Differential Characteristics of Viral siRNAs between Leaves and Roots of Wheat Plants Naturally Infected with Wheat Yellow Mosaic Virus, a Soil-Borne Virus

Abstract

RNA silencing is an important innate antiviral defense in plants. Soil-borne plant viruses naturally infect roots via soil-inhabiting vectors, but it is unclear how antiviral RNA silencing responds to virus infection in this particular tissue. In this study, viral small interfering RNA (siRNA) profiles from leaves and roots of wheat plants naturally infected with a soil-borne virus, wheat yellow mosaic virus (WYMV, genus Bymovirus), were analyzed by deep sequencing. WYMV siRNAs were much more abundant in roots than leaves, which was positively correlated with the accumulation of viral RNA. WYMV siRNAs in leaves and roots were predominantly 21- and 22-nt long and equally derived from the positive- and negative-strands of the viral genome. WYMV siRNAs from leaves and roots differed in distribution pattern along the viral genome. Interestingly, compared to siRNAs from leaves (and most other reports), those from roots obviously had a lower A/U bias at the 5'-terminal nucleotide. Moreover, the expression of Dicer-like genes upon WYMV infection were differently regulated between leaves and roots. Our data suggest that RNA silencing in roots may operate differently than in leaves against soil-borne virus invasion.

Keywords: antiviral RNA silencing; deep sequencing; soil-borne plant viruses; viral small interfering RNA; wheat yellow mosaic virus.

FIGURE 1

Abundance of WYMV RNA and siRNAs in leaves and roots of wheat plants. (A) RT-qPCR analysis of WYMV RNA accumulation. (B) Abundance of WYMV siRNAs. (C,D) Size distribution of WYMV siRNAs. “-” and “+” indicate siRNAs derived respectively from the complementary (negative) or positive viral genomic strands. Asterisk indicates significant difference at P < 0.01 (one-way ANOVA).

FIGURE 2

Distribution of WYMV siRNAs (21 and 22 nt) along the viral genome. (A) A schematic presentation of the WYMV genome. Boxes indicate the open reading frame for the polyproteins. P3, P3 protein; 7k, 7 kDa protein; CI, cylindrical inclusion protein; 14k, 14 kDa protein; VPg, genome-linked viral protein; NIa-Pro, nuclear inclusion protein a-proteinase; Nib, nuclear inclusion protein b; CP, coat protein; A(n), polyA. (B,C) Distribution of WYMV siRNAs (21 and 22 nt) from leaf and root samples (three replicates of each) along the WYMV genome. Color coding indicates vsiRNAs derived, respectively, from the positive (+) and negative viral genomic strands (–). All siRNA reads in this analysis were redundant and normalized.

FIGURE 3

5′-Terminal nucleotide profile of WYMV siRNAs. (A,B) Distribution patterns of the 5′-terminal nucleotide of WYMV siRNAs. (C) Sequence logo analysis of WYMV siRNAs. 21- and 22-nt WYMV siRNAs corresponding to RNA1 were separately analyzed, and the 4 nt proximal to the 5′ and 3′ ends of the siRNAs in the viral genome sequence were included in the analysis. The overall height of the stack indicates the sequence conservation at that position, while the height of characters within the stack indicates the relative frequency of nucleotide at that position.

FIGURE 4

Relative transcript expressions of wheat AGO1, AGO2, AGO4, DCL2, and DCL4 upon WYMV infection. Three biological replicates were performed in this experiment. Data are means ± SD (n = 3). Asterisk indicates significant difference at P < 0.01 (one-way ANOVA).