The MAP kinase Bbslt2 controls growth, conidiation, cell wall integrity, and virulence in the insect pathogenic fungus Beauveria bassiana

Entomopathogenic fungi, such as Beauveria bassiana, are key environmental pathogens of insects that have been exploited for biological control of insect pests. Mitogen-activated protein (MAP) kinases play crucial roles in regulating fungal development, growth, and pathogenicity, mediating responses to the environment. Bbslt2, encoding for an Slt2 family MAPK, was isolated and characterized from B. bassiana. Gene disruption of Bbslt2 affected growth, caused a significant reduction in conidial production and viability, and increased sensitivity to Congo Red and fungal cell wall degrading enzymes. ΔBbslt2 mutants were altered in cell wall structure and composition, which included temperature dependent chitin accumulation, reductions in conidial and hyphal hydrophobicity, and alterations in cell surface carbohydrate epitopes. The ΔBbslt2 strain also showed hypersensitivity to heat shock and altered trehalose accumulation, which could only be partially attributed to changes in the expression of trehalase (ntl1). Insect bioassays revealed decreased virulence in the ΔBbslt2 strain using both topical and intrahemoceol injection assays. These results indicate that Bbslt2 plays an important role in conidiation, viability, cell wall integrity and virulence in B. bassiana. Our findings are discussed within the context of the two previous MAP kinases characterized from B. bassiana.     

Functional analysis of the superoxide dismutase family in Aspergillus fumigatus

Reactive oxidant species produced by phagocytes have been reported as being involved in the killing of Aspergillus fumigatus. Fungal superoxide dismutases (SODs) that detoxify superoxide anions could be putative virulence factors for this opportunistic pathogen. Four genes encoding putative Sods have been identified in the A. fumigatus genome: a cytoplasmic Cu/ZnSOD (AfSod1p), a mitochondrial MnSOD (AfSod2p), a cytoplasmic MnSOD (AfSod3p) and AfSod4 displaying a MnSOD C‐terminal domain. During growth, AfSOD1 and AfSOD2 were highly expressed in conidia whereas AfSOD3 was only strongly expressed in mycelium. AfSOD4 was weakly expressed compared with other SODs. The deletion of AfSOD4 was lethal. Δsod1 and Δsod2 mutants showed a growth inhibition at high temperature and a hypersensitivity to menadione whereas the sod3 mutant had only a slight growth delay at high temperature. Multiple mutations had only an additive effect on the phenotype. The triple sod1/sod2/sod3 mutant was characterized by a delay in conidial germination, a reduced conidial survival during storage overtime, the highest sensitivity to menadione and an increased sensitivity to killing by alveolar macrophage of immunocompetent mice. In spite of these phenotypes, no significant virulence difference was observed between the triple mutant and parental strain in experimental murine aspergillosis models in immunocompromised animals.     

Additive roles of two TPS genes in trehalose synthesis,conidiation, multiple stress responses and host infection of a fungal insect pathogen

Intracellular trehalose accumulation is relevant to fungal life and pathogenicity. Trehalose-6-phosphate synthase (TPS) is known to control the first step of trehalose synthesis, but functions of multiple TPS genes in some filamentous fungi are variable. Here, we examined the functions of two TPS genes (tpsA and tpsB) in Beauveria bassiana, a fungal insect pathogen widely applied in arthropod pest control. Intracellular TPS activity and trehalose content decreased by 71–75 and 72–80% in ΔtpsA, and 21–30 and 15–45% in ΔtpsB, respectively, and to undetectable levels in ΔtpsAΔtpsB, under normal and stressful conditions. The three mutants lost 33, 50, and 98% of conidiation capacity in standard cultures. Conidial quality indicated by viability, density, intracellular trehalose content, cell wall integrity, and hydrophobicity was more impaired in ΔtpsA than in ΔtpsB and mostly in ΔtpsAΔtpsB, which was also most sensitive to nutritional, chemical, and environmental stresses and least virulent to Galleria mellonella larvae. Almost all of phenotypic defects in ΔtpsAΔtpsB approached to the sums of those observed in ΔtpsA and ΔtpsB and were restored by targeted gene complementation. Altogether, TpsA and TpsB play complementary roles in sustaining trehalose synthesis, conidiation capacity, conidial quality, multiple stress tolerance, and virulence, highlighting a significance of both for the fungal adaptation to environment and host.

     

Contribution of the gas1 gene of the entomopathogenic fungus Beauveria bassiana, encoding a putative glycosylphosphatidylinositol-anchored beta-1,3-glucanosyltransferase, to conidial thermotolerance and virulence

Beauveria bassiana is a mycoinsecticide alternative to chemicals for use in biological pest control. The fungus-insect interaction is also an emerging model system to examine unique aspects of the development, pathogenesis, and diversity of fungal lifestyles. The glycoside hydrolase 72 (GH72) family includes β-1,3-glucanosyltransferases that are glycosylphosphatidylinositol (GPI)-anchored cell wall-modeling enzymes affecting fungal physiology. A putative B. bassiana GPI-anchored β-1,3-glucanosyltransferase (Bbgas1) was isolated and characterized. B. bassiana targeted gene knockouts lacking Bbgas1 were affected in Congo red and salt sensitivity but displayed minor growth defects in the presence of sorbitol, SDS, or calcofluor white. Lectin and antibody mapping of surface carbohydrates revealed increased exposure of carbohydrate epitopes, including β-1,3-glucans, in the ΔBbgas1 strain. Transmission electron micrographs revealed localized destabilization of the cell wall in ΔBbgas1 conidia, in which fraying of the outer cell wall was apparent. Heat shock temperature sensitivity profiling showed that in contrast to the wild-type parent, ΔBbgas1 conidial spores displayed decreased germination after 1 to 4 h of heat shock at temperatures >40°C, and propidium iodide exclusion assays revealed decreased membrane stability in the knockout strain at temperatures >50°C. The ΔBbgas1 knockout showed reduced virulence in Galleria mellonella insect bioassays in both topical and intrahemocoel-injection assays. B. bassiana ΔBbgas1 strains complemented with the complete Bbgas1 open reading frame were indistinguishable from the wild-type parent in all phenotypes examined. The Bbgas1 gene did not complement the phenotype of a Saccharomyces cerevisiae β-1,3-glucanosyltransferase Δgas1 mutant, indicating that this family of enzymes likely possess discrete cellular functions.     

Two phosphodiesterase genes, PDEL and PDEH, regulate development and pathogenicity by modulating intracellular cyclic AMP levels in Magnaporthe oryzae

Cyclic AMP (cAMP) signaling plays an important role in regulating multiple cellular responses, such as growth, morphogenesis, and/or pathogenicity of eukaryotic organisms such as fungi. As a second messenger, cAMP is important in the activation of downstream effector molecules. The balance of intracellular cAMP levels depends on biosynthesis by adenylyl cyclases (ACs) and hydrolysis by cAMP phosphodiesterases (PDEases). The rice blast fungus Magnaporthe oryzae contains a high-affinity (PdeH/Pde2) and a low-affinity (PdeL/Pde1) PDEases, and a previous study showed that PdeH has a major role in asexual differentiation and pathogenicity. Here, we show that PdeL is required for asexual development and conidial morphology, and it also plays a minor role in regulating cAMP signaling. This is in contrast to PdeH whose mutation resulted in major defects in conidial morphology, cell wall integrity, and surface hydrophobicity, as well as a significant reduction in pathogenicity. Consistent with both PdeH and PdeL functioning in cAMP signaling, disruption of PDEH only partially rescued the mutant phenotype of ΔmagB and Δpka1. Further studies suggest that PdeH might function through a feedback mechanism to regulate the expression of pathogenicity factor Mpg1 during surface hydrophobicity and pathogenic development. Moreover, microarray data revealed new insights into the underlying cAMP regulatory mechanisms that may help to identify potential pathogenicity factors for the development of new disease management strategies.     

Protein kinase FgSch9 serves as a mediator of the target of rapamycin and high osmolarity glycerol pathways and regulates multiple stress responses and secondary metabolism in Fusarium graminearum

Saccharomyces cerevisiae protein kinase Sch9 is one of the downstream effectors of the target of rapamycin (TOR) complex 1 and plays multiple roles in stress resistance, longevity and nutrient sensing. However, the functions of Sch9 orthologs in filamentous fungi, particularly in pathogenic species, have not been characterized to date. Here, we investigated biological and genetic functions of FgSch9 in Fusarium graminearum. The FgSCH9 deletion mutant (ΔFgSch9) was defective in aerial hyphal growth, hyphal branching and conidial germination. The mutant exhibited increased sensitivity to osmotic and oxidative stresses, cell wall‐damaging agents, and to rapamycin, while showing increased thermal tolerance. We identified FgMaf1 as one of the FgSch9‐interacting proteins that plays an important role in regulating mycotoxin biosynthesis and virulence of F. graminearum. Co‐immunoprecipitation and affinity capture‐mass spectrometry assays showed that FgSch9 also interacts with FgTor and FgHog1. More importantly, both ΔFgSch9 and FgHog1 null mutant (ΔFgHog1) exhibited increased sensitivity to osmotic and oxidative stresses. This defect was more severe in the FgSch9/FgHog1 double mutant. Taken together, we propose that FgSch9 serves as a mediator of the TOR and high osmolarity glycerol pathways, and regulates vegetative differentiation, multiple stress responses and secondary metabolism in F. graminearum.     

Oxidative stress response in eukaryotes: effect of glutathione, superoxide dismutase and catalase on adaptation to peroxide and menadione stresses in Saccharomyces cerevisiae

Aiming to clarify the mechanisms by which eukaryotes acquire tolerance to oxidative stress, adaptive and cross-protection responses to oxidants were investigated in Saccharomyces cerevisiae. Cells treated with sub-lethal concentrations of menadione (a source of superoxide anions) exhibited cross-protection against lethal doses of peroxide; however, cells treated with H2O2 did not acquire tolerance to a menadione stress, indicating that menadione response encompasses H2O2 adaptation. Although, deficiency in cytoplasmic superoxide dismutase (Sod1) had not interfered with response to superoxide, cells deficient in glutathione (GSH) synthesis were not able to acquire tolerance to H2O2 when pretreated with menadione. These results suggest that GSH is an inducible part of the superoxide adaptive stress response, which correlates with a decrease in the levels of intracellular oxidation. On the other hand, neither the deficiency of Sod1 nor in GSH impaired the process of acquisition of tolerance to H2O2 achieved by a mild pretreatment with peroxide. Using a strain deficient in the cytosolic catalase, we were able to conclude that the reduction in lipid peroxidation levels produced by the adaptive treatment with H2O2 was dependent on this enzyme. Corroborating these results, the pretreatment with low concentrations of H2O2 promoted an increase in catalase activity.     

Involvement of a putative response regulator FgRrg-1 in osmotic stress response, fungicide resistance and virulence in Fusarium graminearum

Response regulator (RR) proteins are core elements of the high-osmolarity glycerol (HOG) pathway, which plays an important role in the adaptation of fungi to a variety of environmental stresses. In this study, we constructed deletion mutants of two putative RR genes, FgRRG-1 and FgRRG-2, which are orthologues of Neurospora crassa RRG-1 and RRG-2, respectively. The FgRRG-1 deletion mutant (ΔFgRrg1-6) showed increased sensitivity to osmotic stress mediated by NaCl, KCl, sorbitol or glucose, and to metal cations Li(+) , Ca(2+) and Mg(2+) . The mutant, however, was more resistant than the parent isolate to dicarboximide and phenylpyrrole fungicides. Inoculation tests showed that the mutant exhibited decreased virulence on wheat heads. Quantitative real-time polymerase chain reaction assays indicated that the expression of FgOS-2, the putative downstream gene of FgRRG-1, was decreased significantly in ΔFgRrg1-6. All of the defects were restored by genetic complementation of ΔFgRrg1-6 with the wild-type FgRRG-1 gene. Different from the FgRRG-1 deletion mutant, FgRRG-2 deletion mutants were morphologically indistinguishable from the wild-type progenitor in virulence and in sensitivity to the dicarboximide fungicide iprodione and osmotic stresses. These results indicate that the RR FgRrg-1 of F. graminearum is involved in the osmotic stress response, pathogenicity and sensitivity to dicarboximide and phenylpyrrole fungicides and metal cations.