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.

The authors have addressed most of my previous concerns.

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.

Author response:

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

eLife assessment

The study reports on a previously unrecognized function of ATG6 in plant immunity. The work is valuable because it proposes a direct interaction between ATG6 and a well-studied salicylic acid receptor protein, NPR1, which may interest researchers investigating plant immunity regulation. While the data presented are compelling, more information regarding the specificity of ATG6's role would improve the overall impact of the study, especially with an eye towards consistency with prior work.

We also genuinely thank the editor and reviewers for the constructive and helpful suggestions and comments. These comments have greatly improved the quality and thoroughness of our manuscript. We have carefully studied these comments and have made the appropriate changes as far as possible. Additionally, some minor errors were also corrected during the revision process. New text is shown in blue in the revised manuscript. Our responses to the reviewer's comments are provided below each respective comment.

Public Reviews:

Reviewer #1 (Public Review):
Summary:
The authors showed that autophagy-related genes are involved in plant immunity by regulating the protein level of the salicylic acid receptor, NPR1.
Strengths:
The experiments are carefully designed and the data is convincing. The authors did a good job of understanding the relationship between ATG6 and NRP1.

Thank you very much for recognizing our research.

Weaknesses:
- The authors can do a few additional experiments to test the role of ATG6 in plant immunity.
I recommend the authors to test the interaction between ATGs and other NPR1 homologs (such as NPR2).

Thanks to your valuable feedback, it was discovered that the Arabidopsis NPRs family comprises six members: NPR1, NPR2, NPR3, NPR4, NPR5/PETIOLE 1 (BOP1), and NPR6/BOP2. NPR3/4 function in tandem as negative regulators to modulate SA signaling and plant immune responses (Ding et al., 2018). Similar to NPR1, NPR2 acts as a positive regulator of SA signaling (Castello et al., 2018). NPR5/BOP1 and NPR6/BOP2 primarily participate in the regulation of plant growth and development (McKim et al., 2008). This study specifically investigates the correlation between ATG6 and NPRs in plant resistance to pathogenic bacteria. Consequently, we experimentally confirmed the interaction between ATG6 and NPR1, NPR3, and NPR4 (Fig. 1 and Fig. S1 in the revised manuscript). It would be intriguing to further explore the interactions between ATG6 and other NPRs in the context of regulating plant growth and development in future research endeavors.

-The concentration of SA used in the experiment (0.5-1 mM) seems pretty high. Does a lower concentration of SA induce ATG6 accumulation in the nucleus?

Thank you for pointing this out. The NPR1 protein is known to be unstable and prone to degradation through the 26S proteasome pathway (Spoel et al., 2009; Saleh et al., 2015). Consequently, to investigate the function of NPR1, many scientists and research groups typically employ higher concentrations of SA (e.g., 0.5 mM, 1 mM, or even 5 mM) to elucidate its role (Spoel et al., 2009; Fu et al., 2012; Lee et al., 2015; Saleh et al., 2015; Skelly et al., 2019; Zavaliev et al., 2020; Chen et al., 2021a). In our study, we observed an interaction between ATG6 and NPR1. To enhance the detection of the NPR1 protein, we standardized the SA concentration (Arabidopsis was treated with 0.5 mM SA; Tobacco was treated with 1 mM SA) used in our experiments. Subsequently, we analyzed the nuclear accumulation ATG6 or NPR1 using a relatively high SA concentration (Arabidopsis was treated with 0.5 mM SA; Tobacco was treated with 1 mM SA), consistent with concentrations used in previous studies (Spoel et al., 2009; Lee et al., 2015; Saleh et al., 2015; Skelly et al., 2019; Zavaliev et al., 2020; Chen et al., 2021a).

-Does the silencing of ATG6 affect the cell death (or HR) triggered by AvrRPS4?

Thank you for pointing this out. In this study, we examined changes in Pst DC3000/avrRps4-induced cell death in Col, amiRNAATG6 # 1, amiRNAATG6 # 2, npr1, NPR1-GFP, ATG6-mCherry and ATG6-mCherry × NPR1-GFP plants. The results of Taipan blue staining showed that Pst DC3000/avrRps4-induced cell death in npr1, amiRNAATG6 # 1 and amiRNAATG6 # 2 was significantly higher compared to Col (Fig. S15 in the revised manuscript). Conversely, Pst DC3000/avrRps4-induced cell death in ATG6-mCherry, NPR1-GFP and ATG6-mCherry × NPR1-GFP was significantly lower compared to Col. Notably, Pst DC3000/avrRps4-induced cell death in ATG6-mCherry × NPR1-GFP was significantly lower compared ATG6-mCherry and NPR1-GFP (Fig. S15 in the revised manuscript). These results suggest that ATG6 and NPR1 cooperatively inhibit Pst DC3000/avrRps4-induced cell dead. The relevant description can be found in lines 394-404 of the revised manuscript.

-SA and NPR1 are also required for immunity and are activated by other NLRs (such as RPS2 and RPM1). Is ATG6 also involved in immunity activated by these NLRs?

Thank you for your valuable comments. The most notable event in the NLR-mediated ETI immune response is the induction of hypersensitive response-programmed cell death (HR-PCD) (Jones and Dangl, 2006; Yuan et al., 2021). SA plays a dual role in the ETI response. On one hand, the accumulation of SA during the R gene-mediated ETI defense response is directly linked to the onset of HR-PCD (Nawrath and Metraux, 1999). SA and NPR1 can enhance the ETI response by regulating the expression of downstream target genes (Falk et al., 1999; Feys et al., 2001; Ding et al., 2018; Liu et al., 2020). On the other hand, the activation of SA signaling can have a negative regulatory effect on HR-PCD during the ETI response. High levels of SA have been shown to significantly inhibit HR-PCD triggered by the avrRpt2 effector (Rate and Greenberg, 2001; Devadas and Raina, 2002; Jurkowski et al., 2004). Rate et al. discovered that the inhibition of HR-PCD by SA relies on NPR1 (Rate and Greenberg, 2001).

Arabidopsis AtATG6 or its homologs in other species (such as NbBECLIN1, TaATG6s, etc.) have been identified as positive regulators in plant immunity, playing a crucial role in inhibiting cell death and preventing invasion by pathogenic microorganisms (Liu et al., 2005; Patel and Dinesh-Kumar, 2008; Yue et al., 2015). Patel et al. demonstrated that, akin to autophagy-deficient mutants previously documented, AtATG6 antisense (AtATG6-AS) plants treated with Pst DC3000/avrRpm1 exhibited diffuse cell death, indicating the necessity of ATG6 in restricting cell death (Patel and Dinesh-Kumar, 2008). In tobacco, deficiencies in BECLIN 1 result in the onset of diffuse HR-PCD, underscoring the essential role of BECLIN 1 in limiting HR-PCD (Liu et al., 2005). Despite the genetic evidence supporting the critical function of ATG6 in plant immunity, the precise molecular mechanisms through which ATG6 impedes the invasion of pathogenic microorganisms remain elusive.

In our study, we uncovered that ATG6 interacts with NPR1 to hinder pathogen invasion and inhibit the initiation of cell death. In animals, members of the NLR family have been observed to interact with the autophagy-related protein LC3 to inhibit the survival of pathogen (Zhang et al., 2019). Similar mechanisms may exist in plants. However, it remains to be explored whether NLR directly induces the activation of ATG6 through interaction or the relationship between NPR1-ATG6 interactions and NLR-mediated plant immunity, necessitating further investigation.

Reviewer #2 (Public Review):

Summary:

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.

However, the overall conclusions of the study are not well supported experimentally. The significance of the findings is low because of their mostly correlational nature, and lack of consistency with earlier reports on the same protein.

Thank you for your valuable and constructive suggestions. In this article, we unveil a novel relationship in which ATG6 positively regulates NPR1 in plant immunity (Fig. 8 in the revised manuscript). ATG6 interacts with NPR1 to synergistically enhance plant resistance by regulating NPR1 protein levels, stability, nuclear accumulation, and formation of SINCs-like condensates. This may be of interest to researchers studying the regulation of plant immunity. While there may be minor flaws in our current study, the significance of these findings cannot be overstated, as they have the potential to redirect scientific attention towards uncovering novel functions for autophagy genes.

Based on the integrity and quality of the data as well as the depth of analysis, it is not yet clear if ATG6 is a specific regulator of NPR1 or if it is affecting NPR1's stability indirectly, through inducing an elevation of SA levels in plants. As such, the current study demonstrates a correlation between overexpression of ATG6, SA accumulation, and NPR1 stability, however, whether and how these components work together is not yet demonstrated.

Thanks to your valuable feedback. Although as the reviewer said there may be some flaws in our data from the current results, scientific research is an ongoing process and I am confident that future studies will be even better. From the results given to us at the moment at least this study reports a previously undiscovered function of ATG6 in plant immunity. We propose a direct interaction between ATG6 and a well-studied salicylic acid receptor protein, NPR1. We unveil a novel relationship in which ATG6 positively regulates NPR1 in plant immunity (Fig. 8 in the revised manuscript). ATG6 interacts with NPR1 to synergistically enhance plant resistance by regulating NPR1 protein levels, stability, nuclear accumulation, and formation of SINCs-like condensates. This may be of interest to researchers studying the regulation of plant immunity.

Based on the provided biochemical data, it is not yet clear if the ATG6 functions specifically through NPR1 or through its paralogs NPR3 and NPR4, which are negative regulators of immunity. It is quite possible that interaction with NPR1 (or any NPR) is not the major regulatory step in the activity of ATG6 in plant immunity. The effect of ATG6 on NPR1 could well be indirect, through a change in the SA level and redox environment of the cell during the immune response. Both SA level and redox state of the cell were reported to induce accumulation of NPR1 in the nucleus and increase in stability.

Thanks to your valuable feedback. In this study, we validated the interaction between ATG6 and NPR1 through various approaches and identified the key regions mediating their interaction. Our findings indicate that ATG6 interacts with NPR1 to synergistically enhance plant resistance by regulating NPR1 protein levels, stability, nuclear accumulation, and the formation of SINC-like condensates. These results clearly demonstrate the involvement of ATG6 in the regulation of NPR1.Furthermore, we also found that ATG6 interacts with NPR3/4 (Fig. S1 in the revised manuscript). This is particularly relevant given that NPR3 and NPR4 have been shown to act as adaptors for the ubiquitin E3 ligase Cullin 3 (CUL3) to regulate the degradation of NPR1. Therefore, whether ATG6 regulates NPR1 through its interactions with NPR3/4 is an intriguing question worth exploring in future studies. We appreciate the reviewer's concerns and are committed to addressing them in our future research to further elucidate the complex regulatory mechanisms involving ATG6, NPR1, and other key players in plant immunity.

Another major issue is the poor quality of the subcellular analyses. In contradiction to previous studies, ATG6 in this study is not localized to autophagosome puncta, which suggests that the soluble localization pattern presented here does not reflect the true localization of ATG6. Even if the authors propose a novel, non-canonical nuclear localization for ATG6, they still should have detected the canonical autophagy-like localization of this protein.

Thanks to your valuable feedback. We conducted predictions at NLS Mapper (https://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi) and identified two bipartite NLSs in ATG6, with the sequences "MRKEEIPDKSRTIPIDPNLPKWVCQNCHHS" and "DPNLPKWVCQNCHHS LTIVGVDSYAGKFFNDP". To further elucidate the nuclear localization of ATG6, we introduced Agrobacterium tumefaciens carrying ATG6-GFP into nls-mCherry tobacco leaves through transient transformation. Subsequently, we observed the localization of ATG6-GFP, along with the canonical autophagy-like patterns. Our findings revealed fluorescence signals of ATG6-GFP in both the cytoplasm and nuclei (Figure 2b). The nuclear-localized ATG6-GFP overlapping with the nuclear-localized marker, nls-mCherry (indicated by white arrows). Additionally, we observed punctate patterns indicative of canonical autophagy-like localization of ATG6-GFP fluorescence signals (indicated by red circles). Based on these results, we are more confident about the authenticity of ATG6's nuclear localization. The revised manuscript includes clearer images to support our observations.

Recommendations for the Authors:

Reviewer #2 (Recommendations For The Authors):

The duration and concentration of SA treatments are quite variable between experiments which makes comparisons difficult.

Thank you for pointing this out. The NPR1 protein is known to be unstable and prone to degradation through the 26S proteasome pathway (Spoel et al., 2009; Saleh et al., 2015). Consequently, to investigate the function of NPR1, many scientists and research groups typically employ higher concentrations of SA (e.g., 0.5 mM, 1 mM, or even 5 mM) to elucidate its role (Spoel et al., 2009; Fu et al., 2012; Lee et al., 2015; Saleh et al., 2015; Skelly et al., 2019; Zavaliev et al., 2020; Chen et al., 2021a). In our study, we observed an interaction between ATG6 and NPR1. To enhance the detection of the NPR1 protein, we standardized the SA concentration used in our experiments. In this study, for the treatment of Arabidopsis, we followed the protocols outlined in Saleh et al. and Spoel et al., utilizing 0.5 mM SA (Spoel et al., 2009; Saleh et al., 2015). For tobacco treatment, we adopted the methodology described in the study by Zavaliev et al., administering 1 mM SA (Zavaliev et al., 2020).

The methods section does not explain some of the essential experimental conditions and reagents used in the study.

Thank you for pointing this out. Due to word limitations we have placed the detailed experimental methods and reagents in Supplemental Data 1. In Supplemental Data 1, we provide a comprehensive overview of the experimental flow and conditions employed in our study.

Lines 62-63: the C-terminal domain of all NPRs has a name (already defined as SA-binding domain (SBD)). Also, it would be worth referring to the structure of NPR1 (Kumar et al 2022, Nat) as the source of information about its domains.

Thank you for pointing this out, we have changed this description in the revised manuscript (lines 62-63).

Lines 66-69: NPR1 doesn't form monomers. A recent study showed that the basic functional unit of NPR1 is a dimer (Kumar et al 2022, Nat).

Thank you for pointing this out. In the revised manuscript (line 67) " monomers " has been changed to “dimer”.

Lines 89-95 and elsewhere: the term "invasion" has a very specific meaning and it doesn't necessarily refer to disease. A pathogen can invade the plant but cause no disease (e.g. ETI). Most plant genetic immune mechanisms act after pathogen invasion, not before it. Those cited works reported the disease resistance, not the invasion resistance.

Thank you for pointing this out. We've changed the incorrect description in the revised manuscript (line 91).

Lines 113-119: the truncation at the aa328 includes half of the ANK domain (repeats 1 and 2), not just BTB. The C-terminal truncation variant contains the other half (repeats 3 and 4) of the ANK domain, not the entire ANK domain. It also contains the SBD, not just the NLS. So, this kind of analysis cannot determine the role of ANK domain in the interaction, nor it can conclusively determine if the interaction is through SBD. The interaction should be tested with the SBD domain only in order to make this conclusion.

Thank you for pointing this out, we have removed the inappropriate description and made the appropriate changes in the revised manuscript (lines 114 and 115).

In Figure S1, the equally strong interaction of atg6 is found for NPR3/NPR4. Does that mean that atg6 functions also through these other NPRs? What's the significance of these data compared to NPR1-ATG6 interaction? This is especially important, because both NPR3 and NPR4 are predominantly nuclear proteins, and they are unlikely to significantly overlap with autophagy components in the cytoplasm.

NPR1 and its paralogues NPR3/NPR4, which frequently interact with other proteins to regulate plant immune responses (Backer et al., 2019; Chen et al., 2019). To identify ATGs that interact with NPRs, we performed yeast two-hybrid (Y2H) screens using NPRs as bait. Interestingly, ATG6 interacted with NPR1, NPR3 and NPR4, respectively, and different concentrations of SA treatment did not significantly affect their interaction (Fig. S1a). NPR1 is an important positive regulator of the plant immune response (Chen et al., 2021b). In Arabidopsis and N. benthamian, ATG6 or its homologues was reported to act as a positive regulator to enhance plant disease resistance to P. syringae pv. tomato (Pst) DC3000 and Pst DC3000/avrRpm1 bacteria (Patel and Dinesh-Kumar, 2008), N. benthamiana mosaic virus (TMV) (Liu et al., 2005). Therefore, in this study we focused on investigating the biological significance of the interaction between ATG6 and NPR1. Whether the interaction between ATG6 and NPR3/4 also has an effect on plant immunity is a question that remains to be explored in future studies.

In Figure 1c and elsewhere: why not use the anti-mCherry antibody to detect atg6-mcherry? Are we seeing the correct protein band of atg6-mcherry? Also, it is not clear what antibodies they used throughout the study: the sources and specificities of antibodies are not provided.

Thank you for pointing this out. We initially synthesized the ATG6 antibody (anti-ATG6, 1:200, peptide, C-KEKKKIEEEERK, Abmart) in order to detect the endogenous ATG6 protein, and we also tested the specificity and potency of the ATG6 antibody (results are shown in Fig. S17). Additionally, in order to determine the location of the ATG6-mCherry bands, we also detected ATG6-mCherry in ATG6-mCherry Arabidopsis using the ATG6 antibody, and we also used Col as a control (results are shown in Fig. S4). These results show that our synthesized ATG6 antibody can effectively and clearly immunize to both ATG6 and ATG6-mCherry. Therefore, in this study, we used the ATG6 antibody to analyze both ATG6-mCherry and endogenous ATG6. Detailed antibody information is presented in Supplementary Data 1, table S4

In Figures 1d, 2a, and 2b, the subcellular localization pattern of atg6 contradicts what was published before (Fujiki et al 2007, Plant Phys; Liu et al 2018, FPlS; Xu et al 2017, Autophagy; Li et al 2018, Nat. Comm.). As an autophagy protein, atg6 was shown to localize to cytoplasmic puncta (autophagosomes), like atg8. No nuclear localization was found in those studies. The lack of puncta and the strong nuclear accumulation are signs that the localization of atg6 reported here has to be interpreted with caution. With the data provided, I am not convinced yet that we are looking at the correct ATG6 subcellular localization. Even if the authors propose a novel, non-canonical localization for atg6, they still should have detected the canonical autophagy-like localization of this protein.

Thanks to your valuable feedback. To further elucidate the nuclear localization of ATG6, we introduced Agrobacterium tumefaciens carrying ATG6-GFP into nls-mCherry tobacco leaves through transient transformation. Subsequently, we observed the localization of ATG6-GFP, along with the canonical autophagy-like patterns. Our findings revealed fluorescence signals of ATG6-GFP in both the cytoplasm and nuclei (Figure 2b). The nuclear-localized ATG6-GFP overlapping with the nuclear-localized marker, nls-mCherry (indicated by white arrows). Additionally, we observed punctate patterns indicative of canonical autophagy-like localization of ATG6-GFP fluorescence signals (indicated by red circles). Based on these results, we are more confident about the authenticity of ATG6's nuclear localization. The revised manuscript includes clearer images to support our observations.

It would make more sense to include the BiFC data (fig. S2) in the main figure, instead of the co-localization (fig. 1d) which cannot serve as evidence for interaction.

Thank you for the feedback. We accept your suggestion. In Fig.1, we have replaced the co-localization image with a BiFC (Bimolecular Fluorescence Complementation) image to better illustrate the interaction.

In Figure S2, the bifc signals have to be quantified to qualify as evidence for interaction. also, a subcellular marker has to be used (e.g. nuclear mcherry). From the current poor-quality images, one cannot determine where in the cell the presumed interaction takes place, nucleus or cytoplasm, or both. Also, no puncta are seen in these images.

Thank you for pointing this out. Despite the lack of clarity in the images we provided, our BiFC results unequivocally demonstrate the interaction between ATG6 and NPR1 in both the cytoplasm and nucleus. Notably, as the reviewer pointed out, punctate signals were not observed in our images. This lack of punctate signals is consistent with previous studies (Figure 2) that have also shown BiFC results between autophagy-associated proteins ATG8s and their interacting partners. For instance, Fig 1G (Marshall et al. 2019, Cell), Fig 2F (Marshall et al. 2019, Cell), Fig 4B (Macharia et al. 2019, BMC Plant Biology), and Fig 3 (Zhou et al. 2018, Autophagy) all did not exhibit punctate signals, aligning closely with our findings.

In Figure S3a, the nuclear localization is shown for stomata. It is known that stomata are especially strong expressors of the transgenes, and localization there could be an artefact of overaccumulation of the fusion protein. Also, why do they present the localization of atg6-gfp, if the analysis and the cross were made with atg6-mcherry?

Thank you for pointing this out. In our previous experiments, we observed the localization of ATG6 in the nucleus of Arabidopsis thaliana plants overexpressing ATG6-GFP (Fig. S3a). To clearly visualize the location of the nucleus, we used the cytosolic DAPI dye, which readily stained the nuclei of the stomatal guard cells. This allowed us to easily identify the nuclear regions for our observations. Additionally, in Fig. 2a and Fig.S3b, we detected the fluorescence signal of ATG6-mCherry within the nucleus, further confirming the nuclear localization of ATG6. Moreover, the nuclear and cytoplasmic fractions were separated. Under SA treatment, ATG6-mCherry and ATG6-GFP were detected in the cytoplasmic and nuclear fractions in N. benthamiana (Fig. 2c and d). Similarly, ATG6 was also detected in the nuclear fraction of UBQ10::ATG6-GFP and UBQ10::ATG6-mCherry overexpressing plants (Fig. 2e and f).

In Figure S3b, the images are low resolution and of poor quality. Why atg6-mcherry is expressed in a single cell if these are transgenic plants? The nuclear co-localization with npr1-gfp has to be shown more clearly with high res. images and also be quantified, because the expression of atg6-mcherry is not as uniform as npr1-gfp.

Thank you for pointing this out. Contrary to the reviewer's assertion, the ATG6-mCherry fluorescence signal depicted in Figure S3b was not exclusive to a single cell. In fact, this fluorescence was also evident in other cells, albeit with relatively weaker intensity. This disparity in fluorescence intensity may be attributed to the irregularities in leaf structure at the time of image capture using the microscope. To bolster our conclusion, we further examined the fluorescence signals in the cells of the root elongation zone in ATG6-mCherry x NPR1-GFP, as depicted in the figure below. Our observations revealed that the fluorescence signals of ATG6-mCherry exhibited uniform distribution, with detection in both the cytoplasm and nucleus. We have replaced the original unclear image with a high-quality image.

Lines 138-143: In fig. S3d, it would make more sense to show the WB on the hybrid npr1-gfp/atg6-mcherry plants with both anti-gfp and anti-mcherry antibodies to detect the free mcherry/gfp. Since the analysis of the level of free FP is done, then why didn't they test the free mcherry levels in Figure S4a? This would be more important than testing the free GFP in ATG6-GFP plants, because the imaging of atg6-mcherry was done in the hybrid plants (fig. S3b).

Thank you for pointing this out. We initially synthesized the ATG6 antibody (anti-ATG6, 1:200, peptide, C-KEKKKIEEEERK, Abmart) in order to detect the endogenous ATG6 protein, and we also tested the specificity and potency of the ATG6 antibody (results are shown in Fig. S17). Additionally, in order to determine the location of the ATG6-mCherry bands, we also detected ATG6-mCherry in ATG6-mCherry Arabidopsis using the ATG6 antibody, and we also used Col as a control (results are shown in Fig. S4). These results show that our synthesized ATG6 antibody can effectively and clearly immunize to both ATG6 and ATG6-mCherry. Therefore, in this study, we used the ATG6 antibody to analyze both ATG6-mCherry and endogenous ATG6. Detailed antibody information is presented in Supplementary Data 1, table S4. In the previous experiments, we procured the mCherry antibody (mCherry-Tag Monoclonal Antibody(6B3), BD-PM2113, China) to immunolabel ATG6-mCherry. However, we encountered challenges with the potency of this mCherry antibody, and considering our budget constraints, as well as the availability of our self-synthesized ATG6 antibody, we chose not to pursue the purchase of another antibody from a different company for the continuation of the Western Blot experiment.

In Figure 2c, there's no atg6-mcherry detected at time 0, in either cytoplasm or nucleus, yet the microscope images in panel a show strong accumulation in both compartments.

Thank you for pointing this out. Previous studies ATG6 can also be degraded via the 26s proteasome pathway (Qi et al., 2017). We speculate that this phenomenon might be attributed to the rapid turnover of ATG6 at time 0.

Lines 156-160: this statement is unsupported by the data. In fig. S5, the bands for native atg6 in the nuclear fraction are extremely weak, and they do not show the reverse pattern of change along the time points compared to the cytoplasmic fraction, which would indicate that the nuclear fraction is complementary to the cytoplasmic pool of the protein. The result more likely suggests that the majority of the ATG6 is in the cytoplasm, and that the weak bands detected in the nucleus are either background signal, or a contamination from the cytoplasmic pool. At this low protein level or poor immuno-detection the background signal is inevitable due to overexposure. Even though the actin marker is not detected in the nuclear fraction, it doesn't necessarily mean that there's no contamination from the cytoplasm in the nuclear fraction. The actin is just too abundant and can be detected at lower exposure.

Thank you for pointing this out. In Fig. S5, we detected the subcellular localization of endogenous ATG6, although the image quality was somewhat low. Nevertheless, the cytosolic and nuclear localization of ATG6 could be clearly observed. In addition to this, we also verified the cytosolic and nuclear localization of ATG6 in Arabidopsis using confocal fluorescence microscopy and nucleoplasmic separation experiments. Actin and H3 were used as cytoplasmic and nucleus internal reference, respectively. (Fig. 2e and f). Furthermore, we observed the cytosolic and nuclear localization of ATG6 when we expressed ATG6-GFP or ATG6-mCherry in tobacco leaves through cis-transfection experiments (Fig. 2a-d). These results are consistent with the prediction of the subcellular location of ATG6 in the Arabidopsis subcellular database (https://suba.live/) (Fig. S3c). The reviewer's feedback has been valuable in helping us present these findings more clearly. We acknowledge the limitations in the image quality for the endogenous ATG6 localization, but we believe the combination of multiple experimental approaches, including the use of fluorescent protein fusions, provides robust evidence for the cytosolic localization of ATG6 in plant cells. Moving forward, we will continue to investigate the significance of ATG6's subcellular distribution and its potential dual roles in both the nucleus and the cytosol, particularly in the context of its interaction with the key immune regulator NPR1. We appreciate the reviewer's constructive comments, as they will help us strengthen the presentation and interpretation of our findings.

In Figure 3a the images are of too low resolution to see the co-localization. The focal planes of the top and bottom panels are quite different: the top is focused on stomata, the bottom - on pavement cells. So, the number of the NPR1-GFP nuclei between these two focal planes is dramatically different. Also, it looks like the atg6-mcherry in these plants are predominantly in the cytoplasm, not the nucleus as the authors claim. A higher resolution and higher quality of images are required to determine this.

Thank you for pointing this out. To ensure the clarity and accuracy of our confocal images, we have supplied a clearer image as supplementary evidence. The Bright images distinctly show that both sets of images are in the same plane of focus. Furthermore, in the figure (third one in the fourth column), the nucleus localization of ATG6-mCherry is clearly visible, and that ATG6-mCherry is co-localized with NPR1-GFP in the nucleus, as indicated by the white arrow.

In Figure 3b, it is not indicated what exactly was measured and in what condition, mock or SA. If these are numbers of nuclei, then it should be indicated what size of the area was sampled, not just "section", and both mock and SA should be included in the measurements. Also, how many independent images have been sampled? what does the error bar represent? What does "normal" mean? Shouldn't this be a mock treatment?

Thank you for pointing out this. The term "Normal" in this context refers to mock treatment, and we have revised the description for clarity. In Figure 3b, the graph illustrates the count of nuclear localizations of NPR1-GFP in ATG6-mCherry × NPR1-GFP and NPR1-GFP Arabidopsis plants following SA treatment. Statistical data were obtained from three independent experiments, each comprising five individual images, resulting in a total of 15 images analyzed for this comparison. Detailed descriptions were also added to the revised manuscript (Lines 568-570, 800-804).

Lines 167-168: the proposed increase of NPR1-GFP in the nucleus could be simply due to a higher accumulation of SA in the hybrid plants, not because of the direct interaction of atg6.

Thank you for pointing out this. Our results confirmed that ATG6 overexpression significantly increased nuclear accumulation of NPR1 (Fig. 3). Notably, the ratio (nucleus NPR1/total NPR1) in ATG6-mCherry × NPR1-GFP was not significantly different from that in NPR1-GFP, and there is a similar phenomenon in N. benthamiana (Fig. 3c-f). These results suggested that the increased nuclear accumulation of NPR1 by ATG6 might result from higher levels and more stable NPR1, rather than the enhanced nuclear translocation of NPR1 facilitated by ATG6. Furthermore, we found that under SA treatment, the protein levels of NPR1 were significantly higher in the ATG6-mCherry × NPR1-GFP line compared to the NPR1-GFP line (Fig. 5a). Notably, even in the absence of differences in SA levels between the two lines, we observed that ATG6 could delay the degradation of NPR1 under normal conditions (Fig. 6). These findings suggest that ATG6 employs both SA-dependent and SA-independent mechanisms to maintain the stability of the key immune regulator NPR1. In summary, we therefore suggest that the increased nuclear accumulation in NPR1 cells is a dual effect of SA and ATG6.

Lines 202-204: "Increased nuclear accumulation" implies increased translocation. However, they found that the ratio of NPR1-GFP does not change (Figure 3), so the reason for higher nuclear accumulation is not translocation, but abundance.

Thank you for pointing out this. Our results confirmed that ATG6 overexpression significantly increased nuclear accumulation of NPR1 (Fig. 3). ATG6 also increases NPR1 protein levels and improves NPR1 stability (Fig. 5 and 6). Therefore, we consider that the increased nuclear accumulation of NPR1 in ATG6-mCherry x NPR1-GFP plants might result from higher levels and more stable NPR1 rather than the enhanced nuclear translocation of NPR1 facilitated by ATG6. To verify this possibility, we determined the ratio of NPR1-GFP in the nuclear localization versus total NPR1-GFP. Notably, the ratio (nucleus NPR1/total NPR1) in ATG6-mCherry × NPR1-GFP was not significantly different from that in NPR1-GFP, and there is a similar phenomenon in N. benthamiana (Fig. 3c-f). These results suggested that the increased nuclear accumulation of NPR1 by ATG6 might result from higher levels and more stable NPR1, rather than the enhanced nuclear translocation of NPR1 facilitated by ATG6. Further we analyzed whether ATG6 affects NPR1 protein levels and protein stability. Our results show that ATG6 increases NPR1 protein levels under SA treatment and ATG6 maintains the protein stability of NPR1 (Fig. 5 and 6). These results suggested that the increased nuclear accumulation of NPR1 by ATG6 result from higher levels and more stable NPR1. The corresponding description is shown in revised manuscript (lines 338~352).

Lines 204-205: the co-localization in Figure 1d cannot be interpreted as interaction.

Thank you for the feedback. We have replaced the co-localization image with a BiFC (Bimolecular Fluorescence Complementation) image to better illustrate the interaction in Fig 1d.

What age of plants were used for the analysis in Figures 4 and S7? The age of the plant might significantly affect the free SA levels under control conditions.

Thank you for the feedback. In Figures 4 and S7, 3-week-old plants were used to determine salicylic acid (SA) levels and the expression of target genes. Figures 4 and S7 figure notes provide detailed descriptions (lines 818-819).

In Figure 5a they treat with SA, but the analysis in Figure S10 is done with the pathogen, so how can these data be correlated?

Thank you for pointing out this. Previous studies have demonstrated that pathogen infestation rapidly increases the salicylic acid (SA) content in plants, and the elevated SA then activates plant immune responses. Therefore, both pathogen treatment and direct SA treatment can activate SA-dependent plant immune responses. The NPR1 protein is known for its instability. In Figure 5a, we utilized a 0.5 mM SA treatment to assess the changes in NPR1 protein levels, as the impact of SA treatment is more immediate and pronounced.

Lines 241-242: In Figure 5b, it is not clear why there's no detection of NPR1-GFP and atg6-mcherry at time 0?? The levels of proteins in the transient assay are sufficiently high for detection by WB.

Thank you for pointing this out. The NPR1 protein is known to be unstable and prone to degradation through the 26S proteasome pathway (Spoel et al., 2009; Saleh et al., 2015). In addition, previous studies ATG6 can also be degraded via the 26s proteasome pathway (Qi et al., 2017). We speculate that this phenomenon might be attributed to the rapid turnover of NPR1 and ATG6 at time 0.

In Figures 5c-d, the quality of these images is very poor, and they do not clearly show the signs. What structure was exactly measured in these images? There are so many fluorescent bodies there, that it is not clear what are we looking at. Also, it is not clear why they did not show the mcherry channel? It would be important to see if the bodies in SA-treated plants show co-localization with atg6-mcherry autophagosomes (if these exist at all).

Thank you for pointing this out. Interestingly, similar to previous reports (Zavaliev et al., 2020), SA promoted the translocation of NPR1 into the nucleus, but still a significant amount of NPR1 was present in the cytoplasm (Fig. 3c and e). Previous studies have shown that SA increased NPR1 protein levels and facilitated the formation of SINCs in the cytoplasm, which are known to promote cell survival (Zavaliev et al., 2020). We therefore observed the fluorescence signal of SINCs-like condensates in the cytoplasm of tobacco leaves. After 1mM SA treatment, more SINCs-like condensates fluorescence were observed in N. benthamiana co-transformed with ATG6-mCherry + NPR1-GFP compared to mCherry + NPR1-GFP (Fig. 5c-d and Supplemental movie 1-2). We have a clearer demonstration in the supplemental video movie 1-2. Additionally, we observed that SINCs-like condensates signaling partial co-localized with certain ATG6-mCherry autophagosomes fluorescence signals.

Lines 245-247: so, is it atg6 or SA that increases the NPR1 levels? If this is due to SA, then the whole study doesn't have novelty, because we already know from previous works that SA increases the stability of npr1.

Thank you for pointing this out. Indeed, previous studies have shown that salicylic acid (SA) increases NPR1 levels and protein stability (Spoel et al., 2009; Saleh et al., 2015). In our experiments, we found that under SA treatment, the protein levels of NPR1 were significantly higher in the ATG6-mCherry × NPR1-GFP line compared to the NPR1-GFP line (Fig. 5a). Additionally, free SA levels were also significantly elevated in the ATG6-mCherry × NPR1-GFP line under pathogen challenge (Pst DC3000/avrRps4), but not under normal conditions (Fig. 4a). Furthermore, even in the absence of differences in SA levels between the two lines, we observed that ATG6 could delay the degradation of NPR1 under normal conditions (Fig. 6). These findings represent one of our new discoveries. These findings suggest that ATG6 employs both SA-dependent and SA-independent mechanisms to maintain the stability of the key immune regulator NPR1.

Lines 313-316: npr1 and atg6 can function independently from each other, so the term "jointly" is misleading. Based on the overall data provided in this manuscript it cannot be concluded that the two proteins work in one complex to control plant immunity.

Thank you for pointing this out. In the revised manuscript "jointly" has been changed to “cooperatively”.

Lines 369-374: this speculation is beyond the main hypothesis claiming that atg6 functions through npr1. If atg6 can activate the transcription alone, then what is the significance of its activation of npr1? How can one distinguish between the two?

Thank you for pointing this out. Transcription activation by transcription factors typically requires at least two conserved structural domains: a transcription activation domain and a DNA-binding domain. However, ATG6 does not possess these two typical conserved structural domains found in canonical transcription factors. Given this structural context, it is unlikely that ATG6 would be able to directly activate transcription on its own. The lack of the canonical transcription factor domains in ATG6 suggests that it may not be able to function as a direct transcriptional activator. Previous studies have shown that acidic activation domains (AADs) in transcriptional activators (such as Gal4, Gcn4 and VP16) play important roles in activating downstream target genes. Acidic amino acids and hydrophobic residues are the key structural elements of AAD (Pennica et al., 1984; Cress and Triezenberg, 1991; Van Hoy et al., 1993). Chen et al. found that EDS1 contains two ADD domains and confirmed that EDS1 is a transcriptional activator with AAD (Chen et al., 2021a). Here, we also have similar results that ATG6 overexpression significantly enhanced the expression of PR1 and PR5 (Fig. 4b-c and S9), and that the ADD domain containing acidic and hydrophobic amino acids is also found in ATG6 (148-295 AA) (Fig. S14). We speculate that ATG6 might act as a transcriptional coactivator to activate PRs expression synergistically with NPR1.

Lines 389-400: the cell death due to AvrRPS4 in Col-0 ecotype is extremely weak as there's no complete receptor complex for this effector. So, one has to use a very high dose to induce cell death in Col-0, certainly higher than the one used for bacterial growth. The authors used the same dose in both assays, so it is likely that what we see as "cell death" is not an effector-triggered response, but rather symptom-associated for the virulent pathogen.

Thank you for pointing this out. Indeed, as the reviewer pointed out, most cell death assays use higher concentrations of Pst DC3000/avrRps4 or Pst DC3000/avrRpt2, but they typically treat Arabidopsis for a relatively short period, usually less than 1 day(Hofius et al., 2009; Zavaliev et al., 2020). In this study, although we used relatively low Pst DC3000/avrRps4 (0.001) injections, we detected cell death under a relatively long period of Pst DC3000/avrRps4 infestation (3 days). Pst DC3000/avrRps4-infested plants multiply significantly in host cells, and therefore we assumed that the propagated pathogens after 3 days of incubation would be sufficient to induce intense cell death. Consequently, we chose this concentration of Pst DC3000/avrRps4 for the experiment.

Lines 407-416: why do you expect "delay of degradation" with autophagy inhibitor? Shouldn't it be the opposite? In Figure S14, if we compare the bands between 120min and 120min+ConA+WM, the effect of autophagy inhibitors is actually quite strong (0.47 vs 0.22), with about 50% more degradation of NPR1 in their presence. So, the conclusion that the degradation of NPR1 is autophagy-independent is wrong according to this result.

Thank you for pointing this out. We have revised the inaccurate description, as outlined in the revised manuscript (lines 413-425).

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