Transcriptomic investigation reveals a physiological mechanism for Beauveria bassiana to survive under linoleic acid stress

Jia Hou; Hao Zhang; Jin-Li Ding; Ming-Guang Feng; Sheng-Hua Ying

doi:10.1016/j.isci.2023.106551

. 2023 Apr 1;26(4):106551. doi: 10.1016/j.isci.2023.106551

Transcriptomic investigation reveals a physiological mechanism for Beauveria bassiana to survive under linoleic acid stress

Jia Hou ¹, Hao Zhang ¹, Jin-Li Ding ¹, Ming-Guang Feng ¹, Sheng-Hua Ying ^1,^2,^∗

PMCID: PMC10123340 PMID: 37102147

Summary

In integrated pest management program (IPM), the compatibility of mycoinsecticides with bioactive fungicides [e.g., unsaturated fatty acids (UFAs)] has attracted more and more attention; however, the mechanisms underlying fungal resistance to UFAs remain largely unknown. In this study, Beauveria bassiana, an entomopathogenic fungus, was used to explore fungal responses to linoleic acid (LA). Genome-wide expression revealed the transcriptomic responses of fungal cells to LA in a stress-intensity-dependent manner. Enrichment analyses indicated that the up-regulated differentially expressed genes (DEGs) are associated with the metabolism of lipid and fatty acids. Notably, a lipid-droplet protein (BbLar1) maintains the intracellular homeostasis of fatty acids and is crucial to fungal tolerance to LA stress, which significantly contributes to fungal compatibility with UFAs. Additionally, BbLar1 links the lipid droplets to global expression profiles in B. bassiana under LA stress. Our investigations provide an initial framework for improving the efficacy of insect pathogenic fungi in practical application.

Subject areas: Molecular microbiology, Interaction of plants with organisms, Transcriptomics

Graphical abstract

Highlights

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Linoleic acid induces a comprehensive transcriptome in Beauveria bassiana
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BbLar1 is associated with lipid droplets
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BbLar1 contributes to fungal tolerance to linoleic acid
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BbLar1 links lipid droplet to fungal transcriptome under linoleic acid stress

Molecular microbiology; Interaction of insects with organisms; Transcriptomics

Introduction

Fungal entomopathogens (e.g., B. bassiana and Metarhizium anisopliae) are natural enemies of arthropods in ecosystems and have been widely developed into mycoinsecticides for the biological control of insect pests.¹ Under natural conditions, the infectious propagules of entomopathogenic fungi penetrate through the host cuticle and propagate in the host hemoceol via dimorphic transition from mycelia into in vivo hyphal bodies. Ultimately, the pathogenic fungus kills the hosts and generates numerous conidia on cadavers for the initiation of the follow-up infection cycles.² Insect hosts accumulate a plethora of fatty acids in cuticle and hemolymph which play important roles in inhibiting and killing fungal pathogens.³^,⁴ In integrated pest management (IPM) program, the compatibility of mycoinsecticides with fungicides has attracted more and more attention.⁵^,⁶ Unsaturated FAs (UFA) and their derivatives (e.g., linoleic acid and methyl linoleate) have been considered as bioactive fungicides widely used in control of phytopathogenic fungi (e.g. Fusarium oxysporum and Magnaporthe oryzae).⁷^,⁸^,⁹ Similarly, UFAs inhibit spore germination, hyphal growth, pathogenicity of the entomopathogenic fungus (e.g., B. bassiana and Conidiobolus coronatus).¹⁰^,¹¹ This line of evidences indicates that entomopathogenic fungi must withstand the anti-fungal activities of UFAs in pest management practices, and their resistance to UFAs is essential for the pathogenesis and biocontrol efficacy of mycoinsecticides.

Diverse UFAs have been also widely applied to control human pathogenic fungi (e.g., Candida albicans).¹² In yeast C. albicans, UFAs have multiple effects on fungal physiologies and inhibit biofilm development by influencing the attachment to surface, cell-membrane fluidity, biosynthesis of extracellular polysaccharide, hyphal formation, and quorum sensing system.¹³ However, the exact targets and the mechanisms involved in fungal resistance to UFAs remain largely enigmatic in most fungi. In Saccharomyces cerevisiae, linoleic acid performs cell toxicity by inducing apoptosis and repressing metabolic enzyme activities.¹⁴ The very long-chain fatty acids elongases play important roles in yeast resistance to cytotoxicity induced by oleic acid through balancing the FA composition in biomembranes.¹⁵ The pathways for triacylglycerol and steryl ester synthesis are required for the elimination of lipotoxicity of UFAs (e.g., linoleic acid) in baker’s yeast.¹⁶ There is few report about the mechanism underlying resistance to stress caused by UFAs in filamentous fungi. Our recent investigation has revealed that in addition to its conserved roles in fatty-acid oxidization, peroxisome functions as an important metabolic center for eliminating cellular lipotoxicity caused by UFAs.¹⁷ Although the metabolic pathway has been implicated in fungal tolerance to UFA stress, a more comprehensive genetic network involved in fungal response to lipotoxicity is still lacking in filamentous fungi.

We speculate that entomopathogenic fungi might have evolved a distinct mechanism to overcome lipotoxicity owing to their unique lifecycles involving the fungus-host interaction.² To address these questions, herein, B. bassiana, a well-investigated filamentous entomopathogenic fungus,¹⁸ was used as a representative to decipher the mechanistic insights into the cellular response to lipotoxicity caused by linoleic acid in filamentous fungi. Comparative transcriptomics analyses indicated that linoleic-acid stress performed a comprehensive effect on global expression. Many differentially expressed genes (DEGs) were uncovered to be involved in metabolism, cellular transportation, cell rescue, and so forth. Notably, an up-regulated DEG, BbLAR1 (BBA_01631), encodes a lipid-droplet protein that plays an important in fungal resistance to linoleic-acid toxicity. Comparative transcriptomic analyses between the wild-type and disruptant indicated that during LA stress, Bblar1 mediates transcriptional responses of genes associated with the integral component of the membrane, metabolism of lipid/fatty acid, and so forth. Our work highlights the complexity of transcriptional profiling in fungi under UFA stress and a physiological pathway involved in fungal response to lipotoxicity.

Results

Dose-dependent transcriptomes of fungal responses to linoleic acid stress

To explore global expression profiles of B. bassiana responses to LA, eight cDNA libraries (four concentration × two replicates) were constructed from mycelia stressed by different concentrations [0.005% (LC), 0.05% (MC), 0.1% (HC)] of LA as well as the control (without stress). The data features for each library were summarized in Table S1. All libraries had a Q20 value of >97.7%, and all Q30 values were greater than 92.0%. Among all libraries, the percentage of mapped reads to total reads ranged from 74.84 to 91.67%.

To identify the differentially expressed genes (DEGs) after LA stress, the pairwise comparison was conducted between the stressed sample and control. Relative to control, genes with FDR ≤0.05 and |log₂Ratio| ≥1 were considered as DEGs. Our data indicated that there were 53, 349, and 222 genes that were significantly regulated in B. bassiana at LC, MC, and HC, respectively (Figure 1A, Table S2). A Venn diagram showed that there are 24 DEGs overlapped among three concentrations, whereas 17, 262, and 143 DEGs appeared only at LC, MC, and HC, respectively (Figure 1B).

Summary for the linoleic acid-induced transcriptome in *B. bassiana*

The wild-type strain was cultured in the media included with 0.005, 0.05, and 0.1% linoleic acid (LA), using the media without LA as control. Transcriptome was revealed via RNA-seq, and comparison was paired between the indicated LA concentration and the control.

(A) Gene counts for the up- (UR) and down-regulated (DR) genes at the indicated LA concentration. The number on the column indicates the total counts for differentially expressed genes (DEGs).

(B) Venn diagram showing the relationship among three transcriptomes induced with different concentrations of LA.

Functional enrichment analyses of differentially expressed genes

On basis of GO annotation, DEGs were enriched to different functional catalogs belonging to biological process (BP), cellular component (CC), and molecular function (MF) categories (Table S3). As shown in Figure 2A, the number of enriched DEGs varied dynamically with the increased LA concentrations. The changing trends were similar between BP and MF categories. Up-regulated DEGs were significantly enriched at three concentrations, whereas the down-regulated DEGs were enriched at LC. However, in CC categories, the up- and down-regulated DEGs were consistently enriched at LC, and no significant enrichment was observed at MC and HC. The enriched terms significantly differed among different LA concentrations. For example, in BP, the enriched DEGs at LC were associated with seven categories (e.g., fatty acid metabolic process and protein metabolic process); two categories (oxidation-reduction process and response to stress) were enhanced at MC; also, two categories (oxidation-reduction process and response to stress) were enriched at HC.

Enrichment analyses for differentially expressed genes (DEGs)

Three methods were applied to sort DEGs induced at 0.005, 0.05, and 0.1% LA.

(A) Gene Ontology (GO) annotation. All DEGs were sorted to Biological Process (BP), Molecular Functions (MF), Cellular Components (CC). (A) FunCat analyses. All the DEGs were enriched at different functional categories; however, no category was over-presented in the transcriptome induced by 0.005% LA.

(C) k-means cluster analysis was used to sort DEGs, which deciphering various expression patterns dependent on LA concentrations. Hotmap is used to show the DEGs enhanced by 0.05 and 0.1% LA, in which several DEGs are associated with lipid/fatty acid metabolism.

The FunCat classification system sorted DEGs into different functional categories pathways (Figure 2B, Table S4). At LC, no terms were significantly enriched. At MC, the up-regulated DEGs were significantly enriched in the terms of cellular transportation and cell rescue, whereas the down-regulated DEGs were enriched in the categories of metabolism, cellular transport, and interaction with the environment. The enriched up-regulated DEGs were associated with electron transport and heat shock response. At HC, the up-regulated DEGs were significantly enriched in five terms (e.g., metabolism and energy), and no terms were overrepresented for the down-regulated DEGs. The up-regulated DEGs associated with metabolism included many genes involved in lipid/fatty acid metabolism, oxidation of fatty acid, cheolesterol homeostasis, and so on.

All DEGs were pooled together, and their expression modes were analyzed with k-means clustering method. The results indicated that all DEGs were classified into 10 clusters (Table S5). In cluster 9 (Figure 3A), 44 DEGs were significantly up-regulated at MC and HC, in which some genes were involved in the metabolism of lipid/fatty acid (e.g, fatty acid-binding protein, Cytochrome P450, and caleosin). Notably, a protein (locus tag: BBA_01631) is currently annotated as a Zn(II)₂Cys₆ transcription factor-like protein owing to its low similarity with Zn(II)₂Cys₆ transcription factor in C. albicans, which is named as War1 protein that contributes to resistance to weak acid sorbate¹⁹ (Figure S1). The gene BBA_01631 displayed response to linoleic acid and revised as BbLAR1. BbLar1 has no GAL4-like C6 zinc binuclear cluster DNA-binding domain, which was same in the homologs in the related entomopathogenic fungi (Cordyceps militaris and Metarhizium species). Additionally, the homologs from other filamentous fungi contain the GLA4 domain, and no homolog was characterized in S. cerevisiae. BbLar1 was considered a representative downstream gene influenced by linoleic acid stress, and its physiological role was validated in the subsequent study.

Sub-cellular localization of BbLar1 in *B. bassiana*

The *BbLAR1* was fused to the GFP gene, and the resultant hybrid was transformed into the wild-type strain. Conidial suspension of the transformant was inoculated onto the cellophane on water agar plates. At the indicated time point, fungal cells were stained with Nile red. Fluorescent signals were examined under a confocal laser scanning microscope. Bars: 5 μm.

Localization of BbLar1 in B. bassiana and gene target replacement

As illustrated in Figure 3, sub-cellular localization of BbLar1 in B. bassiana was validated by fusing the coding sequence to the N-terminus of GFP. Nile red was used to indicate lipid droplets by emitting red fluorescence. Globular signals were obviously seen in fungal cells (germlings and mycelia), and the red and green signals were co-localized well. This suggests that BbLar1 is associated with lipid droplets.

To explore the physiological functions of BbLar1 in B. bassiana, gene target disruption was accomplished through homologous recombination (Figure S2A). To rescue the gene loss, the entire BbLAR1 together with its promoter region was transformed into the gene disruption mutant strain by ectopic insertion. All gene disruption and complementation mutant strains were first screened by PCR and further verified with fluorescence reporter assay (Figure S2B).

BbLar1 plays a minor role in fungal growth and development

Vegetative growth rates were examined on different media. Compared with the wild-type strain, no significant difference was observed in the growth rate of ΔBblar1 mutant on most media (Figure 4A). However, a slight reduction (28%) in growth was seen on medium supplemented with olive oil as carbon source. The effects of osmotic (NaCl, sorbitol), oxidative (H₂O₂, menadione), and cell wall (Congo red, SDS) stressors were examined on CZA plates containing the indicated chemical (Figure 4B). No significant difference in colony diameter was observed between the wild-type and gene disruption mutant strains. As for conidiation on the aerial plate (Figure 4C), ΔBblar1 mutant generated 4.79 ± 0.07 × 10⁸ conidia/cm² (mean ± standard deviation (SD)), which was similar to that of the wild-type strain (4.96 ± 0.36 × 10⁸ conidia/cm²). Under submerged condition (Figure 4D), the blastospore yield of ΔBblar1 mutant was significantly reduced, producing only 1.10 ± 0.10 × 10⁸ spores/ml, whereas the wild type produced 1.53 ± 0.09 × 10⁸ spores/ml. These results suggest that BbLar1 slightly contributes to fungal growth and development.

Fungal radial growth and development

Fungal strains were incubated on SDAY plates for 7 days at 25°C, and the resultant conidia were used as initial inocula.

(A) Vegetative growth on various nutrients. Sucrose and NaNO₃ of CZA plates were replaced with carbon (C) and nitrogen (N) sources, respectively. SDAY was used as the control of the rich medium.

(B) Fungal growth under stresses. Various chemicals were included in CZA plate to establish stressful conditions. CZA plate was used as control. SDS: sodium dodecyl sulfate; CFW: calcofluor white. Fungal strains (1 μL, 10⁶ conidia/ml) were inoculated on the plates and cultured at 25°C, and colony diameter was examined at 7 days post-incubation.

(C) Conidial yield. Fungal strains (1 μL, 10⁷ conidia/ml) were smeared on SDAY plates and cultured at 25°C, and conidial quantity in mycelia was shown as 10⁸ conidia per square centimeter.

(D) Blastospore production. Fungal strains were inoculated into SDB (SDAY without agar) and cultured for 3 days at 25°C. Spore yield was calculated as 10⁸ cells per milliliter. The statistical difference was determined with Student’s t test (∗: p < 0.05). Error bars: standard deviation (SD). Data are represented as mean ± SD.

BbLar1 significantly contributes to lipid biology

As the LA concentration increases from 0 to 0.1%, the wild-type strain did not exhibit a significant change in the morphology of lipid droplets. In ΔBblar1 mutant, lipid droplets became significantly enlarged at LA concentrations of 0.05 and 0.1% (Figure 5A). This result indicated that gene loss led to abnormal size of lipid droplet in mycelia at high concentration of LA. Disruption of BbLAR1 resulted in a significant reduction in the content of four fatty acids (FAs) (Figure 5B). The decreases for linoleic (LA) and oleic (OA) acids were approximately 30% and 24%, respectively; whereas the reductions for stearic (SA) and palmitic (PA) acids were approximately 24% and 43%, respectively. Exogenous LA stress did not cause a significant change in the contents of SA, PA, and OA of the wild-type strain, but significantly increased its LA content. As for ΔBblar1 mutant, the contents for OA, SA, and PA were not significantly changed after LA stress; however, the LA stress only resulted in a slight increase in the intracellular level of LA which was only approximately 50% of that in the wild type. These data suggested that the ablation of BbLAR1 significantly reduced the FA content in mycelia; particularly, LA stress significantly exacerbated the intracellular homeostasis of LA.

BbLar1 contributes to fungal lipid biology

(A) Morphologies of lipid droplets. The indicated strain was grown in TPB at 25°C for 2 days, and the resultant culture was stressed with different LA concentrations (0, 0.005, 0.05, and 0.1%) for 1 day. Resultant mycelia were stained with BODIPY493/503, and fluorescence was examined under a laser scanning confocal microscope. Bar: 10 μm.

(B) Fatty-acid (FA) content in mycelia. Conidia were cultured in TPB supplemented with 0.1% linoleic acid (TL) for 2 days at 25°C, using TPB as control. Free FAs in mycelia were quantified and calculated as mg/g biomass. FAs included stearic (SA), palmitic (PA), oleic (OA), and linoleic (LA) acids. The numbers on the column indicate the relative level of LA for Bblar1 versus wild type.

(C) Fungal tolerance to LA stress. LA was included in TPA and CZA plates, and stressful intensity was established by different concentrations of LA. Conidial suspension (1 μL, 10⁶ conidia/ml) was inoculated onto plates and cultured at 25°C, and colony diameter was examined at 7 days post-incubation. The statistical difference between the wild-type and Δ*Bblar1* was determined with Student’s t test (∗: p < 0.05; ∗∗: p < 0.01; ∗∗∗: p < 0.001). Error bars: standard deviation (SD). Data are represented as mean ± SD.

Fungal tolerance to LA stress was examined on two types of media (Figure 5C). On TPA plates, the colony diameter for the wild-type strain reduced when the LA concentrations increased. ΔBblar1 mutant strain exhibited dramatic reduction in colony size when LA concentration increased, and did not generate an obvious colony on the plates with 0.1% LA (Figure S3). Similar varying trends were also observed on CZA plates. Notably, the radial growth of ΔBblar1 was completely inhibited by 0.05% LA. No significant difference in response trend was observed between the wild-type and complementation mutant strains. Thus, BbLar1 is involved in fungal resistance to LA stress, which is influenced by the availability of ambient nutrients.

BbLar1 is essential for biological compatibility with linoleic acid during infecting hosts

Conidial compatibility with LA during attacking hosts was determined with topical infection bioassay (Figure 6A). Without LA, all three strains killed all bioassay insects in eight days, and no significant difference in the host survival was observed between the WT and ΔBblar1 mutant strains (p > 0.05). The survival curve of the ΔBblar1 strain was significantly different with that of the wild-type strain (p < 0.0001) when using 0.1% LA (LA diluted in liquid paraffin) and LA after the topical inoculation of conidia to the insect hosts. Liquid paraffin did not cause any negative effects on the bioassay insects. As for the wild-type strain, 0.1% LA did not result in a significant change in median lethal time (LT₅₀), and LA only resulted in a slight delay in LT₅₀ value (Figure 6B). The treatment of 0.1% LA resulted in a slight delay in LT₅₀ of ΔBblar1 mutant strain, but statistical significance was detected. Without LA treatment, there was no significant difference in LT₅₀ between the wild-type and gene disruption mutant strains; however, both 0.1% LA and LA treatments led to the significant delay in LT₅₀ of ΔBblar1 mutant strain when compared with that of the wild type. These results suggested that the BbLar1 loss impaired fungal entomopathogenicity under LA treatment.

Fungal virulence in the insect model

Fungal strains were incubated on SDAY plates for 7 days at 25°C, and the resultant conidia were used as infectious propagules. In bioassay, the insect hosts were immersed in conidial suspension (10⁷ conidia/ml). After air drying, the inoculated insects were sprayed with 0.1% and pure LA. The group of insects only treated with conidia was used as control (CK). The resultant insects were reared at 25°C, and accumulative mortality was recorded daily.

(A) Survival percentage was plotted against the time post-infection, and statistical difference between the paired curves was analyzed with log rank test.

(B) Median lethal time (LT₅₀) was calculated with Kaplan-Meier analyses. The statistical difference in the paired comparison was determined with Student’s t test (ns: no significance; ∗: p < 0.05; ∗∗∗: p < 0.001; ∗∗∗∗: p < 0.0001).

Global expression changes as a result of BbLar1 loss

In order to explore the BbLar1-mediated global expression profiles under LA stress, two sets of genome-wide expression analysis using high-throughput sequencing (RNA-seq) were performed; i.e. comparative analysis of ΔBblar1 mutant to wild type stressed with 0.05% (set 1) and 0.1% (set 2) LA, respectively. At 0.05% LA (MC), loss of BbLAR1 resulted in expression changes of 3,462 genes, with 2,316 up-regulated (22.3% of the genome) and 1,326 down-regulated (12.8% of the genome) genes in the mutant strain when compared to the wild type. At 0.1% LA (HC), ablation of BbLAR1 caused the expression profiles of 3,307 differentially changed, with 1,978 up-regulated (19.1% of the genome) and 1,329 down-regulated (12.8% of the genome) as compared to the wild type (Figure 7A, Table S6). In addition, 2,009 DEGs were overlapped between two comparison sets (Figure 7B).

RNA-seq for the BbLar1-mediated transcriptome

The wild-type and Bblar1 mutant strains were cultured in the media included with 0.05 and 0.1% linoleic acid (LA). Comparative transcriptomic analyses were conducted between the wild-type and disruption mutant strains at the indicated LA concentration.

(A) Gene counts for the up- (UR) and down-regulated (DR) genes at the indicated LA concentration, and the total number of differentially expressed genes (DEGs) is shown in the column.

(B) Venn diagram showing the relationship between above two sets of BbLar1-mediated DEGSs.

(C) Gene Ontology (GO) annotation and enrichment analyses were used to functionally sort the overlapping DEGs in (B). All DEGs were sorted to Biological Process (BP), Molecular Functions (MF), and Cellular Components (CC), in which gene counts differed with LA concentrations.

Following GO annotation, DEGs were overrepresented in different functional terms included in BP, CC, and MF (Table S7). As shown in Figure 7C, the number of enriched DEGs varied with LA concentrations. In BP, only DEGs at MC were enriched in categories of translation (e.g., a set of ribosomal proteins) and ATP synthesis coupled proton transport (e.g., a set of ATP synthase subunits). In CC, at MC, the up-regulated DEGs were enriched in the categories of ribonucleoprotein complex (e.g., small nuclear ribonucleoprotein Prp4) and ribosome (e.g., a set of ribosomal proteins); whereas the down-regulated DEGs were enriched in the category of integral component of membrane, including a number of transporter genes (e.g., ABC transporter, amino acid permease, and copper transport protein). In MF, the up-regulated DEGs at MC were enriched in the functional term of the structural constituent of ribosome, including a number of ribosomal proteins (e.g., 40S ribosomal protein S11 and mitochondrial ribosomal protein S16), and the up-regulated DEGs at HC were enriched in the functional term of oxidoreductase activity, in which many genes were involved in lipid/fatty acid metabolism (e.g, fatty acid hydroxylase, 3-hydroxyacyl-CoA dehydrogenase, and short chain dehydrogenase). FunCat enrichment analyses indicated only the up-regulated DEGs at MC were overrepresented in the functional category of protein synthesis, including a set of genes crucial for ribosome biogenesis and translation (e.g., ribosomal protein and translation initiation factor), and no other DEGs were sorted in functional terms (Table S8).

The overlapped DEGs between two comparison sets were analyzed with k-means clustering method and classified into 40 clusters (Table S9). Most DEGs repressed at both MC and HC were sorted into eight clusters (e.g., clusters 11 and 12), in which cluster 11 was the biggest group involving 355 DEGs. In this cluster, DEGs were involved in various physiologies in B. bassiana. For instance, some genes were involved in the metabolism of lipid/fatty acid, including fatty acid hydroxylase, lipase, thioesterase, and so on. Some genes were involved in stress response, including heat shock protein 70, 90 and so on. As for organellar components, peroxin 11 and 19 were observed. These results suggested that the BbLar1 loss performed comprehensive influences on the genome-wide expression profiles of B. bassiana. Notably, a DEG (BBA_08327) encoded the proteins contain common in fungal extracellular membrane (CFEM) domain, and another CFEM domain-containing protein gene (BBA_07758) was repressed in ΔBblar1 mutant strain at HC.

Phenotypic evaluation of two common in fungal extracellular membrane domain-containing proteins in fungal tolerance to linoleic acid

The products of two genes (BBA_07758 and BBA_08327) have been designated as BbCfem7 and BbCfem8 in B. bassiana, respectively. BbCfem7 is a membrane-associated protein, and BbCfem8 localizes in cytoplasm.²⁰ In the present study, the localization of BbCfem8 was further determined by expressing the fusion gene BbCFEM8-GFP in the wild-type strain. The lipid droplets in mycelia were indicated by the red fluorescent signals of Nile red. Dual-fluorescence assay showed that most green signals were consistent with red ones, which indicated that BbCfem8 localizes in lipid droplets (Figure S4).

Two mutant strains ΔBbcfem7 and ΔBbcfem8 exhibited similar responses to LA stress (Figure 8). On TPA plates, the colony diameter for the wild-type strain reduced when the LA concentrations increased. Both mutant strains displayed the dramatic reduction in colony size when LA concentration increased, and generated very small colony on the plates with 0.1% LA (Figure S3). On CZA plates, the radial growth of these two disruptants was completely inhibited by 0.05% LA. No significant difference in response trends was observed between the wild-type and complementation mutant strains. Thus, both BbCfem7 and BbCfem8 are involved in fungal tolerance to LA stress, which is also dependent on the nutrient availability in media.

The roles of BbCfem7 and BbCfem8 in fungal tolerance to LA stress

LA was included in TPA and CZA plates with gradient concentrations. Conidia were harvested from fungal culture on the SDAY plate. Conidial suspension (1 μL, 10⁶ conidia/ml) was inoculated onto plates and cultured at 25°C, and colony diameter was examined at 7 days post-incubation. The statistical difference between the wild-type and gene disruption mutant strains was determined with Student’s t test (∗: p < 0.05; ∗∗: p < 0.01; ∗∗∗: p < 0.001). Error bars: standard deviation (SD). Data are represented as mean ± SD.

Discussion

Unsaturated FAs (UFA) (e.g., linoleic acid) have been widely used in control of phytopathogenic fungi.⁹ UFAs inhibit spore germination, hyphal growth, and pathogenicity of the entomopathogenic fungus.¹⁰^,¹¹ Therefore, the efficacy of entomopathogenic fungi would be greatly influenced by UFAs in the integrated management of the phytopathogenic fungi and insect pests. In this study, we revealed a physiological pathway involved in B. bassiana responses to linoleic acid (LA) stress through transcriptomic analyses coupled with functional characterization.

To our knowledge, this study presents the first transcriptomic profile in filamentous fungi under the UFA stress. The current study reiterates that UFA performs inhibitory effects on conidial germination and hyphal growth in B. bassiana,²¹ and LA stress triggers a dynamic transcriptomic response in the stress-intensity-dependent manner. Under mild stress, the enhanced physiological processes are associated with fatty acid metabolic process, oxygen transport, protein metabolic process and so on. In bacteria, LA has inhibitory effects on growth, which resulting in a global metabolic stress response that reprograms the metabolic process.²² These findings suggest metabolic reprogramming is conserved among microorganisms. In addition, LA also enhanced the expression of the genes in respiratory chain. In yeast, the complex III in electron transmission chain (ETC) is crucial to maintain mitochondrial membrane potential and a low rate of ROS production. Excess UFA results in the dysfunction and electron leakage in ETC.²³ In B. bassiana, ETC plays an important role in maintaining mitochondrial functionality, which is critical for fungal stress response, development, and pathogenicity.²⁴ This implies that UFAs have an adverse impact on mitochondrial activity, and B. bassiana activates the transcription of the genes in ETC for compensation. LA stress represses ribosome biogenesis and RNA processing in B. bassiana. In fungi, the up-regulation of ribosomal protein genes is associated with rapid growth under optimal conditions.²⁵ This suggests UFAs inhibit the growth of B. bassiana via reducing protein biogenesis. Under moderate and severe stresses, the enhanced physiological processes are associated with oxidation-reduction process and response to stress. In yeasts, UFAs induce the accumulation of intracellular reactive oxygen species (ROS).²⁶ Undoubtedly, UFA stress activates the mechanisms involved in fungal response to stress. In terms of physiological function, B. bassiana responds to LA stress by adopting the mechanisms mainly involved in metabolism, stress tolerance, and so on. In particular, we focus on the LA-enhanced genes involved in fatty acid metabolism.

Among a group of genes enhanced by moderate and severe stresses, we found a lipid-droplet protein (BbLar1) that contributes to fungal tolerance to LA toxicity in B. bassiana. This protein has been annotated as a Zn(II)2Cys6 transcription factor-like protein owing to its low similarity with a Zn(II)₂Cys₆ transcription factor (War1) in C. albicans.¹⁹ However, BbLar1 has no the GAL4-like C6 zinc binuclear cluster DNA-binding domain and is associated with lipid droplets. Exogenous LA stress increases the intracellular levels of LA. Under stressful conditions, Rhodotorula yeast strains synthesize more UFAs, especially linoleic and linolenic acids.²⁷ Evidently, endogenous LA functions as a general molecule for stress response, which may be attributed to the enhancement of the membrane fluidity.²⁸ BbLar1 maintains the morphologies of lipid droplets and the homeostasis of FAs in B. bassiana. In B. bassiana, perilipin and caleosin contribute to lipid storage and lipid-droplet homeostasis, but play a slight role in fungal virulence.²⁹^,³⁰ This suggests that different proteins in lipid droplets perform diverse functions. Lipid droplets are cytoplasmic organelles that store neutral lipids and play important roles in metabolism, energy supply, stress response, and so on.³¹ In yeast, a very long-chain fatty acid elongase balances the FA composition in biomembranes to relieve the cytotoxicity induced by oleic acid.¹⁵ Therefore, BbLar1 contributes to lipid/fatty acid metabolism in lipid droplets to supply the endogenous FA; particularly LA, which offers an explanation to the resultant growth defect of the gene disruption mutant under the stress caused by exogenous LA. A recent study indicated that the peroxisomal metabolism of FAs is involved in fungal tolerance to exogenous LA.¹⁷ On the contrary, yeast eliminates the lipotoxicity of UFAs through the synthesis of triacylglycerol and steryl esters.¹⁶ These discoveries suggest the pathways for lipid/fatty metabolism are significantly involved in the detoxification of lipotoxicity in fungi. As for philological functions, BbLar1 does not play significant roles in fungal growth and virulence in B. bassiana, but significantly contributes to fungal survival in the ambient environment with UFAs (e.g., using UFAs to control phytopathogenic fungi). Thus, BbLar1 contributes to the compatibility of B. bassiana with UFAs and their derivatives in the practical application. BbLar1 might be used as a molecular marker to evaluate the fungal tolerance to UFA stress and biocontrol potential.

Notably, BbLar1 links the homeostasis of lipid/fatty acid to global expression in B. bassiana under LA stress. In terms of physiological function, BbLar1 mediates a transcriptome mainly involved in protein synthesis, energy generation, integral component of membrane, and so on. Peroxisomes are single membrane-bound organelles that contribute to the assimilation of fatty acids. In Arthrobotrys oligospora (a nematode-trapping fungus), mutation of peroxisome biogenesis proteins results in fungal defects in fatty acid utilization, which ultimately performs the significant influences on fungal transcriptome.³² Apparently, intracellular homeostasis of fatty acids is critical for the global gene expression in fungi. Among the repressed differentially expressed genes, we uncover two genes coding CFEM-domain protein (BbCfem7 and BbCfem8) that contributes to fungal resistance to LA stress. BbCfem7 and BbCfem8 are associated with cytomembranes²⁰ and lipid droplets, respectively. In yeast, plasma membrane acts as a blocking-up mechanism in relieving squalene lipotoxicity.³³ BbCfem7 may be attributed to plasma membrane permeability, which contributes to reducing the influx of LA outside cell. BbCfem8 may have a role similar to that of BbLar1 owing to their association with lipid droplets. BbLar1 mediates the transcription of BbCFEM7 and BbCFEM8, but relationship between the BbLar1 and BbCfem7/8 might be indirect. These two proteins also significantly contribute to iron acquisition in B. bassiana.²⁰ The CFEM domain is specifically present in fungal proteins, and the CFEM-containing proteins are involved in differentiation, stress response, and pathogenicity in filamentous fungi.³⁴^,³⁵^,³⁶ This study provides evidences for the new physilological functions of CFEM-containing proteins in fungi. As for practical application, BbCfem7/8 might be used as a molecular marker in screening the strain with high tolerance to UFA stress and a molecular target in strain improvement by genetic modification.

In summary, we have uncovered a physiological pathway as the resistance mechanism of B. bassiana under LA stress. Upon stress, B. bassiana dynamically activates the comprehensive genes involved in metabolism, stress response, cellular functionality, and so on. Notably, a lipid-droplet protein (BbLar1) maintains the morphologies of lipid droplets and the intracellular homeostasis of fatty acids. BbLar1 is crucial to fungal tolerance to LA stress, which significantly contributes to conidial compatibility with fungicides using UFAs as active ingredients. Interestingly, BbLar1 links the lipid droplet to global expression profiles in B. bassiana under LA stress. Our studies highlight lipid droplets function as the key organelles in fungal resistance to UFAs, but also provide a valuable framework for improving the compatibility of entomopathogenic fungi with anti-fungal reagents in the integrated management of insect and pathogenic fungi in crop production.

Limitations of the study

B. bassiana has been widely developed into mycoinsecticides for integrated pest management program. In this study, we revealed a physiological mechanism for this fungus to survive under the stress caused by unsaturated fatty acids through comparative transcriptomic analyses. First of all, the transcriptional regulation of lipid-droplet protein BbLar1 gene is lacking in the present study. Future study is needed to exlpore the transcription factor for the activation of BbLAR1. In addition, the BbLar1-meditaed pathway is generated from the transcriptomic data. Therefore, the detailed mechanisms underlying the involvement of lipid droplets in transcriptional regulation deserve more comprehensive investigations.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Bacterial and virus strains

Escherichia coliDH5a	Takara	Cat#9057

Chemicals, peptides, and recombinant proteins

Sucrose	Sigma	Cat#V900116
Trehalose	Sigma	Cat#V900932
Peptone	GIBCO	Cat#211677
Yeast extract	GIBCO	Cat#212750
Linoleic acid	MedChemExpress	Cat#HY-N0729/CS-0009742
Sorbitol	Solarbio	Cat#S8090
NaCl	Solarbio	Cat#S8210
SDS	Solarbio	Cat#S8010
Congo red	Solarbio	Cat#C8450
Calcofluor white (CFW)	Sigma	Cat#910090
Menadione	Sigma	Cat#M5750
H₂O₂	Sinopharm Chemical Reagent Co., Ltd	Cat#10011218
Nile Red	Sigma	Cat#N3013
BODIPY493/503	Thermo Fisher Scientific	Cat#D3922
Fatty acid methyl ester	Sigma	Cat#47885U

Critical commercial assays

ClonExpress II One Step Cloning Kit	Vazyme Biotech	Cat#C112-02

Experimental models: Organisms/strains

Beauveria bassiana	U.S. Department of Agriculture Entomopathogenic Fungus Collection	ARSEF2860
Galleria mellonella	Pet food company	N/A

Deposited data

Transcriptome under linoleic stress	This study	GSE218933
The BbLar1-mediated transcriptome	This study	GSE220873

Oligonucleotides

All primers for PCR	This study	Table S10

Software and algorithms

SMART	Letunic et al., 2021³⁷	http://smart.embl-heidelberg.de/
ImageJ	Schneider et al., 2012³⁸	https://ImageJ.net/Welcome
Prism 8	GraphPad Software	https://www.graphpad.com/

Open in a new tab

Resource availability

Lead contact

Further information and requests for resources and reagent should be directed to and will be fulfilled by the Lead Contact, Sheng-Hua Ying (yingsh@zju.edu.cn).

Material availability

This study did not generate new unique reagents.

Experimental model and subject details

Fungal strain

The wild-type (WT) strain of B. bassiana ARSEF2860 (Bb2860) was obtained from the U.S. Department of Agriculture Entomopathogenic Fungus Collection (Ithaca, NY, USA). Sabouraud dextrose agar with yeast extract (SDAY) (4% glucose, 1% peptone, and 1.5% agar plus 1% yeast extract) was prepared for routine maintenance of fungal strain at 25°C. Conidia were used as initial inocula in subsequent experiments, which were obtained by cultivating fungal strain on SDAY at 25°C for 7 days. Czapek-Dox agar (CZA) (3% glucose, 0.3% NaNO₃, 0.1% K₂HPO₄, 0.05% KCl, 0.05% MgSO₄, and 0.001% FeSO₄ plus 1.5% agar) was used as the synthetic medium. Trehalose-Peptone agar (TPA) was prepared by replace the carbon and nitrogen sources in CZA with trehalose (3%) and peptone (0.5%), respectively. In phenotypic assays, above three kinds of media were used as necessary.

Bacterial strain

Escherichia coli DH5α was used for propagation of constructs by culturing them in Luria-Bertani (LB) broth supplemented with necessary antibiotics at 37 °C.

Bioassay insects

The larvae of Galleria mellonella were feed with artificial diet and reared as previously described.³⁹ Last-instar larvae (∼300 mg in weight) were used in this study.

Method details

Illumina sequencing the transcriptomic responses of B. bassiana to LA stress

Conidial suspension (0.5 mL, 10⁸ conidia/ml) of the WT strain was inoculated into 150-mL flasks containing 50 mL TPB (TPA without agar) supplemented with LA. LA concentrations (v/v) were adjusted to 0, 0.005, 0.05, and 0.1%. After an incubation of 3 days at 25°C with consistent shaking, the resultant mycelia were collected through vacuum filtration.

Total RNA was extracted from mycelia cultured in media as described above, resulting in four RNA libraries, i.e., WTCK, WTLC, WTMC, and WTHC, respectively. Molecules of mRNA were further enriched and constructed into library which was sequenced on platform of Illumina NovaSeq 6000 at Anhui Microanaly Genetech Co., Ltd. (Anhui, China). Sequencing data have been deposited in NCBI’s Gene Expression Ominibus and are accessible through GEO Series Accession No. GSE218933. All samples were replicated two times independently.

All clean reads were mapped onto the genome database of Bb2860¹⁸ using the HISAT program.⁴⁰ All retrieved genes were normalized in terms of the expected number of fragments per kilobase of transcript sequence per million base pairs sequenced (FKPM), using Cufflinks software.⁴¹ The differentially expressed genes (DEGs) between the paired comparisons (the indicated concentration vs. control) were analyzed with the Cuffdiff method.⁴² The DEGs were screened when the q-value was less than 0.05 (5% false discovery rate), and the absolute value of log₂ ratio (fold change) was greater than 1. The resultant DEGs were subjected to enrichment analyses via the online FungiFun2 portal (https://elbe.hki-jena.de/fungifun/).⁴³ The threshold of the corrected p value was set at 0.05.

Sequence analyses of gene locus BBA_01631 in B. bassiana

Gene expression of BBA_01631 was enhanced at LA concentrations of 0.05 and 0.1%. This gene was proposed to be involved in fungal response to LA stress and analyzed in the follow-up investigation. Its homologs in other fungi were searched via online BLAST analyses in NCBI. Fungal species included Aspergillus nidulans FGSC A4, A. niger CBS 513.88, A. fumigatus Af293, C. albicans SC5314, C. militaris CM01, Fusarium graminearum PH-1, M. oryzae 70-15, Metarhizium acridum CQMa 102, M. robertsii ARSEF 23, Penicillium rubens Wisconsin 54-1255, S. cerevisiae S288C, and Yarrowia lipolytica CLIB122. Domain analyses were performed through an online portal SMART (http://smart.embl-heidelberg.de/).³⁷

Cellular localizations of the product of BBA_01631 were determined using green fluorescent protein as a reporter.⁴⁴ All primers were shown in Table S1. The coding sequence was amplified with primers PL1 and PL2 using cDNA as template. The amplified fragment was fused with 5′-flank in the green fluorescent protein gene (GFP) of pBMGB. The expression construct was introduced into the genome of the WT strain. The resultant transformant with bar-resistance was screened and further grown in SDB medium (SDAY plate without agar) at 25°C for 2 days. To view intracellular lipid droplets, the mycelia were stained with Nile red. The fluorescent signals in mycelia were examined under a laser scanning confocal microscope (LSM 710, Carl Zeiss Microscopy GmbH, Jena, Germany) and analyzed with the ImageJ software (https://imagej.net/Welcome).³⁸

Gene disruption and complementation

Gene disruption and complementation were conducted with homologous replacement and ectopical insertion, respectively.²⁰ All primers were listed in Table S10. The up- and downstream flanking sequences for the indicated gene were amplified with the primer pairs P1/P2 and P3/P4, respectively. The disruption vector was constructed by ligating the resulting fragments into the EcoRI/BamHI and XbaI/HpaI sites of p0380-bar with the ClonExpress II One Step Cloning Kit (Vazyme Biotech, Nanjing, China). The gene disruption vector was transformed into the WT strain with the Agrobacterium-mediated transformation method. The transformants were screened on CZA plates supplemented with glufosinate ammonium (200 μg/mL). All recombination events for gene disruption were screened by PCR with the primer pair P5/P6, and the disruptants were further verified with the fluorescence-coupled double screening method. To construct the gene complemented strain, the primer pair P7/P8 was used to amplify the entire ORF with the promoter which was cloned into the plasmid p0380-sur-gateway. The complementation vector was inserted into the genome in the disruptant. The transformants for gene complementation were grown on CZA plates included with chlorimuron ethyl (15 μg/mL). The complementation mutant strains were also screened by PCR with the primer pair P5/P6.

Quantification of the intracellular fatty acids

Conidia were inoculated into TPB with 0.1% LA and cultured at 25°C under submerged condition. After a 3-day incubation, the resultant mycelia were collected and lyophilized. Fatty acids (FA) were extracted and quantified as described previously with some modifications.⁴⁵ In brief, mycelia (100 mg) were suspended in extraction solvent consisting of chloroform and methanol. Fatty acid methyl ester (FAME) was prepared via derivative reaction and examined on a Focus series gas chromatograph (Thermo Scientific), using a mixture of FAMEs (47885U) (Sigma) as the internal standards. Mycelia grown in TPB were used as the control group.

Morphological view of lipid droplets

Conidia were grown in TPB at 25°C for 2 days, and the resultant culture was stressed with different concentrations of LA (final concentration: 0, 0.005, 0.05, and 0.1%) for 1 day. The treated mycelia were rinsed to remove the nutrient residues and stained with BODIPY493/503 (Thermo Fisher Scientific). The fluorescence in mycelia was examined under a laser scanning confocal microscope (LSM 710, Carl Zeiss Microscopy GmbH, Jena, Germany).

Assays for fungal growth and development

To test vegetative growth on various nutrients, the carbon and nitrogen sources in CZA were replaced as required. Carbon sources included glucose (3%), sucrose (3%), trehalose (3%), and olive oil (0.3%). Nitrogen sources (0.5%) included NH₄Cl, urea, gelatin, peptone, and chitin. SDAY was used as the control of nutrient-rich medium. To examine fungal response to various abiotic stresses, CZA plates supplemented with chemical stressors, including 0.5 M NaCl, 1 M sorbitol, 2 mM H₂O₂, 0.02 mM menadione, 3 μg/mL Congo red, and sodium dodecyl sulfate (SDS). CZA plate was used as a control. Conidial suspension (1 μL, 10⁶ conidia/ml) was inoculated on plates and incubated at 25°C. The colony diameter was measured at 7 days post incubation.

Fungal development under aerial and submerged conditions was determined on SDAY and SDB media, respectively. Conidial suspensions (100 μL 10⁷ cells/ml) on SDAY plates were incubated for 7 days at 25°C. Conidial yield was calculated as conidial number per square centimeter. The blastospore yield was determined by culturing fungal strain in SDB for 3 days at 25°C with constant aeration and shown as spore number per milliliter of broth.

Fungal sensitivity to linoleic acid (LA)

Fungal responses to LA were examined among the WT, ΔBblar1, and complemented mutant strains as described previously with three replicates.¹⁷ Assays were performed on two types of media (CZA and TPA). In CZA, LA concentration (v/v) were adjusted to 0.002, 0.005, 0.01, 0.02, 0.04, and 0.05%. In TPA, an additional concentration of 0.1% was included, and the concentration of 0.04% was omitted. Plates without FAs were used as controls.

Fungal entomopathogenicity

Conidial pathogenicity was determined via bioassay on G. mellonella larvae with topical infection method. Each treatment included 30–35 larvae which were immersed in conidial suspension (10⁷ conidia/ml) for 10 s. After air-drying, the infected larvae were transferred into a plastic box [13.5 cm (L) × 8.0 cm (W) × 4.5 cm (H)] and sprayed with 0.25 mL 0.1% LA (LA diluted in liquid paraffin) and LA for determination of the LA effect on fungal virulence. The bioassay insects were reared at 25°C, and mortality was recorded daily. The Kaplan-Meier method was used to calculate median lethal time (LT₅₀), and log rank test was used to determine the difference between the paired survival trends.

Effects of the BbLAR1 loss on fungal transcriptome

In B. bassiana, the gene BBA_01631 is the first gene found to be essentially for fungal resistance to linoleic acid (LA) stress, and this gene is designated as BbLAR1. In order to probe the BbLar1-mediated transcriptome under LA stress, genome-wide expression analysis was performed on the wild-type and the ΔBblar1 mutant strains as described previously.³⁸ Conidial suspension of mutant strain was inoculated in TPB included with 0.05 and 0.1% LA (final concentration) and cultured at 25°C for 3 days with aeration. The resultant mycelia were collected for library construction, generating two libraries, i.e., Lar1MC and Lar1HC. Each library included two independent biological replicates.

All methods for library construction, RNA-sequencing, and bioinformatic analyses were as same as those used in the section of “illumina sequencing the transcriptomic responses of B. bassiana to LA stress”. Sequencing data have been deposited in NCBI’s Gene Expression Ominibus and are accessible through GEO Series Accession No. GSE220873. In order to detect the BbLar1-mediated DEGs, the comparison was conducted between paired libraries, i.e., Lar1MC/WTMC and Lar1HC/WTHC.

Among the BbLar1-mediated DEGs, two genes coding common in fungal extracellular membrane (CFEM) domain proteins (BbCfem 7 and BbCfem8) were significantly repressed in ΔBblar1 mutant strain. In B. bassiana, BbCfem 7 is associated with cytomembrane, and BbCfem8 is observed in cytoplasm.²⁰ In this study, the localization of BbCfem8 was further determined by staining the wild type transformed with the fusion gene BbCFEM8-GFP with Nile red, a selective fluorescent stain for intracellular lipid droplets. Fungal resistance to LA stress was examined as same as those used for ΔBblar1 mutant.

Quantification and statistical analysis

Each experiment was conducted with three parallel replicates, and the data were shown as mean ± standard deviation (SD). One- and two-way analyses of variance (ANOVA) were applied in statistical analysis of multiple measurements for all strains, and the significance was determined by Tukey’s honest significance test (Tukey’s HSD). The paired measurements were subjected to Student’s t test, and the significance was determined when p is less than 0.05. Statistical analyses were performed with the software of GraphPad Prism 8 (GraphPad Software, Boston, MA, USA).

Acknowledgments

We acknowledge the excellent technical assistance provided by Dr. She-Long Zhang (Equipment and Technology Service Platform, College of Life Sciences, Zhejiang University) in the manipulation of fluorescence microscope. This study was supported by National Natural Science Foundation of China (32170027 and 31972331).

Author contributions

J.H.: Formal analysis, Investigation, Validation, Visualization, Writing - original draft. H.Z: Formal analysis, Investigation. J.L.D.: Formal analysis, Methodology. M.G.F.: Writing - review & editing. S.H.Y.: Conceptualization, Funding acquisition, Project administration, Supervision, Writing - review & editing.

Declaration of interests

The authors declare no competing interests.

Published: April 1, 2023

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2023.106551.

Supplemental information

Document S1. Figures S1-S4 and Tables S1, S8, S10

mmc1.pdf^{(395.4KB, pdf)}

Table S2 Dose dependent transcriptomes of Beauveria bassiana under linoleic acid stress, Related to Figure 1

mmc2.xlsx^{(78.1KB, xlsx)}

Table S3 Enrichment analyses of Gene ontology (GO) assignments for the differentially expressed genes (DEGs) of Beauveria bassiana stressed by linoleic acid, Related to Figure 2

mmc3.xlsx^{(13.8KB, xlsx)}

Table S4 FunCat analyses for the differentially expressed genes (DEGs) of Beauveria bassiana stressed by linoleic acid, Related to Figure 2

mmc4.xlsx^{(14.5KB, xlsx)}

Table S5 Clustering analyses for the differentially expressed genes of Beauveria bassiana stressed by linoleic acid, Related to Figure 2

mmc5.xlsx^{(66.6KB, xlsx)}

Table S6 The differentially expressed genes in ΔBblar1 mutant strain under different concentrations of linoleic acid, Related to Figure 7

mmc6.xlsx^{(749.8KB, xlsx)}

Table S7 Enrichment analyses of Gene ontology (GO) assignments for the differentially expressed genes (DEGs) in ΔBblar1 mutant strain stressed by linoleic acid, Related Figure 7

mmc7.xlsx^{(13.8KB, xlsx)}

Table S9 Clustering analyses for the overlapped differentially expressed genes in ΔBblar1 mutant stressed by two concentrations of linoleic acid, Related to Figure 7

mmc8.xlsx^{(236.6KB, xlsx)}

Data and code availability

•
All data and methods necessary to reproduce this study are included in the manuscript and Supplemental Information.
•
This paper does not report original code.
•
RNA-seq data of this study have been deposited in NCBI’s Gene Expression Ominibus under accession number GSE218933 and GSE220873.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1-S4 and Tables S1, S8, S10

mmc1.pdf^{(395.4KB, pdf)}

Table S2 Dose dependent transcriptomes of Beauveria bassiana under linoleic acid stress, Related to Figure 1

mmc2.xlsx^{(78.1KB, xlsx)}

Table S3 Enrichment analyses of Gene ontology (GO) assignments for the differentially expressed genes (DEGs) of Beauveria bassiana stressed by linoleic acid, Related to Figure 2

mmc3.xlsx^{(13.8KB, xlsx)}

Table S4 FunCat analyses for the differentially expressed genes (DEGs) of Beauveria bassiana stressed by linoleic acid, Related to Figure 2

mmc4.xlsx^{(14.5KB, xlsx)}

Table S5 Clustering analyses for the differentially expressed genes of Beauveria bassiana stressed by linoleic acid, Related to Figure 2

mmc5.xlsx^{(66.6KB, xlsx)}

Table S6 The differentially expressed genes in ΔBblar1 mutant strain under different concentrations of linoleic acid, Related to Figure 7

mmc6.xlsx^{(749.8KB, xlsx)}

Table S7 Enrichment analyses of Gene ontology (GO) assignments for the differentially expressed genes (DEGs) in ΔBblar1 mutant strain stressed by linoleic acid, Related Figure 7

mmc7.xlsx^{(13.8KB, xlsx)}

Table S9 Clustering analyses for the overlapped differentially expressed genes in ΔBblar1 mutant stressed by two concentrations of linoleic acid, Related to Figure 7

mmc8.xlsx^{(236.6KB, xlsx)}

Data Availability Statement

•
All data and methods necessary to reproduce this study are included in the manuscript and Supplemental Information.
•
This paper does not report original code.
•
RNA-seq data of this study have been deposited in NCBI’s Gene Expression Ominibus under accession number GSE218933 and GSE220873.

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PERMALINK

Transcriptomic investigation reveals a physiological mechanism for Beauveria bassiana to survive under linoleic acid stress

Jia Hou

Hao Zhang

Jin-Li Ding

Ming-Guang Feng

Sheng-Hua Ying

Summary

Graphical abstract

Highlights

Introduction

Results

Dose-dependent transcriptomes of fungal responses to linoleic acid stress

Figure 1.

Functional enrichment analyses of differentially expressed genes

Figure 2.

Figure 3.

Localization of BbLar1 in B. bassiana and gene target replacement

BbLar1 plays a minor role in fungal growth and development

Figure 4.

BbLar1 significantly contributes to lipid biology

Figure 5.

BbLar1 is essential for biological compatibility with linoleic acid during infecting hosts

Figure 6.

Global expression changes as a result of BbLar1 loss

Figure 7.

Phenotypic evaluation of two common in fungal extracellular membrane domain-containing proteins in fungal tolerance to linoleic acid

Figure 8.

Discussion

Limitations of the study

STAR★Methods

Key resources table

Resource availability

Lead contact

Material availability

Experimental model and subject details

Fungal strain

Bacterial strain

Bioassay insects

Method details

Illumina sequencing the transcriptomic responses of B. bassiana to LA stress

Sequence analyses of gene locus BBA_01631 in B. bassiana

Gene disruption and complementation

Quantification of the intracellular fatty acids

Morphological view of lipid droplets

Assays for fungal growth and development

Fungal sensitivity to linoleic acid (LA)

Fungal entomopathogenicity

Effects of the BbLAR1 loss on fungal transcriptome

Quantification and statistical analysis

Acknowledgments

Author contributions

Declaration of interests

Footnotes

Supplemental information

Data and code availability

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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