Differential Synergistic Interactions Among Four Different Wheat-Infecting Viruses

Abstract

Field-grown wheat (Triticum aestivum L.) plants can be co-infected by multiple viruses, including wheat streak mosaic virus (WSMV), Triticum mosaic virus (TriMV), brome mosaic virus (BMV), and barley stripe mosaic virus (BSMV). These viruses belong to four different genera in three different families and are, hence, genetically divergent. However, the impact of potential co-infections with two, three, or all four of them on the viruses themselves, as well as the wheat host, has yet to be examined. This study examined bi-, tri-, and quadripartite interactions among these viruses in wheat for disease development and accumulation of viral genomic RNAs, in comparison with single virus infections. Co-infection of wheat by BMV and BSMV resulted in BMV-like symptoms with a drastic reduction in BSMV genomic RNA copies and coat protein accumulation, suggesting an antagonism-like effect exerted by BMV toward BSMV. However, co-infection of either BMV or BSMV with WSMV or TriMV led to more severe disease than singly infected wheat, but with a decrease or no significant change in titers of interacting viruses in the presence of BMV or BSMV, respectively. These results were in stark contrast with exacerbated disease phenotype accompanied with enhanced virus titers caused by WSMV and TriMV co-infection. Co-infection of wheat by WSMV, TriMV, and BMV or BSMV resulted in enhanced synergistic disease accompanied by increased accumulation of TriMV and BMV but not WSMV or BSMV. Quadripartite interactions in co-infected wheat by all four viruses resulted in very severe disease synergism, leading to the death of the most infected plants, but paradoxically, a drastic reduction in BSMV titer. Our results indicate that interactions among different viruses infecting the same plant host are more complex than previously thought, do not always entail increases in virus titers, and likely involve multiple mechanisms. These findings lay the foundation for additional mechanistic dissections of synergistic interactions among unrelated plant viruses.

Keywords: Triticum mosaic virus; barley stripe mosaic virus; brome mosaic virus; co-infection; synergistic interaction; virus–virus interaction; wheat; wheat streak mosaic virus.

FIGURE 1

Bipartite synergistic interactions among brome mosaic virus (BMV), barley stripe mosaic virus (BSMV), wheat streak mosaic virus (WSMV), and Triticum mosaic virus (TriMV) in wheat cv. Arapahoe. (A) Wheat leaves showing symptoms elicited by BMV, BSMV, WSMV, or TriMV in singly or doubly infected wheat at 10 and 30 days post-inoculation (dpi). Note that co-infection of wheat by BMV and BSMV elicited symptoms similar to those of BMV. (B–E) Absolute quantification of genomic RNA copies of BMV (B), BSMV (C), WSMV (D), and TriMV (E) in singly or doubly infected wheat at 10, 20, and 30 dpi by real-time RT-PCR. Histograms indicate accumulation of viral genomic RNA copies in 2.5 ng total RNA with standard error. Note that accumulation of BSMV in BMV + BSMV infected wheat was reduced at 20 and 30 dpi compared to BSMV-infected wheat. The same letters above the histograms indicate non-significant, whereas different letters indicate significance at p = 0.05. M, Mock-inoculation; BM, BMV; BS, BSMV; W, WSMV; T, TriMV.

FIGURE 2

The phenotypic effect of single and double infections of BMV, BSMV, WSMV, and TriMV on wheat cv. Arapahoe at 30 dpi. Phenotypic effects of singly and doubly infected wheat cv. Arapahoe by BMV and BSMV (A), WSMV and TriMV (B), WSMV and BMV (C), TriMV and BMV (D), WSMV and BSMV (E), and TriMV and BSMV (F) at 30 dpi. M, mock-inoculation; BM, BMV; BS, BSMV; W, WSMV; T, TriMV.

FIGURE 3

Brome mosaic virus repressed accumulation of BSMV in co-infected wheat. (A) Coomassie brilliant blue stained SDS-PAGE gel of total proteins from singly and doubly infected wheat at 10, 14, and 21 dpi. (B,C) Western blot analysis of total proteins extracted from wheat leaves infected by BMV, BSMV, or both at 10, 14, and 21 dpi with BMV- (B) and BSMV-specific (C) polyclonal antibodies. Lower panels (B,C) are Coomassie blue-stained SDS-PAGE gels showing the large subunit of wheat RuBisCo protein as a loading control for total protein loaded per well for immunoblot assay. Lanes 1, 8, and 12: mock-inoculated wheat; lanes 2, 5, and 9: BMV-infected wheat; lanes 3, 6, and 10: BSMV-infected wheat; and lanes 4, 7, and 11: BMV + BSMV-infected wheat. Lanes 1–4: 10 dpi; lanes 5–8: 14 dpi; and lanes 9–12: 21 dpi. Location of BMV and BSMV CPs were indicated with arrows and arrowheads, respectively. *The nature of this protein in “C” with BSMV antiserum from BMV-infected wheat is not known.

FIGURE 4

Tri- and tetrapartite synergistic interactions among WSMV, TriMV, BMV, and BSMV in wheat cv. Arapahoe. (A) Wheat leaves showing the symptom phenotype of individual and co-infections by WSMV, TriMV, and BMV or BSMV or both on wheat at 10, 20, and 30 dpi. Note that co-infection of wheat by BMV or BSMV with WSMV and TriMV elicited more severe symptoms compared to individual virus infections. Co-infection of wheat by all four viruses induced severe chlorosis and bleaching of leaves. (B–D) Effect of co-infection of wheat by triple [WSMV + TriMV + BMV (B) and WSMV + TriMV + BSMV (C)] or quadruple (D) viruses on wheat phenotype compared to singly infected plants. Wheat seedlings at the single leaf stage were inoculated singly or with a combination of viruses, and wheat plants at 30 dpi were uprooted and photographed. M, mock inoculation; W, WSMV; T, TriMV; BM, BMV; BS, BSMV.

FIGURE 5

Effect of co-infection of wheat by three (WSMV, TriMV, and BMV or BSMV) or four viruses (WSMV, TriMV, BMV, and BSMV) on genomic RNA accumulation of interacting viruses at 10, 20, and 30 dpi. Absolute quantification of genomic RNA copies of BMV (A), BSMV (B), WSMV (C), and TriMV (D) were determined by real-time RT-PCR. Histograms indicate accumulation of viral genomic RNA copies in 2.5 ng total RNA with standard error. The same letters above the histograms indicate non-significant, whereas different letters indicate significance at p = 0.05. BM, BMV; BS, BSMV; W, WSMV; T, TriMV.

FIGURE 6

Brome mosaic virus and BSMV affected the expression of WSMV and TriMV in co-infected wheat. Wheat seedlings at the single-leaf stage were inoculated with WSMV-RFP, TriMV-GFP, WSMV-RFP + TriMV-GFP, WSMV-RFP + BMV, WSMV-RFP + BSMV, TriMV-GFP + BMV, or TriMV-GFP + BSMV. Fluorescent images of wheat leaves infected singly or doubly in combination with WSMV-RFP or TriMV-GFP were observed with an RFP filter (A) or GFP filter (B), respectively, under a Stereo Discovery V12 fluorescent microscope at 14 days post-inoculation. W-R, RFP-tagged WSMV (WSMV-RFP); T-G, GFP-tagged TriMV (TriMV-GFP); BM, BMV; BS, BSMV. Note reduced fluorescent protein in wheat co-infected by BMV and WSMV-RFP or TriMV-GFP. Compare images W-R + BM with W-R + T-G and T-G + BM with T-G + W-R. Bars represent 500 μm.