The Limits of Cross-Species WGCNA: Library Imbalance and Signal Dilution Constrain Effector Gene Recovery in Dual-Organism RNA-seq

Dual-organism RNA sequencing (RNA-seq) experiments, in which the transcriptomes of a host and a microbe are sequenced simultaneously, are increasingly used to study plant–microbe interactions. A central analytical goal is identifying effector proteins and their host targets through gene co-expression. Weighted Gene Co-expression Network Analysis (WGCNA) is the dominant tool for gene co-expression analyses, yet its ability to recover interaction-interface genes from a merged dual-organism matrix has not been systematically characterised. Here we present a simulation framework using real gene models from Hordeum vulgare (barley) and Blumeria graminis f. sp. Hordei M.Liu & Hambl (powdery mildew) to evaluate single-network WGCNA across a gradient of plant-to-fungal library size ratios (1:1–20:1), three levels of co-expression signal strength, and three WGCNA network construction types (signed, unsigned, signed hybrid). We embed 20 model effector genes (bridge genes) driven by a mixed host–pathogen eigengene and evaluate recovery using four metrics aligned with the biological objective: cross-species hub rank, top-decile hub enrichment, bridge gene detection rate, and bridge co-separation (the fraction of effector–target pairs co-assigned to the same detected module). Across 225 simulation runs (15 conditions × 5 replicates × 3 network types), bridge genes are robustly identifiable as cross-species connectivity hubs (mean rank 0.92 versus 0.50 for module genes) but co-assignment of effector–target pairs to the same module fails in 41% of runs due to scale-free topology collapse. Signal strength ( η ² = 0.12) and library ratio ( η ² = 0.22) are the primary determinants of co-separation, while network type choice accounts for less than 2%. A read-depth bias systematically inflates pathogen gene hub ranks relative to host genes at high ratios. These results establish that the method can identify effector candidates as cross-species hubs under a broad range of conditions, but reliable co-assignment requires adequate pathogen read depth and strong co-expression signal—properties that experimental design, not analytical parameterisation, must provide.