Dynamics of small RNA profiles of virus and host origin in wheat cultivars synergistically infected by Wheat streak mosaic virus and Triticum mosaic virus: virus infection caused a drastic shift in the endogenous small RNA profile

Abstract

Co-infection of wheat (Triticum aestivum L.) by Wheat streak mosaic virus (WSMV, a Tritimovirus) and Triticum mosaic virus (TriMV, a Poacevirus) of the family Potyviridae causes synergistic interaction. In this study, the effects of the synergistic interaction between WSMV and TriMV on endogenous and virus-derived small interfering RNAs (vsiRNAs) were examined in susceptible ('Arapahoe') and temperature-sensitive resistant ('Mace') wheat cultivars at 18°C and 27°C. Single and double infections in wheat caused a shift in the profile of endogenous small RNAs from 24 nt being the most predominant in healthy plants to 21 nt in infected wheat. Massive amounts of 21 and 22 nt vsiRNAs accumulated in singly and doubly infected Arapahoe at both temperatures and in Mace at 27°C but not 18°C. The plus- and minus-sense vsiRNAs were distributed throughout the genomic RNAs in Arapahoe at both temperature regimens and in Mace at 27°C, although some regions served as hot-spots, spawning an excessive number of vsiRNAs. The vsiRNA peaks were conserved among cultivars, suggesting that the Dicer-like enzymes in susceptible and resistant cultivars similarly accessed the genomic RNAs of WSMV or TriMV. Accumulation of large amounts of vsiRNAs in doubly infected plants suggests that the silencing suppressor proteins encoded by TriMV and WSMV do not prevent the formation of vsiRNAs; thus, the synergistic effect observed is independent from RNA-silencing mediated vsiRNA biogenesis. The high-resolution map of endogenous and vsiRNAs from WSMV- and/or TriMV-infected wheat cultivars may form a foundation for understanding the virus-host interactions, the effect of synergistic interactions on host defense, and virus resistance mechanisms in wheat.

Figure 1. Accumulation of virus-derived small interfering RNAs (vsiRNAs) of plus polarity in WSMV-, TriMV- or WSMV plus TriMV-infected wheat cultivars Arapahoe and Mace at 18°C and 27°C.

WSMV and TriMV minus-strand RNA-specific DIG-labeled riboprobes complementary to nucleotides 9384 to 6303 and 10,266 to 6401, respectively, were used for Northern blot hybridization. Riboprobes hydrolyzed to ∼50 nt were used for Northern hybridization of small RNAs. Ethidium bromide stained TBE-urea polyacrylamide gels showing low molecular weight (LMW) RNAs as sample loading control are presented under the Northern blots. Ar: Wheat cv. Arapahoe; Ma: Wheat cv. Mace; WS: WSMV; Tri: TriMV; Bu: buffer.

Figure 2. Size distribution of 20 to 24 nt virus- and host-specific small RNAs from WSMV-, TriMV-, or WSMV plus TriMV-infected wheat cvs. Arapahoe and Mace at 18°C and 27°C.

A dramatic shift in accumulation of 24 nt endogenous small RNAs (sRNAs) as predominant in healthy samples to 21 nt in singly and doubly infected wheat cvs. Arapahoe at 18°C and 27°C and Mace at 27°C is evident. No shift in predominant sRNA accumulation was observed in wheat cv. Mace at 18°C. SI: single infection; DI: Double infection; A: Arapahoe at 27°C; B: Arapahoe at 18°C; C: Mace at 27°C; D: Mace at 18°C.

Figure 3. Percent polarity distribution of 21 and 22 nt virus-derived small interfering RNAs (vsiRNAs) in WSMV-, TriMV-, and WSMV plus TriMV-infected wheat cvs. Arapahoe and Mace at 18°C and 27°C.

SI: single infection; DI: double infection; A: Arapahoe at 27°C; B: Arapahoe at 18°C; C: Mace at 27°C; D: Mace at 18°C.

Figure 4. Histograms showing the 5′-terminal nucleotide of 21- and 22 nt redundant (R) and nonredundant (NR) vsiRNAs in WSMV-, TriMV-, and WSMV plus TriMV-infected wheat cvs. Arapahoe and Mace at 18°C and 27°C.

SI: single infection; DI: double infection. A: Arapahoe at 27°C; B: Arapahoe at 18°C; C: Mace at 27°C; D: Mace at 18°C.

Figure 5. Distribution of 21 to 24 nt redundant vsiRNAs throughout the genomes of WSMV and TriMV in wheat cv. Arapahoe and Mace at 18°C and 27°C.

The vsiRNA reads greater than 50,000 were considered as hot-spots. Note that a different scale was used for vsiRNAs in Mace at 18°C.