Comparative Transcriptome Profiling of <i>Nicotiana benthamiana</i> Plants Infected with Potato Mop-Top Virus and Its Mutant Lacking a Gene for the 8K Protein Underlines the Role of Chloroplasts During Infection

Potato mop-top virus (PMTV) is a significant pathogen causing potato "spraing" disease worldwide. The PMTV 8K protein functions as a weak viral suppressor of RNA silencing (VSR), has viroporin activity and plays a role in pathogenicity by promoting viral long-distance movement and modulating host responses. Uniquely, PMTV can establish systemic infection in the absence of the 8K protein, though the infection is slightly delayed. To elucidate the molecular mechanisms underlying PMTV-host interactions, we conducted comprehensive RNA-seq analysis comparing wild-type PMTV with a mutant lacking the <i>8K</i> gene (PMTV-Δ8K) in <i>Nicotiana benthamiana</i>. Our transcriptomic analysis shows that wild-type PMTV and PMTV-Δ8K elicit largely distinct transcriptional responses in the host, with more unique than shared differentially expressed genes. The analysis also revealed extensive reprogramming of metabolic pathways, stress responses, and defense mechanisms. Notably, wild-type PMTV induced more defense-related transcription factors, including 27 <i>WRKY</i> genes compared to 8 in PMTV-Δ8K infections. RNA silencing pathway genes displayed distinct expression patterns, with <i>AGO2, RDR1,</i> and <i>AGO-MEL1</i> showing notably enhanced upregulation (up to 9.7-fold) in PMTV-Δ8K infections. Functional analysis identified chloroplast-associated genes <i>GNS2, CHUP1,</i> and <i>KIN5l</i> as host restriction factors. Virus-induced gene silencing experiments confirmed that GNS2 and CHUP1 restrict viral accumulation under both infection scenarios (wild-type PMTV and PMTV-Δ8K), while localization studies revealed that TGB2 protein and GNS2 co-localize at chloroplast structures. These findings provide insights into PMTV pathogenesis, suggest that 8K is a multifunctional protein operating through diverse mechanisms, and advance understanding of viral suppression strategies.