The soybean aphid (Aphis glycines) and soybean cyst nematode (Heterodera glycines) are major aboveground and belowground pests of soybean (Glycine max) in the U.S. Midwest, but the molecular basis of their combined effects on soybean defense remains poorly understood. This study examines how soybean genotypes influence demographic and root-transcriptomic responses to single and combined pest infestation. Soybean cyst nematode reproduction increased under combined infestation in the susceptible cultivar but remained unchanged in the resistant cultivar, whereas soybean aphid populations declined when plants were also infested with nematodes. Root RNA-seq revealed strong time-dependent transcriptional responses, with substantially more differentially expressed genes at 30 days post-infestation than at 5 days post-infestation. Co-expression and enrichment analyses showed that early responses were associated with defense signaling, plant–pathogen interaction, and cutin, suberin, and wax biosynthesis, whereas later responses involved redox processes, isoflavonoid biosynthesis, phenylpropanoid metabolism, and one-carbon metabolism. Several differentially expressed soybean genes co-localized with known soybean cyst nematode resistance quantitative trait loci, including genes near the rhg1 region. Together, these results suggest that soybean genotypes strongly influence soybean aphid–soybean cyst nematode interactions and identify candidate genes and pathways that may contribute to durable resistance against interacting aboveground and belowground pests.

Soybean aphid (SBA), Aphis glycines Matsumura (Hemiptera: Aphididae), and soybean cyst nematode (SCN), Heterodera glycines Ichinoe (Tylenchida: Heteroderidae), are major pests of soybean, Glycine max L. Merr., in the U.S. Midwest. This study examined three-way interactions among soybean, SBA, and SCN using demographic and transcriptomic analyses. SCN-resistant and SCN-susceptible cultivars were evaluated under three treatments (SBA, SCN, SCN+SBA) in a randomized complete block design with six replicates, repeated eight times in greenhouse cone-tainers. Plants were infested with 2,000 SCN eggs at planting or 15 SBA at the V2 stage. Aphid populations were counted at 5-, 15-, and 30-days post-infestation (dpi), and SCN eggs sampled at 30 dpi. SCN egg density increased significantly in the susceptible cultivar but remained unchanged in the resistant cultivar in the presence of SBA, while SBA populations declined under SCN infestation. RNA-seq identified 4,637 differentially expressed genes (DEGs) at 5 dpi and 19,032 DEGs at 30 dpi. Analyses focused on DEGs shared across treatments but discordantly expressed in resistant cultivars during SBA–SCN interactions. Weighted Gene Co-expression Network Analysis revealed seven and nine modules at 5 and 30 dpi, respectively. Enrichment analyses identified ‘Plant–Pathogen Interaction’ and ‘Cutin, Suberin, and Wax Biosynthesis’ at 5 dpi, and ‘Isoflavonoid Biosynthesis’ and ‘One-Carbon Pool by Folate’ at 30 dpi. Several DEGs overlapped with SCN resistance QTLs, identifying candidate genes for cross-resistance breeding.