Fungal co-expression network analyses identify pathogen gene modules associated with host insect invasion

Abstract

The broad host range fungal insect pathogen, Beauveria bassiana, has been commercialized as an alternative to chemical insecticides for pest control worldwide. B. bassiana represents a unique model system with which to examine host-pathogen interactions, and a wide range of genes and processes have been studied. However, significant aspects of virulence, particularly on the genomic scale, remain poorly studied. Here, we have combined available transcriptomes with three newly generated data sets for a combined total analysis of 76 deep-sequenced samples covering growth, development, stress responses, and infection during the life cycle of B. bassiana. Co-expression network analyses resulted in the identification of gene modules enriched during two critical stages of the infection process, namely (i) cuticle penetration and (ii) in vivo hyphal body (dimorphic transition) growth capable of avoiding innate and humoral immune defenses. These analyses identify unique signatures of metabolism, signaling, secondary metabolite production, host defense suppression, membrane reorganization, effector production, and secretion for each stage, including genetic regulators and epigenetic patterns. These data provide a comprehensive framework for understanding and probing fungal adaptations to its pathogenic life cycle and expand the candidate repertoire for continued dissection of the host-pathogen interaction. IMPORTANCE Insect fungal pathogens have evolved unique strategies for overcoming host structural and immunological defenses that span from the sclerotized cuticle to innate and humoral cellular responses. Two critical stages of the infection process involve (i) cuticle penetration and (ii) immune evasion within the insect hemocoel. A set of 76 global transcriptomic data for B. bassiana that include the cuticle penetration and hemocoel growth stages were analyzed for patterns (gene modules) of expression, yielding unique insights into these different life stages. These analyses integrate gene networks involved in fungal development, stress response and pathogenesis to further the systematic understanding of the global processes integral to the unique adaptation employed by fungal pathogens of insects.

Keywords: Beauveria bassiana; co-expression; entomopathogenic fungi; host-pathogen interaction; infection-associated modules; transcriptome.

Fig 1

Working pipeline and sample collections for identification of infection-associated gene modules. RNA-seq samples derived from different cell types in the B. bassiana life cycle. (A) Overview of bioinformatic analysis workflow. (B) Physiological niches of collected RNA-seq samples. Fungal cell types were annotated as: AH, aerial hyphae; CP, cuticle-penetrated cells; HB, hyphal bodies; and LH, Liquid hyphae. Nutrition bases include SDA, SDB, GB (germination broth), CZA, NLB and chitin, trehalose, glucose-supplemented CZA or CZB medium for CZA-chitin, CZB_TRE, and CZB_GLU, respectively. The proportion of each sample type was plotted accordingly.

Fig 2

Correlations of RNA-Seq samples within or between growth conditions. (A) Correlation matrix of normalized RNA samples for FPKM (down-left) and LogCLR (up-right), respectively. The bars on the left and top of the matrix are labeled according to growth condition: CP, cuticle penetrations; HB, hyphal bodies; LH, liquid hyphae; AH, aerial hyphae. Sample correlation intensity was colored as bars. (B) Principal component analysis (PCA) of RNA-seq samples. Transcriptomes from different groups were decorated and enclosed in different colors as indicated. (C) Distribution of PCCs of RNA samples inside given groups. The mean PCC value was connected as a red dashed line, and sample groups were decorated accordingly.

Fig 3

Discovery and illustration of infection-associated modules. (A) Correlation matrix of WGCNA generated co-expression modules with physiological traits. Correlation coefficients of PCC were listed and decorated red to green for positive and negative correlations in the up-left triangle in each correlation box, and correlation P-value was marked and decorated accordingly in the down-right panel. (B) B. bassiana GGM gene co-expression network. Only modules with 100+ genes were shown due to the limited space. Different modules were color-coded as indicated in the legend. Nodes refer genes, co-expressed genes were interconnected with line color similar to each modules; the size of node represents connection counts. Genes with PCC threshold of 0.6 were used for presentation. (C) Expression profile of infection-associated module genes detected by Coseq, mean expression value of module genes in the given condition was linked with solid red. Cell types were color-coded as indicated. The number on top refers to the module rank number in the analysis.

Fig 4

Global expression and co-expression networks of infection-associated modules. Two infection modules were separated into four subgroups: HB-Up and HB-Dn refer to the subgroup module genes that are mainly expressed or depressed in hyphal bodies, respectively; CP-Up and CP-Dn represent CP-associated module subgroups that were overexpressed or silent in CP trait. Expression profile of genes in subgroups HB-Up (A), HB-Dn (B), CP-Up (C), and CP-Dn (D) in four conditions were plotted and color-coded accordingly. Mean expression value of module genes in each condition was linked in solid red. (E) Co-expression network of four module subgroup genes associated with infection. Positive gene-gene PCC correlations of over 0.7 were used for illustration, size of node represents connection counts, and connections between genes indicate a co-expression relationship.

Fig 5

The RT-qPCR validation of the expression profile of 10 genes that are mainly expressed in HB condition. Samples were divided into saprophytic and infection groups and marked as bars in different colors. Trehalose, sucrose, glucose, and cellulose refer to CZB broth supplemented with carbohydrates previously listed as a solo carbon source [3% (vol/wt)]. Cuticle indicates fungal cells incubated with silkworm cuticle; HB refers to hyphal bodies collected from B. bassiana-infected G. mellonella. Relative expression refers to z-score normalized expression between samples.

Fig 6

Enrichment analysis of infection-associated module genes. Four infection-related subgroups were identified and categorized as detailed in the experimental protocols and results section. GO analysis of module genes in subsets of HB-Up (A), HB-Dn (B), CP-Up (C), CP-Dn (D) was shown, and enriched GO catalogs were selected for presentation. The size of the symbols in the plots reflects the relative number of module genes in the indicated category. Confidence intervals (P-values) of assignments are coded in color from green to red as indicated. (E) Subcellular localization of three infection-associated module genes (HB-Up, HB-Dn, and CP-Up). The number of module genes that fell into eight cellular compartments predicted by deeploc2 was calculated and illustrated. Cyto, cytoplasm; Memb, membrane; Extr, extracellular; ER, endoplasmic reticulum; Pero, peroxisome; Golgi, golgi apparatus; Nuclear, cell nucleus. (F) Enrichment of module genes in Carbohydrate-Active enZYmes (CZAY) Database. The size of symbols indicates enriched gene number, groups were decorated in three shapes: triangle (HB-Up), circle (HB-Dn), and square (CP-Up). Confidence intervals (P-values) were synergistic to color bar from green to red, as indicated.

Fig 7

Metabolic pathway analysis of infection-associated module genes. Module genes were mapped to yeast metabolic pathways and pathways in the Metacyc database and overrepresented pathways were selected for illustration. The partially activated pathways of fatty acid degradation (A), GTP synthesis (B), and glycolysis (C) in HB-Up subgroup were illustrated. The active pathways in cuticle penetration (CP-Up) were shown as indicated. (D) Glutamic acid degradation pathway. (E) Pyruvate metabolic pathway. (F) Tyrosine degradation pathway. (G) Coenzyme biosynthesis pathway. The known homologs of module genes in yeast were listed as indicated or as sole B. bassiana genes after being mapped to Metacyc. Abbreviations of chemicals listed were annotated in Metacyc databases. Multiple reactions in between substrates were linked with a dotted arrow, while reversible reactions were shown as double arrows.

Fig 8

Analysis of transporters and mitochondrial function in infection-associated modules. Module genes were mapped to Transporter Classification Database (TCDB), and over-presented transporter catalogs were selected for enrichment illustration: (A) HB-Up, (B) HB-Dn, and (C) CP-Up. The size of nodes reflects the number of genes enriched in given categories. Confidence intervals (P-values) were colored from blue to red as indicated. (D) Enrichment of transporter substrates in three infection correlated modules. Node size refers to the number of enriched module genes. AA, amino acids; FAA, fatty acids; ION, ions; Hydron, hydrogen ion; TOX, toxins. (E) Active MT genes during hemolymph colonization. Yeast homologs of MT genes in the HB-Up module were used for illustration, including members in electron transfer chain, MT ribosome, and inner membrane transporters.

Fig 9

Global expression profile of two secondary metabolite biosynthesis clusters and co-expression networks of regulators in infection-associated modules. (A) Oosporein biosynthesis gene cluster. (B) A putative siderophore biosynthesis gene cluster. Normalized LogCLR values were illustrated as expression values. Samples were clustered as condition of CP, HB, LH, and AH. (C) Co-expression network of transcription factors and kinases in four infection-associated modules. Known/homologous genes were annotated as indicated.

Fig 10

Overview of metabolic and signal transduction processes during infection by entomopathogenic fungi. Insect cuticle comprised of epicuticle, procuticle, and epidermis. Fungal cells attached to the epicuticle germinated to form appressorium that secretes suites of cuticle-degrading enzymes (lipases, chitinases, and peptidases). To overcome barriers of insect cuticle, B. bassiana stimulated sets of cellular metabolisms: amino acid degradation, secondary metabolites biosynthesis, detoxification of ROS and cuticle toxins, e.g., quinones, and assumption of nutrition including ions and amino acids, which were orchestrated by the series regulators functions in transcription or epigenetic processes. After penetration into insect hemocoel, fungal cells evade humoral defenses and differentiate in vivo into hyphal bodies, which proliferate and secrete metabolites and enzymes (peptidases, lipases, and SSPs) to counter host responses and facilitate nutrition uptake. In hyphal bodies, fungal mRNA/ncRNA biogenesis machinery, members of transcription/epigenetic regulation, and signal sensors of G-protein complex were mobilized, and metabolic processes including fatty acid degradation, glycolysis/TCA cycle, mitochondrial proliferation/metabolism as well as membrane-mediated nutrition transportation (ions, FAA) were activated.