Figures and data
Physical interaction between NPR1 and ATG6.
(a). Interaction of NPR1 with ATG6 in yeast. The CDS of ATG6, NPR1, NPR1-N (0-328AA) and NPR1-C (328-594AA), SnRK2.8 were fused to pGADT? (AD) and pGBKT? (BO), respectively. Co-transformation of NPR1-BD + AD, BO+ ATG6-AD, BO+ SnRK2.8-AD, NPR1-N-BD + AD, NPR1-C-BD + AD were used as negative controls. The interaction of NPR1-BD and SnRK2.8-AD was used as a positive control. Yeast growth on SD/-Trp-Leu-His-Ade media represents interaction. Numbers represent the dilution fold of yeast. 0, -1 (10 fold dilution), -2 (100 fold dilution) and -3 (1000 fold dilution). (b). In vitro pull-down assays of NPR1-His with GST-ATG6 fusion protein. NPR1-His prokaryotic proteins were incubated with GST-tag Purification Resin conjugated with GST-ATG6, GST and GST-SnRK2.8. Western blotting analysis with anti-GST and anti-His. Black asterisk(*) indicate GST-SnRK2.8 bands. Red asterisk(*) indicate GST-ATG6 bands. (c). Co-immunoprecipitation of NPR1 with ATG6 in vivo. Total protein was extracted from N. benthamiana transiently transformed with ATG6-mCherry + GFP and ATG6- mCherry + NPR1-GFP, followed by IP with GFP-Trap. Western blots analysis with ATG6 and GFP antibodies. (d). Bimolecular fluorescence complementation assay of NPR1 with ATG6 in Nicotiana benthamiana leaves. Agrobacterium carrying ATG6-nYFP and NPR1-cYFP was co-expressed in leaves of Nicotiana benthamiana for 3 days. As a positive control, NPR1-nYFP and SnRK2.8-cYFP were co-expressed. As negative controls, nYFP and ATG6-cYFP, NPR1-nYFP and cYFP, nYFP and SnRK2.8-cYFP were co-expressed. Confocal images were obtained from chloroplast (Chi), YFP, Bright field. Scale bar= 20 µm. (e). Relative fluorescence intensity of YFP in (d) using image J software, ND means not detected, n = 15 independent images were analyzed to quantify YFP fluorescence. All experiments were performed with three biological replicates.
ATG6 is localized in the cytoplasm and nucleus.
(a). The nuclear localization of ATG6-mCherry in N. benthamiana. Scale bar, 100 µm. (b). Co-localization of ATG6-GFP and nls-mCherry in N. benthamiana. Scale bar, 50 µm. (c). ATGs protein nuclear localization sequence analysis using the online INSP (Identification of Nuclear Signal Peptide) prediction software. (d). Subcellular fractionation of ATG6-mCherry in N. benthamiana after 1 mM SA treatment. Black asterisk(*) indicate ATG6-mCherry bands. (e). Subcellular fractionation of ATG6- GFP in N. benthamiana after 1 mM SA treatment. Black asterisk(*) indicate ATG6-GFP bands. (f). Subcellular fractionation of ATG6-GFP in ATG6-GFP Arabidopsis after 0.5 mM SA treatment. (g). Subcellular fractionation of ATG6-mCherry in ATG6-mCherry Arabidopsis after 0.5 mM SA treatment. In (d-g), ATG6-mCherry (d and g) and ATG6-GFP (e and f) were detected using ATG6 or GFP antibody. Actin and H3 were used as cytoplasmic and nucleus internal reference, respectively. Numerical values indicate quantitative analysis of ATG6-mCherry and
ATG6 increases the nuclear accumulation of NPR1 under SA treatment.
(a). Confocal images of NPR1-GFP nuclear localization in 7-day-old seedlings of NPR1-GFP and ATG6-mCherry x NPR1-GFP under normal and 0.5 mM SA spray for 3 h. Scale bar, 50 µm. (b). The count of nuclear localizations of NPR1-GFP in ATG6-mCherry x NPR1-GFP and NPR1-GFP Arabidopsis plants following SA treatment. in (a), Statistical data were obtained from three independent experiments, each comprising five individual images, resulting in a total of 15 images analyzed for this comparison. ** indicates that the significant difference compared to the control is at the level of 0.01 (Student t test p value,** p< 0.01). (c). Subcellular fractionation of NPR1-GFP in 7-day-old seedlings of NPR1-GFP and ATG6-mCherry x NPR1-GFP after 0.5 mM SA treatment for 0, 3 and 6 h. (d). The ration of NPR1 in the nucleus/total NPR1 in (c), ns indicates no significant difference. (e). Subcellular fractionation of NPR1-GFP in N. benthamiana after 1 mM SA treatment for 0, 8 and 20 h. Black asterisk(*) indicate ATG6-mCherry bands. (f). The ration of NPR1 in the nucleus/total NPR1 in (e), ns indicates no significant difference. In (c and e), cytoplasmic and nuclear proteins were extracted from Arabidopsis or N. benthamiana. NPR1-GFP were detected using GFP antibody. Actin and H3 were used as cytoplasmic and nucleus internal reference, respectively. Numerical values indicate quantitative analysis of NPR1-GFP using
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 x 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 x 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.
ATG6 increases the NPR1 protein levels and the formation of SINCs-like condensates.
(a). NPR1-GFP protein levels in 7-day-old seedlings of NPR1-GFP and ATG6- mCherry x NPR1-GFP after 0.5 mM SA treatment for 0, 3, 6 and 9 h. Numerical values indicate quantitative analysis of NPR1-GFP protein using image J. (b). NPR1-GFP protein levels in N. benthamiana. ATG6-mCherry + NPR1-GFP, NPR1-GFP + mCherry were co-expressed in N. benthamiana. After 2 days, leaves were treated with 1 mM SA for 8, 20, 24 h. Total proteins were extracted and analyzed. Numerical values indicate quantitative analysis of NPR1-GFP protein using image J. (c). ATG6 promotes the formation of SINCs-like condensates. ATG6-mCherry + NPR1- GFP, NPR1-GFP + mCherry were co-expressed in N. benthamiana. After 2 days, leaves were treated with 1 mM SA for 24 h. Confocal images obtained at excitation with wavelengths of 488 nm, scale bar= 50 µm. (d). SINCs-like condensates numbers of per section in (c), n > 10 sections. ** indicates that the significant difference compared to the control is at the level of 0.01 (Student t test p value,** p< 0.01). All experiments were performed with three biological replicates.
ATG6 improves the protein stability of NPR1.
(a). NPR1-GFP degradation assay in Arabidopsis. Total proteins from 7-day-old seedlings of NPR1-GFP and ATG6-mCherry x NPR1-GFP were extracted, using Actin as an internal reference. “M” indicates 100 µM MG 115 treatment. (b). Quantification of NPR1-GFP degradation rates in (a) using Image J. In (a and b), the extracts were incubated for 0 ∼ 180 min at room temperature (25°C), the degradation rate of NPR1-GFP was analyzed. (c). NPR1-GFP protein turnover. 7-day-old NPR1-GFP and ATG6-mCherry x NPR1-GFP seedlings were treated with 100 µM cycloheximide (CHX) for different times. Total proteins were analyzed, Actin was used as an internal reference. (d). Quantification of NPR1-GFP protein turnover rates in (c) using Image J. (e). NPR1-GFP protein turnover.7-day-old NPR1-GFP and NPR1-GFP/atg5 seedlings were treated with 100 µM cycloheximide (CHX) for different times. 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.
ATG6 and NPR1 cooperatively inhibit the growth of Pst DC3000/avrRps4.
(a). Expression of ATG6 under Pst DC3000/avrRps4 injection in 3-week-old Col leaves. (b). Expression of ATG6 in the presence of 0.5 mM SA in 3-week-old Col leaves. (c). The protein levels of ATG6 after 0.5 mM SA in 3- week-old Col leaves. Total leaf proteins from Arabidopsis were analyzed, Actin was used as an internal reference. Numerical values indicate quantitative analysis of ATG6 protein using image J. (d). Growth of Pst DC3000/avrRps4 in Col/silencing ATG6 and Col/negative control (NC). (e). Phenotypes of 16-day old amiRNAATG6# 1 and amiRNAATG6# 2. Bar, 1 cm. (f). Phenotypes of 23-day-old amiRNAATG6# 1 and amiRNAATG6# 2. Bar, 3 cm. (g). Expression of ATG6 in Col, amiRNAATG6# 1 and amiRNAATG6# 2 under injection treatment of 100 µM -estradiol. (h). Growth of Pst DC3000/avrRps4 in Arabidopsis leaves of amiRNAATG6# 1, amiRNAATG6# 2 and Col. (i).Growth of Pst DC3000/avrRps4 in NPR1-GFP/silencing ATG6 and NPR1-GFPINC. (j). Growth of Pst DC3000/avrRps4 in Arabidopsis leaves of Col, amiRNAATG6# 1, amiRNAATG6# 2, npr1, NPR1-GFP, ATG6-mCherry and ATG6-mCherry x NPR1-GFP. In (d, h-j), a low dose of Pst DC3000/avrRps4 (OD600 = 0.001) was infiltrated. After 3 days, the growth of Pst DC3000/avrRps4 was counted. *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.
Working model for NPR1 regulation by ATG6.
ATG6 interacts directly with NPR1 to increase NPR1 protein level and stability, thereby promoting the formation of SINCs-like condensates and increasing the nuclear accumulation of NPR1. ATG6 synergistically activates PRs expression with NPR1 to cooperatively enhance resistance to inhibit Pst DC3000/avrRps4 invasion in Arabidopsis.
