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

Autophagy-related gene 6 (ATG6) plays a crucial role in plant immunity. Nonexpressor of pathogenesis-related genes1 (NPR1) acts as a signaling hub of plant immunity. However, the relationship between ATG6 and NPR1 is unclear. Here, we find that ATG6 directly interacts with NPR1. ATG6 overexpression significantly increased nuclear accumulation of NPR1. Furthermore, we demonstrate that ATG6 increases NPR1 protein levels and improves its stability. Interestingly, ATG6 promotes the formation of SINCs (SA-induced NPR1 condensates)-like condensates. Additionally, ATG6 and NPR1 synergistically promote the expression of pathogenesis-related genes. Further results showed that silencing ATG6 in NPR1-GFP exacerbates Pst DC3000/avrRps4 invasion, while double overexpression of ATG6 and NPR1 synergistically inhibits Pst DC3000/avrRps4 invasion. In summary, our findings unveil an interplay of NPR1 with ATG6 and elucidate important molecular mechanisms for enhancing plant immunity. Highlight We unveil a novel relationship in which ATG6 positively regulates NPR1 in plant immunity.


Introduction
Plants are constantly challenged by pathogens in nature.In order to survive and reproduce, plants have evolved complex mechanisms to cope with attack by pathogens (Jones and Dangl, 2006).Nonexpressor of pathogenesisrelated genes 1 (NPR1) is a key regulator of plant immunity (Chen et al., 2021b).It contains the BTB/POZ (Broad Compex, Tramtrack, and BricaBrac/Pox virus and Zinc finger) domain in the N-terminal region, the ANK (Ankyrin repeats) domain in the middle region, and SA-binding domain (SBD) and the nuclear localization sequence (NLS) in the C-terminal region (Cao et al., 1997;Rochon et al., 2006;Kumar et al., 2022).NPR1 is a receptor of SA (salicylic acid) mainly localized as an oligomer in the cytoplasm and sensitive to the surrounding redox state (Tada et al., 2008;Wu et al., 2012).SA mediates the dynamic oligomer to dimer response of NPR1 (Tada et al., 2008) and promotes translocation of NPR1 into the nucleus, which increases plant resistance to pathogens by activating the expression of immune-related genes (Kinkema et al., 2000;Chen et al., 2021b).
Here, we show that ATG6 and NPR1 synergistically enhance Arabidopsis resistance to Pst DC3000/avrRps4 infiltration.We discover that ATG6 increases NPR1 protein levels and nuclear accumulation of NPR1.Moreover, ATG6 can stabilize NPR1 and promote the formation of SINCs (SA-induced NPR1 condensates)-like condensates.Our study revealed a unique mechanism in which NPR1 cooperatively increases plant immunity with ATG6.

NPR1 physically interacts with ATG6 in vitro and in vivo
To examine the relationship between ATGs and NPRs, we predicted that some ATGs might interact with NPRs.In a yeast two-hybrid (Y2H) screen, we identified that NPR1, NPR3 and NPR4 could interact with ATG6 and several ATG8 isoforms (Fig. S1 and Results S1 in the Supplemental Data 1).In this study, we mainly investigated the relationship between ATG6 and NPR1 during the process of plant immune response.Firstly, the NPR1 truncations NPR1-N (1-328AA, containing the BTB/POZ domain, ANK1, ANK2,) and NPR1-C (328-594AA, containing the ANK3, ANK4, SA-binding domain (SBD) and NLS) were used to identify the interaction domains between NPR1 and ATG6.The results showed that NPR1-C interacted with full-length ATG6 in yeast (Fig. 1a, line 3).The interaction between NPR1 and SnRK2.8 was used as a positive control (Lee et al., 2015).Secondly, pull-down assays were performed in vitro.NPR1-His bound GST-ATG6, but not GST (Fig. 1b).
Furthermore, co-immunoprecipitation (Co-IP) assays were performed in N. benthamiana, as shown in Fig. 1c, ATG6-mCherry was coimmunoprecipitated with NPR1-GFP.In Fig. S2, fluorescence signals of NPR1-GFP and ATG6-mCherry were co-localized in the nucleus under normal and 1 mM SA treatment conditions.The interaction between ATG6 and NPR1 was also verified by a bimolecular fluorescence complementation (BiFC) assay (Fig. 1d and e).These results demonstrate that ATG6 interacts with NPR1 both in vitro and in vivo.

ATG6 co-localized with NPR1 in the nucleus
Remarkably, under normal and SA treatment conditions, we found that ATG6 is localized in the cytoplasm and nucleus, and it co-localized with NPR1 in the nucleus (Fig. S2).Nuclear localization of ATG6 was also observed in N. benthamiana transiently transformed with ATG6-mCherry and ATG6-GFP under normal and SA treatment conditions (Fig. 2a and b).ATG6-GFP colocalizes with the nuclear localization marker nls-mCherry (indicated by white arrows) (Fig. 2b).Additionally, we observed punctate patterns indicative of canonical autophagy-like localization of ATG6-GFP fluorescence signals (indicated by red circles) (Fig. 2b).The nuclear localization signal of ATG6 was also observed in UBQ10::ATG6-GFP overexpressing Arabidopsis (Fig. S3a).To exclude the possibility that the observed localization of ATG6-GFP is due to free GFP.The protein levels of ATG6-GFP and free GFP in UBQ10::ATG6-GFP Arabidopsis and N.benthamiana were detected before and after SA treatment.Notably, no free GFP was detected and this means that the fluorescence signal observed by confocal microscopy is ATG6-GFP, not free GFP (Fig. S3d and e within the ATG6 protein, spanning from the 217th to the 223rd amino acid (Fig. 2c).We also utilized INSP to investigate the NLS of various ATG proteins (TaATG6a (Yue et al., 2015), TaATG6b (Yue et al., 2015), TaATG6c (Yue et al., 2015), SlATG8h (Li et al., 2020)) 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 (Fig. 2c).
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. 2d and e).
However, in N. benthamiana, we observed that ATG6-mCherry was not detected in the nuclear fractions under normal conditions, which differents with the results shown in Fig. 2a.We suspect that this discrepancy may be due to the fluorescence signal in Fig. 2a primarily arising from free mCherry rather than the ATG6-mCherry fusion.ATG6 was also detected in the nuclear fraction of UBQ10::ATG6-GFP and UBQ10::ATG6-mCherry overexpressing plants, and SA promoted both cytoplasm and nuclear accumulation of ATG6 (Fig. 2f and g).Additionally, we obtained ATG6 and NPR1 double overexpression of Arabidopsis UBQ10::ATG6-mCherry × 35S::NPR1-GFP (ATG6-mCherry ×NPR1-GFP) by crossing and screening (Fig. S4a).In ATG6-mCherry × NPR1-GFP, we observed co-localization of ATG6-mCherry with NPR1-GFP in the nucleus (Fig. S3b).These results are consistent with the prediction of the subcellular location of ATG6 in the Arabidopsis subcellular database (https://suba.live/) (Fig. S3c).Additionally, we have conducted an investigation into the localization of endogenous ATG6 in Col. Our results demonstrate that endogenous ATG6 is present in both the nucleus and cytoplasm, and we have observed that SA treatment promotes the accumulation of ATG6 in the nucleus (Fig. S5).Together, these findings suggest that ATG6 is localized to both cytoplasm and nucleus, and colocalized with NPR1 in the nucleus.

ATG6 overexpression increased nuclear accumulation of NPR1
Previous studies have shown that the nuclear localization of NPR1 is essential for improving plant immunity (Kinkema et al., 2000;Chen et al., 2021b).We observed that a stronger nuclear localization signal of NPR1-GFP in ATG6-mCherry × NPR1-GFP leaves than that in NPR1-GFP under normal condition and 0.5 mM SA treatment for 3 h (Fig. 3a-b and Fig. S6).These findings indicate that ATG6 might increase nuclear accumulation of NPR1.To exclude the possibility that the observed localization of NPR1-GFP is due to free GFP, we detected the levels of NPR1-GFP and free GFP in ATG6-mCherry x NPR1-GFP plants before and after SA treatment.Only ~ 10 % of free GFP was detected in ATG6-mCherry x NPR1-GFP plants before and after SA treatment, confirming that the observed localization of NPR1-GFP is not due to free GFP (Fig. S4b).Furthermore, the nuclear and cytoplasmic fractions of ATG6-mCherry × NPR1-GFP and NPR1-GFP were separated.
Under normal conditions, the nuclear fractions NPR1-GFP in ATG6-mCherry × NPR1-GFP and NPR1-GFP were relatively weaker (Fig. 3c), which differs from the above observation (Fig. 3a).We speculate that this phenomenon might be attributed to the rapid turnover of NPR1 in the nucleus (Spoel et al., 2009;Saleh et al., 2015).Consistent with the fluorescence distribution results, the nuclear fractions of NPR1-GFP in ATG6-mCherry × NPR1-GFP were significantly higher than those in NPR1-GFP under 0.5 mM SA treatment for 3 and 6 h (Fig. 3c and Fig. S7).Furthermore, Agrobacterium harboring ATG6-mCherry and NPR1-GFP were transiently transformed to N. benthamiana leaves.After 1 d, the leaves were treated with 1 mM SA for 8 and 20 h.
Subsequently nucleoplasmic separation experiments were performed.Similar to Arabidopsis, increased nuclear accumulation of NPR1 was found when ATG6 was overexpressed (Fig. 3e and Fig. S7).Notably, we found that the ratio (nucleus NPR1/total NPR1) in ATG6-mCherry × NPR1-GFP was not significantly different from that in NPR1-GFP after SA treatment, and a similar phenomenon was observed in N. benthamiana (Fig. 3d, 3f and Fig. S7).
These results suggested that the increased nuclear accumulation of NPR1 in ATG6-mCherry x NPR1-GFP plants might attributed to higher levels and more stable NPR1 rather than the enhanced nuclear translocation of NPR1 facilitated by ATG6.Furthermore, we validated the functionality of the ATG6-GFP and ATG6-mCherry fusion proteins utilized in this study by examining the phenotypes of ATG6-GFP and ATG6-mCherry Arabidopsis plants under carbon starvation conditions (Fig. S8 and Results S2 in the Supplemental Data 1).

ATG6 increases endogenous SA levels and promotes the expression of NPR1 downstream target genes
NPR1 localized in the nucleus is essential for activation of immune gene expression (Kinkema et al., 2000;Chen et al., 2021b).In our study, we observed that ATG6 overexpression increased nuclear accumulation of NPR1 (Fig. 3) and demonstrated an interaction between ATG6 and NPR1 in the nucleus (Fig. 1d).Therefore, we speculate that ATG6 might regulate NPR1 transcriptional activity.Notably, the expression level of ICS1 in ATG6-mCherry × NPR1-GFP seedlings was significantly higher than that in NPR1-GFP under normal and SA treatment conditions (Fig. S9).Free SA levels in ATG6-mCherry × NPR1-GFP were also significantly higher compared to NPR1-GFP under Pst DC3000/avrRps4 treatment.While there was no significant difference was observed under normal condition (Fig. 4a), this may be related to free SA consumption, as it can be converted to bound SA (Ding and Ding, 2020).In addition, the expression of PR1 (pathogenesis-related gene) and PR5 in ATG6-mCherry × NPR1-GFP was significantly higher than that of NPR1-GFP under normal and SA treatment conditions (Fig. 4b and c).The expression of PR1 and PR5 in ATG6-mCherry was significantly higher than that of Col under Pst DC3000/avrRps4 treatment (Fig. S10).These results support the role of ATG6 in facilitating the expression of NPR1 downstream PR1 and PR5 genes.

ATG6 increases NPR1 protein levels and the formation of SINCs-like condensates
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).In our experiments, we observed that under SA treatment, the protein levels of NPR1 in ATG6-mCherry × NPR1-GFP was significantly higher than that in NPR1-GFP (Fig. 5a).To further support our conclusions, we proceeded to silence ATG6 in NPR1-GFP (NPR1-GFP/silencing ATG6) and subsequently assessed the protein level of NPR1-GFP before and after SA treatment.Our findings revealed that the protein level of NPR1-GFP in NPR1-GFP/silencing ATG6 under SA treatment was notably lower than that in the NPR1-GFP/Negative control (Fig. S11).Under SA treatment for 8 h, the protein levels of NPR1-GFP in N. benthamiana co-transformed with ATG6-mCherry + NPR1-GFP was also significantly higher than that of mCherry + NPR1-GFP (Fig. 5b).While there was a slight increase at 20 h, a minor decrease was observed at 24 h, suggesting that the rise in NPR1 protein levels induced by ATG6 was transient.We also detected the expression of NPR1 was detected.It is worth noting that NPR1 up-regulation was more obvious in Col after 3 h treatment with Pst DC3000/avrRps4.After 6 h treatment with Pst DC3000/avrRps4, there was no significant difference in the expression of NPR1 between Col and ATG6-mCherry (Fig. S12).These results suggest that ATG6 increases NPR1 protein levels.After 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).Additionally, we observed that SINCs-like condensates signaling partial co-localized with certain ATG6-mCherry autophagosomes fluorescence signals (Fig. S13).
Taken together, these results suggest that ATG6 increases the protein levels of NPR1 and promotes the formation of SINCs-like condensates, possibly caused by ATG6 increasing SA levels in vivo.

ATG6 maintains the protein stability of NPR1
Maintaining the stability of NPR1 is critical for enhancing plant immunity (Skelly et al., 2019).To further verify whether ATG6 regulates NPR1 stability, we co-transfected NPR1-GFP with ATG6-mCherry or mCherry in N.
benthamiana and performed cell-free degradation assays.Our results showed that NPR1-GFP degradation was significantly delayed when ATG6 was overexpressed (Fig. S14).A similar trend was observed in Arabidopsis, where the NPR1-GFP protein in ATG6-mCherry × NPR1-GFP showed a slower degradation rate compared to NPR1-GFP during 0~180 min time period in a cell-free degradation assay (Fig. 6a and b).Moreover, when Arabidopsis seedlings were treated with cycloheximide (CHX) to block protein synthesis, we found that NPR1-GFP in NPR1-GFP was degraded after CHX treatment for 3~9 h and the half-life of NPR1-GFP is ~3 h, while the half-life of NPR1-GFP in ATG6-mCherry × NPR1-GFP is ~9 h (Fig. 6c and d).In addition, we also analyzed the degradation of NPR1-GFP in NPR1-GFP and NPR1-GFP/atg5 following 100 μM cycloheximide (CHX) treatment.The results show that the degradation rate of NPR1-GFP in NPR1-GFP/atg5 plants was similarly to that in NPR1-GFP plants (Fig. 6e and f).These results indicate that ATG6 plays a role in maintaining the stability of NPR1, which may also be related to the fact that ATG6 promotes an increase in free SA in vivo, since SA has the function of increasing NPR1 stability (Ding et al., 2016;Skelly et al., 2019).

ATG6 and NPR1 cooperatively inhibit invasion of Pst DC3000/avrRps4
The mRNA expression levels of ATG6 in Col were significantly increased after 6 h, 12 h and 24 h under Pst DC3000/avrRps4 treatment (Fig. 7a).Similarly, both the ATG6 gene and protein were significantly up-regulated under 0.5 mM SA treatment (Fig. 7b and c).These results suggest that the expression of ATG6 could be induced by Pst DC3000/avrRps4 and 0.5 mM SA treatment.

These results confirm that ATG6 and NPR1 cooperatively enhance
Arabidopsis resistance to inhibit Pst DC3000/avrRps4 invasion.Together, these results suggest that ATG6 improves plant resistance to pathogens by regulating NPR1.

Discussion
Although SA signaling and autophagy are related to the plant immune system (Yoshimoto et al., 2009;Munch et al., 2014;Wang et al., 2016), the connection of these two processes in plant immune processes and their interaction is rarely reported.Previous studies have shown that unrestricted pathogen-induced PCD requires SA signalling in autophagy-deficient mutants.
SA and its analogue benzo (1,2,3) thiadiazole-7-carbothioic acid (BTH) induce autophagosome production (Yoshimoto et al., 2009).Moreover, autophagy has been shown to negatively regulates Pst DC3000/avrRpm1-induced PCD via the SA receptor NPR1 (Yoshimoto et al., 2009), implying that autophagy regulates SA signaling through a negative feedback loop to limit immunerelated PCD.Here, we demonstrated that ATG6 increases NPR1 protein levels and nuclear accumulation (Fig. 3 and 5).Additionally, ATG6 also maintains the stability of NPR1 and promotes the formation of SINCs-like condensates (Fig. 5 and 6).These findings introduce a novel perspective on the positive regulation of NPR1 by ATG6, highlighting their synergistic role in enhancing plant resistance.
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).
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.
NPR1 is an important signaling hub of the plant immune response.
Nuclear localization of NPR1 is essential to enhance plant resistance (Kinkema et al., 2000;Chen et al., 2021b), it interacts with transcription factors such as TGAs in the nucleus to activate expression of downstream target genes (Chen et al., 2019;Chen et al., 2021a).A recent study showed that nuclear-located ATG8h recognizes C1, a geminivirus nuclear protein, and promotes C1 degradation through autophagy to limit viral infiltration in solanaceous plants (Li et al., 2020).Here, we confirmed that ATG6 is also distributed in the nucleus and ATG6 is co-localized with NPR1 (Fig. 1d and 2), suggesting that ATG6 interact with NPR1 in the nucleus.ATG6 synergistically inhibits the invasion of Pst DC3000/avrRps4 with NPR1.Chen et al. found that in the nucleus, NPR1 can recruit enhanced disease susceptibility 1 (EDS1), a transcriptional coactivator, to synergistically activate expression of downstream target genes (Chen et al., 2021a).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 Fig. S10), and that the ADD domain containing acidic and hydrophobic amino acids is also found in ATG6 (148-295 AA) (Fig. S15).We speculate that ATG6 might act as a transcriptional coactivator to activate PRs expression synergistically with NPR1.
A recent study showed that SA not only enhances plant resistance by increasing NPR1 nuclear import and transcriptional activity, but also promotes cell survival by coordinating the distribution of NPR1 in the nucleus and cytoplasm (Zavaliev et al., 2020).Notably, NPR1 accumulated in the cytoplasm recruits other immunomodulators (such as EDS1, PAD4 etc) to form SINCs to promote cell survival (Zavaliev et al., 2020).Similarly, we also found that NPR1 accumulated abundantly in the cytoplasm after SA treatment and that ATG6 significantly increased NPR1 protein levels (Fig. 3c, 3e and 5a-b).Obviously, the accumulation of NPR1 in the cytoplasm may be related to ATG6 synergizing with NPR1 to enhance plant resistance.Interestingly, ATG6 overexpression significantly increased the formation of SINCs-like condensates (Fig. 5c-d and Supplemental movie 1-2), which should also be a way for ATG6 and NPR1 to synergistically resist invasion of pathogens.We consider that ATG6 promotes the formation of SINCs-like condensates through the dual action of endogenous and exogenous SA.Considering that ATG6 promotes SINCs-like condensates formation, we further examined changes in cell death in Col, amiRNA ATG6 # 1, amiRNA ATG6 # 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, amiRNA ATG6 # 1 and amiRNA ATG6 # 2 was significantly higher compared to Col (Fig. S16).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. S16).These results suggest that ATG6 and NPR1 cooperatively inhibit Pst DC3000/avrRps4-induced cell dead.
ATG6 is a common and required subunit of PtdIns3K lipid kinase complexes, which regulates autophagosome nucleation in Arabidopsis (Qi et al., 2017;Bozhkov, 2018).In this study, we also found that ATG6 can maintain the stability of NPR1.Thus, to confirm whether the regulation of NPR1 protein stability by ATG6 is autophagy-dependent, we used autophagy inhibitors (Concanamycin A, ConA and Wortmannin, WM) to detect the degradation of NPR1-GFP.Cell-free degradation assays showed that 100 μM MG115 treatment significantly inhibited the degradation of NPR1-GFP.
However, 5 μM concanamycin A treatment did not significantly delay NPR1 degradation (Fig. S17).Remarkably, treatment with 30 μM Wortmannin resulted in a slight acceleration of NPR1 degradation, while the combined treatment of ConA and WM significantly expedited the degradation of NPR1 (Fig. S17).This may be related to crosstalk between autophagy and 26S Proteasome.It has been demonstrated that autophagy directly regulates the activity of the 26S proteasome under normal conditions or treatment with Pst DC3000 (Marshall et al., 2015;Ustun et al., 2018).Marshall et al. found that the 26S proteasome subunits (RPN1, RPN3, RPN5, RPN10, PAG1, PBF1) are significantly enriched in autophagy-deficient mutantsunder normal growth conditions (Marshall et al., 2015).Treatment with concanamycin A (ConA), an inhibitor of vacuolar-type ATPase, increased the level of the 20S proteasome subunit PBA1 under treatment with Pst DC3000 (Ustun et al., 2018).In addition, we also analyzed the degradation of NPR1-GFP in NPR1-GFP and NPR1-GFP/atg5 following 100 μM cycloheximide (CHX) treatment.The results show that the degradation rate of NPR1-GFP in NPR1-GFP/atg5 plants was similarly to that in NPR1-GFP plants (Fig. 6e and f).These results suggest that deletion of ATG5 do not affect the protein stability of NPR1.
An increasing number of studies have shown that ATGs differentially affect plant immunity.Deletion of ATGs (ATG5, ATG7, ATG10 etc.) leads to reduced resistance of plants to necrotrophic pathogens (Lai et al., 2011;Lenz et al., 2011;Minina et al., 2018).ATGs can directly interact with other proteins to positively regulate plant immunity.In N. benthamiana, ATG8f interacts the effector protein βC1 of the cotton leaf curl multan virus and promotes its degradation to limit pathogen invasion (Haxim et al., 2017).Notably, ATG18a can interact with WRKY33 transcription factor to synergistically against Botrytis invasion (Lai et al., 2011).Our evidence shows that ATG6 interacts with NPR1 and works together to counteract pathogen invasion by positively regulating NPR1 and SA levels in vivo.In conclusion, we unveil a novel relationship in which ATG6 positively regulates NPR1 in plant immunity (Fig. 8).ATG6 interacts with NPR1 to synergistically enhance plant resistance by regulating NPR1 protein levels, stability, nuclear accumulation, and formation of SINCs-like condensates.

Plasmid construction
Details of plasmid construction methods are listed in Methods S1 of Supplemental Data 1, primer used are listed in Table S1 and S2 of conditions (Jiao et al., 2019).

Treatment conditions Treatment of 7-day-old seedlings
For SA treatment 7-day-old Arabidopsis seedlings were transferred to 1/2 MS liquid medium containing 0.5 mM SA for 0, 3 and 6 h, respectively.The corresponding results are shown in Fig. 2f, g, 3c, d and 5a.

For cycloheximide (CHX) treatment
Seedlings of Arabidopsis (7 days) were transferred to 1/2 MS liquid medium containing 100 μM CHX for 0, 3, 6 and 9 h, respectively.The corresponding results are shown in Fig. 6c and e.
For β-estradiol treatment 100 μM β-estradiol was infiltrated to treat 3-week-old Arabidopsis leaves.After 24 h of treatment with β-estradiol, Pst DC3000/avrRps4 was infiltrated and then growth of Pst DC3000/avrRps4 was detected after 3 d.

For Pst DC3000/avrRps4 infiltration
Infiltration with Pst DC3000/avrRps4 was performed as previously described (Wang et al., 2016;Skelly et al., 2019).Full description of the Pst DC3000/avrRps4 culture is included in Methods S3 of Supplemental Data 1.

For SA treatment
For 3-week-old Col, 0.5 mM SA was infiltrated into the leaves for 0, 2, 4, 6 and 8 h.The corresponding results are shown in Fig. 7b and c.

Yeast two-hybrid assay
Yeast two-hybrid experiments were performed according to the previously described protocol (Fu et al., 2012).Full description of Yeast two-hybrid is included in Methods S4 of Supplemental Data 1.

Pull down assays in vitro
500 μL of GST, GST-ATG6, SnRK2.8-GST were incubated with GST-tag Purification Resin (Beyotime, P2250) for 2 h at 4°C.The mixture was then centrifuged at 1500 g for 1 min at 4°C, and the resin was washed three times with PBS buffer.Next, the GST-tag purification resin was incubated with the NPR1-His for 2 h at 4°C.After washing three times with PBS buffer, 2 × sample buffers were added to the resin and denatured at 100 °C for 10 mins.
The resulting samples were then used for western blotting analysis.Full description of prokaryotic proteins expression is included in Methods S5 of Supplemental Data 1.
After incubation, the GFP-Trap magnetic Agarose beads were washed three times with cold wash buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA) and denatured at 75°C for 10 minutes after adding 2 × sample buffer.
Western blotting was performed with antibodies to ATG6 and GFP.

Nuclear and cytoplasmic separation
Nuclear and cytoplasmic separation were performed according to the previously described method (Kinkema et al., 2000).Full description of nuclear and cytoplasmic separation is given in Methods S6 of Supplemental Data 1.

Protein degradation in vitro
Protein degradation assays were performed according to a previously described method (Spoel et al., 2009;Saleh et al., 2015).Full description of protein degradation is included in Methods S7 of Supplemental Data 1.

Protein Extraction and Western Blotting Analysis
Protein extraction and western blotting were performed as previously described (Lei et al., 2020;Zhang et al., 2022).Protein was denatured at 100°C for 10 mins.NPR1 protein was denatured at 75°C for 10 mins (Lei et al., 2020).Full description is included in Methods S8 of Supplemental Data 1.
Antibody information is presented in Table S4 of Supplemental Data 1.

Confocal microscope observation
For nuclear localization of NPR1-GFP observation 7-day-old seedlings of NPR1-GFP and ATG6-mCherry × NPR1-GFP were sprayed with 0.5 mM SA for 0 and 3 h.For the Bimolecular Bluorescence Complementation assay (BiFC) Agrobacterium was infiltrated into N. benthamiana as previously described (Jiao et al., 2019).Fluorescence signals were observed after 3 days.The full description of BiFC is contained in Methods S9 and S10 of Supplemental Data 1.

For the observation of SINCs-like condensates
Agrobacterium was infiltrated into N. benthamiana.After 2 days, the leaves were treated in 1 mM SA solution for 24 h, and then fluorescence signals were observed.At least 20 image sets were obtained and analyzed.A full description of SINCs-like condensates observation is included in Methods S11 of Supplemental Data 1.

For growth of Pst DC3000/avrRps4
A low dose (OD 600 = 0.001) of Pst DC3000/avrRps4 was used for the infiltration experiments.After 3 days, the colony count was counted according to a previous description (Wang et al., 2016;Lei et al., 2020).Full description of the growth of Pst DC3000/avrRps4 is given in Methods S12 of Supplemental Data 1.

Free SA measurement
Free SA was extracted from 3-week-old Arabidopsis using a previously described method (Wang et al., 2016;Gong et al., 2020).Free SA was measured by High-performance liquid chromatography (Shimadzu LC-6A, Japan).Detection conditions: 294 nm excitation wavelength, 426 nm emission wavelength.

Real-Time Quantitative PCR (RT-qPCR)
Total RNA was extracted from Arabidopsis (100 mg) using Trizol RNA reagent (Invitrogen, 10296-028, Waltham, MA, USA).RT-qPCR assays were performed as previously described (Zhang et al., 2018;Zhang et al., 2022).All primers for RT-qPCR are listed individually in Table S5 of Supplemental Data 1.Full description of RT-qPCR is included in Methods S13 of Supplemental Data 1.

Statistical Analysis
All quantitative data in this study were presented as mean ± SD.Quantification of NPR1-GFP protein turnover rates in (c) using Image J. (e).
Total proteins were analyzed, Actin was used as an internal reference.(f).
Quantification of protein levels of NPR1-GFP in (e) using Image J.All experiments were performed with three biological replicates.
).Furthermore, we analyzed the putative nuclear localization signal (NLS) in the ATG6 protein sequence using the online //www.csbio.sjtu.edu.cn/bioinf/INSP/).The prediction results indicated the presence of a potential nuclear localization sequence "FLKEKKKKK" GFP and mCherry fluorescence signals in leaves were observed under the confocal microscope (Zeiss LSM880).Statistical data were obtained from three independent experiments, each comprising five individual images, resulting in a total of 15 images analyzed for this comparison.

Figure 4 .
Figure 4. ATG6 increases endogenous SA levels and promotes the expression of NPR1 downstream target genes (a).Level of free SA in 3-week-old NPR1-GFP and ATG6-mCherry × NPR1-GFP after Pst DC3000/avrRps4 for 12 h.(b-c).Expression of PR1 (b) and PR5 (c) in 3-week-old NPR1-GFP and ATG6-mCherry × NPR1-GFP under normal and SA treatment conditions, values are means ± SD (n = 3 biological replicates).The AtActin gene was used as the internal control.* or ** indicates that the significant difference compared to the control is at the level of 0.05 or 0.01 (Student t test p value, * p< 0.05 or ** p< 0.01).All experiments were performed with three biological replicates.

Fig
Fig. S7 ATG6 increases the nuclear accumulation of NPR1 under SA treatment.Fig. S8 Overexpression of ATG6 delayed dark-induced leaf senescence.

Fig. S9
Fig. S9 Expression of ICS1 under normal and SA treatment conditions.

Fig. S15
Fig. S15 Structural analysis of acidic activation domains in ATG6.

Fig
Fig. S17 NPR1-GFP degradation assay in ATG6-mCherry x NPR1-GFP The experimental data were analyzed by a two-tailed Student's t-test.Significance was assigned at P values < 0.05 or < 0.01.nanoparticles carrying exogenous gene in periwinkle mesophyll cells and its practice in huanglongbing research.iScience 25, 104479.