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

Lin KY, Cheng CP, Chang BC, Wang WC, Huang YW, Lee YS, Huang HD, Hsu YH, Lin NS - PLoS ONE (2010)

Bottom Line: 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.DCL2 producing 24-nt siRNAs under biotic stresses may play a vital role in the antiviral mechanism in N. benthamiana.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biotechnology, National Cheng Kung University, Tainan, Taiwan.

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.

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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 (+).
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pone-0011928-g008: 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 (+).

Mentions: The abundant regions of siRNAs derived from BaMV were further confirmed by probes hybridizing to sense and anti-sense ORF1 and CP regions of the viral genome, respectively. In this experiment, RNA samples of BaMV-infected or BaMV- and satBaMV-co-infected systemic leaves of N. benthamiana at 20 dpi and inoculated leaves of A. thaliana at 7 dpi were hybridized with different probes by northern blot analyses. Although different probes may result in different intensities, the signals obtained in the northern hybridization (Figure 8) and the abundance of BaMV siRNAs (Figure 5) showed the same trend. In the systemic leaves of N. benthamiana, the CP probes showed stronger signals than did the ORF1 probes, and the CP (+) probe detected more than did the CP (−) probe, which indicates that vsiRNAs derived from the (−) strand of the CP region were indeed more abundant. All these results matched the findings for the siRNAs analyzed along the genome (Figure 5A, BaMV and BaMV+F4). As well, the CP probes detected fewer siRNAs than did the ORF1 probes in A. thaliana inoculated leaves. This finding agreed well with the deep sequencing results, whereby most of the BaMV siRNAs were located at the ORF1 region in A. thaliana (Figures 7 and 8).


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

Lin KY, Cheng CP, Chang BC, Wang WC, Huang YW, Lee YS, Huang HD, Hsu YH, Lin NS - PLoS ONE (2010)

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 (+).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2914070&req=5

pone-0011928-g008: 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 (+).
Mentions: The abundant regions of siRNAs derived from BaMV were further confirmed by probes hybridizing to sense and anti-sense ORF1 and CP regions of the viral genome, respectively. In this experiment, RNA samples of BaMV-infected or BaMV- and satBaMV-co-infected systemic leaves of N. benthamiana at 20 dpi and inoculated leaves of A. thaliana at 7 dpi were hybridized with different probes by northern blot analyses. Although different probes may result in different intensities, the signals obtained in the northern hybridization (Figure 8) and the abundance of BaMV siRNAs (Figure 5) showed the same trend. In the systemic leaves of N. benthamiana, the CP probes showed stronger signals than did the ORF1 probes, and the CP (+) probe detected more than did the CP (−) probe, which indicates that vsiRNAs derived from the (−) strand of the CP region were indeed more abundant. All these results matched the findings for the siRNAs analyzed along the genome (Figure 5A, BaMV and BaMV+F4). As well, the CP probes detected fewer siRNAs than did the ORF1 probes in A. thaliana inoculated leaves. This finding agreed well with the deep sequencing results, whereby most of the BaMV siRNAs were located at the ORF1 region in A. thaliana (Figures 7 and 8).

Bottom Line: 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.DCL2 producing 24-nt siRNAs under biotic stresses may play a vital role in the antiviral mechanism in N. benthamiana.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biotechnology, National Cheng Kung University, Tainan, Taiwan.

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.

Show MeSH
Related in: MedlinePlus