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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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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).
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pone-0011928-g001: 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).

Mentions: Viruses can induce post-transcriptional gene silencing (PTGS) as the antiviral mechanism in plants [1], [3], [38], [39], and subviral agents, such as viroids or satRNAs, can also trigger PTGS to produce small RNAs [40], [41]. N. benthamiana is highly susceptible to BaMV and satBaMV, which can move systemically and cause mosaic symptoms. To understand the association of the accumulation of BaMV and satBaMVs and their derived small RNAs, N. benthamiana was mechanically inoculated with BaMV and a noninterfering satBaMV, BSF4, or an interfering satBaMV, BSL6. The inoculated and systemic leaves were harvested at 8 and 20 days post-inoculation (dpi), respectively, for detection of the level of BaMV and satBaMV RNAs, vsiRNAs and satsiRNAs by northern blot analysis. As shown in Figure 1A and previously [35], [36], [42], BaMV accumulation was slightly reduced in the inoculated leaves of N. benthamiana on co-inoculation with non-interfering BSF4 satBaMV; however, BaMV accumulation was greatly reduced on co-inoculation with interfering BSL6 satBaMV. In BaMV-inoculated or BSF4 co-inoculated N. benthamiana, BaMV siRNAs were barely detected in the inoculated leaves, and BaMV siRNAs were not detected in the BSL6 co-inoculated leaves (Figure 1A). In contrast, a significant amount of BaMV siRNAs were detected, and the level was associated with the accumulation of BaMV, in the systemic leaves of BaMV-infected or BSF4-co-infected N. benthamiana (Figure 1B). The level of siRNAs derived from BSF4 was substantial in the co-inoculated and systemic leaves; however, neither BaMV nor satBaMV siRNAs could be detected in BaMV-infected and BSL6-co-infected inoculated or systemic leaves of N. benthamiana (Figures 1A and 1B), probably because of the low accumulation of both BaMV and satBaMV in the BSL6-co-inoculated plants. These results show that the levels of vsiRNAs and satsiRNAs are well associated with BaMV and satBaMV accumulation in inoculated and systemic leaves of infected N. benthamiana.


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)

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).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2914070&req=5

pone-0011928-g001: 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).
Mentions: Viruses can induce post-transcriptional gene silencing (PTGS) as the antiviral mechanism in plants [1], [3], [38], [39], and subviral agents, such as viroids or satRNAs, can also trigger PTGS to produce small RNAs [40], [41]. N. benthamiana is highly susceptible to BaMV and satBaMV, which can move systemically and cause mosaic symptoms. To understand the association of the accumulation of BaMV and satBaMVs and their derived small RNAs, N. benthamiana was mechanically inoculated with BaMV and a noninterfering satBaMV, BSF4, or an interfering satBaMV, BSL6. The inoculated and systemic leaves were harvested at 8 and 20 days post-inoculation (dpi), respectively, for detection of the level of BaMV and satBaMV RNAs, vsiRNAs and satsiRNAs by northern blot analysis. As shown in Figure 1A and previously [35], [36], [42], BaMV accumulation was slightly reduced in the inoculated leaves of N. benthamiana on co-inoculation with non-interfering BSF4 satBaMV; however, BaMV accumulation was greatly reduced on co-inoculation with interfering BSL6 satBaMV. In BaMV-inoculated or BSF4 co-inoculated N. benthamiana, BaMV siRNAs were barely detected in the inoculated leaves, and BaMV siRNAs were not detected in the BSL6 co-inoculated leaves (Figure 1A). In contrast, a significant amount of BaMV siRNAs were detected, and the level was associated with the accumulation of BaMV, in the systemic leaves of BaMV-infected or BSF4-co-infected N. benthamiana (Figure 1B). The level of siRNAs derived from BSF4 was substantial in the co-inoculated and systemic leaves; however, neither BaMV nor satBaMV siRNAs could be detected in BaMV-infected and BSL6-co-infected inoculated or systemic leaves of N. benthamiana (Figures 1A and 1B), probably because of the low accumulation of both BaMV and satBaMV in the BSL6-co-inoculated plants. These results show that the levels of vsiRNAs and satsiRNAs are well associated with BaMV and satBaMV accumulation in inoculated and systemic leaves of infected N. benthamiana.

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