The clover cutworm, Scotogramma trifolii Rottemberg (Lepidoptera: Noctuidae), is a globally distributed polyphagous pest causing significant economic losses to agricultural crops. RT-qPCR is a gold-standard technique for gene expression analysis, yet its accuracy depends critically on stable reference genes for data normalization. To address the lack of validated reference genes in S. trifolii, we evaluated six candidate genes (β-actin, RPL9, GAPDH, RPL10, EF1-α, and TUB) across four developmental stages (egg, larva, pupa, and adult) and six adult tissues (head, thorax, abdomen, wings, legs, and antennae) using geNorm, NormFinder, BestKeeper, and RefFinder algorithms. Stability analysis identified β-actin, RPL9, and GAPDH as the most reliable reference genes for developmental stage normalization, while RPL10, GAPDH, and TUB were validated for adult tissues. Functional validation using the odorant receptor gene StriOR20 revealed significant discrepancies in relative expression levels when normalized with unstable reference genes (TUB and RPL9), emphasizing the necessity of rigorous reference gene selection. This study establishes the first comprehensive reference gene panel for S. trifolii, providing a robust foundation for gene expression studies in this agriculturally important pest.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), poses a severe threat to global wheat production. The adaptor protein complex AP-1 plays a crucial role in vesicular trafficking, yet its function in rust fungi remains poorly understood. In this study, a gene encoding an AP-1 σ subunit, designated PsAP1, was identified in Pst. The expression of PsAP1 was highly induced during the early infection stage. Heterologous expression of PsAP1 in a Fusarium graminearum mutant partially restored its pathogenic defects. Subcellular localization analysis revealed that PsAP1 localizes to the plasma membrane, cytoplasm, and nucleus. Silencing PsAP1 in wheat using Barley stripe mosaic virus-mediated host-induced gene silencing (BSMV-HIGS) significantly attenuated Pst pathogenicity, reducing hyphal growth by 6.7% (colony diameter), sporulation by 61.6% (lesion length), and pathogen biomass by 66%, along with enhanced accumulation of host reactive oxygen species. Transcriptomic analysis further demonstrated that silencing PsAP1 disrupted multiple pathways, including MAPK signaling, glutathione metabolism, and carbohydrate metabolism. These findings indicate that PsAP1 facilitates Pst infection by modulating vesicular trafficking, suppressing host immunity, and reprogramming host metabolism. This study provides novel insights into the pathogenic mechanisms of rust fungi and suggests a potential target for disease control.