A viral protein promotes host SAMS1 activity and ethylene production for the benefit of virus infection

1) What is the mechanism of a potential effect on ethylene production?

Virus infection appears to result in the increase in SAM, ACC, and ethylene (Figure 6B). However, whether this is due to the interaction of PNS11 with the SAM synthase OsSAMS1 has not been addressed.

To show the causal relationship the authors should use their Ossams1 KO lines (Figure 4), and show that there is no longer an effect on the ethylene pathway when infection occurs.

We thank this reviewer for the insightful comments. To address this concern, we first carried out experiments to confirm the loss of Pns11-OsSAMS1 interaction in Ossams1 KO lines, with or without RDV infection. This is done through a pull-down assay, the results of which are described in subsection “Ethylene is induced by viral infection andethylene accumulation increases host susceptibility” of the revised manuscript, and shown in Figure 6—figure supplement 2A. Specifically, mock-inoculated WT and Ossams1 KO plants, as well as RDV-infected WT and Ossams1 KO plants, were subjected to protein extraction, followed by immune pull-down with an anti-OsSAMS1 antibody. Pns11 was pulled down from RDV-infected WT extracts, but not Ossams1 KO extracts.

We then measured the SAM, ACC and ethylene levels in the same set of plants, and found that the RDV-induced increase of SAM, ACC and ethylene levels disappeared in Ossams1 KO plants (Figure 6—figure supplement 2B-D). These results are exactly as predicted by the reviewer, thus addresses his/her concern.

2) Is there an interaction between Pns11 and osSAMS1 during RDV infection? This needs to be addressed using a time course. The necessary antibody reagents seem to be available for performing these experiments.

This concern is addressed in part by the pull-down assay described above. We further bolstered this conclusion by performing the time course pull-down experiments recommended by this reviewer, which showed a consistent OsSAMS1-Pns11 interaction starting three weeks post inoculation (subsection “Ethylene is induced by viral infection andethylene accumulation increases host susceptibility” in the revised manuscript and Figure 6—figure supplement 1).

3) Are there gene expression changes that reflect activation of the ethylene pathway? This could be addressed using RNA-seq data from osSAMS1 and Pns11 transgenic plants. Changes in gene expression in the ethylene pathway may show a similar pattern as that seen during the virus infection. If not, the model should be revised.

RNA-seq experiments have now been carried out and the results of which incorporated in the revised manuscript (See subsection “Ethylene is induced by viral infection andethylene accumulation increases host susceptibility” in the revised manuscript). Specifically, the gene expression profiles of RDV-infected rice, OsSAMS1 OX lines, Ossams1 KO lines and S11 OX lines were analyzed to identify differentially expressed genes in all comparable pairs (See Supplementary file 3). To determine whether the ethylene pathway was activated by RDV infection, Gene Ontology (GO) was used for analysis (See Figure 6—figure supplement 3). Known ethylene-activated pathway genes were highly enriched in both RDV-infected and OsSAMS1 OX transgenic rice, and depleted in Ossams1 KO plants. Interestingly, although genes in the hormone mediated signaling pathway category were enriched in S11 OX transgenic lines, those in the ethylene-activated pathway were not. This is consistent with the observation that Pns11 overexpression enhances OsSAMS1 activity without up-regulating its mRNA. Additionally, relative to the Pns11 expression level in RDV infection, the transgenically expressed Pns11 level in S11 OX lines was probably low and insufficient to induce significant changes in ethylene pathway genes. Nevertheless, these results indicate that the ethylene pathway genes are regulated by RDV infection, most likely through Pns11-OsSAMS1 interaction. However, additional mechanisms involving other RDV proteins cannot be ruled out at this point.

4) The authors repeatedly cite Satoh et al., (2011) as showing that ethylene related genes are induced following RDV infection (Introduction, Abstract). But that cited paper actually says the opposite: "However, the genes for ET and SA synthesis were not strongly activated by RDV infection." The authors finding that SAMS1 is regulated post-transcriptionally is consistent with the lack of transcriptional changes noted by Satoh et al.,. The rationale for stating that Satoh et al., demonstrate the induction of ethylene related genes needs to be justified (GO analysis of microarray data?) or else interpretation of these earlier results revised and discussed accordingly.

We are sorry for misquoting the published work by Satoh et al., (2011). We have revised the phrase “RDV infection induced expression of ethylene-related genes” into “RDV infection perturbed the expression of several ethylene response genes like ERFs (Ethylene response factors)” in the revised manuscript, please see Introduction and subsection “SAM, ACC, and ethylene contents increased in S11and OsSAMS1overexpression lines and decreased in OsSAMS1 CRISPR/Cas9 knockout and RNAi lines”

Satoh et al., (2011) stated “[…] the genes for ET and SA synthesis were not strongly activated by RDV infection”. We also found no strong induction of ET synthesis genes in our own RNA-seq dataset (See Supplementary file 3). Instead, OsSAMS1, the main subject of the current study, is post-transcriptionally modified by Pns11 to become enzymatically more active, leading to increased production of ethylene. However, we did detect a significant transcriptional change in a set of genes that respond to increase ethylene availability (see above).

5) The authors cannot state that virus accumulation 'significantly' increased (subsection “RDV infection”) as they do not provide statistics. Also, they cannot state "the infection rates were much higher in the S11 OX lines compared to Wt (Figure 1D)" as the difference is actually rather small, and line OX#3 is the same as Wt.

We apologize for the misleading statements. We have corrected these in the revised manuscript. Please see subsection “Overexpression of Pns11in rice enhances susceptibility to RDV infection”.

6) Supplementary file 2A data suggests that the effect is observed before 2-3 wpi; but at later time points there is no difference between wild type and OX plants. Authors should comment on this; because it is not providing complete resistance to RDV, it is providing tolerance to the RDV suggesting ethylene is not the only factor required for RDV pathogenesis.

Thank you for your comments. We have addressed this issue and discussed in the revised manuscript in the Discussion section as follows:

“We didn’t find a strong difference between WT and S11 OX#3 (Figure 1), especially in virus accumulation and infection rate. This is probably due to the relatively low expression level of Pns11 in this particular line (Figure 1—figure supplement 1A, B), which may be insufficient to induce a significant increase in ACC and ethylene production (Figure 3B, C). Furthermore, the hyper-susceptibility to RDV in Pns11 overexpressing plants was more prominent prior to 3 wpi (Supplementary file 2A). This is easily explained by the fact that enhanced susceptibility allowed more Pns11 transgenic plants to show more conspicuous symptoms at earlier time points. This however does not prevent WT plants from becoming symptomatic at later time points, thus catching up with the transgenic plants in the rate of infected plants.”

7) Figure legends – lack details on timing of tissue collection and days after infection, etc.

Thank you for your comments. We have added details like timing of tissue collection and days after infection etc. in the figure legends.

8) Text in terms resistance should be toned down; it is really tolerance, not resistant. "Inhibits RDV infection" – is misleading because it reduces infection compared to the wild type plants. subsection “Ethylene is induced by viral infection and ethylene accumulation increases host susceptibility”: "RDV infection triggers ethylene synthesis and accumulation through the interaction of Pns11 and OsSAMS1, and resultant activation of OsSAMS1". Although there is indirect data for this conclusion in the paper, there is no direct evidence that this happens during RDV infection.

Thank you for your comments. We have replaced “resistance” with “tolerance”; “inhibits RDV infection” with “reduces infection”.

We have performed a new assay (See subsection “Ethylene is induced by viral infection andethylene accumulation increases host susceptibility” in the revised manuscript) to confirm that Pns11 interacted with OsSAMS1 during RDV infection (See Figure 6—figure supplement 1) but not in RDV-infected Ossams1 KO lines (See Figure 6—figure supplement 2A). We also measured the contents of SAM, ACC and ethylene in RDV-infected Ossams1 KO lines and mock infected Ossams1 KO lines, and found that the RDV-induced increase SAM, ACC and ethylene disappeared in these lines (See Figure 6—figure supplement 2B-D). These data indicated that RDV infection triggers ethylene synthesis and accumulation through the interaction of Pns11 and OsSAMS1 and resultant activation of OsSAMS1.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

You should revise the text (subsection “OsSAMS1 enzymatic activity”). It is misleading to indicate that proteins present in the same fraction means they are interacting. Your BiFC data and Figure 6—figure supplement 1 does show complex formation. Please be sure to carefully revise the text and be sure to note what each experiment really shows.

We apologize for the misleading statements describing the gel-filtration result and have replaced the gel-filtration experiment with IP western experiment (See Figure 2D and subsection “OsSAMS1 enzymatic activity “in the revised manuscript). For the BiFC data, we have conducted all the positive and negative controls to confirm the interaction between Pns11 and OsSAMS1. For the in vivo time course pull-down assay (Figure 6—figure supplement 1), we have chosen one time point experiment (4wpi) to show the interaction between Pns11 and OsSAMS1 during RDV infection (See Figure 2D).

Reviewer #2:

Authors have addressed most of the issues raised in the last reviews. However, authors fail to respond to this comment:

– Figure 2D – This reviewer fail to understand how the authors conclude interaction based on fractionation? This should be a simple IP experiment considering the authors have antibodies to Pns11 and OsSASM1. IP with OsSAMS1 antibody and probe for Pns11, in mock and RDV infected tissues. Alternatively, IP with Pns11 antibody and probe for OsSAMS1.

Thank you for your comments. We indeed have data showing the interaction between Pns11 and OsSAMS1 in RDV-infected rice by in vivo time course pull-down assay using OsSAMS1 antibody. We apologize for the misleading statements and have replaced the gel-filtration experiment with one time point IP western experiment (See Figure 2Dand subsection “OsSAMS1 enzymatic activity “in the revised manuscript) as described above.