ATG6 interacting with NPR1 increases Arabidopsis thaliana resistance to Pst DC3000/avrRps4 by increasing its nuclear accumulation and stability

Reviewer #1 (Public Review):

The authors showed that autophagy-related genes are involved in plant immunity by regulating the protein level of the salicylic acid receptor, NPR1.

The experiments are carefully designed and the data is convincing. The authors did a good job of understanding the relationship between ATG6 and NRP1.

Comments on latest version:

The authors have already addressed all my comments. I have no further issues with the manuscript.

Reviewer #2 (Public Review):

The manuscript by Zhang et al. explores the effect of autophagy regulator ATG6 on NPR1-mediated immunity. The authors propose that ATG6 directly interacts with NPR1 in the nucleus to increase its stability and promote NPR1-dependent immune gene expression and pathogen resistance. This novel role of ATG6 is proposed to be independent of its role in autophagy in the cytoplasm. The authors demonstrate through biochemical analysis that ATG6 interacts with NPR1 in yeast and very weakly in vitro. They further demonstrate using overexpression transgenic plants that in the presence of ATG6-mcherry the stability of NPR1-GFP and its nuclear pool is increased.

Comments on latest version:

The term "invasion" has to be replaced with infection, as it doesn't have much meaning to this particular study. I already explained this point in the first review, but authors did not address it throughout the manuscript.

In fig. 1e there's no statistical analysis. How can one show measurements from multiple samples without statistical analysis? All the data points have to be shown in the graph and statistics performed. In the arg6-npr1 and snrk-npr1 pairs no nuclear marker is included. How can one know where the nucleus is, particularly in such poor quality low res. images? The nucleus marker has to be included in this analysis and shown. This is an important aspect of the study as nuclear localization of ATG6 is proposed to be essential for its new function. Co-localization provided in the fig. S2 cannot complement this analysis, particularly since no cytoplasmic fraction is present for NPR1-GFP in fig. S2.

In the alignment in fig 2c, it is not explained what are the species the atg6 is taken from. The predicted NLS has to be shown in the context of either the entire protein sequence alignment or at least individual domain alignment with the indication of conserved residues (consensus). They have to include more species in the analysis, instead of including 3 proteins from a single species. Also, the predicted NLS in atg6 doesn't really have the classical type architecture, which might be an indication that it is a weak NLS, consistent with the fact that the protein has significant cytoplasmic accumulation. They also need to provide the NLS prediction cut-off score, as this parameter is a measure of NLS strength.

Line 150: the NLS sequence "FLKEKKKKK" is a wrong sequence.

In fig. 3d no explanation for the error bars is included, and what type of statistical analysis is performed is not explained.

Author response:

The following is the authors’ response to the previous reviews.

Public Reviews:

Reviewer #1 (Public Review):

The authors showed that autophagy-related genes are involved in plant immunity by regulating the protein level of the salicylic acid receptor, NPR1.
The experiments are carefully designed and the data is convincing. The authors did a good job of understanding the relationship between ATG6 and NRP1.

The authors have addressed most of my previous concerns.

Thank you so much for acknowledging our research. It is incredibly rewarding to see our work recognized. We hope that our findings will inspire new perspectives and foster further exploration in this area.

Reviewer #2 (Public Review):

The manuscript by Zhang et al. explores the effect of autophagy regulator ATG6 on NPR1-mediated immunity. The authors propose that ATG6 directly interacts with NPR1 in the nucleus to increase its stability and promote NPR1-dependent immune gene expression and pathogen resistance. This novel role of ATG6 is proposed to be independent of its role in autophagy in the cytoplasm. The authors demonstrate through biochemical analysis that ATG6 interacts with NPR1 in yeast and very weakly in vitro. They further demonstrate using overexpression transgenic plants that in the presence of ATG6-mcherry the stability of NPR1-GFP and its nuclear pool is increased.

Comments on revised version:

The authors demonstrate the correlation between overexertion of atg6 and higher stability and activity of npr1. They claim a novel activity of atg6 in the nucleus.

Overall, the experimental scope of the study is solid, however, the over-interpretation of the results substantially reduces the significance and value of this study for the target plant immunity readership.

Thank you very much for you constructive and insightful comments, as well as for acknowledging the experimental scope of this study. In addition, we have made every effort to address the over-interpretation of the results, as per your comments, ensuring they are more accurate and concise. In the revised version, the modified content has been highlighted in blue to clearly indicate the changes made.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

The authors have addressed most of my concerns. I have no further comments.

Thank you so much for acknowledging our research. It is incredibly rewarding to see our work recognized. We hope that our findings will inspire new perspectives and foster further exploration in this area.

Reviewer #2 (Recommendations For The Authors):

As I previously commented, in fig. 2a and c, the discrepancy between levels of atg6-mcherry in microscope image vs WB has to be explained. The explanation provided by the authors is incomplete and may mislead. The most likely reason for the difference is that the fluorescence signal in fig. 2a is predominantly from free mCherry, rather than the atg6-mcherry fusion. This has to be included in the main text to avoid misleading the reader.

Thank you very much for you constructive and insightful comments, in response to your comments, we have incorporated the necessary explanations into the revised manuscript (lines 160-164).

In fig. 1B, the PD fraction has to show the size range of free GST. Also, please use "anti" to indicate that these are immunoblots,.

Thank you for pointing this out. In the revised manuscript, we identified the range of free GST and used "anti:GST and anti:His" to indicate that these are immunoblots.

In fig 1C, the WB has to show the free GFP band in the input and IP fractions together with NPR1, rather than in separate blots.

Thank you for bringing this to our attention. Fig. 1c has been replaced, and the updated image now shows the free GFP band in the input and IP fractions together with NPR1-GFP.

In fig. 1d, the bifc signal has to be quantified from multiple images across the biological repeats. Also, there's no significance in showing the chlorophyll autofluorescence. What is the purpose of this? They need to use a nuclear marker instead.

Thank you for your suggestion. Based on your input, we utilized ImageJ software to quantify the YFP fluorescence signal. A total of n = 15 independent images were analyzed, and the corresponding results have been added to Figure 1e. Monitoring chlorophyll autofluorescence serves as a useful background signal, aiding in the distinction between the fluorescence signal of the target protein and background noise. This approach helps reduce potential signal overlap or interference during the experiment, thereby enhancing the reliability of the results.

Please provide a sequence alignment with multiple ATGs to show the conservation of the presumed bipartite NLS. This information has to be included in the main data.

Thank you very much for your constructive and insightful comments. We analyzed the putative nuclear localization signal (NLS) in the ATG6 protein sequence using the online INSP (Identification of Nuclear Signal Peptide) prediction software (http://www.csbio.sjtu.edu.cn/bioinf/INSP/). The prediction results indicated the presence of a potential nuclear localization sequence "FLKEKKKKK" within the ATG6 protein, spanning from the 217th to the 223rd amino acid. Additionally, we utilized INSP to investigate the nuclear localization sequences of various ATG proteins (TaATG6a [1], TaATG6b [1], TaATG6c [1], SlATG8h [2]) that have been previously reported to localize in the nucleus. This analysis revealed a relatively conserved NLS sequence motif: "E/K-K/E-K-K-L/K-K" in these ATG proteins. In line with your suggestion, the results of this sequence comparison have been incorporated into the revised manuscript as Figure 2c. The revised manuscript includes a description of the corresponding results. (lines 146-156).

Fig. 3d and f, how many blots are used for this quantification? Please include all the individual analyzed blots in the supplementary data. In addition, if you present such quantification with error bars, then statistical analysis is required.

Thank you for pointing this out. In Figure 3d, three independent blots were utilized for this quantification. In Figure 3f, two independent blots were used. The individual analyzed blots have been included in the supplementary Figure 7. We also conducted a statistical analysis as shown in Fig 3d and f, with a detailed description included in the legend section (lines 858 and 861).

In fig. 4, please indicate what is the normalizing gene. Also, what are the error bars?

Thank you for pointing this out. In Fig.4, values are means ± SD (n = 3 biological replicates). The AtActin gene was used as the internal control. We have included a detailed description in the figure notes

In fig. 4b the labeling is missing.

Thank you for bringing this to our attention. We have included the labeling for Fig. 4 in the revised manuscript.

Lines 236-239: this statement contradicts the data in fig. 5b: the levels of NPR1-GFP are actually reduced in the presence of atg6 at 24h. So, this result has to be described more accurately by stating that the increase is transient, and it is evident more at 8h, but not at 20-24h.

Thank you very much for you constructive and insightful comments. We have revised the description of this section to provide a more accurate account of the results (lines 253-258).

Reference

(1) Yue J, Sun H, Zhang W, et al. Wheat homologs of yeast ATG6 function in autophagy and are implicated in powdery mildew immunity. BMC Plant Biol. 2015;15:95.

(2) Li F, Zhang M, Zhang C, et al. Nuclear autophagy degrades a geminivirus nuclear protein to restrict viral infection in solanaceous plants. New Phytol. 2020;225:1746-1761.