Global analyses of small interfering RNAs derived from Bamboo mosaic virus and its associated satellite RNAs in different plants

Abstract

Background: Satellite RNAs (satRNAs), virus parasites, are exclusively associated with plant virus infection and have attracted much interest over the last 3 decades. Upon virus infection, virus-specific small interfering RNAs (vsiRNAs) are produced by dicer-like (DCL) endoribonucleases for anti-viral defense. The composition of vsiRNAs has been studied extensively; however, studies of satRNA-derived siRNAs (satsiRNAs) or siRNA profiles after satRNA co-infection are limited. Here, we report on the small RNA profiles associated with infection with Bamboo mosaic virus (BaMV) and its two satellite RNAs (satBaMVs) in Nicotiana benthamiana and Arabidopsis thaliana.

Methodology/principal findings: Leaves of N. benthamiana or A. thaliana inoculated with water, BaMV alone or co-inoculated with interfering or noninterfering satBaMV were collected for RNA extraction, then large-scale Solexa sequencing. Up to about 20% of total siRNAs as BaMV-specific siRNAs were accumulated in highly susceptible N. benthamiana leaves inoculated with BaMV alone or co-inoculated with noninterfering satBaMV; however, only about 0.1% of vsiRNAs were produced in plants co-infected with interfering satBaMV. The abundant region of siRNA distribution along BaMV and satBaMV genomes differed by host but not by co-infection with satBaMV. Most of the BaMV and satBaMV siRNAs were 21 or 22 nt, of both (+) and (-) polarities; however, a higher proportion of 22-nt BaMV and satBaMV siRNAs were generated in N. benthamiana than in A. thaliana. Furthermore, the proportion of non-viral 24-nt siRNAs was greatly increased in N. benthamiana after virus infection.

Conclusions/significance: The overall composition of vsiRNAs and satsiRNAs in the infected plants reflect the combined action of virus, satRNA and different DCLs in host plants. Our findings suggest that the structure and/or sequence demands of various DCLs in different hosts may result in differential susceptibility to the same virus. DCL2 producing 24-nt siRNAs under biotic stresses may play a vital role in the antiviral mechanism in N. benthamiana.

Figure 1. Accumulation of BaMV, satBaMV, and siRNAs in leaves of N. benthamiana and A. thaliana.

Total RNA of 2.5 µg from N. benthamiana or 5 µg from A. thaliana were analyzed by northern blot analysis. The BaMV genomic (6.4 Kb), two subgenomic RNAs (2.0 and 1.0 Kb) and vsiRNAs were detected by a BaMV-specific probe and satRNA and satsiRNAs by a satBaMV-specific probe. M: mock (water) inoculation; −: BaMV alone. F4: noninterfering BSF4 satBaMV; L6: interfering BSL6 satBaMV. All films were exposed overnight except for detection of BaMV in N. benthamiana systemic leaves infected with BaMV or co-infected with BSF4 and BSL6 (3-hr exposure).

Figure 2. Size distribution of siRNAs derived from BaMV or satBaMV-co-inoculated N. benthamiana.

The total siRNAs were isolated from BaMV-inoculated (I) and systemic (S) leaves, BaMV and BSF4 co-inoculated (I) and systemic (S) leaves, and BaMV and BSL6 inoculated leaves (I). (A) siRNAs matched to positive-strand (+) BaMV (left panel) or negative-strand (−) BaMV (right panel). (B) siRNAs matched to satBaMV (+) (left panel) or satBaMV (−) (right panel). Y axis represents the counts of siRNAs; X axis represents the length of siRNAs. The relative percentages of siRNAs of 21 and 22 nt to total siRNAs are shown above the bars.

Figure 3. Size distribution of total small RNAs isolated from N. benthamiana.

Total sRNAs ranging from 17 to 28 nt are shown on the X axis and relative percentages are shown on the Y axis. Small RNAs were collected from mock (⧫), BaMV-inoculated leaves (▪) and systemic leaves (▴), BaMV and BSF4 co-inoculated leaves (×) and systemic leaves (*), and BaMV and BSL6 co-inoculated leaves (•) and systemic leaves (+). Inoculated (I) and systemic (S) leaves were harvested at 8 dpi and 20 dpi, respectively.

Figure 4. Comparison of the small RNAs between healthy and BaMV infected N. benthamiana.

Small RNAs were isolated from healthy (H) or BaMV infected (I) leaves and 5′-end labeled with [γ-³²P]-ATP. The 5′-end labeled small RNAs were separated by electrophoresis through a 10% acrylamide gel containing 7 M urea. The lower panel shows the quantity of equal loading by the separation of small RNAs on 1% agarose gel and stained with ethidium bromide. The positions of 22- and 24- nt RNAs are indicated by arrows.

Figure 5. The abundant distribution of siRNAs on BaMV and satBaMV genomes from infected N. benthamiana.

siRNAs derived from the viral genome of BaMV (A) and satBaMV (B) are shown in red above (positive strand) or green below (negative strand) the horizontal line. X axis represents the length of the genome, and Y axis the counts of the siRNAs.

Figure 6. Size distribution of total small RNAs isolated from A. thaliana.

Total sRNAs ranging from 17 to 28 nt are shown on the X axis, and relative percentages are shown on the Y axis. The percentages of sRNAs of different lengths of mock (♦), BaMV (■), and BaMV co-inoculated with BSF4 (▲) or BSL6 (×) plants are shown.

Figure 7. The distribution of siRNAs on the BaMV (A) and satBaMV (B) genome from A. thaliana.

The siRNAs are shown in red above (positive strand) or in green below (negative strand) the horizontal line. X axis represents the length of the genome, and Y axis represents the counts of the siRNAs.

Figure 8. Northern blot analyses of siRNAs derived from BaMV in N. benthamiana and A. thaliana.

Detection of BaMV siRNA to confirm the abundant regions in BaMV or satBaMV-co-inoculated N. benthamiana and A. thaliana by different probes as indicated. Total RNA of 25 µg from BaMV-inoculated or satBaMV-co-inoculated N. benthamiana or 45 µg from BaMV-inoculated or satBaMV-co-inoculated A. thaliana were loaded onto 19% acrylamide/7 M urea gel. The same blot was used for detection by different probes. All films were processed overnight, except A. thaliana probe with CP (+).

Figure 9. The distribution of siRNAs from sense and antisense BSF4 5′ (A) and 3′ (B) UTRs.

The 21, 22 and 24 nt siRNAs derived from the genome of satBaMV BSF4 are shown in yellow, green and orange above (positive strand) or below (negative strand) the horizontal line, respectively. X axis represents the length of the genome, and Y axis represents the counts of the siRNAs.