Plant virus mediated induction of mir168 is associated with repression of ARGONAUTE1 accumulation

Transcription

1 Manuscript EMBO Plant virus mediated induction of mir168 is associated with repression of ARGONAUTE1 accumulation Éva Várallyay, Anna Válóczi, Ákos Ágyi, József Burgyán and Zoltan Havelda Corresponding author: Zoltan Havelda, Agricultural Biotechnology Center Review timeline: Submission date: 11 February 2010 Editorial Decision: 11 March 2010 Revision received: 08 June 2010 Editorial Decision: 28 June 2010 Revision received: 09 August 2010 Editorial Decision: 12 August 2010 Accepted: 12 August 2010 Transaction Report: (Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity, letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this compilation.) 1st Editorial Decision 11 March 2010 Thank you for submitting your manuscript to the EMBO Journal. Your study has now been seen by three referees and their comments to the authors are provided below. As you can see all three referees find the work interesting. However, it is also clear that further work would be needed in order to consider publication here and in particular further support for that mir168 triggers translational repression of AGO1 mrna is needed. Should you be able to address the concerns raised in full then we would be happy to consider a revised version. I should remind you that it is EMBO Journal policy to allow a single round of revision only and that it is therefore important to address the concerns raised at this stage. When preparing your letter of response to the referees' comments, please bear in mind that this will form part of the Review Process File, and will therefore be available online to the community. For more details on our Transparent Editorial Process initiative, please visit our website: Thank you for the opportunity to consider your work for publication. I look forward to your revision. Yours sincerely, Editor The EMBO Journal REFEREE REPORTS European Molecular Biology Organization 1

2 Referee #1 (Remarks to the Author): This article by Varallyay et al describes how virus infection in plants induces the mir168 and its target - the mrna for the silencing effector protein AGO1. The mir168 induction is associated with increased accumulation of the intermediate fragments in the processing of its precursor and it is likely to be due to transcriptional activation. The large increase in the AGO1 mrna in virus infected plants is not however associated with an increase in the amount of the AGO1 protein - there may even be a decrease in certain circumstances. These two responses to virus infection can be separated by the use of a mutant virus that stimulates expression of the mrna but not the mirna. The authors conclude that these two responses represent aspects of defense and counter defense: defense would be associated with stimulation of the AGO mrna whereas counterdefense may be associated with stimulation of the mir168. So far so good although I do not think the proposed translational effect of the mirna is supported fully by the presented data. It could well be that there is increased degradation of the mrna without a corresponding increase in the level of the 3' cleavage products. The next part of the paper is quite complicated. First it shows in a straightforward manner that the mir168 is induced by transient expression of the P19 viral suppressor of silencing although it is not clear whether this effect is direct or indirect. Thus the counter defense role of P19 is both through binding to sirnas and through induction of mir168. Other data show that in virus-infected (at least CyRSV-infected) plants the viral sirnas are predominantly bound to p19 rather than to AGO and that mir168 also accumulates predominantly in a form that is in a non bound form rather than being bound to AGO1. These additional data are a distraction from the main thrust of the paper and they are not incorporated into the model: perhaps they could be left out if they cannot be incorporated into the main model? The authors do not comment on the high abundance of mir168* in Figure 3 - could this observation help reconcile other data from figure 6 into a coherent model? The authors do not consider the possibility that mir168 is silenced or blocked by a mechanism that can be suppressed by P19 or the silencing suppressors produced by the various viruses used in this paper. The discussion is rather rambling and ill-structured: it could be cut down to half length and be easier to read. Referee #2 (Remarks to the Author): In plants, virus infection induce mir168 and AGO1 mrna accumulation (Csorba et al, 2007; Havelda et al, 2008; Zhang et al, 2006). In this manuscript, V rallyay et al. shows that viral suppressors of RNA silencing are responsible for mir168 induction, which in turn down-regulates AGO1 protein levels, probably by inhibiting AGO1 mrna translation. This hypothesis is very interesting. However, it is mandatory that the authors directly test this hypothesis by infecting mutants impaired in mirna-mediated translational repression. Such mutants are viable and publicly available. Detailed comments: The authors show that virus-infected plants have elevated levels of mir168 and AGO1 mrna but reduced levels of AGO1 protein. They conclude that virus-mediated mir168 over-accumulation triggers translational repression of AGO1 mrna. This is a reasonable hypothesis, consistent with papers showing association of AGO1 and mature mir168 with active polysomes (Lanet et al, 2009), and translational repression of AGO1 by mir168 (Mallory et al, PLoS Genetics, 2009). Nevertheless, the authors' hypothesis need to be directly tested by analyzing mutants impaired in translation repression, which are viable and available (ago10, ktn1, vcs). If the authors' hypothesis is right, these mutants should not show AGO1 protein down-regulation after virus infection. European Molecular Biology Organization 2

3 The authors also show that mir168-mediated AGO1 mrna cleavage products do not accumulate, which allow them to conclude that virus-mediated mir168 over-accumulation triggers translational repression of AGO1 mrna instead of cleavage. For this to happen, not only mir168 must be able to mediate translational repression of AGO1 mrna, but mir168-directed cleavage activity must be inhibited. How does this happen? Referee #3 (Remarks to the Author): In this manuscript the authors present evidence suggesting an interesting viral defence-counter defence mechanism in plants through the induction of AGO1 (for viral defence) and mir168 (for viral counter defence). They show that viral infection is associated with the induction of both mir168 and its target AGO1 mrna - such induction of mir168 and AGO1 by viral infection or by expression of viral suppressors has been previously reported. What is new here are the observations that i) induction of mirnas is specific to mir168, with the expression of several other mirnas tested being unaffected by viral infection (this is in contrast to previous reports by Zhang et al. and Csorba et al. showing general induction of many mirnas by viral infections or expression of viral suppressors), ii) AGO1 expression remains unchanged or is markedly reduced at the protein level despite the increased mrna abundance, and iii) the induction of mir168 and its target AGO1 mrna can be uncoupled, with the former requiring the silencing suppressor p19 while the latter being independent of p19. The finding that AGO1 protein is downregulated is particularly interesting as it supports the ideas of the authors that the induction of mir168 is a viral counterdefence mechanism, and that mir168 inhibits AGO1 expression by translational repression. The paper would be significantly strengthened if evidence is provided showing a direct causal link between the induction of mir168 and downregulation of AGO1 protein (i.e. to show that the downregulation of AGO1 protein is indeed due to translational repression by mir168). One possible experiment to address this is to introduce into plants a recombinant copy of the AGO1 gene with a mutated mir168 target sites (so it can not be targeted by mir168), and examine if AGO1 protein expression is no longer downregulated by the induction of mir168 upon viral infection. The significant downregulation of AGO1 protein accumulation in the presence of increased abundance of AGO1 mrna appears to suggest that translational repression is the predominant mechanism of mir168-mediated repression - does this have anything to do with viral infections or viral suppressors? If so, translational repression could also be enhanced with other mirnas and their targets by viral infection - this could be interesting to test. Extra care is needed when using abundance of AGO1 cleavage product as a basis to judge if mir168 functions via cleavage or translational repression (Figure 3A). This is because i) the putative cleavage fragments of AGO1 have not been characterized and ii) in many cases real mirna cleavage products are undetectable by northern hybridization presumably due to rapid degradation. A minor point: It would help if more explanations are given to the in situ hybridization result in Figure 2A. For someone who has no experience with the experiment, it is hard to understand what the 3 arrows point to and where the mir168 hybridizing signals are etc. 1st Revision - Authors' Response 08 June 2010 Answers to Referee #1 1. I do not think the proposed translational effect of the mirna is supported fully by the presented data. It could well be that there is increased degradation of the mrna without a corresponding increase in the level of the 3' cleavage products. We accept that it can not be excluded that mir168 directed cleavage of AGO1 mrna can result in the very fast degradation of cleavage products evading the successful detection by northern blot. To further analyse the mir168 mediated cleavage of mir168 degradation we investigated the accumulation of potential cleavage products by northern blot analyses in xrn4 mutants where the possible over accumulation cleavage product can be expected. We did not detect the accumulation European Molecular Biology Organization 3

4 of 3' cleavage products in these plants (see Supplementary data Figure S3 and revised text ). We also used quantitative RT PCR approach, as more sensitive technology, to search for 3' cleavage products in virus infected plants and in transient p19 expression studies. These experiments demonstrated that virus infected plants AGO1 mrna accumulates predominantly in full length intact form and no enhanced accumulation of 3' cleavage product was found by this approach plants (see Supplementary data Figure S1 and revised text). Moreover, no AGO1 mrna decrease was observed during transient p19 expression mediated down regulation of AGO1. (see Figure 7A and revised text). Although, these results can not exclude the very rapid degradation of 3' cleavage products but indicates that in the investigated time points the reduced accumulation of AGO1 protein is associated with the presence of full length intact AGO1 mrna. To further test the potential role of translational inhibition we used mutant plants previously demonstrated to be inhibited in translational activities. Since AGO10 was previously shown to be involved in the regulation of AGO1 protein accumulation we focused our research on this mutant. Using crtmv infected zll-3 mutants we showed that AGO1 repression in these plants was inhibited in spite of the induction of mir168 and AGO1 mrna (see Figure 8C and revised text). These finding reinforce our hypothesis that AGO1 mrna indeed is under translational control. Moreover, this data also suggest that AGO10 is one of the key components regulating AGO1 protein expression. We also investigated vcs-7 heterozygotes for the crtmv mediated regulation of AGO1 and found also moderate inhibition of AGO1 protein repression (see Supplementary data Figure S2 and revised text). 2. First it shows in a straightforward manner that the mir168 is induced by transient expression of the P19 viral suppressor of silencing although it is not clear whether this effect is direct or indirect. To test direct role of mir168 in this process we mimicked p19 expression induced accumulation of mir168 by transiently expressing the MIR168a precursor. Transient expression of the precursor resulted in the enhanced accumulation of mature mir168 and in the down regulation of AGO1 indicating the induction of mature mir168 in the infiltrated patch is able to mediate the down regulation of AGO1 protein in the absence of p19 (see Figure 7C and revised text). Moreover, coexpression of a mir168 target mimicry construct with the MIR168a precursor resulted in the inhibition of AGO1 protein repression further confirming the direct role of mature mir168 in this process (see Figure 7D and revised text). To further test the direct role of mir168 in the investigated process we used 4mAGO1 plants (generously provided by Herve Vaucheret), which contains four silent mutations in the mir168 target site rendering the mrna to be resistant for mir168 mediated control. CrTMV infection of mutant plant resulted in the inhibition of AGO1 protein down regulation demonstrating the direct activity of mir168 mediated control in A. thaliana (see Figure 8B and revised text). 3. Other data show that in virus-infected (at least CyRSV-infected) plants the viral sirnas are predominantly bound to p19 rather than to AGO and that mir168 also accumulates predominantly in a form that is in a non bound form rather than being bound to AGO1. These additional data are a distraction from the main thrust of the paper and they are not incorporated into the model: perhaps they could be left out if they cannot be incorporated into the main model? Since p19 has a strong small RNA duplex binding ability, we think that it is important to show that p19 is not able to effectively bind to mir168/mir68* duplexes (especially since mir168* accumulate to extremely high level compared to other conservative mirnas (see later). The ability of p19 to bind mir168/mir68* duplexes could result in the accumulation of both mir168 and mir68* but in inactive form (bound to p19). We considered this finding to be important to show demonstrating that virus infection induced mir168 is present in unbound form capable for carrying out its regulatory function. 4. The authors do not comment on the high abundance of mir168* in Figure 3 - could this observation help reconcile other data from figure 6 into a coherent model? European Molecular Biology Organization 4

5 Accumulation of mir168 during the virus infection process is accompanied with the high level accumulation of mir168*. To test the nature of the high level accumulation of mir168* we compared the relative accumulation of mir168, mir168*, mir171 and mir171* in RNA samples originating from different organs and virus infected plants (see Supplementary data Figure S2 and revised text ). We found that in contrast to mir171* which was hardly detectable after long exposure time mir168* accumulated to high level not only in virus infected but also in other samples originating from different organs. Based on this results we concluded that high level of mir168* accumulation is part of the normal maturation process MIR168 precursor and is not a specific phenomenon associate with virus infections. We also incorporated data into the Figure 6 demonstrating that mir168* is present predominantly in unbound form similarly to mir168. We also incorporated mir168* data in experiments showing the effect of transient expression of p19 and MIR168 precursor (see Figure 7A and C). 5. The authors do not consider the possibility that mir168 is silenced or blocked by a mechanism that can be suppressed by P19 or the silencing suppressors produced by the various viruses used in this paper. We now consider this possibility in the text (see "Induction of mir168 spatially overlaps with virus accumulation and linked to enhanced accumulation of pre-mir168a loop intermediate." part of text). 6. The discussion is rather rambling and ill-structured: it could be cut down to half length and be easier to read. We rewrote and restructured the discussion section. Hopefully, it is now more concise and understandable. Answers to Referee #2 1. Nevertheless, the authors' hypothesis need to be directly tested by analyzing mutants impaired in translation repression, which are viable and available (ago10, ktn1, vcs). If the authors' hypothesis is right, these mutants should not show AGO1 protein down-regulation after virus infection. Since AGO10 was previously shown to be involved in the regulation of AGO1 protein accumulation we focused our research on this mutant. Using crtmv infected zll-3 mutants we showed that AGO1 repression in these plants was inhibited in spite of the induction of mir168 and AGO1 mrna (see Figure 8C and revised text). These finding reinforce our hypothesis that AGO1 mrna indeed is under translational control. Moreover, this data also suggest that AGO10 is one of the key components regulating AGO1 protein expression. We also investigated vcs-7 heterozygotes for the crtmv mediated regulation of AGO1 and found also moderate inhibition of AGO1 protein repression (see Supplementary data Figure S2 and revised text). 2. The authors also show that mir168-mediated AGO1 mrna cleavage products do not accumulate, which allow them to conclude that virus-mediated mir168 over-accumulation triggers translational repression of AGO1 mrna instead of cleavage. For this to happen, not only mir168 must be able to mediate translational repression of AGO1 mrna, but mir168-directed cleavage activity must be inhibited. How does this happen? The coexistence of high level of mir168 and AGO1 mrna indicates that in contrast to the translational repression the cleavage activity of mir168 excess is inhibited. One possible explanation for this phenomenon can be the different activities of AGO proteins. It is possible that virus infection induced mir168 is sorted mainly into ZWILLE/PINHEAD/AGO10, which exerts its European Molecular Biology Organization 5

6 activity at the level of translational repression while mir168 incorporated into AGO1 mediates cleavage of AGO1 mrna. However, the exact nature of factors responsible for determining the slicing or translational inhibitory activities of mir168 and the role of translational repression during the developmental processes remains to be investigated. This hypothesis is now introduced into the discussion part of the revised manuscript (see revised text). Answers to Referee #3 1. induction of mirnas is specific to mir168, with the expression of several other mirnas tested being unaffected by viral infection (this is in contrast to previous reports by Zhang et al. and Csorba et al. showing general induction of many mirnas by viral infections or expression of viral suppressors Virus infection, depending on the particular viruses and host plants, can induce various changes in expression pattern of endogenous mirnas. What we intended to demonstrate here that independently of the host plants and the various ability of the particular viruses to interfere with endogenous mirna accumulation (for example CymRSV induce only very mild changes at mirna expression levels while TEV or tobamoviruses have more profound impact on the accumulation of mirnas) the induction of mir168 was always consistently drastic in all of the investigated plant virus interactions. We now tried to make clear this point in revised text. 2. The paper would be significantly strengthened if evidence is provided showing a direct causal link between the induction of mir168 and downregulation of AGO1 protein (i.e. to show that the downregulation of AGO1 protein is indeed due to translational repression by mir168). One possible experiment to address this is to introduce into plants a recombinant copy of the AGO1 gene with a mutated mir168 target sites (so it can not be targeted by mir168), and examine if AGO1 protein expression is no longer downregulated by the induction of mir168 upon viral infection. To test the direct role of mir168 in the investigated process we used 4mAGO1 plants (generously provided by Herve Vaucheret), which contains four silent mutations in the mir168 target site rendering the mrna to be resistant for mir168 mediated control. CrTMV infection of mutant plant resulted in the inhibition of AGO1 protein down regulation demonstrating the direct activity of mir168 mediated control in A. thaliana (see Figure 8B and revised text). To further test the direct role of mir168 in this process we mimicked p19 expression induced accumulation of mir168 by transiently expressing the MIR168a precursor. Transient expression of the precursor resulted in the enhanced accumulation of mature mir168 and in the down regulation of AGO1 indicating the induction of mature mir168 in the infiltrated patch is able to mediate the down regulation of AGO1 protein in the absence of p19 (see Figure 7C and revised text). Moreover, co-expression of a mir168 target mimicry construct with the MIR168a precursor resulted in the inhibition of AGO1 protein repression further confirming the direct role of mature mir168 in this process (see Figure 7D and revised text). 3. The significant downregulation of AGO1 protein accumulation in the presence of increased abundance of AGO1 mrna appears to suggest that translational repression is the predominant mechanism of mir168-mediated repression - does this have anything to do with viral infections or viral suppressors? If so, translational repression could also be enhanced with other mirnas and their targets by viral infection - this could be interesting to test. To test whether virus infection also modulates the translational regulation of other mirna targets, we investigated the accumulation of CCS1 protein which has been recently showed that in addition to cleavage it is also under mirna directed translational control (Beauclair et al). We found that CCS1 protein level did not change in crtmv infected wild type A. thaliana plants indicating that the observed down regulation of AGO1 protein is not a part of general enhancement of translational repression in the infected plants (see in Figure 8A and revised text). European Molecular Biology Organization 6

7 4. Extra care is needed when using abundance of AGO1 cleavage product as a basis to judge if mir168 functions via cleavage or translational repression (Figure 3A). This is because i) the putative cleavage fragments of AGO1 have not been characterized and ii) in many cases real mirna cleavage products are undetectable by northern hybridization presumably due to rapid degradation. We accept that it can not be excluded that mir168 directed cleavage of AGO1 mrna can result in the very fast degradation of cleavage products evading the successful detection by northern blot and AGO1 mrna cleavage products are not well characterized. To further analyse the mir168 mediated cleavage of mir168 degradation we investigated the accumulation of potential cleavage products by northern blot analyses in xrn mutants where the possible over accumulation cleavage product can be expected. We did not detect the accumulation of 3' cleavage products in these plants (see Supplementary data Figure S3 and revised text ). We also used quantitative RT PCR approach, as more sensitive technology, to search for 3' cleavage products in virus infected plants and in transient p19 expression studies. These experiments demonstrated that virus infected plants AGO1 mrna accumulates predominantly in full length intact form and no enhanced accumulation of 3' cleavage product was found by this approach plants (see Supplementary data Figure S1 and revised text). Moreover, no AGO1 mrna decrease was observed during transient p19 expression mediated down regulation of AGO1. (Figure 7A and revised text). Although, these results can not exclude the very rapid degradation of 3' cleavage products but indicates that in the investigated time points the reduced accumulation of AGO1 protein is associated with the presence of full length intact AGO1 mrna. To further test the potential role of translational inhibition we used mutant plants previously demonstrated to be inhibited in translational activities. Since AGO10 was previously shown to be involved in the regulation of AGO1 protein accumulation we focused our research on this mutant. Using crtmv infected zll-3 mutants we showed that AGO1 repression in these plants was inhibited in spite of the induction of mir168 and AGO1 mrna (see Figure 8C and revised text). These finding reinforce our hypothesis that AGO1 mrna indeed is under translational control. Moreover, this data also suggest that AGO10 is one of the key components regulating AGO1 protein expression. We also investigated vcs-7 heterozygotes for the crtmv mediated regulation of AGO1 and found also moderate inhibition of AGO1 protein repression (see Supplementary data Figure S2 and revised text). 5. A minor point: It would help if more explanations are given to the in situ hybridization result in Figure 2A. For someone who has no experience with the experiment, it is hard to understand what the 3 arrows point to and where the mir168 hybridizing signals are etc. We reformat the Figure 2A and added more detailed explanation to the Figure legend. Hopefully this figure is now more understandable. 2nd Editorial Decision 28 June 2010 Thank you for submitting your revised manuscript to the EMBO Journal. I asked the original referees #2 and 3 to review the revised manuscript and their comments are provided below. As you can see both referees appreciate the carried out revisions. Referee #2 has a few remaining issues concerning figure 8 that should be resolved before acceptance here. Given these comments I would like to ask you to respond to the remaining concerns in a final round of revision. When you send us your revision, please include a cover letter with an itemised list of all changes made, or your rebuttal, in response to comments from review. When preparing your letter of response to the referees' comments, please bear in mind that this will form part of the Review Process File, and will therefore be available online to the community. For more details on our Transparent Editorial Process initiative, please visit our website: European Molecular Biology Organization 7

8 I look forward to seeing the final version. Yours sincerely, Editor The EMBO Journal REFEREE REPORTS Referee #2 (Remarks to the Author): The authors performed additional experiments to address the reviewers' concerns. Unfortunately, the data are presented in a way that does not allow determining if they support the authors' conclusions. In particular, the added Figure 8 has several issues: - Wildtype and mutants are presented in separate boxes but it is not known if they can be directly compared one to another. Were they on the same blot? Were the blots exposed the same length of time? - Even if all boxes are comparable, there is an ecotype issue in this experiment. 4mAGO1 and ago1-25 are in Col while zll-3 is in Ler, and there is only one wildtype control. In which ecotype is this control? Col or Ler? In any case, one control is missing. This is particularly important because Mallory et al, 2009 showed that ZLL is involved in translation repression in Ler but not in Col (zll has an effect in Col only when AGO1 is partially compromised). Thus, it is very difficult to interpret these data. And the vcs-7 heterozygote analysis is inconclusive. The authors should perform their analysis using null alleles in the same ecotype. - What happens to AGO1 mrna in ago1-25? It looks degraded and/or shorter whereas the protein is detectable (although it is unclear whether is has a wildtype size because the blots has been cut into boxes). - The authors propose that during virus infection mir168 is sorted mainly by ZLL to trigger AGO1 translation repression. However, Figure 8 reveals an over-accumulation of mir168 in the infected zll-3 mutant. Because AGO1 level is unaffected by virus infection in zll-3, this suggests that mir168 can be sorted by other AGO, reinforcing the necessity to analyze zll mutants in the same ecotype as ago1 and 4mAGO1. Minor comments The legends of FigureS4 is missing Referee #3 (Remarks to the Author): The revised version is significantly improved over the original version. The evidence that mir168 regulates AGO1 expression via translational repression in viral infected plants has become more compelling with the added data involving the use of 4mAGO1 mutant plants, although Figure 8B appears to imply that the 4-base silent mutation may have not completely disrupted binding between mir168 and the 4mAGO1 transcript (AGO1 protein levels would have been higher). The English of the newly added texts may need to be edited. 2nd Revision - Authors' Response 09 August 2010 Referee # 2 - Wildtype and mutants are presented in separate boxes but it is not known if they can be directly compared one to another. Were they on the same blot? Were the blots exposed the same length of time?- We were not able to directly compare the wild type and mutant plants since the mutant plants grew more slowly, display strong mutant phenotypes (different leaf and tissue organization) and produce various amount of experimental material. These alterations of mutant plants can result in the changes of the basal AGO1 protein level. Consequently, the data presented in the boxes are not directly comparable because every box represents independent experiments. However, in the European Molecular Biology Organization 8

9 experiments we always compared the virus infection induced changes to the parallel control group of mock inoculated test plants of the same genotype. We think that this approach can provide reliable data revealing the specific answers of mutant plants to virus infections. - Even if all boxes are comparable, there is an ecotype issue in this experiment. 4mAGO1 and ago1-25 are in Col while zll-3 is in Ler, and there is only one wildtype control. In which ecotype is this control? Col or Ler? In any case, one control is missing. This is particularly important because Mallory et al, 2009 showed that ZLL is involved in translation repression in Ler but not in Col (zll has an effect in Col only when AGO1 is partially compromised). Thus, it is very difficult to interpret these data. And the vcs-7 heterozygote analysis is inconclusive. The authors should perform their analysis using null alleles in the same ecotype. - We now added the Ler wild type control panel to Figure 8. The Ler wild type plants behaved similarly in virus infection studies compared to Col wt plants. We detected the induction of AGO1 mrna and mir168 as a result of virus infection and in parallel the inhibition of AGO1 protein accumulation. In zll-3 plants the down regulation of AGO1 was inhibited supporting the hypothesis that AGO10 is involved in the regulation of AGO1. These experiments revealed that in Ler background, AGO10, a factor which has been previously shown to be responsible for translational control mediated by mirnas, is necessary for the observed down regulation of AGO1 accumulation. Based on this finding we can hypothesize that the similar factors are responsible for the control of AGO1 accumulation in Col background. We made it clear now that the AGO10 experiments has been carried out in Ler background. Unfortunately, we were not able to carry out our analyzes in vcs-7 homozygous plants since this mutant is seedling lethal and does not reach the suitable size for virus infection. - What happens to AGO1 mrna in ago1-25? It looks degraded and/or shorter whereas the protein is detectable (although it is unclear whether is has a wildtype size because the blots has been cut into boxes). - Since the technical quality of the ago1-25 northern blot was indeed bad we remade the northern blot from the ago1-25 RNA samples and incorporated the new blot into the Figure 8. There is no alteration in the size of AGO1 protein in ago1-25 mutant plants it runs at the size of wild type AGO1 protein(ago1-25 is a point mutant). - The authors propose that during virus infection mir168 is sorted mainly by ZLL to trigger AGO1 translation repression. However, Figure 8 reveals an over-accumulation of mir168 in the infected zll-3 mutant. Because AGO1 level is unaffected by virus infection in zll-3, this suggests that mir168 can be sorted by other AGO, reinforcing the necessity to analyze zll mutants in the same ecotype as ago1 and 4mAGO1. - We used Ler ecotype in zll-3 experiments since this mutant, in contrast to Col ecotype ago10 mutant, shows strong developmental defects indicating the importance of AGO10 activity. Moreover, Ler ecotype ago10 mutant was also used by Brosdersen et al. (2008) to demonstrate the role of AGO10 in the translational control mediated by some mirnas. Since the virus infected wt Ler plants shows similar responses to virus infection as the Col wt plants we think that this system is suitable to investigate the role of AGO10 in this process. The abundant accumulation of mir168 in zll-3 plants show that this process is independent of the activity of AGO10. Our gel infiltration experiments are showed that majority of mir168 are in unbound form in mock and also in virus infected plants indicating that mir168 can be stable without incorporating into AGO proteins. We can not exclude the possibility that mir168 is also incorporated into other AGO proteins. Minor comments We introduced the FigureS4 legends. Referee # 3 - The revised version is significantly improved over the original version. The evidence that mir168 regulates AGO1 expression via translational repression in viral infected plants has become more compelling with the added data involving the use of 4mAGO1 mutant plants, although Figure 8B appears to imply that the 4-base silent mutation may have not completely disrupted binding between mir168 and the 4mAGO1 transcript (AGO1 protein levels would have been higher). - The 4mAGO1 plant has been constructed by transforming the wild type plant with an expression cassette containing the promoter and the mutant version of AGO1. This means that in this mutant the European Molecular Biology Organization 9

10 wild type and mutant version of AGO1 mrna are expressed in parallel. Because of this mixed expression we can expect here limited up regulation of AGO1, since the mock signal is produced by the additional expression of mutant and wild type mrnas while during virus infection the translational capacity of the wild type mrnas are efficiently inhibited and AGO1 expression can mainly derive from the induced mutant mrna. However, we can not exclude the possibility that the introduced mutations did not completely abolish the regulatory capacity of mir168. -The English of the newly added texts may need to be edited. - A native speaker corrected the text and the correction has been incorporated into the text. 3rd Editorial Decision 12 August 2010 Thank you for submitting your revised manuscript to the EMBO Journal. I asked the original referee #2 to take a final look at the paper and I have now received the comments back from this referee. As you can see below, this referee is satisfied with the revised version. I am therefore very pleased to proceed with the acceptance of the paper for publication here. You will receive the formal acceptance letter shortly. Thank you for submitting your interesting study for publication here. Sincerely Editor The EMBO Journal Referee #2 The authors have correctly addressed my comments. European Molecular Biology Organization 10