Juan-Juan Wang

Three α-1,2-mannosyltransferases contribute differentially to conidiation, cell wall integrity, multistress tolerance and virulence of Beauveria bassiana

Members of α-1,2-mannosyltransferase (Ktr) family are required for protein O-mannosylation for the elongation of Ser/Thr mannose residues in yeasts but functionally unknown in most filamentous fungi. Here we characterized the functions of the Ktr orthologues Ktr1, Ktr4 and Kre2/Mnt1 in Beauveria bassiana, a filamentous enotmopathogen, and found that they were positive, but differential, mediators of many biological traits. Inactivation of Ktr4 and Kre2 resulted in 92% reduction of conidial yield on a standard medium and growth defects on substrates with altered carbon or nitrogen sources and availability, accompanied with reduced conidial size and complexity. This contrasts to the dispensability of Ktr1 for fungal growth and conidiation. More cell wall damage occurred in Δktr4 and Δkre2 than in Δktr1, including altered contents of the cell wall components mannoproteins, α-glucans and chitin, more carbohydrate epitopes changed on conidial surfaces, much lower conidial hydrophobicity, and thinner cell walls. Consequently, Δktr4 and Δkre2 became more sensitive to oxidation and cell wall perturbation than Δktr1 during colony growth or conidial germination despite less difference in their sensitivities to two osmotic agents. Conidial thermotolerance, UV-B resistance and virulence were all lowered greatly in Δktr4 and Δkre2 but only the thermotolerance decreased in Δktr1. All the phenotypical changes were well restored to wild-type levels by the complementation of each target gene. Our results indicate that Ktr4 and Kre2 contribute more to the biocontrol potential of B. bassiana than Ktr1 although all of them are significant contributors.

Unveiling equal importance of two 14-3-3 proteins for morphogenesis, conidiation, stress tolerance and virulence of an insect pathogen.

Two conserved 14-3-3 proteins orthologous to Saccharomyces cerevisiae Bmh1/2 are poorly understood in filamentous fungi. Here we show that Bmh1 and Bmh2 contribute equally to the fundamental biology and physiology of Beauveria bassiana by targeting many sets of proteins/enzymes. Single Bmh deletion caused similar upregulation of another. Excellent knockdown (∼91%) expressions of Bmh1 in ΔBmh2 and Bmh2 in ΔBmh1 resulted in equally more severe multiphenotypic defects than the single deletions, including G2 /M transition, blastospore size, carbon/nitrogen utilization, conidiation, germination and conidial tolerances to high osmolarity, oxidation, cell wall stress, high temperature and UV-B irradiation. All the deletion and deletion/knockdown mutants showed similar defects in blastospore yield and density, hyphal septation and cell size, hyphal responses to most chemical stresses and virulence. All the defects were evident with altered transcripts of phenotype-related genes and well restored by each Bmh complementation. Our Bmh1- and Bmh2-specific transcriptomes generated under osmotic and oxidative stresses revealed up to 6% genes differentially expressed by at least twofold in the fungal genome. Many of those were greatly depressed or co-depressed in ΔBmh1 and ΔBmh2. Our findings provide a thorough insight into the functions and complementary effects of the two 14-3-3 proteins in the filamentous entomopathogen.

Wee1 and Cdc25 control morphogenesis, virulence and multistress tolerance of Beauveria bassiana by balancing cell cycle‐required cyclin‐dependent kinase 1 activity

Modification of cell cycle in entomopathogenic fungi is likely crucial for host infection and environmental adaptation. Here we show that Wee1 and Cdc25 can balance cell cycle-required cyclin-dependent kinase 1 (Cdk1) activity in Beauveria bassiana. The Cdk1 phosporylation signal was strong in Δcdc25 but very weak in Δwee1 and absent in Δwee1Δcdc25. Consequently, cell cycles, septation patterns and many septation-dependent gene transcripts of these mutants were reversely changed. Hyphal cells were short in Δwee1, slender in Δcdc25 and short and swollen in Δwee1Δcdc25. Conidiation was most defective in Δwee1, followed by Δcdc25. Their conidia and yeast-like blastospores also altered antagonistically in both size and complexity, accompanied with abnormally branched germlings in Δwee1 and Δwee1Δcdc25. Conidial thermotolerance and UV-B resistance decreased much more in Δwee1Δcdc25 than in Δwee1 but significantly increased in Δcdc25. The double deletion and the point mutation Cdk1(T14A/P15F) for inhibitory phosphorylation caused most defective virulence, followed by wee1 deletion. All the changes were restored by ectopic gene complementation. Virulence changes in all the mutants and control strains were highly correlated to those in blastospore size or complexity. Taken together, Wee1 and Cdc25 control cell cycle, morphogenesis, asexual development, stress tolerance and virulence of B. bassiana by balancing the Cdk1 activity.

Phytochrome controls conidiation in response to red/far‐red light and daylight length and regulates multistress tolerance in Beauveria bassiana

Phytochromes (Phy) in filamentous fungi are Group VIII histidine kinases that share a unique N-terminal photosensory core, but their functions are largely unknown. Here we show that Beauveria bassiana Phy (Bbphy) is functionally vital for growth, conidiation and multistress tolerance of the fungal entomopathogen lacking sexual stage. Colony growth of ΔBbphy was significantly slower in a nutrition-rich medium but faster in several minimal media. Conidial yield of ΔBbphy in the rich medium increased at the fitted rate of 3.4 × 10(7) conidia h(-1) white light in the light/dark cycles of 0:24 to 16:8 h, decreased greatly in the short-, long- and full-day cycles of red/far-red light, but was unaffected under full-day blue light. Moreover, ΔBbphy showed higher osmosensitivity, increased antioxidant capability, and decreased conidial thermotolerance and UV-B resistance, accompanied with downregulation of Hog1 phosphorylation and of four Hog1-related genes under osmotic stress, and upregulation of five superoxide dismutases and four catalases under oxidative stress. All the changes were restored by the gene complementation. Taken together, Bbphy controls conidiation by responding to daylight length and red/far-red light and regulates multistress responses perhaps because of an involvement in Hog1 pathway. Our findings highlight diverse functions of Bbphy in B. bassiana.

Transcriptional control of fungal cell cycle and cellular events by Fkh2, a forkhead transcription factor in an insect pathogen

Transcriptional control of the cell cycle by forkhead (Fkh) transcription factors is likely associated with fungal adaptation to host and environment. Here we show that Fkh2, an ortholog of yeast Fkh1/2, orchestrates cell cycle and many cellular events of Beauveria bassiana, a filamentous fungal insect pathogen. Deletion of Fkh2 in B. bassiana resulted in dramatic down-regulation of the cyclin-B gene cluster and hence altered cell cycle (longer G2/M and S, but shorter G0/G1, phases) in unicellular blastospores. Consequently, ΔFkh2 produced twice as many, but smaller, blastospores than wild-type under submerged conditions, and formed denser septa and shorter/broader cells in aberrantly branched hyphae. In these hyphae, clustered genes required for septation and conidiation were remarkedly up-regulated, followed by higher yield and slower germination of aerial conidia. Moreover, ΔFkh2 displayed attenuated virulence and decreased tolerance to chemical and environmental stresses, accompanied with altered transcripts and activities of phenotype-influencing proteins or enzymes. All the changes in ΔFkh2 were restored by Fkh2 complementation. All together, Fkh2-dependent transcriptional control is vital for the adaptation of B. bassiana to diverse habitats of host insects and hence contributes to its biological control potential against arthropod pests.

The connection of protein O-mannosyltransferase family to the biocontrol potential of Beauveria bassiana, a fungal entomopathogen

O-Mannosylation dependent on the protein O-mannosyltransferase (Pmt) family is an essential post-translational modification process in eukaryotes, but their connection to the biocontrol potential of a filamentous entomopathogen against arthropod pests is not understood. Here, we characterized the functions of three Pmt orthologues (Pmt1, Pmt2 and Pmt4) in the Pmt family of Beauveria bassiana and found that they were positive, but differential, regulators of the fungal growth, conidiation, multi-stress tolerance and virulence. Three Pmt2 knockdown mutants (ΔPmt2 was lethal), ΔPmt1 and ΔPmt4 grew 20-79% slower on nutrition-rich and limited media. Their conidial yields on a standard medium were reduced by 17-62%, accompanied with delayed germination. All the mutants became significantly less tolerant to most stresses of cell wall perturbation, high osmolarity, oxidation, wet heat and UV-B irradiation during colony growth and conidial germination and lost virulence by 53-62% via cuticle infection, although their virulence via hemocoel injection was not affected. Strikingly, these phenotypic defects were accompanied with remarkable cell wall damage, including thinner cell wall, lower conidial hydrophobicity and altered cell wall composition. All the changes were well restored to wild-type levels by targeted Pmt1 or Pmt4 complementation. Our results indicate for the first time that Pmt1, Pmt2 and Pmt4 are all required for the full biocontrol potential of B. bassiana despite differential contributions.

Gcn5-dependent histone H3 acetylation and gene activity is required for the asexual development and virulence of Beauveria bassiana : Gcn5 required for B. bassiana

Gcn5 is a core histone acetyltransferase that catalyzes histone H3 acetylation on N-terminal lysine residues in yeasts and was reported to catalyze H3K9/K14 acetylation required for activating asexual development in Aspergillus. Here, we report a localization of Gcn5 ortholog in the nucleus and cytoplasm of Beauveria bassiana, a fungal insect pathogen. Deletion of gcn5 led to hypoacetylated H3 at K9/14/18/27 and 97% reduction in conidiation capacity as well as severe defects in colony growth and conidial thermotolerance. Two master conidiation genes, namely brlA and abaA, were transcriptionally repressed to undetectable level in Δgcn5, but sharply upregulated in wild-type, at the beginning time of conidiation. Based on chromatin immunoprecipitation, both DNA and acetylation levels of the distal and proximal fragments of the brlA promoter bound by acetylated H3K14 alone were upregulated in wild-type, but not in Δgcn5, at the mentioned time. In Δgcn5, normal cuticle infection was abolished while virulence through cuticle-bypassing infection was greatly attenuated, accompanied by drastically reduced activities of putative cuticle-degrading enzymes, retarded dimorphic transition and transcriptional repression of associated genes. These findings unveil a novel mechanism by which Gcn5 activates asexual development pathway by acetylating H3K14 and regulates the virulence-related cellular events in B. bassiana.

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.

Rtt109-dependent histone H3 K56 acetylation and gene activity are essential for the biological control potential of Beauveria bassiana : Role of Rtt109 in sustaining pest control potential of Beauveria bassiana

BACKGROUND Rtt109 is a histone acetyltransferase that catalyzes histone H3K56 acetylation required for genomic stability, DNA damage repair and virulence-related gene activity in yeast-like human pathogens but remains functionally unknown in fungal insect pathogens. This study seeks to elucidate the catalytic activity of a Rtt109 orthologue and its possible role in sustaining the biological control potential of Beauveria bassiana, a fungal entomopathogen. RESULTS Deletion of rtt109 in B. bassiana abolished histone H3K56 acetylation and triggered histone H2A-S129 phosphorylation. Consequently, the deletion mutant showed increased sensitivity to the stresses of DNA damage, oxidation, cell wall perturbation, high osmolarity and heat shock during colony growth, severe conidiation defects under normal culture conditions, reduced conidial hydrophobicity, decreased conidial UV-B resistance, and attenuated virulence through normal cuticle infection. These phenotypic changes correlated well with reduced transcript levels of many genes that encode the families of H2A-S129 dephosphorylation-related protein phosphatases, DNA damage-repairing factors, antioxidant enzymes, heat-shock proteins, key developmental activators, hydrophobins and cuticle-degrading Pr1 proteases respectively. CONCLUSION Rtt109 can acetylate H3K56 and dephosphorylate H2A-S129 in direct and indirect ways respectively, and hence has an essential role in sustaining the genomic stability and global gene activity required for conidiation capacity, environmental fitness and pest control potential in B. bassiana.