In vivo transcriptome analysis of the entomopathogenic fungus Beauveria bassiana strain GHA during infection against the beneficial predator tobacco leaf bug, Nesidiocoris tenuis (Hemiptera: Miridae)

Technical Summary: In Vivo Transcriptome Analysis of Beauveria bassiana Strain GHA During Infection of Nesidiocoris tenuis

Problem Statement
The entomopathogenic fungus Beauveria bassiana strain GHA is a commercial mycoinsecticide widely used for pest control. However, a significant limitation of this strain is its high virulence toward beneficial predatory insects, specifically the tobacco leaf bug (Nesidiocoris tenuis, TLB), which is utilized in agriculture for biological pest management. While the general infection mechanism of B. bassiana (cuticle penetration followed by hemocoel proliferation) is understood, the specific molecular pathogenicity mechanisms and virulence genes responsible for the high mortality observed in TLBs remain uncharacterized. Identifying these specific virulence factors is essential for developing strategies to mitigate the strain's non-target effects on beneficial predators while maintaining efficacy against pests.

Methodology
The study employed a multi-stage approach to characterize the infection process and identify stage-specific gene expression in B. bassiana strain GHA infecting TLBs:

1. Infection Kinetics and Microscopy:

   • Inoculation: TLBs were inoculated with conidial suspensions at two concentrations ($5 \times 10^6$ and $5 \times 10^7$ conidia/mL).
   • Morphological Observation: Scanning electron microscopy (SEM) and light microscopy were used to observe conidial germination, appressorium formation, and hyphal invasion at 12, 18, 24, and 36 hours post-inoculation (hpi). Paraffin-embedded sections stained with Grocott Methenamine Silver were examined at 24, 36, 48, 72, and 96 hpi to track internal fungal growth.
   • Quantitative Growth Monitoring: Genomic quantitative PCR (qPCR) targeting the rDNA IGS region of strain GHA was performed on host DNA extracted at various time points to quantify fungal biomass accumulation within the host.

2. Transcriptomic Analysis (RNA-seq):

   • Sampling: Based on the kinetic data, two critical time points were selected for RNA extraction: 36 hpi (representing the cuticle penetration stage) and 72 hpi (representing the hemocoel proliferation stage).
   • Sequencing: Total RNA was extracted from pooled surviving TLBs, and RNA-seq was performed. Reads were mapped to the B. bassiana ARSEF 2860 reference genome.
   • Differential Expression Analysis: Gene expression levels between the 36 hpi and 72 hpi stages were compared using TMM normalization and statistical testing (edgeR) to identify differentially expressed genes (DEGs).
   • Functional Annotation: Gene enrichment analysis (Gene Ontology) and protein localization predictions (Deep-Loc 2.1) were conducted to characterize the functional roles of the identified DEGs.

Key Results

• Infection Progression:

  • Conidial germination and appressorium formation were confirmed by 12 hpi.
  • Invasive hyphae were first detected penetrating the cuticle at 36 hpi.
  • By 48 hpi, filamentous hyphae and hyphal bodies were observed near the cuticle, expanding to a wide area of the body cavity by 72 hpi.
  • qPCR data confirmed a gradual increase in fungal biomass, with a sharp rise occurring after 48 hpi in the lower-dose group, validating 36 hpi and 72 hpi as appropriate time points for analyzing the penetration and proliferation phases, respectively.

• Differential Gene Expression:

  • A total of 182 genes showed significant differential expression between the two stages (adjusted $p < 0.05$).
  • 36 hpi (Penetration Stage): 83 genes were upregulated (DEGs36h). These were enriched for extracellular proteins and hydrolases. Gene Ontology (GO) analysis highlighted terms related to "Glucosidases," "Signaling receptor binding," and "Response to cytokine."
  • 72 hpi (Proliferation Stage): 99 genes were upregulated (DEGs72h). These genes spanned a wider range of functional categories without significant GO term enrichment, suggesting dispersion across various metabolic pathways.

• Identification of Virulence Candidates:

  • The study identified 16 previously reported virulence genes among the DEGs.
  • DEGs36h included homologs associated with cuticle adhesion (Mad1), appressorium formation (Gas1), immune evasion (Bps8), cuticle degradation (ChiV4), and infection processes (Bbnmo5, BbNoxA, cfp).
  • DEGs72h included genes involved in oosporein synthesis (OpS1, OpS2, OpS4–7), a transcription factor regulating morphological switching (BbHCR1), a perilipin-like gene (Mpl1), and a peroxin gene (BbPex1).
  • Notably, while Mpl1 is known for lipid metabolism in appressorium formation in Metarhizium robertsii, its homolog showed high expression at 72 hpi in this study, suggesting a potential alternative function in B. bassiana.

Significance and Claims
The authors claim this study as the first to describe the infection process of B. bassiana strain GHA against TLBs and to identify genes with expression patterns that vary in vivo during specific infection stages in this host.

The primary significance lies in demonstrating the efficacy of comparative in vivo transcriptomic analysis for selecting virulence genes. The findings suggest that while the functional categories of genes expressed during the penetration stage in TLBs are analogous to those observed in mosquitoes (a previous model for B. bassiana infection), the specific gene expression profiles differ at the individual gene level. This indicates that virulence mechanisms may be host- or strain-specific.

The paper concludes that these identified genes represent potential targets for future research aimed at selectively reducing the virulence of B. bassiana toward beneficial predators like TLBs without compromising its efficacy against pest species, thereby addressing a critical weakness in current biological control strategies. The authors explicitly state that their future goal is to validate these candidates by knocking them out to achieve selective virulence reduction.