In China, wheat yellow mosaic disease is mostly caused by wheat yellow mosaic virus (WYMV) and Chinese wheat mosaic virus (CWMV). If wheat is co-infected with these two viruses, it can cause severe yellow mosaic symptoms and yield losses. Early detection of viruses is crucial for preventing disease in the field. In this study, we optimized a sensitive, specific reverse transcription recombinase polymerase amplification (RT-RPA) detection method for two viruses, WYMV and CWMV. Two sets of primers were designed based on the capsid protein (CP)-encoding genes of the two viruses, and the reaction conditions were determined. The RT-RPA method, which amplified the target amplicon by a handheld reaction mixture for 20 min, was more sensitive than PCR-CP in the detection of WYMV. Finally, the RT-RPA method was performed on 110 randomly selected field samples, demonstrating its applicability to samples from different regions and specificity for co-infected samples. This study not only describes an improved method for detecting WYMV and CWMV using RT-RPA but also demonstrates the potential of this method, which could be applied under field conditions.

Glycosylation, a dynamic modification prevalent in viruses and higher eukaryotes, is principally regulated by uridine diphosphate (UDP)-glycosyltransferases (UGTs) in plants. Although UGTs are involved in plant defense responses, their responses to most pathogens, especially plant viruses, remain unclear. Here, we aimed to identify UGTs in the whole genome of Nicotiana benthamiana (N. benthamiana) and to analyze their function in Chinese wheat mosaic virus (CWMV) infection. A total of 147 NbUGTs were identified in N. benthamiana. To conduct a phylogenetic analysis, the UGT protein sequences of N. benthamiana and Arabidopsis thaliana were aligned. The gene structure and conserved motifs of the UGTs were also analyzed. Additionally, the physicochemical properties and predictable subcellular localization were examined in detail. Analysis of cis-acting elements in the putative promoter revealed that NbUGTs were involved in temperature, defense, and hormone responses. The expression levels of 20 NbUGTs containing defense-related cis-acting elements were assessed in CWMV-infected N. benthamiana, revealing a significant upregulation of 8 NbUGTs. Subcellular localization analysis of three NbUGTs (NbUGT12, NbUGT16 and NbUGT17) revealed their predominant localization in the cytoplasm of N. benthamiana leaves, and NbUGT12 was also distributed in the chloroplasts. CWMV infection did not alter the subcellular localization of NbUGT12, NbUGT16, and NbUGT17. Transient overexpression of NbUGT12, NbUGT16, and NbUGT17 enhanced CWMV infection, whereas the knockdown of NbUGT12, NbUGT16 and NbUGT17 inhibited CWMV infection in N. benthamiana. These NbUGTs could serve as potential susceptibility genes to facilitate CWMV infection. Overall, the findings throw light on the evolution and function of NbUGTs.

Reactive oxygen species (ROS), as signaling molecules, play a crucial role in the plant immune response. However, the mechanism(s) by which viruses affect ROS metabolism remain largely unexplored. Here, we found that wheat yellow mosaic virus (WYMV)-encoded P1 is a pathogenic protein. Transcriptomic and proteomic integrative analyses were performed on WYMV-infected overexpressing-P1 wheat and wild-type plants. A total of 9245 differentially expressed genes (DEGs) and 1383 differentially expressed proteins (DEPs) were identified in the transcriptome and proteome, respectively. At their intersection, 373 DEGs/Ps were identified. Enrichment analysis revealed that the expression of genes related to the ROS metabolism pathway in overexpressed P1 transgenic wheat (OE-P1) plants significantly increased during WYMV infection. We screened peroxidase (TaPOD) and thioredoxin reductase (TaTrxR) as they showed the most significant differences in expression. The silencing of TaPOD and TaTrxR revealed that they positively regulate WYMV infection by reducing ROS accumulation. Furthermore, hydrogen peroxide treatment induced WYMV resistance in wild-type wheat plants and OE-P1 transgenic plants. This study provides a theoretical basis for the role of P1 in plant viral infection.

Although RNA cytosine-5 methylation (m⁵C) is an important post-transcriptional regulatory mechanism, its contribution to plant antiviral immunity remains unclear. In this study, we identified Thiamine thiazole synthase 2 (TaTHI2) as a host mRNA target of the wheat m⁵C methyltransferase TaNSUN2 during infection by Chinese wheat mosaic virus (CWMV), a soil-borne virus that poses a major threat to wheat production. TaNSUN2 contributes to the m⁵C modification of TaTHI2 transcripts, enhancing mRNA stability and sustaining TaTHI2 accumulation. The disruption of a key m⁵C site markedly reduced methylation, weakened TaNSUN2–RNA binding, and accelerated transcript decay, leading to the compromised production of reactive oxygen species (ROS) and increased viral infection. Mechanistically, the TaNSUN2-dependent m⁵C modification stabilized TaTHI2 mRNA, thereby promoting ROS-mediated antiviral defense. Collectively, our results establish the m⁵C modification of TaTHI2 mRNA as a critical post-transcriptional control point in CWMV resistance and highlight TaNSUN2-dependent RNA methylation as an integral component of host antiviral immunity.