Plant virus-mediated induction of miR168 is associated with repression of ARGONAUTE1 accumulation
2010; Springer Nature; Volume: 29; Issue: 20 Linguagem: Inglês
10.1038/emboj.2010.215
ISSN1460-2075
AutoresÉva Várallyay, Anna Válóczi, Ákos Ágyi, József Burgyán, Zoltán Havelda,
Tópico(s)Plant tissue culture and regeneration
ResumoArticle7 September 2010free access Plant virus-mediated induction of miR168 is associated with repression of ARGONAUTE1 accumulation Éva Várallyay Éva Várallyay Agricultural Biotechnology Center, Plant Virology and Bioinformatics Group, Gödöllő, Hungary Search for more papers by this author Anna Válóczi Anna Válóczi Department of Stem Cell Biology, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Ákos Ágyi Ákos Ágyi Agricultural Biotechnology Center, Plant Virology and Bioinformatics Group, Gödöllő, Hungary Search for more papers by this author József Burgyán József Burgyán Agricultural Biotechnology Center, Plant Virology and Bioinformatics Group, Gödöllő, Hungary Istituto di Virologia Vegetale, CNR Strada delle Cacce, Torino, Italy Search for more papers by this author Zoltán Havelda Corresponding Author Zoltán Havelda Agricultural Biotechnology Center, Plant Virology and Bioinformatics Group, Gödöllő, Hungary Search for more papers by this author Éva Várallyay Éva Várallyay Agricultural Biotechnology Center, Plant Virology and Bioinformatics Group, Gödöllő, Hungary Search for more papers by this author Anna Válóczi Anna Válóczi Department of Stem Cell Biology, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Ákos Ágyi Ákos Ágyi Agricultural Biotechnology Center, Plant Virology and Bioinformatics Group, Gödöllő, Hungary Search for more papers by this author József Burgyán József Burgyán Agricultural Biotechnology Center, Plant Virology and Bioinformatics Group, Gödöllő, Hungary Istituto di Virologia Vegetale, CNR Strada delle Cacce, Torino, Italy Search for more papers by this author Zoltán Havelda Corresponding Author Zoltán Havelda Agricultural Biotechnology Center, Plant Virology and Bioinformatics Group, Gödöllő, Hungary Search for more papers by this author Author Information Éva Várallyay1, Anna Válóczi2, Ákos Ágyi1, József Burgyán1,3 and Zoltán Havelda 1 1Agricultural Biotechnology Center, Plant Virology and Bioinformatics Group, Gödöllő, Hungary 2Department of Stem Cell Biology, University of Heidelberg, Heidelberg, Germany 3Istituto di Virologia Vegetale, CNR Strada delle Cacce, Torino, Italy *Corresponding author. Plant Virology and Bioinformatics Group, Agricultural Biotechnology Center (ABC), Szent Györgyi A. ut 4., Gödöllő H-2100, Hungary. Tel.: +36 28526155; Fax: +36 25526145; E-mail: [email protected] The EMBO Journal (2010)29:3507-3519https://doi.org/10.1038/emboj.2010.215 Correction(s) for this article Plant virus-mediated induction of miR168 is associated with repression of ARGONAUTE1 accumulation01 June 2017 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Virus infections induce the expression of ARGONAUTE1 (AGO1) mRNA and in parallel enhance the accumulation of miR168 (regulator of AGO1 mRNA). Here, we show that in virus-infected plants the enhanced expression of AGO1 mRNA is not accompanied by increased AGO1 protein accumulation. We also show that the induction of AGO1 mRNA level is a part of the host defence reaction, whereas the induction of miR168, which overlaps spatially with virus-occupied sectors, is mediated mainly by the Tombusvirus p19 RNA-silencing suppressor. The absence of p19 results in the elimination of miR168 induction and accompanied with the enhanced accumulation of AGO1 protein. In transient expression study, p19 mediates the induction of miR168 and the down-regulation of endogenous AGO1 level. P19 is not able to efficiently bind miR168 in virus-infected plants, indicating that this activity is uncoupled from the small RNA-binding capacity of p19. Our results imply that plant viruses can inhibit the translational capacity of AGO1 mRNA by modulating the endogenous miR168 level to alleviate the anti-viral function of AGO1 protein. Introduction During virus infection, siRNAs are generated from viral double-stranded RNA products and secondary RNA structures by the activity of RNase-III ribonuclease Dicer-like (DCL) proteins (Molnar et al, 2005; Ding and Voinnet, 2007). Virus-specific siRNAs are then incorporated into the RNA-induced-silencing complex (RISC) determining its specificity and bringing about the degradation of complementary viral RNAs (Pantaleo et al, 2007). RNA-silencing suppressors of viruses can inhibit this mechanism at different steps by biding to viral siRNAs, double-stranded RNAs or directly interacting with AGO1 (Merai et al, 2006; Burgyan, 2008; Mlotshwa et al, 2008). Therefore, elimination of RNA-silencing suppressors from the virus-infection process can result in decreased virus accumulation and the development of the recovery phenotype (Qiu et al, 2002; Qu and Morris, 2002; Szittya et al, 2002). Plant miRNAs represent another RNA-silencing pathway and are indispensable for the control of wide variety of biological functions, including development, hormone responses, feedback mechanisms and biotic and abiotic stresses (Voinnet, 2009). MiRNAs are generated by sequential processing of genome-coded long single-stranded RNA precursor molecules possessing specific secondary structures by DCL1 and other factors. DCL1 processing liberates the miRNA/miRNA* duplex and the selected strand (the miRNA) is exported to the cytoplasm, whereas the miRNA* strand is usually degraded. In contrast, the generation of virus-specific siRNAs is mediated mainly by DCL4 and also by DCL2 (Ding and Voinnet, 2007). Small RNAs generated by the activity of DCL enzymes are incorporated into ARGONAUTE (AGO) proteins, which are the central components of the RISC complex. AGO1, the most important AGO protein in the miRNA pathway, is responsible for the cleavage or translational inhibition of target RNAs determined by the loaded miRNA (Mallory and Bouche, 2008; Mallory et al, 2008). As a feedback mechanism, AGO1 homeostasis itself is controlled by coordinated action of miR168 (Rhoades et al, 2002; Vaucheret et al, 2004) and AGO1-derived siRNAs (Mallory and Vaucheret, 2009) on AGO1 mRNA. Moreover, additional components of the miRNA-mediated feedback regulation of AGO1 have been also described involving the AGO1-mediated post-transcriptional stabilization of miR168 and the co-regulated expression of AGO1 and MIR168 genes (Vaucheret et al, 2006). These results show the existence of a complex refined feedback regulatory loop, which balances AGO1 and miR168 accumulation. Analyses of affinity-purified AGO1 revealed that it is preferably associated with miRNAs and mediates target mRNA cleavage (Baumberger and Baulcombe, 2005; Qi et al, 2005; Mi et al, 2008). However, AGO1 also recruits virus-specific siRNAs and is involved in RNA-silencing-mediated defence mechanisms (Zhang et al, 2006). It was also shown that virus-specific siRNAs of Cymbidum ringspot virus (CymRSV) and miRNAs co-fractionate with large protein complexes, which contain AGO1 likely corresponding to RISC complexes (Pantaleo et al, 2007; Csorba et al, 2010). Moreover, it was shown that cucumber mosaic virus (CMV) 2b RNA-silencing suppressor directly interacts with siRNA-loaded AGO1 inhibiting its slicing activity (Zhang et al, 2006). P0 RNA-silencing suppressor of a Polerovirus mediates the targeted degradation of AGO1 (Pazhouhandeh et al, 2006; Baumberger et al, 2007; Bortolamiol et al, 2007; Csorba et al, 2010). In line with these observations, it was found that ago1 hypomorphic mutant was more susceptible to CMV infection (Morel et al, 2002). This mutant showed also increased susceptibility to coat protein (CP) deletion mutant turnip crinkle virus (TCV), which is compromised also in RNA-silencing suppressor function (Qu et al, 2008). Plant virus infections are often associated with changes in endogenous miRNA levels (Bazzini et al, 2007; Csorba et al, 2007). RNA-silencing suppressors of plant viruses have been shown to be responsible for altering endogenous miRNA levels inducing changes also in target mRNA accumulations (Kasschau et al, 2003; Dunoyer et al, 2004; Zhang et al, 2006). Moreover, several recent works described the enhanced expression of miR168 and AGO1 mRNA in virus-infected plants (Zhang et al, 2006; Csorba et al, 2007; Havelda et al, 2008). As AGO1 is one of the central components of RNA-silencing-mediated host defence, we investigated the regulation of AGO1 and miR168 expressions and their function during virus infections. Here, we show the specific induction of miR168 accumulation in CymRSV-infected plants, which spatially overlaps with virus-occupied sectors. The enhanced accumulation of miR168 was also characteristic for all the other plant–virus interactions investigated in this study, indicating its importance in virus-infection process. Moreover, we show that in virus-infected plants the elevated level of miR168 is associated with the induced AGO1 mRNA expression but reduced AGO1 protein accumulation, indicating that miR168-mediated translational repression mechanism might be responsible for the control of AGO1 level. We also show that p19 RNA-silencing suppressor of Tombusviruses is responsible for the specific induction of miR168 and this activity is independent from the RNA-binding capacity of p19. On the basis of our results, we propose a new layer in the combat between plants and viruses in which the virus-induced expression of miR168 controls the accumulation of AGO1 limiting the effect of host-mounted RNA-silencing defence. Results Induction of miR168 is ubiquitous in plant–virus interactions Using the CymRSV—Nicotiana benthamiana—system, we investigated the induction of miR168 relative to other miRNAs. Small RNA northern blot analyses of RNA samples derived from symptomatic systemically infected leaves at 4 days post-inoculation (dpi) revealed that CymRSV infections induced elevated accumulation of miR168 compared with the mock-infected plants (Figure 1A). The induction of miR168 was not a part of a general response at the level of miRNA expressions to the virus infection as the level of other miRNAs (miR159, miR171 and miR319) remained unchanged. We experienced similar results using N. benthamiana test plants infected with crucifer-infecting tobacco mosaic virus (crTMV), potato virus X (PVX) and tobacco etch virus (TEV) (Figure 1B). In all cases, we observed markedly increased accumulation of miR168 in virus-infected plants, whereas no or moderate changes occurred in the level of miR159 in the same samples. TEV infection was an exception as miR159 also showed increased accumulation as described previously (Bazzini et al, 2007). Next, we tested several other plant–virus interactions for the accumulation of miR168 (Figure 1C). In Arabidopsis thaliana, both TCV and ribbgrass mosaic virus (RMV) infections induced miR168 expression. Sunn-hemp mosaic virus (SHMV) infection on Medicago truncatula and TMV U1 and PVX infections on Solanum lycopersicum were also accompanied with drastic induction of miR168. The induction of miR168 was not a general unspecific answer to these infections as miR159 level did not change significantly or in line with previous data showed only moderate increases in Tobamovirus (RMV and SHMV)-infected plants (Csorba et al, 2007) in the investigated time points. Figure 1.Induction of miR168 is ubiquitous in plant–virus interactions. Total RNAs were prepared from systemically infected leaves of different host plants or mock-inoculated tissues and used for small RNA northern blot analyses using LNA probes for miRNAs as indicated. (A) N. benthamiana infected with CymRSV. (B) N. benthamiana infected with distinct unrelated viruses. (C) Investigation of various host–virus interactions. Relative gel loadings and viral accumulations are indicated by ethidium bromide staining of ribosomal RNAs (rRNA) and viral genomic RNAs (v). Download figure Download PowerPoint These results show that infection of the investigated viruses is always associated with the enhanced accumulation of miR168 irrespective of their various ability to interfere with the expression of other endogenous miRNAs. The general feature of miR168 over-accumulation implies its important function in the virus-infection processes and/or host defence. Induction of miR168 spatially overlaps with virus accumulation and is linked to enhanced accumulation of pre-miR168a-loop intermediate As virus infections have strong effect on miR168 accumulation, we wanted to know how the accumulation of miR168 is spatially regulated in virus-infected plant. We performed in situ analyses on consecutive sections of young developing symptomatic leaf of CymRSV-infected N. benthamiana to detect the relative accumulation of viral RNA and miR168 (Figure 2A). We revealed that virus accumulation and the increased miR168 expression spatially overlapped. We did not detect any alteration in miR159 accumulation level at the virus-occupied regions showing that the enhanced miR168 level is a specific response to virus infections. This finding indicates that enhanced miR168 accumulation is directly activated by the viral-derived products and does not represent a general whole plant response. Figure 2.Analyses of miR168 expression in virus-infected plants. (A) Spatial analysis of the accumulation of virus-derived RNAs (CymRSV), miR168 and miR159 on consecutive sections (12 μm) of systemically infected N. benthamiana leaves. Cross-sections show the basal part of a young developing apical leaf. Section represented in the upper panel was hybridized with RNA probe detecting viral RNAs (CymRSV). The dark colour indicates the accumulation of viral RNAs showing the virus-occupied sector. Arrowheads point to the front of virus infection. Consecutive section hybridized with LNA probe detecting miR168 displays very similar expression pattern. The enhanced miR168 accumulation spatially overlaps with the virus-occupied zone. Arrowheads point to the border of increased miR168 accumulation. The next section was hybridized with miR159 detecting LNA probe and no altered miR159 expression was detected on the tissue section. Chicken-specific miR449 was used as negative control to detect the possible technical background signals. Bar shows 100 μm. (B) A. thaliana total RNA samples originated from mixed flowers and virus and mock-inoculated leaves were separated on polyacrylamide gel and used for northern blot analyses using RNA probe complementary to MIR168a precursor (upper panel). The membrane was stripped and used for subsequent hybridizations with LNA probes-specific mature miR168 and miR171 or DNA probe detecting U6 snRNA (loading control). Relative gel loadings are also indicated by ethidium bromide staining of ribosomal RNAs (rRNA). Download figure Download PowerPoint There are several possibilities, which can account for the increased accumulation of miR168 in virus-infected plants. Mechanisms such as the increased stability of miR168, transcriptional activation of the MIR168 precursor and/or the more efficient processing of the precursor RNAs can be responsible for enhanced miR168 accumulation. As recent data described the transcriptional activation of the promoter of MIR164a precursor in virus-infected plants (Bazzini et al, 2009), we also tested the accumulation of MIR168a precursor RNA. The MIR168 precursors of N. benthamiana are not known; therefore, we investigated the accumulation MIR168a precursor in virus-infected and -non-infected A. thaliana RNA samples by northern blot analyses (Figure 2B). We detected the same MIR168a precursor processing products, the 104nt stem-loop and 64nt-loop intermediates, which have been described in MIR168a-overexpressing transgenic plants (Vaucheret et al, 2006). We showed that in virus-infected leaves the loop intermediates accumulated to a much higher level than in mock-infected leaves. Surprisingly, the stem-loop intermediate did not show enhanced accumulation in virus-infected leaves. RNA sample originating from the flowers shows higher stem-loop intermediate accumulation level; however, this was not accompanied by proportionally higher accumulation levels of the loop intermediate and mature miR168 in comparison with virus-infected samples (Figure 2B). For example, in TCV-infected sample, less loop intermediate is present compared with flower sample; however, it is associated with higher level of mature miRNA accumulation. These findings suggest that in virus-infected plants the processing of the stem-loop intermediate to loop intermediate and mature miRNA is more efficient than in wild-type flowers in normal conditions. Another possibility is that the RNA-binding activity of RNA-silencing suppressors can interfere with the stability of miR168 precursor intermediates and mature miR168. However, in this case, we would expect a more profound effect on other miRNAs. Indeed, we also showed that p19 was not able to bind efficiently to miR168 (see Figure 6). Alternatively, it is also possible that accumulation of miR168 is controlled by an RNA-silencing-mediated mechanism, which is inhibited by the RNA-silencing suppressor bringing about the increased accumulation of miR168. In the in situ experiments, we showed that in CymRSV-infected plants the miR168 induction shows spatial overlap with viral accumulation, indicating the direct effect of viral-derived products on miR168 accumulation. Moreover, we detected high-level accumulation of processed MIR168a precursor intermediate, suggesting that the activation of the expression of MIR168a precursor and its increased processing rate can be mainly responsible for the enhanced accumulation of miR168 in virus-infected plants. Increased miR168 accumulation is accompanied by AGO1 mRNA induction Several recent publications described that in addition to the increased miR168 accumulation, its target AGO1 mRNA also showed induced expression in virus-infected plants (Zhang et al, 2006; Csorba et al, 2007; Havelda et al, 2008). First, we investigated the relative expression of AGO1 mRNA and miR168 in CymRSV-infected N. benthamaina plants at 4 and 5 dpi using a probe specific for AGO1 mRNA downstream from the miR168 recognition site. The parallel increase of miR168 and AGO1 mRNA was not followed by the marked accumulation of potential 3′ cleavage products detected in both mock- and virus-inoculated samples (Figure 3A). Quantitative RT–PCR analyses of mock-, CymRSV- and Cym19Stop-inoculated plants also revealed that the majority of virus-infection-induced AGO1 mRNA is present in intact form and no significant over-accumulation of 3′ cleavage product is detectable (Supplementary Figure S1). These observations suggested that virus-infection-induced miR168 accumulation does not, or only with very low efficiency, mediate the cleavage of AGO1 mRNA. This finding was surprising as previous studies showed that AGO1 mRNA level is regulated through miR168-mediated cleavage (Vaucheret et al, 2004; Mallory and Vaucheret, 2009). Figure 3.Induction of AGO1 mRNA level is associated with increased miR168 accumulation. (A) Northern blot analyses of CymRSV-infected N. benthamiana plants for AGO1 mRNA and miRNA expressions. AGO1-1 mRNA was detected with a DNA probe specific for region downstream from the miR168 recognition site. LNA probe was used for miR168 detection. The accumulation of the potential 3′ cleavage products are indicated. (B) Northern blot analyses of virus-infected A. thaliana plants for AGO1 mRNA and miRNA expressions. AGO1 mRNA was detected with a DNA probe specific for region downstream from the miR168 recognition site. LNA probes were used for miRNA detections as indicated. Download figure Download PowerPoint Similar results were obtained in RMV-, CMV- and TCV-infected A. thaliana in a time course analyses (Figure 3B). It was found that miR168, miR168* and AGO1 mRNA expressions were always increased in parallel with the advance of virus-infection processes. Previously, it has been described that miR168* also showed enhanced accumulation in virus-infected plants (Zhang et al, 2006; Csorba et al, 2007). We also showed that miR168* accumulated to high levels in RMV, CMV and TCV infections (Figure 3B). We found that high-level miR168* accumulation is a part of the normal miR168a precursor processing, as presence of miR168 in uninfected tissues is also accompanied with high-level miR168* accumulation (Supplementary Figure S2). The expression level of other miRNAs (miR159 and miR171) showed no or only moderate changes. Slightly increased expression of miR159 was detected in RMV- and CMV-infected samples. This simultaneous increase of AGO1 mRNA and miR168 levels can be explained by the previous observation that AGO1 and miR168 possess a co-expressional regulation mechanism (Vaucheret et al, 2006). We were not able to detect the increased accumulation miR168-mediated AGO1 mRNA cleavage products even in a sample in which the parallel accumulation of miR168 and AGO1 mRNA reached an extremely high level (RMV infection 15 dpi.). Moreover, we did not detect the appearance of potential cleavage products in xrn-4 mutant plants, which have been described to be affected in degradation of mRNAs and selected miRNA targets (Gazzani et al, 2004; Souret et al, 2004) (Supplementary Figure S3). Although it cannot be excluded that cleavage products are undetectable because of their rapid degradation, these results suggest that in virus-infected plants the increased expression of AGO1 mRNA and miR168 is not accompanied with drastically enhanced accumulation of miR168-mediated AGO1 mRNA cleavage products. AGO1 accumulation is repressed in virus-infected plants As several recent reports described miRNA-mediated translational control in plants (Aukerman and Sakai, 2003; Chen, 2004; Brodersen et al, 2008; Dugas and Bartel, 2008; Beauclair et al, 2010), we investigated the accumulation of AGO1 protein relative to the expression of miR168 and AGO1 mRNA in virus-infected plants. For this purpose, we infected N. benthamiana plants with CymRSV and investigated the accumulation of AGO1 in systemically infected leaves at 7 dpi using an antibody raised against endogenous N. benthamiana AGO1-1. We found that in virus-infected plants the AGO1 level was maintained close to the normal level in spite of the strongly increased expression of AGO1 mRNA (Figure 4A). This phenomenon was associated with the presence of drastically increased miR168 level. Similar results were observed when FLAG-AGO1 transgenic A. thaliana plants (Baumberger and Baulcombe, 2005) were infected with crTMV and TCV. RNA and protein samples from the symptomatic systemically infected leaves showed that enhanced level of AGO1 mRNA produced less AGO1 protein than in mock-inoculated control plants (Figure 4B). In contrast to CymRSV-infected N. benthamiana plants the down-regulation of AGO1 protein was more profound in A. thaliana plants especially in the case of crTMV. In the investigated samples, we detected again the high-level accumulation of miR168 compared with mock-inoculated sample as well. Figure 4.Reduced accumulation of AGO1 protein in virus-infected plants. Systemically infected leaves of A. thaliana and N. benthamiana plants were homogenized and divided into RNA and protein extractions. The corresponding samples were used for detecting AGO1 mRNA, AGO1 protein and miR168 expressions. (A) Top: northern blot analyses of CymRSV-infected GFP transgenic N. benthamiana plants for AGO1 mRNA accumulation. The membrane was striped and used for hybridization with DNA probe specific for COX mRNA (loading control). Middle: western blot of total protein extract for AGO1 accumulation using AGO1-1 (N. benthamiana) antibodies. Equal loading was verified by detecting GFP accumulation using GFP-specific antibody and Ponceau staining of the membrane after western blotting. Bottom: small RNA northern blot analyses using miR168-specific LNA probe. (B) Top: northern blot analyses of virus-infected FLAG-AGO1 transgenic A. thaliana plants for AGO1 mRNA accumulation. The membrane was striped and used for hybridization with DNA probe specific for GAPDH mRNA (loading control). Middle: western blot of total protein extract for AGO1 accumulation using FLAG antibodies. Equal loading was verified by detecting actin accumulation using actin-specific antibody and Ponceau staining of the membrane after western blotting. Bottom: small RNA northern blot analyses using miR168-specific LNA probe. Download figure Download PowerPoint These results showed that in spite of the induction of AGO1 mRNA in virus-infected plants the level of AGO1 protein remained unchanged or showed down-regulation implying a translational control mechanism on AGO1 mRNA mediated by miR168. Elimination of p19 RNA-silencing suppressors from virus-infection process results in loss of miR168 induction and in parallel-enhanced AGO1 accumulation RNA-silencing suppressors of viruses are important symptom determinants and can interfere with the accumulation and activity of endogenous miRNAs (Kasschau et al, 2003; Dunoyer et al, 2004; Zhang et al, 2006). We used the previously described CymRSV N. benthamiana system to test the function of an RNA-silencing suppressor in the control of miR168 and AGO1 accumulation. CymRSV-encoded p19 protein has been previously characterized as an RNA-silencing suppressor (Silhavy et al, 2002) and also as an important symptom determinant (Scholthof et al, 1995; Burgyan et al, 2000). CymRSV infection induces the necrosis of the systemically infected N. benthamiana leaves, which later culminates in the death of the plant (Burgyan et al, 2000). In contrast, Cym19Stop (p19 defective mutant virus)-infected plants show non-necrotic symptoms on the first systemically infected leaves and the plants grow further exhibiting the development of RNA-silencing-associated recovery phenotype and drastically reduced levels of virus accumulation (Silhavy et al, 2002). RNA and protein samples were taken from N. benthamiana plants infected with either CymRSV or Cym19Stop at 21°C. Northern blot analyses using an AGO1 mRNA-specific probe revealed that in both CymRSV- and Cym19Stop-infected plants the level of AGO1 mRNA significantly increased compared with the mock-inoculated plants (Figure 5A). In line with previous experiments, we found that in CymRSV-infected plants the AGO1 level did not display changes in spite of the strong AGO1 mRNA induction. In contrast, in Cym19Stop-infected plants the comparable level AGO1 mRNA induction was followed by enhanced accumulation of AGO1, indicating that p19 has a central function in the control of AGO1 accumulation. Next, we investigated the miR168 level in the samples. We found that in striking contrast to CymRSV infection, in which miR168 accumulated to extremely high levels, in Cym19Stop-infected plants the miR168 (21 nucleotide in length) level showed no changes. Only the moderate increase of a higher-molecular weight-type miR168 was detected whose origin and biological competence is unknown (Figure 5A). According to a recent study, this higher-molecular weight miR168 species can be the product of MIR168b precursor (Vaucheret, 2009). However, according to the western blot data the moderate increase of this miR168 species cannot efficiently interfere with the translation of AGO1 (Figure 5). The level of miR159 and miR171 remained unaffected in these samples. At 21°C, Cym19Stop accumulation is severely inhibited (compare viral accumulations in Figure 5A), which could account for the loss of increased miR168 expression. To exclude this possibility, we carried out similar experiments at 15°C. It has been described previously that at 15°C the activity of siRNA-mediated RNA silencing is inhibited and the Cym19Stop-infected plants do not show the recovery phenotype as inefficient RNA silencing allows the accumulation of the mutant virus to the wild-type level (Szittya et al, 2003). Our experiments at 15°C showed that the wild-type level accumulation of Cym19Stop did not induce the enhanced accumulation of normal size miR168 (Figure 5B). Moreover, similar to the previous results, the absence of miR168 increase in Cym19Stop-infected plants was associated with the massive accumulation of AGO1 compared with wild-type virus infection (Figure 5B). These findings also show that the inhibited activity of RNA silencing at low temperature is not the outcome of the lack of proper AGO1 amount, suggesting that other molecular step(s) are affected. The level of control miRNAs remained unchanged also in this experiment. Figure 5.P19 RNA-silencing suppressors of CymRSV is responsible for miR168 induction. (A) Systemically infected leaves of CymRSV-, Cym19Stop- and mock-inoculated N. benthamiana plants were homogenized and divided into RNA and protein extractions. The corresponding samples were used for detecting AGO1 mRNA, AGO1 and miRNA expressions. Top: northern blot analyses of virus-infected and mock-inoculated plants for AGO1 mRNA at 21°C. Middle: western blot of total protein extract for AGO1 accumulation using AGO1-1 (N. benthamiana) antibodies. Bottom: small RNA northern blot analyses using miR168-, miR159- and miR171-specific LNA probes. (B) The same experiments at 15°C. CymRSV-infected plants were harvested at 20 dpi, whereas Cym19Stop-infected plants at 25 dpi to allow the mutant virus to accumulate at same level as the wild-type virus. Download figure Download PowerPoint These data show that the induction of AGO1 mRNA accumulation in virus-infected tissue is a part of a host defence reaction, whereas enhanced accumulation of miR168 is primarily mediated by vira
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