Identification of a Novel Quinvirus in the Family Betaflexiviridae That Infects Winter Wheat

Yellow mosaic disease in winter wheat is usually attributed to the infection by bymoviruses or furoviruses; however, there is still limited information on whether other viral agents are also associated with this disease. To investigate the wheat viromes associated with yellow mosaic disease, we carried out de novo RNA sequencing (RNA-seq) analyses of symptomatic and asymptomatic wheat-leaf samples obtained from a field in Hokkaido, Japan, in 2018 and 2019. The analyses revealed the infection by a novel betaflexivirus, which tentatively named wheat virus Q (WVQ), together with wheat yellow mosaic virus (WYMV, a bymovirus) and northern cereal mosaic virus (a cytorhabdovirus). Basic local alignment search tool (BLAST) analyses showed that the WVQ strains (of which there are at least three) were related to the members of the genus Foveavirus in the subfamily Quinvirinae (family Betaflexiviridae). In the phylogenetic tree, they form a clade distant from that of the foveaviruses, suggesting that WVQ is a member of a novel genus in the Quinvirinae. Laboratory tests confirmed that WVQ, like WYMV, is potentially transmitted through the soil to wheat plants. WVQ was also found to infect rye plants grown in the same field. Moreover, WVQ-derived small interfering RNAs accumulated in the infected wheat plants, indicating that WVQ infection induces antiviral RNA silencing responses. Given its common coexistence with WYMV, the impact of WVQ infection on yellow mosaic disease in the field warrants detailed investigation.

Integrated Proteomics and Transcriptomics Analyses Reveal the Transcriptional Slippage of P3N-PIPO in a Bymovirus

P3N-PIPO (P3 N-terminal fused with Pretty Interesting Potyviridae ORF), the movement protein of potyviruses, is expressed as a translational fusion with the N-terminus of P3 in potyviruses. As reported in previous studies, P3N-PIPO is expressed via transcriptional slippage at a conserved G2A6 slippery site in the genus Potyvirus. However, it is still unknown whether a similar expression mechanism of P3N-PIPO is used in the other genera of the family Potyviridae. Moreover, due to the extremely low expression level of P3N-PIPO in natural virus-infected plants, the peptides spanning the slippery site which provide direct evidence of the slippage at the protein level, have not been identified yet. In this study, a potato virus X (PVX)-based expression vector was utilized to investigate the expression mechanism of P3N-PIPO. A high expression level of the P3N-PIPO(WT) of turnip mosaic virus (TuMV, genus Potyvirus) was observed based on the PVX expression vector. For the first time, we successfully identified the peptides of P3N-PIPO spanning the slippery site by mass spectrometry. Likewise, the P3N-PIPO(WT) of wheat yellow mosaic virus (WYMV, genus Bymovirus) was also successfully expressed using the PVX expression vector. Integrated proteome and transcriptome analyses revealed that WYMV P3N-PIPO was expressed at the conserved G2A6 site through transcriptional slippage. Moreover, as revealed by mutagenesis analysis, Hexa-adenosine of the G2A6 site was important for the frameshift expression of P3N-PIPO in WYMV. According to our results, the PVX-based expression vector might be used as an excellent tool to study the expression mechanism of P3N-PIPO in Potyviridae. To the best of our knowledge, this is the first experimental evidence for the expression mechanism of P3N-PIPO in the genus Bymovirus, the only genus comprising bipartite virus species in the family Potyviridae.

Transcriptome-Wide N6-Methyladenosine (m6A) Profiling of Susceptible and Resistant Wheat Varieties Reveals the Involvement of Variety-Specific m6A Modification Involved in Virus-Host Interaction Pathways

N6-methyladenosine (m6A) methylation is the most prevalent internal modification of post-transcriptional modifications in mRNA, tRNA, miRNA, and long non-coding RNA in eukaryotes. m6A methylation has been proven to be involved in plant resistance to pathogens. However, there are no reports on wheat (Triticum aestivum) m6A transcriptome-wide map and its potential biological function in wheat resistance to wheat yellow mosaic virus (WYMV). To the best of our knowledge, this study is the first to determine the transcriptome-wide m6A profile of two wheat varieties with different resistances to WYMV. By analyzing m6A-sequencing (m6A-seq) data, we identified 25,752 common m6A peaks and 30,582 common m6A genes in two groups [WYMV-infected resistant wheat variety (WRV) and WYMV-infected sensitive wheat variety (WSV)], and all these peaks were mainly enriched in 3′ untranslated regions and stop codons of coding sequences. Gene Ontology analysis of m6A-seq and RNA-sequencing data revealed that genes that showed significant changes in both m6A and mRNA levels were associated with plant defense responses. Kyoto Encyclopedia of Genes and Genomes analysis revealed that these selected genes were enriched in the plant–pathogen interaction pathway. We further verified these changes in m6A and mRNA levels through gene-specific m6A real-time quantitative PCR (RT-qPCR) and normal RT-qPCR. This study highlights the role of m6A methylation in wheat resistance to WYMV, providing a solid basis for the potential functional role of m6A RNA methylation in wheat resistance to infection by RNA viruses.

Molecular Characterization of a Novel Wheat-infecting Virus in Family Betaflexiviridae

Wheat plants showing yellow mosaic in leaves and stunting were observed and collected from wheat fields in the Henan Province, China. Analyses of these plants through transmission electron microscopy showed that these plants contained two filamentous virus-like particles of 200–500 nm and 1000–1300 nm long, respectively. RNA-seq result unveiled a co-infection of wheat yellow mosaic virus (WYMV) and an unknown wheat-infecting virus. The complete genome sequence of the unknown virus is 8,410 nucleotide long, excluding its 3’ end poly (A) tail. This unknown virus has six open reading frames (ORFs). The ORF1 encodes a putative viral replication-associated protein, and the ORF2, 3 and 4 encode the triple gene block (TGB) proteins. The ORF5 and 6 encode the capsid protein (CP) and a protein with unknown function, respectively. Phylogenetic relationship analyses showed that this novel virus is evolutionarily related to viruses in the subfamily Quinvirinae, family Betaflexiviridae. It is, however, distinctly different from the viruses in other genera. Based on the species and genus demarcation criteria set by the International Committee on Taxonomy of Viruses (ICTV), we tentatively name this novel virus as wheat yellow stunt-associated betaflexivirus (WYSaBV). We also propose WYSaBV as a new member in a new genus in the family Betaflexiviridae.

A novel type of IRES having variable numbers of eIF4E-binding site and synergistic effect in RNA2 of WYMV isolates

Some viral proteins were translated in cap-independent manner via internal ribosome entry site (IRES), which ever maintained conservative characteristic among different isolates of same species of virus. However, IRES activity presented 7-fold of variance in RNA2 of wheat yellow mosaic virus (WYMV) HC and LYJN isolates. Based on RNA structure probing and mutagenesis assay, the loosened middle stem of H1 and hepta-nucleotide top loop of H2 in LYJN isolate synergistically ensured the higher IRES activity than that in HC isolate. In addition, the conserved top loop of H1 ensured basic IRES activity in HC and LYJN isolates. RNA2 5′-UTR specifically interacted with the wheat eIF4E, which was accomplished by the top loop of H1 in HC isolate or the top loop of H1 and H2 in LYJN isolate. Different IRES activity of WYMV RNA2 was regulated by different numbers of eIF4E-binding site and their synergistic effect, which was accomplished by the proximity of H1 and H2 due to the flexibility of middle stem in H1. It is represented a novel evolution pattern of IRES.

Genome-Wide Identification and Expression Analysis of the Histone Deacetylase Gene Family in Wheat (Triticum aestivum L.)

Histone acetylation is a dynamic modification process co-regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Although HDACs play vital roles in abiotic or biotic stress responses, their members in Triticumaestivum and their response to plant viruses remain unknown. Here, we identified and characterized 49 T. aestivumHDACs (TaHDACs) at the whole-genome level. Based on phylogenetic analyses, TaHDACs could be divided into 5 clades, and their protein spatial structure was integral and conserved. Chromosomal location and synteny analyses showed that TaHDACs were widely distributed on wheat chromosomes, and gene duplication has accelerated the TaHDAC gene family evolution. The cis-acting element analysis indicated that TaHDACs were involved in hormone response, light response, abiotic stress, growth, and development. Heatmaps analysis of RNA-sequencing data showed that TaHDAC genes were involved in biotic or abiotic stress response. Selected TaHDACs were differentially expressed in diverse tissues or under varying temperature conditions. All selected TaHDACs were significantly upregulated following infection with the barley stripe mosaic virus (BSMV), Chinese wheat mosaic virus (CWMV), and wheat yellow mosaic virus (WYMV), suggesting their involvement in response to viral infections. Furthermore, TaSRT1-silenced contributed to increasing wheat resistance against CWMV infection. In summary, these findings could help deepen the understanding of the structure and characteristics of the HDAC gene family in wheat and lay the foundation for exploring the function of TaHDACs in plants resistant to viral infections.