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

Abstract

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.

Keywords: Betaflexiviridae; bymovirus; quinvirus; soil borne; variants; virome; wheat; yellow mosaic disease.

Figure 1

Reverse transcriptase PCR (RT-PCR) detection of viruses in wheat leaf RNA samples. Sample names are shown in the right-hand box. Lane 1 (pool-18L) and lane 11 (pool-19L) are two pooled RNA samples subjected to NGS analysis. Lanes 10 and 18 are RNA samples of mock-inoculated plants. KTH, cv. Kitahonami; KTN, cv. Kitanokaori; YM, cv. Yumechikara; and Flu, fluazinam treatment to the soil. The wheat 18S ribosomal RNA (rRNA) gene was used as an endogenous RNA reference. The primers used in the RT-PCR analyses are listed in Supplementary Table S1. Selected PCR products were sequenced to confirm the amplification of the virus targets. The PCR products were analyzed in 1.5% agarose gel electrophoresis. The gel was stained with ethidium bromide and viewed under UV light.

Figure 2

Virus sequences of novel quinvirus (wheat virus Q, WVQ) strains identified from the RNA sequencing (RNA-seq) of wheat leaves. The black lines in the genomic RNA show ORFs. The conserved domains in the WVQ replicase are shown (Mtr, methyltransferase; Pep, Peptidase_C23 superfamily; HEL, RNA helicase; and RdRp_2, RNA-dependent RNA polymerase). The entire genomic sequence of the WVQ strains (a and b) was obtained from the overlapping RT-PCR products and the 5ꞌ and 3ꞌ RACE products (see the double-arrowed or arrowed lines below each schematic genome of the WVQ strains for RT-PCR target regions). A genomic region of WVQ, as indicated by the double-arrowed red lines, was used for strain-specific detection by RT-PCR in Figures 1, 6. The primers used in the RT-PCR and RACE analyses are listed in Supplementary Table S1. Virus-sequence contigs from two separate total RNA pools of wheat leaf samples (18NGS, pool-18L; 19NGS: pool-19L) are shown. The lines marked with a black filled-circle are the contigs listed in Supplementary Table S2, and they were used for constructions of the WVQ genomic sequences. Some unlisted contigs in Supplementary Table S2 were also used for the construction of strain a sequence. The lines marked with an open circle were potentially derived from the variant(s) of WVQ strain a that have sequences slightly different from the reference strain a. The read depth coverage throughout the WVQ genomes (strains a and b) is presented in Supplementary Figure S2C.

Figure 3

Pairwise comparison of the replicase (RdRP) encoded by quinviruses or their candidates. The results of pairwise comparisons are shown as a heatmap with each pairwise amino acid identity calculated using SDT ver. 1.2. The virus names with asterisks are representative members of the viral species.

Figure 4

Phylogenetic relationships of the quinviruses and other unassigned related viruses. A maximum likelihood phylogenetic tree was constructed using a MAFFT alignment of the full-length replicase amino acid sequences. Ambiguously aligned sequences were removed using Gblocks with the stringency levels lowered for all parameters. A model LG + I + G + F was selected as a best-fit model for the alignment (the 1,235 positions remaining in the input dataset). The tree with the highest log likelihood (−43,188,80) is shown. The tree was drawn using the midpoint rooting method. The virus names referring to plant viruses [genera Foveavirus, Carlavirus (selected), Robigovirus, and floating (proposed genera “Banmivirus” and “Sustrivirus”) or unassigned members of subfamily Quinvirinae, family Betaflexiviridae] are followed by the GenBank accession or Ref-seq numbers of their sequences. Virus names with asterisks show representative members of the viral species. The two betaflexiviruses – apple chlorotic leaf spot virus (genus Trichovirus) and apple stem grooving virus (genus Chapillovirus) – of subfamily Trivirinae (family Betaflexiviridae) were used as the outgroups. The scale bar represents amino acid distances. The numbers at the nodes in the tree are bootstrap support values following 100 iterations.

Figure 5

Viral-derived small RNA profiles of WYMV and WVQ in the wheat leaves (the pooled KTH-18-1 and −2 samples). (A) Proportion of plus (+)- and minus (−)-strand small RNAs sizes, 15–32 nt, are shown. (B) Proportions of 5ꞌ-terminal nucleotide sequences of viral-derived small RNAs in the wheat leaves. The composite bar graphs represent the percentage of the 5ꞌ-terminal nucleotide sequences for each size-class (20–24 nt). Other profiles of the viral-derived small RNAs are presented in Supplementary Figure S6.

Figure 6

(A) Transmission assay of WVQ. RT-PCR detection of viruses in the roots of wheat plants planted in WVQ and wheat yellow mosaic virus (WYMV)-infested soil. Wheat grains were sown in pots containing the field soil and grown under the growth-cabinet condition (below 16°C) or the greenhouse condition (non-controlled temperature). Three pots for each treatment were used. Total RNA fractions from the root samples were pooled according to the treatment and subjected to RT-PCR analysis with a common primer set for the WVQ strains. (B) RT-PCR detection of WVQ and WYMV in leaves or roots of wheat or rye plants. Sample names and their respective tissue origin are shown below the panel or in the bottom right. Lanes 3 and 8 are RNA samples of mock-inoculated plants. KTH, cv. Kitahonami; rye, cv. Fuyumidori. The wheat 18S rRNA gene was used as an endogenous RNA reference. The primers used in the RT-PCR analyses are listed in Supplementary Table S1. The PCR products were analyzed in 1.5% agarose gel electrophoresis. The gel was stained with ethidium bromide and viewed under UV light. Selected PCR products were sequenced to confirm the amplification of the virus targets.