps). Here, we characterized the function of AoMedA and three central developmental regulators (AoBrlA, AoAbaA, and AoWetA) in A. oligospora by gene disruption, phenotypic comparison, and multi-omics analyses, as these regulators are required for conidiation and play divergent roles in mycelial development, trap formation, lipid droplet accumulation, vacuole assembly, and secondary metabolism. A combined analysis of phenotypic traits and transcriptome showed that AoMedA and AoWetA are involved in the regulation of peroxisome, endocytosis, and autophagy. Moreover, yeast one-hybrid analysis showed that AoBrlA can regulate AoMedA, AoAbaA, and AoWetA, whereas AoMedA and AoAbaA can regulate AoWetA. Our results highlight the important roles of AoMedA, AoBrlA, AoAbaA, and AoWetA in conidiation, mycelia development, trap formation, and pathogenicity of A. oligospora and provide a basis for elucidating the relationship between conidiation and trap formation of NT fungi. IMPORTANCE Conidiation is the most common reproductive mode for many filamentous fungi and plays an essential role in the pathogenicity of fungal pathogens. Nematode-trapping (NT) fungi are a special group of filamentous fungi owing to their innate abilities to capture and digest nematodes by producing traps (trapping devices). Sporulation plays an important role in the growth and reproduction of NT fungi, and conidia are the basic components of biocontrol reagents for controlling diseases caused by plant-parasitic nematodes. Arthrobotrys oligospora is a well-known NT fungus and is a routinely used model fungus for probing the interaction between fungi and nematodes. In this study, the functions of four key regulators (AoMedA, AoBrlA, AoAbaA, and AoWetA) involved in conidiation were characterized in A. oligospora. A complex interaction between AoMedA and three central regulators was noted; these regulators are required for conidiation and trap formation and play a pleiotropic role in multiple intracellular activities. Our study first revealed the role of AoMedA and three central regulators in conidiation, trap formation, and pathogenicity of A. oligospora, which contributed to elucidating the regulatory mechanism of conidiation in NT fungi and helped in developing effective reagents for biocontrol of nematodes.

BCB1, a member of the acyl-coenzyme A synthetase family, regulates the morphogenesis and pathogenicity of Botrytis cinerea.

Botrytis cinerea is a non-host-specific phytopathogenic fungus capable of infecting numerous cash crops. Here, we analyzed the functions of the Bcb1 gene in B. cinerea, which encodes a membrane protein belonging to the acyl-coenzyme A synthase family. Compared to the wild type, Bcb1-deletion mutants exhibited obvious morphological abnormalities, including slower vegetative growth and reduced melanin production. The absence of Bcb1 causes B. cinerea to form only small and incompletely developed infection cushions and fail to produce spores. The Bcb1 mutants displayed hypersensitivity to the membrane stressor SDS, the cell wall stressor Congo red, and the oxidative stressor H2O2 and increased resistance to intracellular osmotic stress caused by KCl compared to the wild-type strain. However, there were no differences in tolerance to extracellular osmotic stress caused by NaCl. The deletion of Bcb1 also caused a reduction in pathogenicity. The qRT‒PCR results showed that the genes Bcpks12 and Bcpks13, which are related to melanin biosynthesis, and Bcpg2, BcBOT2, and cutA, which are related to virulence, were downregulated in ∆Bcb1. These data suggest that BCB1 is important for conidial morphogenesis, and pathogenesis in B. cinerea.

Arginine metabolism governs microcycle conidiation by changing nitric oxide content in Metarhizium acridum.

Microcycle conidiation commonly exists in filamentous fungi and has great potential for mass production of mycoinsecticides. L-Arginine metabolism is essential for conidiation and conditional growth and virulence, but its role in microcycle conidiation has not been explored. Here, a unique putative arginase (MaAGA) was characterized in the entomopathogenic fungus Metarhizium acridum. Conidial germination and thermotolerance were facilitated by the disruption of MaAGA. Despite little impact on fungal growth and virulence, the disruption resulted in normal conidiation after a 60-h incubation on microcycle conidiation medium (SYA) under normal culture conditions. In the MaAGA-disruption mutant (ΔMaAGA), intracellular arginine accumulation was sharply increased. Replenishment of the direct metabolites of arginase, namely ornithine and/or urea, was unable to restore the disruption mutant's microcycle conidiation on SYA. Interestingly, nitric oxide synthase (NOS) activity and nitric oxide (NO) levels of the ΔMaAGA strain were markedly decreased in the 60-h-old SYA cultures. Finally, adding Nω-nitro-L-arginine, an inhibitor of NOS, into the SYA converted the microcycle conidiation of the wild-type strain to normal conidiation. In contrast, adding sodium nitroprusside, an NO donor, into the SYA recovered the mutant's microcycle conidiation. The results indicate that arginine metabolism controls microcycle conidiation by changing the content of NO. KEY POINTS: • The MaAGA-disruption led to normal conidiation on microcycle conidiation medium SYA. • Nitric oxide (NO) level of the ΔMaAGA strain was markedly decreased. • Adding an NO donor into the SYA recovered the microcycle conidiation of ΔMaAGA.

Tetracarboxylic acid transporter regulates growth, conidiation, and carbon utilization in Metarhizium acridum.

Carbon sources and their utilization are vital for fungal growth and development. C4-dicarboxylic acids are important carbon and energy sources that function as intermediate products of the tricarboxylic acid cycle. Transport and regulation of C4-dicarboxylic acid uptake are mainly dependent on tetracarboxylic acid transporters (Dcts) in many microbes, although the roles of Dct genes in fungi have only been partially characterized. Here, we report on the functions of two Dct genes (Dct1 and Dct2) in the entomopathogenic fungus Metarhizium acridum. Our data showed that loss of the MaDct1 gene affected utilization of tetracarboxylic acids and other carbon sources. ΔMaDct1 mutants showed larger colony sizes with extensive mycelial growth but were delayed in conidiation with decreased conidia yield as compared to the wild-type parental strain. On the nutrient-deficient medium, SYA, the wild-type strain produced microcycle conidia, whereas the ΔMaDct1 mutant produced (normal) aerial conidia. In addition, ΔMaDct1 had decreased tolerance to cell wall perturbing agents, but increased tolerances to UV-B radiation and osmotic stress. Insect bioassays indicated that loss of MaDct1 did not affect pathogenicity. In contrast, no distinct phenotypic change was observed for the MaDct2 mutant in terms of growth and biocontrol characteristics. Transcriptomic profiling between wild type and ΔMaDct1 showed that differentially expressed genes were enriched in carbohydrate and amino acid metabolism, transport and catabolism, and signal transduction. These results demonstrate that MaDct1 regulates the conidiation pattern shift and mycelial growth by affecting utilization of carbon sources. These findings are helpful for better understanding the effect of intermediates of carbon metabolism on fungal growth and conidiation. KEY POINTS: • MaDct1 influences fungal growth and conidiation by affecting carbon source utilization. • MaDct1 regulates conidiation pattern shift under nutrient deficiency condition. • MaDct1 is involved in stress tolerance and has no effect on virulence. • MaDct2 has no effect on growth and biocontrol characteristic.

Kinase POGSK-3ß modulates fungal plant polysaccharide-degrading enzyme production and development.

The filamentous fungus Penicillium oxalicum secretes integrative plant polysaccharide-degrading enzymes (PPDEs) applicable to biotechnology. Glycogen synthase kinase-3ß (GSK-3ß) mediates various cellular processes in eukaryotic cells, but the regulatory mechanisms of PPDE biosynthesis in filamentous fungi remain poorly understood. In this study, POGSK-3ß (POX_c04478), a homolog of GSK-3ß in P. oxalicum, was characterised using biochemical, microbiological and omics approaches. Knockdown of POGSK-3ß in P. oxalicum using a copper-responsive promoter replacement system led to 53.5 - 63.6%, 79.0 - 92.8% and 76.8 - 94.7% decreases in the production of filter paper cellulase, soluble starch-degrading enzyme and raw starch-degrading enzyme, respectively, compared with the parental strain ΔKu70. POGSK-3ß promoted mycelial growth and conidiation. Transcriptomic profiling and real-time quantitative reverse transcription PCR analyses revealed that POGSK-3ß dynamically regulated the expression of genes encoding major PPDEs, as well as fungal development-associated genes. The results broadened our understanding of the regulatory functions of GKS-3ß and provided a promising target for genetic engineering to improve PPDE production in filamentous fungi. KEY POINTS: • The roles of glycogen synthase kinase-3ß were investigated in P. oxalicum. • POGSK-3ß regulated PPDE production, mycelial growth and conidiation. • POGSK-3ß controlled the expression of major PPDE genes and regulatory genes.

The Role of Fatty Acids from Plant Surfaces in the Infectivity of Colletotrichum fioriniae.

Aqueous extracts derived from flowers stimulate germination, secondary conidiation, and appressorial formation of various latent fruit rotting fungi. Even raindrops passing over flowers accumulate sufficient activity to influence the infectivity of fruit rotting fungi. Using a spore germination bioassay, high levels of bioactivity were found in chloroform extracts from plant tissues, implicating the nonpolar components of the cuticle. The fatty acid (FA) and fatty acid methyl ester (FAME) composition (C9-C20) of blueberry and cranberry tissues as well as aqueous flower extracts were characterized using a gas chromatography-mass spectrometry (GC-MS) method. The FAs and FAMEs found in the plant extracts were then tested for bioactivity using a spore germination bioassay. The C16:0 and C18:2 FAs and FAMEs, as well as the C18:0 FAME and the C20:0 FA, all stimulated appressorial formation while the C10:0 FA stimulated secondary conidiation. The C10:0 and C16:0 FAs were the only two bioactive components also identified from the aqueous floral extracts of both blueberry and cranberry and are therefore considered as contributors to the bioactivity observed in these extracts. The aqueous extracts from surfaces other than flowers showed little or no activity, and it is speculated that the movement of FAs may be related to the level of polymerization and cutin polyester development in flowers versus other plant organs. This study highlights the importance of the bloom period for infection and that the apparent effects on host susceptibility may therefore depend on the availability of specific FAs or combinations thereof.

MrCreC, a carbon catabolite repression gene, is required for the growth, conidiation, stress tolerance and virulence of Metarhizium robertsii.

As a key component of carbon source metabolism in fungi, CreC WD40 repeat protein is regulated by carbon catabolite repression (CCR). However, the understanding of the functions of CreC in entomopathogenic fungi is currently limited. Here, CreC in Metarhizium robertsii (MrCreC) was identified, and its roles in fungal development, conidiation, environmental stress response, and insecticidal virulence were explored. MrCreC is localized to cytoplasm, and MrCreC deletion affects fungal growth on various nutrients. Compared to the wild type, the sporulation of ΔMrCreC strain was significantly decreased by 60.3%. Further qPCR analysis found that deletion of MrCreC resulted in repression of sporulation-related genes such as AbaA, FlbA, Flbc, MedA, FlbD, FluG, and wetA. In addition, MrCreC loss did not alter heat stress tolerance but resulted in enhanced tolerance to UV-B. Interestingly, bioassays showed that the virulence following exposures to topical applications or injection of conidial suspensions of both infection and injection was impaired compared with that of the wild type. Further analysis showed that the adhesion and cuticle penetration genes in ΔMrCreC was down-regulated during infection, and the appressorial formation rate was significantly reduced. A deletion of MrCreC significantly also reduced immune escape and nutrient utilization genes in insect hemocoel. In conclusion, MrCreC is involved in the growth, development and virulence of M. robertsii. These findings advance our understanding of the function of CCR pathway-related genes.

VmMon1-Ccz1 Complex Is Required for Conidiation, Autophagy, and Virulence in Valsa mali.

Apple Valsa canker caused by Valsa mali is a serious disease in eastern Asia, especially in China. In our previous proteomics study, monensin sensitivity 1 protein in Valsa mali (VmMon1) was identified to be significantly upregulated during V. mali infection. It was reported Mon1 protein formed a heterodimer called MC (Mon1-Ccz1) complex with caffeine, calcium, and zinc sensitivity 1 protein (Ccz1) in yeast. However, Ccz1 had not been identified in plant-pathogenic fungi such as Fusarium graminearum and Magnaporthe oryzae. Here, we identified a Ccz1 ortholog VmCcz1 in V. mali, by using DELTA-BLAST. The interaction of VmMon1 and VmCcz1 were verified using yeast two-hybrid assay, bimolecular fluorescence complementation, and co-immunoprecipitation assays. Further yeast three-hybrid screenings determined that VmRab7 (Ras-related protein in V. mali) interacted with the MC complex. Targeted gene deletion showed that the ∆VmMon1 and ∆VmCcz1 mutants were defective in vegetative growth, conidiation, and pathogenicity. In addition, both mutants were more sensitive to osmotic and oxidative stresses and intracellular protein transport inhibitors. Cytological examination revealed that the ∆VmMon1 and ∆VmCcz1 mutants were impaired in vacuole fusion and autophagy. More importantly, expression of pectinase genes decreased in both mutants compared with those of the wild type during infection. Overall, our study identified Mon1 and Ccz1 genes in V. mali and provided evidence that VmMon1 and VmCcz1 are critical components that modulate vacuole fusion and autophagy, thereby affecting the development, conidiation, and pathogenicity of V. mali. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Dysfunction of Ras-GAP protein AfgapA contributes to hypoxia fitness in Aspergillus fumigatus.

The filamentous fungus Aspergillus fumigatus is the most important pathogenic fungus among Aspergillus species associated with aspergillosis. A. fumigatus must adapt to hypoxic microenvironments to survive and thrive in human lungs. To gain further insights into hypoxic adaptation, we generated a laboratory-evolved strain (Afs35-G20) harboring hypoxia fitness, and identified a nonsense mutation in AfgapA encoding a Ras-GAP protein, which could result in the deletion of 22 amino acids at the C-terminus. We investigated the role of AfgapA in hypoxia fitness by constructing Afs35-G20-AfgapAWT, and ∆AfgapA. Indeed, the hypoxia fitness of Afs35-G20 was reversed by introducing AfgapAWT. ∆AfgapA exhibited greater hypoxia fitness and hypervirulence in the silkworm infection model, indicating that AfgapA is responsible for hypoxia fitness, particularly in liquid cultures. Taken together, the AfgapA dysfunction may lead to the downregulation of its Ras substrate(s), reflecting several phenotypes such as increased hypoxia fitness, hypervirulence, poor conidiation, and conidial pigmentation. Here, we report the function of a Ras-GAP protein AfgapA in A. fumigatus for the first time.