Figure 2A . Confocal microscopy of the association between EGFP - LC3 and GAS strain 188 , 188SLO - ( SLO - ) , 188SLS - ( SLS - ) , or 188SLO - SLS - ( SLO - SLS - ) . Immunofluorescent staining distinguished intracellular ( Alexa - 568 , red ) from extracellular ( Alexa - 568 and Alexa - 660 , red and blue , respectively ) GAS . Scale bar = 10 µm . The percent of intracellular GAS that were associated with EGFP - LC3 at 3 h post - infection is shown for each strain . B . Electron microscopy of GAS strains 188 , 188SLO - , and 188SLO - SLS - at 3 h post - infection . Arrowheads indicate the presence of double or multiple membranes ( 188 and 188SLO - ) or single membranes ( 188SLO - SLS - ) partially or completely surrounding bacteria . Vacuolar compartments associated with 188 and 188SLO - also contain cytosolic material . C . Intracellular survival of GAS strains 188 , 188SLO - , 188SLS - , and 188SLO - SLS - . * , P < 0 . 05 . D . Intracellular survival of 188 or 188SLO - in the presence or absence of Beclin1 knockdown .

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Figure 2A. Confocal microscopy of the association between EGFP-LC3 and GAS strain 188, 188SLO- (SLO-), 188SLS- (SLS-), or 188SLO-SLS- (SLO-SLS-). Immunofluorescent staining distinguished intracellular (Alexa-568, red) from extracellular (Alexa-568 and Alexa-660, red and blue, respectively) GAS. Scale bar = 10 µm. The percent of intracellular GAS that were associated with EGFP-LC3 at 3 h post-infection is shown for each strain.B. Electron microscopy of GAS strains 188, 188SLO-, and 188SLO-SLS- at 3 h post-infection. Arrowheads indicate the presence of double or multiple membranes (188 and 188SLO-) or single membranes (188SLO-SLS-) partially or completely surrounding bacteria. Vacuolar compartments associated with 188 and 188SLO- also contain cytosolic material.C. Intracellular survival of GAS strains 188, 188SLO-, 188SLS-, and 188SLO-SLS-. *, P<0.05.D. Intracellular survival of 188 or 188SLO- in the presence or absence of Beclin1 knockdown.


Figure 3A . Intracellular survival of GAS strains 188 , 188NADase - , and 188NADase ( G330D ) . Deletion of the gene encoding NADase or inactivation of NADase by the amino acid substitution G330D resulted in a 20 - to 40 - fold reduction in intracellular survival compared to parent strain 188 . * , P < 0 . 001 . NADase western blot ( B ) and activity measurements ( C ) of culture supernatants from GAS strains 188 , 188NADase - , and 188NADase ( G330D ) .

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Figure 3A. Intracellular survival of GAS strains 188, 188NADase-, and 188NADase(G330D). Deletion of the gene encoding NADase or inactivation of NADase by the amino acid substitution G330D resulted in a 20- to 40-fold reduction in intracellular survival compared to parent strain 188. *, P<0.001.NADase western blot (B)and activity measurements (C) of culture supernatants from GAS strains 188, 188NADase-, and 188NADase(G330D).


Figure 4A . Confocal microscopy demonstrating the association between ubiquitin ( green ) and intracellular GAS ( red ) at 30 min post - infection in keratinocytes infected with GAS strain 188 , 188SLO - , 188NADase - , or 188SLO ( Y255A ) . Extracellular GAS were stained blue and red for identification as described in Figure 2 . Scale bar = 10 µm . The percentage of intracellular GAS that were associated with ubiquitin is indicated for each strain , based on quantification of at least 100 bacteria in three independent experiments . B . Hemolytic activity of culture supernatants of GAS strain 188 , 188SLO - , and 188SLO ( Y255A ) . The amino acid substitution Y255A in SLO abrogates the ability of SLO to porate the host cell membrane , demonstrated by the failure of culture supernatants from 188SLO ( Y255A ) to lyse erythrocytes despite producing wild - type amounts of the variant SLO protein . Inset , corresponding Western blot for SLO in culture supernatants from these strains . . C . NADase activity in cell culture supernatants and in the cytosol of keratinocytes infected with GAS strain 771 , 771SLO - , 771SLO ( Y255A ) , or 771NADase - . Intracellular NADase activity due to translocation by extracellular GAS was identical between 771 and 771SLO ( Y255A ) , but reduced in 771SLO - . D . The intracellular survival of 188SLO ( Y255A ) was significantly lower than that of its parental strain , 188 , and similar to that of 188SLO - . * , P < 0 . 01 . E . Confocal microscopy demonstrating the association between galectin 8 ( green ) and intracellular GAS ( red ) in keratinocytes infected for 30 min with GAS strain 188 , 188SLO - , or 188 SLO - SLS - . Extracellular GAS ( GAS ( out ) ) were stained blue and red for identification as described in Figure 2 . Scale bar = 10 µm . The percentage of intracellular GAS that were associated with galectin 8 is indicated for each strain , based on quantification of at least 100 bacteria in three independent experiments . 188SLO - SLS - was never associated with galectin 8 ( 0 % ) .

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Figure 4A. Confocal microscopy demonstrating the association between ubiquitin (green) and intracellular GAS (red) at 30 min post-infection in keratinocytes infected with GAS strain 188, 188SLO-, 188NADase-, or 188SLO(Y255A). Extracellular GAS were stained blue and red for identification as described in Figure 2. Scale bar = 10 µm. The percentage of intracellular GAS that were associated with ubiquitin is indicated for each strain, based on quantification of at least 100 bacteria in three independent experiments.B. Hemolytic activity of culture supernatants of GAS strain 188, 188SLO-, and 188SLO(Y255A). The amino acid substitution Y255A in SLO abrogates the ability of SLO to porate the host cell membrane, demonstrated by the failure of culture supernatants from 188SLO(Y255A) to lyse erythrocytes despite producing wild-type amounts of the variant SLO protein. Inset, corresponding Western blot for SLO in culture supernatants from these strains.. C. NADase activity in cell culture supernatants and in the cytosol of keratinocytes infected with GAS strain 771, 771SLO-, 771SLO(Y255A), or 771NADase-. Intracellular NADase activity due to translocation by extracellular GAS was identical between 771 and 771SLO(Y255A), but reduced in 771SLO-.D. The intracellular survival of 188SLO(Y255A) was significantly lower than that of its parental strain, 188, and similar to that of 188SLO-. *, P<0.01.E. Confocal microscopy demonstrating the association between galectin 8 (green) and intracellular GAS (red) in keratinocytes infected for 30 min with GAS strain 188, 188SLO-, or 188 SLO-SLS-. Extracellular GAS (GAS(out)) were stained blue and red for identification as described in Figure 2. Scale bar = 10 µm. The percentage of intracellular GAS that were associated with galectin 8 is indicated for each strain, based on quantification of at least 100 bacteria in three independent experiments. 188SLO-SLS- was never associated with galectin 8 (0%).


Figure 5SLO and NADase inhibit lysosomal fusion to GAS - containing autophagosomes in oropharyngeal keratinocytes . A . Confocal microscopy of keratinocytes infected with GAS strain 188 , 188SLO - , or 188NADase - demonstrating the association of the lysosomal marker LAMP - 1 ( red ) with GAS ( blue ) contained within EGFP - LC3 ( green ) - positive compartments at 1 h , 3 h , and 6 h post - infection . Scale bar = 10 µm . B . Quantification of the percent of GAS within EGFP - LC3 - positive compartments that are co - localized with LAMP - 1 at 1 h , 3 h , and 6 h . Data represent mean values from three independent experiments in which at least 100 intracellular GAS were quantified for each time point in each experiment .

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Figure 5SLO and NADase inhibit lysosomal fusion to GAS-containing autophagosomes in oropharyngeal keratinocytes.A. Confocal microscopy of keratinocytes infected with GAS strain 188, 188SLO-, or 188NADase- demonstrating the association of the lysosomal marker LAMP-1 (red) with GAS (blue) contained within EGFP-LC3 (green)-positive compartments at 1 h, 3 h, and 6 h post-infection. Scale bar = 10 µm. B. Quantification of the percent of GAS within EGFP-LC3-positive compartments that are co-localized with LAMP-1 at 1 h, 3 h, and 6 h. Data represent mean values from three independent experiments in which at least 100 intracellular GAS were quantified for each time point in each experiment.


Figure 1 ( A , B ) Fluorescence confocal microscopy images show TMRE staining and GFP : : MIRO - 1 in WT and fzo - 1 mutants animals . Dashed lines outline regions where fluorescence intensity was quantified . ( C ) Immunoblot shows protein levels of MIRO - 1 in WT and fzo - 1 mutant animals , with tubulin and COX - 4 serving as loading controls . ( D ) Fluorescence confocal images show GFP : : MIRO - 1 and mito : : mKate2 in WT and fzo - 1 mutants . ( E ) Quantification of MIRO - 1 : : GFP levels in WT and fzo - 1 mutants . n ≥ 33 , biological replicates .

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Figure 1(A, B) Fluorescence confocal microscopy images show TMRE staining and GFP::MIRO-1 in WT and fzo-1 mutants animals. Dashed lines outline regions where fluorescence intensity was quantified.(C) Immunoblot shows protein levels of MIRO-1 in WT and fzo-1 mutant animals, with tubulin and COX-4 serving as loading controls.(D) Fluorescence confocal images show GFP::MIRO-1 and mito::mKate2 in WT and fzo-1 mutants. (E) Quantification of MIRO-1::GFP levels in WT and fzo-1 mutants. n≥33, biological replicates.


Figure 2 ( A , B ) Fluorescence confocal microscopy images show TMRE staining and GFP : : MIRO - 1 in WT animals with and without needle wounding . Dashed lines outline regions where fluorescence intensity was quantified . ( C ) Representative confocal images showg GFP : : MIRO - 1 and mito : : mKate2 before and 2 hours after wounding . ( D ) Immunoblot shows protein levels of MIRO - 1 before and 2 hours after wounding , with tubulin and COX - 4 serving as loading controls .

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Figure 2(A, B) Fluorescence confocal microscopy images show TMRE staining and GFP::MIRO-1 in WT animals with and without needle wounding. Dashed lines outline regions where fluorescence intensity was quantified.(C) Representative confocal images showg GFP::MIRO-1 and mito::mKate2 before and 2 hours after wounding.(D) Immunoblot shows protein levels of MIRO-1 before and 2 hours after wounding, with tubulin and COX-4 serving as loading controls.


Figure 3 ( A ) Representative confocal images show the TMRE and mito : : GFP signals in WT , miro - 1 ( tm1966 ) , miro - 1 ( ΔEF ) ( EF - hand domain mutation ) , and miro - 1 ( OE , overexpression ) animals . ( B ) Quantitation of TMRE and mito : : GFP signal ( normalized ratio of TMRE fluorescence to mito : : GFP fluorescence ) . n > 200 mitochondria . ( D ) ATP content of crude isolated mitochondria from WT and miro - 1 ( tm1966 ) mutants as measured with a commercial luminescence kit . n = 3 , biological replicates .

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Figure 3(A) Representative confocal images show the TMRE and mito::GFP signals in WT, miro-1(tm1966), miro-1(ΔEF) (EF-hand domain mutation), and miro-1(OE, overexpression) animals. (B) Quantitation of TMRE and mito::GFP signal (normalized ratio of TMRE fluorescence to mito::GFP fluorescence). n > 200 mitochondria.(D) ATP content of crude isolated mitochondria from WT and miro-1(tm1966) mutants as measured with a commercial luminescence kit. n = 3, biological replicates.


Figure 4 ( D ) Western blot analysis of the interaction between MIRO - 1 and VDAC - 1 by coimmunoprecipitating with GFP nanobody - conjugated beads in GFP : : MIRO - 1 animals and detected by GFP and VDAC - 1 antibodies . ( E ) Western blot analysis of the interaction between MIRO - 1 and VDAC - 1 by coimmunoprecipitating with VDAC - 1 antibody - conjugated beads in GFP : : MIRO - 1 animals and detected by GFP and VDAC - 1 antibodies . ( F and G ) Immunoblot showing the immunoprecipitation of purified GST : : MIRO - 1 and VDAC - 1 : : His with GST - beads and His - beads , respectively . ( H ) BN - PAGE and immunoblot analysis of the total worm lysate of GFP : : MIRO - 1 ( KI ) by GFP and VDAC - 1 antibodies . Green shading highlights the complex that contained MIRO - 1 and VDAC - 1 proteins .

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Figure 4(D) Western blot analysis of the interaction between MIRO-1 and VDAC-1 by coimmunoprecipitating with GFP nanobody-conjugated beads in GFP::MIRO-1 animals and detected by GFP and VDAC-1 antibodies.(E) Western blot analysis of the interaction between MIRO-1 and VDAC-1 by coimmunoprecipitating with VDAC-1 antibody-conjugated beads in GFP::MIRO-1 animals and detected by GFP and VDAC-1 antibodies.(F and G) Immunoblot showing the immunoprecipitation of purified GST::MIRO-1 and VDAC-1::His with GST-beads and His-beads, respectively.(H) BN-PAGE and immunoblot analysis of the total worm lysate of GFP::MIRO-1 (KI) by GFP and VDAC-1 antibodies. Green shading highlights the complex that contained MIRO-1 and VDAC-1 proteins.


Figure 5 ( A ) Schematic of the vdac - 1 transgene by CRISPR - Cas9 method . The images below show the young adult stage worms of WT and vdac - 1 ( zju247 ) mutants . ( B ) Representative confocal images showing TMRE staining and mito : : GFP in L4440 , vdac - 1 RNAi , vdac - 1 ( zju247 ) , and vdac - 1 OE animals . ( E ) ATP content in crude isolated mitochondria of WT animals and vdac - 1 ( zju247 ) mutants . Quantification is conducted with a commercial luminescence kit . n = 3 , biological replicates .

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Figure 5(A) Schematic of the vdac-1 transgene by CRISPR-Cas9 method. The images below show the young adult stage worms of WT and vdac-1(zju247) mutants. (B) Representative confocal images showing TMRE staining and mito::GFP in L4440, vdac-1 RNAi, vdac-1(zju247), and vdac-1 OE animals.(E) ATP content in crude isolated mitochondria of WT animals and vdac-1(zju247) mutants. Quantification is conducted with a commercial luminescence kit. n = 3, biological replicates.


Figure 6 ( B ) Immunoprecipitation of GFP : : MIRO - 1 and GFP : : MIRO - 1 ( E473G ) with GFP nanobody - conjugated beads . ( F ) Immunoprecipitation of GFP : : MIRO - 1 in fzo - 1 mutant with GFP nanobody - conjugated beads . The interaction between GFP : : MIRO - 1 and VDAC - 1 is increased in fzo - 1 mutants . ( G ) BN - PAGE and immunoblot analysis of VDAC - 1 oligomerization crosslinked with BMH in WT and miro - 1 ( tm1966 ) mutants using VDAC - 1 antibody .

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Figure 6(B) Immunoprecipitation of GFP::MIRO-1 and GFP::MIRO-1(E473G) with GFP nanobody-conjugated beads.(F) Immunoprecipitation of GFP::MIRO-1 in fzo-1 mutant with GFP nanobody-conjugated beads. The interaction between GFP::MIRO-1 and VDAC-1 is increased in fzo-1 mutants.(G) BN-PAGE and immunoblot analysis of VDAC-1 oligomerization crosslinked with BMH in WT and miro-1(tm1966) mutants using VDAC-1 antibody.


Figure 1 ( A ) RFP - ATG8g - labeled autophagosomes were quantified from plants syringe infected with mock or Xcv ΔxopQ ( oD600 = 0 . 2 ) at 2 dpi in the presence or absence of ConA ( bars = 20 μm ) . Puncta were calculated from z - stacks ( 15 ) of n = 6 individuals using ImageJ . Central band of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Different letters indicate statistically different groups ( P < 0 . 05 ) as determined by one - way ANOVA . The experiment was repeated twice with similar results . ( B ) Immunoblot analysis of NBR1 and ATG8 protein levels in Xcv ΔxopQ or mock infected N . benthamiana plants at 1 and 2dpi . Agrobacterium - mediated transient expression of AIMp - RFP serves as a control for autophagy suppression . Ponceau Staining ( PS ) served as a loading control . The experiment was repeated three times with similar results . ( C ) RLUC - ATG8a or RLUC - NBR1 constructs were coexpressed with internal control FLUC in N . benthamiana . Xcv ΔxopQ was co - infiltrated with Agrobacteria containing the luciferase reporter constructs . Coexpression of RFP - AIMp serves as a control for autophagy inhibition . Expression of the latter was confirmed with western blot ( inset ) . Renilla ( RLUC ) and Firefly ( FLUC ) luciferase activities were simultaneously measured in leaf extracts at 48 h post - infiltration using the dual - luciferase system ( n = 4 ) . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Statistical significance ( * * * P < 0 . 001 ) was revealed by Student ' s t - test . The experiment was repeated more than 3 times with similar results . ( D ) RT - qPCR analysis of NbATG8 - 1 . 1 / 1 . 2 , NbATG8 - 2 . 1 / 2 . 2 and NbJoka2 transcript levels upon challenge of N . benthamiana plants with Xcv ΔxopQ for 1 and 2 dpi compared to mock infected plants . Values represent expression relative to mock control of respective time point and were normalized to actin . Values are biological replicates ( n = 4 ) . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Statistical significance ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) was revealed by Student ' s t - test . ( E ) Bacterial density in leaves of N . benthamiana infected with Xcv in the presence or absence autophagy suppressor AIMp - RFP . Leaves were syringe - infiltrated with OD600 = 0 . 0004 , and colony - forming units were counted at 6 dpi . Compared to empty vector control ( EV ) , AIMp expressing plants ( n = 6 ) harbour significantly more bacteria . Bacterial growth was repeated with the same result in 12 plants over two independent experiments . Red and yellow data points indicate independent repeats of the experiment . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Statistical significance ( * * * P < 0 . 001 ) was revealed by Student ' s t - test . Expression of RFP - AIMp was verified at 6 dpi with an anti - RFP blot ( inset ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) RFP-ATG8g-labeled autophagosomes were quantified from plants syringe infected with mock or Xcv ΔxopQ (oD600=0.2) at 2 dpi in the presence or absence of ConA (bars = 20 μm). Puncta were calculated from z-stacks (15) of n=6 individuals using ImageJ. Central band of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Different letters indicate statistically different groups (P < 0.05) as determined by one-way ANOVA. The experiment was repeated twice with similar results.(B) Immunoblot analysis of NBR1 and ATG8 protein levels in Xcv ΔxopQ or mock infected N. benthamiana plants at 1 and 2dpi. Agrobacterium-mediated transient expression of AIMp-RFP serves as a control for autophagy suppression. Ponceau Staining (PS) served as a loading control. The experiment was repeated three times with similar results.(C) RLUC-ATG8a or RLUC-NBR1 constructs were coexpressed with internal control FLUC in N. benthamiana. Xcv ΔxopQ was co-infiltrated with Agrobacteria containing the luciferase reporter constructs. Coexpression of RFP-AIMp serves as a control for autophagy inhibition. Expression of the latter was confirmed with western blot (inset). Renilla (RLUC) and Firefly (FLUC) luciferase activities were simultaneously measured in leaf extracts at 48 h post- infiltration using the dual-luciferase system (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (***P<0.001) was revealed by Student's t-test. The experiment was repeated more than 3 times with similar results.(D) RT-qPCR analysis of NbATG8-1.1/1.2, NbATG8-2.1/2.2 and NbJoka2 transcript levels upon challenge of N. benthamiana plants with Xcv ΔxopQ for 1 and 2 dpi compared to mock infected plants. Values represent expression relative to mock control of respective time point and were normalized to actin. Values are biological replicates (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (*P < 0.05, **P < 0.01, *** P < 0.001) was revealed by Student's t-test.(E) Bacterial density in leaves of N. benthamiana infected with Xcv in the presence or absence autophagy suppressor AIMp-RFP. Leaves were syringe-infiltrated with OD600 = 0.0004, and colony-forming units were counted at 6 dpi. Compared to empty vector control (EV), AIMp expressing plants (n=6) harbour significantly more bacteria. Bacterial growth was repeated with the same result in 12 plants over two independent experiments. Red and yellow data points indicate independent repeats of the experiment. Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (***P < 0.001) was revealed by Student's t-test. Expression of RFP-AIMp was verified at 6 dpi with an anti-RFP blot (inset).


Figure 2 ( A ) RLUC - ATG8a or RLUC - NBR1 constructs were coexpressed with internal control FLUC in N . benthamiana . XopL or GFP constructs were co - infiltrated . RLUC and FLUC signals were simultaneously measured in leaf extracts at 48 h post - infiltration using the dual - luciferase system . Values represent the ratio of RLUC - ATG8a and FLUC activities to the mean of control ( n = 4 ) . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Statistical significance ( P < 0 . 01 ) was shown by Student ' s t - test . The experiment was repeated more than 3 times by with similar results . ( B ) Immunoblot analysis of NBR1 and ATG8 protein levels in N . benthamiana plants transiently expressing GFP - XopL or GFP control at 2dpi verified with an anti - GFP antibody ( same blot was split for visualization purpose ) . Ponceau Staining ( PS ) served as a loading control . The experiment was repeated at least three times with similar results . ( C ) Immunoblot analysis of ATG8 protein levels in N . benthamiana plants transiently expressing XopL or GUS control at 2dpi after ConA or DMSO treatment . Expression of GFP - XopL was verified with an anti - GFP antibody , while expression of GUS - HA was confirmed with an anti - HA antibody . Ponceau Staining ( PS ) served as a loading control . The experiment was repeated twice with similar results . ( D ) GFP - ATG8g - labeled autophagosomes were quantified from plants infected with Xcv or Xcv ΔxopL at 2 dpi in the presence or absence of ConA . Puncta were calculated from z - stacks ( 15 ) of n = 12 individuals using ImageJ . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Statistical significance ( * * P < 0 . 01 , * * * P < 0 . 001 ) was determined by one way ANOVA . The experiment was repeated twice with similar results . ( E ) Immunoblot analysis of NBR1 and ATG8 protein levels in Xcv ΔxopQ , Xcv ΔxopQ ΔxopL or mock infected N . benthamiana plants at 2dpi . Ponceau Staining ( PS ) served as a loading control . The experiment was repeated twice with similar results . ( F ) RLUC - ATG8a or RLUC - NBR1 constructs were coexpressed with internal control FLUC in N . benthamiana . Xcv ΔxopQ and Xcv ΔxopQ ΔxopL were co - infiltrated with Agrobacteria containing the respective constructs . RLUC and FLUC activities were simultaneously measured in leaf extracts at 48 h post - infiltration using the dual - luciferase system . Values represent the ratio of RLUC - ATG8a and FLUC activities ( n = 4 ) . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Statistical significance comparing Xcv ΔxopQ and Xcv ΔxopQ ΔxopL values ( * * * P < 0 . 001 ) was revealed by Student ' s t - test . The experiment was repeated 3 times with similar results .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) RLUC-ATG8a or RLUC-NBR1 constructs were coexpressed with internal control FLUC in N. benthamiana. XopL or GFP constructs were co-infiltrated. RLUC and FLUC signals were simultaneously measured in leaf extracts at 48 h post- infiltration using the dual-luciferase system. Values represent the ratio of RLUC-ATG8a and FLUC activities to the mean of control (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (P < 0.01) was shown by Student's t-test. The experiment was repeated more than 3 times by with similar results.(B) Immunoblot analysis of NBR1 and ATG8 protein levels in N. benthamiana plants transiently expressing GFP-XopL or GFP control at 2dpi verified with an anti-GFP antibody (same blot was split for visualization purpose). Ponceau Staining (PS) served as a loading control. The experiment was repeated at least three times with similar results.(C) Immunoblot analysis of ATG8 protein levels in N. benthamiana plants transiently expressing XopL or GUS control at 2dpi after ConA or DMSO treatment. Expression of GFP-XopL was verified with an anti-GFP antibody, while expression of GUS-HA was confirmed with an anti-HA antibody. Ponceau Staining (PS) served as a loading control. The experiment was repeated twice with similar results.(D) GFP-ATG8g-labeled autophagosomes were quantified from plants infected with Xcv or Xcv ΔxopL at 2 dpi in the presence or absence of ConA. Puncta were calculated from z-stacks (15) of n=12 individuals using ImageJ. Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (** P < 0.01, *** P < 0.001) was determined by one way ANOVA. The experiment was repeated twice with similar results.(E) Immunoblot analysis of NBR1 and ATG8 protein levels in Xcv ΔxopQ, Xcv ΔxopQ ΔxopL or mock infected N. benthamiana plants at 2dpi. Ponceau Staining (PS) served as a loading control. The experiment was repeated twice with similar results.(F) RLUC-ATG8a or RLUC-NBR1 constructs were coexpressed with internal control FLUC in N. benthamiana. Xcv ΔxopQ and Xcv ΔxopQ ΔxopL were co-infiltrated with Agrobacteria containing the respective constructs. RLUC and FLUC activities were simultaneously measured in leaf extracts at 48 h post- infiltration using the dual-luciferase system. Values represent the ratio of RLUC-ATG8a and FLUC activities (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance comparing Xcv ΔxopQ and Xcv ΔxopQ ΔxopL values (***P < 0.001) was revealed by Student's t-test. The experiment was repeated 3 times with similar results.


Figure 3 ( A ) Interaction of XopL with SH3P2 in yeast two - hybrid assays . XopL fused to the GAL4 DNA - binding domain was expressed in combination with SH3P2 fused to the GAL4 activation domain ( AD ) in yeast strain Y190 . Cells were grown on selective media before a LacZ filter assay was performed . pSV40 / p53 served as positive control , while the empty AD or BD vector served as negative control . NtSH3P2 = Nicotiana tabacum SH3P2 . - LT = yeast growth on medium without Leu and Trp , - HLT = yeast growth on medium lacking His , Leu , and Trp , indicating expression of the HIS3 reporter gene . LacZ , activity of the lacZ reporter gene . ( B ) Coimmunoprecipitation of GFP - XopL with AtSH3P2 - HA . GFP - XopL or GFP were transiently coexpressed with AtSH3P2 - HA in leaves of N . benthamiana . After 48 h , total proteins ( Input ) were subjected to immunoprecipitation ( IP ) with GFP - Trap beads , followed by immunoblot analysis using either anti - GFP or anti - HA antibodies . GFP blot was split for visualization purpose . AtSH3P2 = Arabidopsis thaliana SH3P2 . Two repetitions with similar results have been conducted . ( C ) Visualization of protein interactions in planta by the bimolecular fluorescence complementation assay . Yellow fluorescent protein ( YFP ) confocal microscopy images show Nicotiana benthamiana leaf epidermal cells transiently expressing VenusN173 - XopL in combination with AtSH3P2 - VenusC155 . A positive control showing the dimerization of fructose - 1 , 6 - bisphosphatase ( FBPase ) within the cytosol . White arrows indicate reconstitution of YFP fluorescence . The red structures indicate autofluorescence of chloroplasts . The combination of VenusN173 - XopL with FBPase - VenusC155 or VenusN173 - FBPase with AtSH3P2 - VenusC155 do not induce YFP fluorescence and serve as negative controls . Bars = 20 μm . ( D ) Colocalization analysis of GFP - XopL with SH3P2 - RFP , RFP - ATG8e and RFP - ATG8g in N . benthamiana leaves . Imaging was performed 2 d after transient expression and images represent single confocal planes from abaxial epidermal cells ( scale bars = 20 μm and 10 μm , lower panel ) . White arrows indicate colocalization of GFP and RFP signals . The experiment was repeated twice with similar results . ( E ) Total proteins were extracted 48 hpi with A . tumefaciens harboring the respective GFP - XopL , HA - XopL and SH3P2 - GFP expression constructs . SH3P2 - GFP protein levels ( lower band ) were detected using an anti - GFP antibody . Expression of the XopL was verified using an anti - HA or anti - GFP antibody . Expression of GUS - HA served as a control . Ponceau S staining serves as a loading control . The experiment was repeated three times with similar results . ( F ) Growth of Xcv and Xcv ΔxopL strains in roq1 N . benthamiana plants silenced for SH3P2 ( pTRV2 - SH3P2 ) compared to control plants ( pTRV2 ) . Leaves were dip - inoculated with a bacteria suspension at OD600 = 0 . 2 and bacteria were quantified at 3 and 6 dpi . Experimental repeats are indicated by data points in red ( n = 8 ) and yellow ( n = 6 ) data points . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Different letters indicate statistically significant differences ( P < 0 . 05 ) as determined by one - way ANOVA .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Interaction of XopL with SH3P2 in yeast two-hybrid assays. XopL fused to the GAL4 DNA-binding domain was expressed in combination with SH3P2 fused to the GAL4 activation domain (AD) in yeast strain Y190. Cells were grown on selective media before a LacZ filter assay was performed. pSV40/p53 served as positive control, while the empty AD or BD vector served as negative control. NtSH3P2 = Nicotiana tabacum SH3P2. -LT = yeast growth on medium without Leu and Trp, -HLT = yeast growth on medium lacking His, Leu, and Trp, indicating expression of the HIS3 reporter gene. LacZ, activity of the lacZ reporter gene.(B) Coimmunoprecipitation of GFP-XopL with AtSH3P2-HA. GFP-XopL or GFP were transiently coexpressed with AtSH3P2-HA in leaves of N. benthamiana. After 48 h, total proteins (Input) were subjected to immunoprecipitation (IP) with GFP-Trap beads, followed by immunoblot analysis using either anti-GFP or anti-HA antibodies. GFP blot was split for visualization purpose. AtSH3P2 = Arabidopsis thaliana SH3P2. Two repetitions with similar results have been conducted.(C) Visualization of protein interactions in planta by the bimolecular fluorescence complementation assay. Yellow fluorescent protein (YFP) confocal microscopy images show Nicotiana benthamiana leaf epidermal cells transiently expressing VenusN173-XopL in combination with AtSH3P2-VenusC155. A positive control showing the dimerization of fructose-1,6-bisphosphatase (FBPase) within the cytosol. White arrows indicate reconstitution of YFP fluorescence. The red structures indicate autofluorescence of chloroplasts. The combination of VenusN173-XopL with FBPase-VenusC155 or VenusN173-FBPase with AtSH3P2-VenusC155 do not induce YFP fluorescence and serve as negative controls. Bars = 20 μm.(D) Colocalization analysis of GFP-XopL with SH3P2-RFP, RFP-ATG8e and RFP-ATG8g in N. benthamiana leaves. Imaging was performed 2 d after transient expression and images represent single confocal planes from abaxial epidermal cells (scale bars = 20 μm and 10 μm, lower panel). White arrows indicate colocalization of GFP and RFP signals. The experiment was repeated twice with similar results.(E) Total proteins were extracted 48 hpi with A. tumefaciens harboring the respective GFP-XopL, HA-XopL and SH3P2-GFP expression constructs. SH3P2-GFP protein levels (lower band) were detected using an anti-GFP antibody. Expression of the XopL was verified using an anti-HA or anti-GFP antibody. Expression of GUS-HA served as a control. Ponceau S staining serves as a loading control. The experiment was repeated three times with similar results.(F) Growth of Xcv and Xcv ΔxopL strains in roq1 N. benthamiana plants silenced for SH3P2 (pTRV2-SH3P2) compared to control plants (pTRV2). Leaves were dip-inoculated with a bacteria suspension at OD600 = 0.2 and bacteria were quantified at 3 and 6 dpi. Experimental repeats are indicated by data points in red (n=8) and yellow (n=6) data points. Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Different letters indicate statistically significant differences (P < 0.05) as determined by one-way ANOVA.


Figure 4 ( A ) SH3P2 - GFP was transiently coexpressed together with GUS - HA and GFP - XopL in N . benthamiana using agroinfiltration . At 42 hpi , 200 μM MG132 was infiltrated into A . tumefaciens - inoculated leaves , and leaf material was collected 48 hpi . Expression of SH3P2 - GFP ( lower band ) and GFP - XopL ( upper band ) was detected using an anti - GFP antibody . GUS - HA expression was confirmed with an anti - HA antibody . Ponceau S staining serves as a loading control . The experiment was repeated three times with similar results . ( B ) In vitro ubiquitination assay reveals ubiquitination of SH3P2 by XopL . GST - XopL , and MBP - SH3P2 were tested using the Arabidopsis His - AtUBA1 and His - AtUBC8 . Lanes 2 to 4 are negative controls . Proteins were separated by SDS - PAGE and detected by immunoblotting using the indicated antibodies . Asterisks indicate bands that are not ubiquitinated an present in controls . The experiment was repeated twice with similar results . ( C ) SH3P2 - GFP was transiently coexpressed together with GFP , GFP - XopL and GFP - XopL ΔE3 in N . benthamiana using agroinfiltration . GFP protein levels were detected with an anti - GFP antibody . Ponceau S staining serves as a loading control . The experiment was repeated three times with similar results . ( D ) Immunoblot analysis of ATG8 protein levels in N . benthamiana plants transiently expressing GFP - XopL , GFP - XopL ΔE3 or GFP control at 2dpi . Expression of binary constructs was verified with an anti - GFP antibody . GFP blot was split for visualization purpose . Ponceau Staining ( PS ) served as a loading control . The experiment was repeated twice with similar results . ( E ) RLUC - ATG8a constructs were coexpressed with internal control FLUC in N . benthamiana . GFP - XopL , GFP - XopL ΔE3 or GFP control were co - infiltrated together with RLUC / FLUC mixture . Renilla and Firefly luciferase activities were simultaneously measured in leaf extracts at 48 hpi using the dual - luciferase system . Values represent the ratio of RLUC - ATG8a and FLUC activities ( n = 4 ) . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Statistical significance ( * P < 0 . 5 , * * P < 0 . 01 ) was revealed by Student ' s t - test . The experiment was repeated 3 times .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) SH3P2-GFP was transiently coexpressed together with GUS-HA and GFP-XopL in N. benthamiana using agroinfiltration. At 42 hpi, 200 μM MG132 was infiltrated into A. tumefaciens-inoculated leaves, and leaf material was collected 48 hpi. Expression of SH3P2-GFP (lower band) and GFP-XopL (upper band) was detected using an anti-GFP antibody. GUS-HA expression was confirmed with an anti-HA antibody. Ponceau S staining serves as a loading control. The experiment was repeated three times with similar results.(B) In vitro ubiquitination assay reveals ubiquitination of SH3P2 by XopL. GST-XopL, and MBP-SH3P2 were tested using the Arabidopsis His-AtUBA1 and His-AtUBC8. Lanes 2 to 4 are negative controls. Proteins were separated by SDS-PAGE and detected by immunoblotting using the indicated antibodies. Asterisks indicate bands that are not ubiquitinated an present in controls. The experiment was repeated twice with similar results.(C) SH3P2-GFP was transiently coexpressed together with GFP, GFP-XopL and GFP-XopL ΔE3 in N. benthamiana using agroinfiltration. GFP protein levels were detected with an anti-GFP antibody. Ponceau S staining serves as a loading control. The experiment was repeated three times with similar results.(D) Immunoblot analysis of ATG8 protein levels in N. benthamiana plants transiently expressing GFP-XopL, GFP-XopL ΔE3 or GFP control at 2dpi. Expression of binary constructs was verified with an anti-GFP antibody. GFP blot was split for visualization purpose. Ponceau Staining (PS) served as a loading control. The experiment was repeated twice with similar results.(E) RLUC-ATG8a constructs were coexpressed with internal control FLUC in N. benthamiana. GFP-XopL, GFP-XopL ΔE3 or GFP control were co-infiltrated together with RLUC/FLUC mixture. Renilla and Firefly luciferase activities were simultaneously measured in leaf extracts at 48 hpi using the dual-luciferase system. Values represent the ratio of RLUC-ATG8a and FLUC activities (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (* P < 0.5, ** P < 0.01) was revealed by Student's t-test. The experiment was repeated 3 times.


Figure 5 ( A ) GFP - XopL was coexpressed with GFP or AIMp - RFP . Proteins were separated by SDS - PAGE and detected by immunoblotting using the indicated antibodies . GFP blot was split for visualization purpose . Ponceau Staining ( PS ) served as a loading control . The experiment was repeated three times with similar results . ( B ) Colocalization of RFP - XopL with GFP - Joka2 in N . benthamiana leaves . Imaging was performed 2 d after transient expression and images represent single confocal planes from abaxial epidermal cells ( bars = 20 μm ) . The experiment was repeated twice with similar results . ( C ) FRET FLIM measurements of GFP - Joka2 and RFP - XopL in N . benthamiana leaves . The freeRFP construct served as a negative control and RFP - ATG8E ( n = 9 ) as a positive control . Scattered points show individual data points , color indicates biological repeats . The lifetime ( in ns ) of GFP - Joka2 ( donor , n = 41 ) was significantly reduced in the presence of RFP - XopL ( n = 40 ) but not in the presence of freeRFP ( n = 35 ) . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Significant differences were calculated using Wilcoxon rank sum test , with significantly different groups denoted by different letters . The experiment was repeated three times with similar results . ( D ) Immunoprecipitation ( IP ) of GFP - XopL reveals association with NBR1 . Immunoblots of input and IP samples from N . benthamiana plants transiently expressing GFP or GFP - XopL were probed with anti - GFP and anti - NBR1 antibodies . GFP blot was split for visualization purpose . ( E ) Immunoprecipitation ( IP ) of GFP - XopL and GFP - XopL ΔE3 reveals association with NBR1 . Immunoblots of input and IP samples from N . benthamiana plants transiently expressing GFP , GFP - XopL and GFP - XopL ΔE3 were probed with anti - GFP and anti - NBR1 antibodies . GFP blot was split for visualization purpose . ( F ) GFP - XopL was transiently expressed in pTRV2 , pTRV2 - Joka2 and N . benthamiana WT plants . Expression of binary constructs was verified with an anti - GFP antibody . Joka2 silencing was verified using an anti - NBR1 antibody . Ponceau Staining ( PS ) served as a loading control . The experiment was repeated twice with similar results . ( G ) Growth of Xcv ΔxopQ in N . benthamiana plants silenced for Joka2 ( pTRV2 - Joka2 ) compared to control plants ( pTRV2 ) . Leaves were dip - inoculated with a bacteria suspension at OD600 = 0 . 2 . and bacteria were quantified at 3 and 6 dpi . Red , blue , and green data points represent repeats of the experiments . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Significant differences were calculated using Student ' s t - test and are indicated by : * * , P < 0 . 01 . The experiment was repeated three times with similar trends . ( H ) GFP - XopL , GFP - XopL ΔE3 were transiently expressed in N . benthamiana . RFP - AIMp was co - infiltrated to stabilize both XopL variants . Samples were taken 48 hpi , and total proteins ( Input ) were subjected to immunoprecipitation ( IP ) with GFP - Trap beads , followed by immunoblot analysis of the precipitates using either anti - GFP or anti - ubiquitin antibodies . GFP served as a negative control . RFP - AIMp expression was verified by an anti - RFP antibody . GFP blot was split for visualization purpose . Asterisk indicates the GFP - XopL full - length protein . The experiment was repeated three times with similar results . ( I ) GFP - XopL was transiently expressed in N . benthamiana . Samples were taken 48 hpi , and total proteins ( Input ) were subjected to immunoprecipitation ( IP ) with the ubiquitin pan selector , followed by immunoblot analysis of the precipitates using either anti - GFP or anti - ubiquitin antibodies . GFP served as a control . Asterisk indicates the GFP - XopL full - length protein . GFP blot was split for visualization purpose . The experiment was repeated two times with similar results . ( J ) Immunoblot analysis GFP - XopL and GFP - XopLK191A at 1 and 2 dpi using an anti - GFP antibody . Ponceau Staining ( PS ) served as a loading control . The experiment was repeated three times with similar results . ( K ) HA - XopL and GFP - Joka2 and its variants were transiently expressed in N . benthamiana . Samples were taken 48 hpi , and total proteins ( Input ) were subjected to immunoprecipitation ( IP ) with GFP - Trap beads , followed by immunoblot analysis of the precipitates using either anti - GFP , anti - ubiquitin and anti - NBR1 antibodies . GFP served as a control .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) GFP-XopL was coexpressed with GFP or AIMp-RFP. Proteins were separated by SDS-PAGE and detected by immunoblotting using the indicated antibodies. GFP blot was split for visualization purpose. Ponceau Staining (PS) served as a loading control. The experiment was repeated three times with similar results.(B) Colocalization of RFP-XopL with GFP-Joka2 in N. benthamiana leaves. Imaging was performed 2 d after transient expression and images represent single confocal planes from abaxial epidermal cells (bars = 20 μm). The experiment was repeated twice with similar results.(C) FRET FLIM measurements of GFP-Joka2 and RFP-XopL in N. benthamiana leaves. The freeRFP construct served as a negative control and RFP-ATG8E (n = 9) as a positive control. Scattered points show individual data points, color indicates biological repeats. The lifetime (in ns) of GFP-Joka2 (donor, n = 41) was significantly reduced in the presence of RFP-XopL (n = 40) but not in the presence of freeRFP (n = 35). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Significant differences were calculated using Wilcoxon rank sum test, with significantly different groups denoted by different letters. The experiment was repeated three times with similar results.(D) Immunoprecipitation (IP) of GFP-XopL reveals association with NBR1. Immunoblots of input and IP samples from N. benthamiana plants transiently expressing GFP or GFP-XopL were probed with anti-GFP and anti-NBR1 antibodies. GFP blot was split for visualization purpose.(E) Immunoprecipitation (IP) of GFP-XopL and GFP-XopL ΔE3 reveals association with NBR1. Immunoblots of input and IP samples from N. benthamiana plants transiently expressing GFP, GFP-XopL and GFP-XopL ΔE3 were probed with anti-GFP and anti-NBR1 antibodies. GFP blot was split for visualization purpose.(F) GFP-XopL was transiently expressed in pTRV2, pTRV2-Joka2 and N. benthamiana WT plants. Expression of binary constructs was verified with an anti-GFP antibody. Joka2 silencing was verified using an anti-NBR1 antibody. Ponceau Staining (PS) served as a loading control. The experiment was repeated twice with similar results.(G) Growth of Xcv ΔxopQ in N. benthamiana plants silenced for Joka2 (pTRV2-Joka2) compared to control plants (pTRV2). Leaves were dip-inoculated with a bacteria suspension at OD600 = 0.2. and bacteria were quantified at 3 and 6 dpi. Red, blue, and green data points represent repeats of the experiments. Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Significant differences were calculated using Student's t-test and are indicated by: **, P < 0.01. The experiment was repeated three times with similar trends.(H) GFP-XopL, GFP-XopL ΔE3 were transiently expressed in N. benthamiana. RFP-AIMp was co-infiltrated to stabilize both XopL variants. Samples were taken 48 hpi, and total proteins (Input) were subjected to immunoprecipitation (IP) with GFP-Trap beads, followed by immunoblot analysis of the precipitates using either anti-GFP or anti-ubiquitin antibodies. GFP served as a negative control. RFP-AIMp expression was verified by an anti-RFP antibody. GFP blot was split for visualization purpose. Asterisk indicates the GFP-XopL full-length protein. The experiment was repeated three times with similar results.(I) GFP-XopL was transiently expressed in N. benthamiana. Samples were taken 48 hpi, and total proteins (Input) were subjected to immunoprecipitation (IP) with the ubiquitin pan selector, followed by immunoblot analysis of the precipitates using either anti-GFP or anti-ubiquitin antibodies. GFP served as a control. Asterisk indicates the GFP-XopL full-length protein. GFP blot was split for visualization purpose. The experiment was repeated two times with similar results.(J) Immunoblot analysis GFP-XopL and GFP-XopLK191A at 1 and 2 dpi using an anti-GFP antibody. Ponceau Staining (PS) served as a loading control. The experiment was repeated three times with similar results.(K) HA-XopL and GFP-Joka2 and its variants were transiently expressed in N. benthamiana. Samples were taken 48 hpi, and total proteins (Input) were subjected to immunoprecipitation (IP) with GFP-Trap beads, followed by immunoblot analysis of the precipitates using either anti-GFP, anti-ubiquitin and anti-NBR1 antibodies. GFP served as a control.


figf1a , The 2 - bp deletion identified in exon 9b of patient 1 . b , The T - to - A nonsense mutation in exon 4 of patient 2 ( stop codon is underlined ) . c , Upper panel , intronic mutation in patient 3 ; lower panel , sequencing of the RT - PCR product in patient 3 , showing exon - 6 skipping . d , Upper panel , The G - to - A point mutation at the splice - donor site in intron 5 of patient 6 ; lower panel , sequencing of the RT - PCR product in patient 6 , showing a 6 - bp insertion at the junction between exons 5 and 6 . The inserted sequence ( underlined ) is derived from the first six nucleotides of intron 5 ( underlined ) , which creates a stop codon ( dotted ) . Most probably , nucleotides 7 and 8 , GT ( double underlined ) , in intron 5 are alternatively recognized as a splice - donor site . e , Upper panel , the 10 - bp deletion in patient 9 . This mutation deletes four nucleotides from intron 5 and six from exon 6 . Lower panel , sequence of the RT - PCR product in patient 9 reveals the deletion of the 10 nucleotides at the 5 ′ end of exon 6 . The ' AG ' ( double underlined ) in exon 6 immediately after the genomic DNA deletion appears to be recognized as an alternative splice - acceptor site .

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figf1a, The 2-bp deletion identified in exon 9b of patient 1. b, The T-to-A nonsense mutation in exon 4 of patient 2 (stop codon is underlined). c, Upper panel, intronic mutation in patient 3; lower panel, sequencing of the RT-PCR product in patient 3, showing exon-6 skipping. d, Upper panel, The G-to-A point mutation at the splice-donor site in intron 5 of patient 6; lower panel, sequencing of the RT-PCR product in patient 6, showing a 6-bp insertion at the junction between exons 5 and 6. The inserted sequence (underlined) is derived from the first six nucleotides of intron 5 (underlined), which creates a stop codon (dotted). Most probably, nucleotides 7 and 8, GT (double underlined), in intron 5 are alternatively recognized as a splice-donor site. e, Upper panel, the 10-bp deletion in patient 9. This mutation deletes four nucleotides from intron 5 and six from exon 6. Lower panel, sequence of the RT-PCR product in patient 9 reveals the deletion of the 10 nucleotides at the 5′ end of exon 6. The 'AG' (double underlined) in exon 6 immediately after the genomic DNA deletion appears to be recognized as an alternative splice-acceptor site.


figf2Figure 2 Representation of the mutant LAMP - 2b . ' Normal ' represents the LAMP - 2b amino - acid sequence . Because patient 1 , who developed full DD symptoms , has a mutation in exon 9b , the LAMP - 2b isoform is most probably responsible for DD . Residue 265 , one of the putative disulphide - bond - creating cysteines , and two glycosylation sites are lost by exon - 6 skipping in patient 3 , which probably results in a significant change in the secondary structure . Black boxes represent the loops created between two cystein residues flanking each of the four loop regions ; Y depicts a glycosylation site ; hatched boxes show altered amino acids caused by frameshift mutations ; dashes represent missing amino acids due to exon - 6 skipping in patient 3 . All of the other mutant proteins lack the transmembrane domain ( grey bar ) because of nonsense or frameshift mutations . Thus , those molecules cannot function as lysosomal ' membrane ' proteins .

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figf2Figure 2 Representation of the mutant LAMP-2b. 'Normal' represents the LAMP-2b amino-acid sequence. Because patient 1, who developed full DD symptoms, has a mutation in exon 9b, the LAMP-2b isoform is most probably responsible for DD. Residue 265, one of the putative disulphide-bond-creating cysteines, and two glycosylation sites are lost by exon-6 skipping in patient 3, which probably results in a significant change in the secondary structure. Black boxes represent the loops created between two cystein residues flanking each of the four loop regions; Y depicts a glycosylation site; hatched boxes show altered amino acids caused by frameshift mutations; dashes represent missing amino acids due to exon-6 skipping in patient 3. All of the other mutant proteins lack the transmembrane domain (grey bar) because of nonsense or frameshift mutations. Thus, those molecules cannot function as lysosomal 'membrane' proteins.


figf3a , Skeletal muscle . Muscle extracts from patients 1 - 6 and 10 and a control were blotted and labelled with antibodies against LAMP - 2 ( top panel ) and β - actin ( bottom panel ) . LAMP - 2 is deficient in all muscles examined except the sample from patient 1 that showed a trace amount of protein , which is probably the LAMP - 2a isoform . In support of this idea , LAMP - 2b is the principal LAMP - 2 species in skeletal muscle6 . Numbers denote patients as in Table 1 ; C , control . b , Heart . Protein was extracted from cardiac muscle of patient 4 and a control , blotted onto a nitrocellulose membrane , and labelled with the antibodies against LAMP - 2 ( too panel ) and β - actin ( bottom panel ) . LAMP - 2 is deficient also in cardiac muscle . C , control ; 4 , patient 4 .

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figf3a, Skeletal muscle. Muscle extracts from patients 1-6 and 10 and a control were blotted and labelled with antibodies against LAMP-2 (top panel) and β-actin (bottom panel). LAMP-2 is deficient in all muscles examined except the sample from patient 1 that showed a trace amount of protein, which is probably the LAMP-2a isoform. In support of this idea, LAMP-2b is the principal LAMP-2 species in skeletal muscle6. Numbers denote patients as in Table 1; C, control. b, Heart. Protein was extracted from cardiac muscle of patient 4 and a control, blotted onto a nitrocellulose membrane, and labelled with the antibodies against LAMP-2 (too panel) and β-actin (bottom panel). LAMP-2 is deficient also in cardiac muscle. C, control; 4, patient 4.


figf4Serial transverse sections of the biopsiedmuscle from six DD patients , and samples from a XMEA patient and a normal control , were immunostained with antibodies against LAMP - 2 and limp - I . LAMP - 2 is absent in all DD muscles ( patient 6 shown here ) , whereas it is present in the XMEA muscle . The antibody against limp - I highlighted vacuoles in both DD and XMEA muscles . LAMP - 1 antibody faintly stained both DD and XMEA muscles , suggesting that the expression level of LAMP - 1 might be lower in skeletal muscle than LAMP - 2 or limp - I ( data not shown ) . The muscle from the patient without a lamp - 2 mutation also showed LAMP - 2 deficiency on immunohistochemistry similar to genetically proven DD patients with primary LAMP - 2 deficiency ( data not shown ) . The immunohistochemical stains of LAMP - 2 , limp - I , and LAMP - 1 in normal muscle shows numerous tiny particles and accentuates type 1 fibres . H & amp ; amp ; E , haematoxylin and eosin , original magnification × 150 .

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figf4Serial transverse sections of the biopsiedmuscle from six DD patients, and samples from a XMEA patient and a normal control, were immunostained with antibodies against LAMP-2 and limp-I. LAMP-2 is absent in all DD muscles (patient 6 shown here), whereas it is present in the XMEA muscle. The antibody against limp-I highlighted vacuoles in both DD and XMEA muscles. LAMP-1 antibody faintly stained both DD and XMEA muscles, suggesting that the expression level of LAMP-1 might be lower in skeletal muscle than LAMP-2 or limp-I (data not shown). The muscle from the patient without a lamp-2 mutation also showed LAMP-2 deficiency on immunohistochemistry similar to genetically proven DD patients with primary LAMP-2 deficiency (data not shown). The immunohistochemical stains of LAMP-2, limp-I, and LAMP-1 in normal muscle shows numerous tiny particles and accentuates type 1 fibres. H&amp;E, haematoxylin and eosin, original magnification ×150.


Figure 2Representative confocal 3D images of LysoTracker ( red ) , DAPI ( blue ) , and BODIPY ( green ) stained ATM subsets obtained from eWAT of HFD ( 16 weeks ) treated mice ( n = 8 mice from two independent experiments ) . The merged figures are the result of the overlap of all three fluorochromes . Scale bar , 10 μm . Mito Stress assay of sorted ATM subsets ( red , MHCIIlow ; green , MHCIIhi ; and blue , CD11c + ) from 16 - week - HFD obese mice showing overall oxygen consumption rate ( OCR ) , maximal respiration , spare capacity respiration , basal respiration , ATP production , and non - mitochondrial respiration . OCR data shown are representative of four independent experiments and expressed as mean ± SD ( n = 4 MHCIIlow ; n = 7 MHCIIhi ; n = 5 CD11c + ) , with statistical significance determined using one - way ANOVA test ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : no significant difference ) .

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Figure 2Representative confocal 3D images of LysoTracker (red), DAPI (blue), and BODIPY (green) stained ATM subsets obtained from eWAT of HFD (16 weeks) treated mice (n = 8 mice from two independent experiments). The merged figures are the result of the overlap of all three fluorochromes. Scale bar, 10 μm.Mito Stress assay of sorted ATM subsets (red, MHCIIlow; green, MHCIIhi; and blue, CD11c+) from 16-week-HFD obese mice showing overall oxygen consumption rate (OCR), maximal respiration, spare capacity respiration, basal respiration, ATP production, and non-mitochondrial respiration. OCR data shown are representative of four independent experiments and expressed as mean ± SD (n= 4 MHCIIlow; n= 7 MHCIIhi; n=5 CD11c+), with statistical significance determined using one-way ANOVA test (*P < 0.05; **P < 0.01; ***P < 0.001; ns: no significant difference).


Figure 3Representative flow cytometric showing the specific depletion efficiency of ATM subsets from eWAT of CD169 - DTR ND - ( A ) or HFD - treated ( 16 weeks ) ( B ) mice . A representative analysis of three independent experiments is shown . eWAT was collected from lean ( C ) and obese ( HFD 16 weeks ) ( D ) WT or CD169 - DTR mice after 12 days DT - mediated ATM depletion . Representative pictures of dissected eWAT are shown on the left , the corresponding eWAT weights expressed as mean ± SD of more than 12 samples are shown in the right , with statistical significance calculated using the Student ' s t - test ( * * P < 0 . 01 and * * * * P < 0 . 0001 ) . The zoomed - in area shows clear , oily lipid accumulation in the absence of ATMs ( lower left part ) . Adipocyte hypertrophy is visible in the absence of ATMs . Representative images of hematoxylin and eosin staining ( H & E ) ( E ) and corresponding adipocyte diameters ( F ) of lean and obese ( HFD 16 weeks ) eWAT from WT or CD169 - DTR mice treated for 7 or 12 days with DT . Scale bar , 50 μm . Each group comprised 3 - 5 mice . Statistical significance was determined using one - way ANOVA test . * * P < 0 . 01 ; * * * * P < 0 . 0001 ; ns : no significant difference . The box and whisker plots show the median value and 10 - 90 percentiles of adipocyte diameters .

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Figure 3Representative flow cytometric showing the specific depletion efficiency of ATM subsets from eWAT of CD169-DTR ND- (A) or HFD-treated (16 weeks) (B) mice. A representative analysis of three independent experiments is shown.eWAT was collected from lean (C) and obese (HFD 16 weeks) (D) WT or CD169-DTR mice after 12 days DT-mediated ATM depletion. Representative pictures of dissected eWAT are shown on the left, the corresponding eWAT weights expressed as mean ± SD of more than 12 samples are shown in the right, with statistical significance calculated using the Student's t-test (**P < 0.01 and ****P < 0.0001). The zoomed-in area shows clear, oily lipid accumulation in the absence of ATMs (lower left part).Adipocyte hypertrophy is visible in the absence of ATMs. Representative images of hematoxylin and eosin staining (H&E) (E) and corresponding adipocyte diameters (F) of lean and obese (HFD 16 weeks) eWAT from WT or CD169-DTR mice treated for 7 or 12 days with DT. Scale bar, 50 μm. Each group comprised 3-5 mice. Statistical significance was determined using one-way ANOVA test. **P < 0.01; ****P < 0.0001; ns: no significant difference. The box and whisker plots show the median value and 10-90 percentiles of adipocyte diameters.


Figure 4qPCR showing the relative expression of genes for adipogenesis ( Pparγ ) , lipolysis ( Lpl ) , and lipogenesis ( Dgat1 ) , plus other lipid receptor / transporters ( Cd36 , Abca1 ) or chaperones ( Fabp4 ) in obese ( HFD 16 weeks ) eWAT of CD169 - DTR ( red bars ) mice and DT - injected WT controls ( blue bars ) ( upper panel ) and obese eWAT of anti - CSF1R antibody - injected mice ( green bars ) mice and isotype control - injected WT controls ( white bars ) ( lower panel ) . Mice were under DT or anti - CSF1R antibody treatment for 12 days . ( n = 5 pooled mice per group , mean ± SD ) . Statistical significance was determined using an unpaired Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : no significant difference .

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Figure 4qPCR showing the relative expression of genes for adipogenesis (Pparγ), lipolysis (Lpl), and lipogenesis (Dgat1), plus other lipid receptor/transporters (Cd36, Abca1) or chaperones (Fabp4) in obese (HFD 16 weeks) eWAT of CD169-DTR (red bars) mice and DT-injected WT controls (blue bars) (upper panel) and obese eWAT of anti-CSF1R antibody-injected mice (green bars) mice and isotype control-injected WT controls (white bars) (lower panel). Mice were under DT or anti-CSF1R antibody treatment for 12 days. (n = 5 pooled mice per group, mean ± SD). Statistical significance was determined using an unpaired Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significant difference.


Figure 5Violin plots depicting the expression of representative marker genes for each CD45 - cell cluster : ASC1 ( Gsn , Pdgfrb , Eln ) ( red ) , ASC2 ( Pi16 , Pdgfra , Cebpd ) ( yellow ) , stromal cells ( Col1a1 , Col1a2 , Myl9 ) ( green ) , VEC1 / VEC2 ( Pecam - 1 , Cldn5 , Aqp1 ) ( purple and blue ) , and mesothelial - like cells ( MLCs ) ( Upk3b , Msln , Acta2 ) ( orange ) .

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Figure 5Violin plots depicting the expression of representative marker genes for each CD45- cell cluster: ASC1 (Gsn, Pdgfrb, Eln) (red), ASC2 (Pi16, Pdgfra, Cebpd) (yellow), stromal cells (Col1a1, Col1a2, Myl9) (green), VEC1/VEC2 (Pecam-1, Cldn5, Aqp1) (purple and blue), and mesothelial-like cells (MLCs) (Upk3b, Msln, Acta2) (orange).


Figure 6Representative 3D fluorescence imaging of lean and obese ( HFD 16 weeks ) eWAT samples stained with anti - MHCII ( green in lean samples ) , anti - CD11c ( green in obese samples ) , anti - CD31 ( blue ) , anti - CD169 ( pink ) , and DAPI ( white ) . The two images at the bottom show the images merged for the lean and obese conditions without DAPI . Scale bar : 100 μm ( lean ) and 70 μm ( obese ) . Higher magnification of specific eWAT areas . Scale bar : 30 μm ( lean ) and 50 μm ( obese ) . ( left panels ) Distance of different ATM subsets to blood vessels . Distances ( in μm ) were calculated using the Imaris software , as described in the Materials and Methods section . The box and whisker plots show the median value and 10 - 90 percentiles of distance values . The right panels show the distribution of ATM subsets to blood vessels within a certain distance ( ≤ 1 μm , 1 - 10 μm , ≥ 10 μm ) . C : CD169 + MHCIIlow and CD169 + MHChi ATMs in lean eWAT . D : CD169 + CD11c - and CD169 + CD11c + ATMs in obese ( HFD 16 weeks ) eWAT . Four mice from two independent experiments were analyzed ( n = 4 - 5 ; mean ± SD ) . Statistical significance was determined using an unpaired Student ' s t - test . * P < 0 . 05 , * * * * P < 0 . 0001 , bars with no asterisks showed no significant difference . Model construction based on 3D reconstructed images showing the relative distance of CD11c + ATMs from traced large blood vessels . Blood vessels larger than 10µm diameter in the image were marked out on the image by tracing ( white filaments ) . CD11c + ATMs are close ( < 10µm ) to the traced large blood vessels ( red ellipsoids ) or far ( > 10 µm ) ( grey ellipsoids ) from the traced blood vessel . Blood vessels are shown in green . Scale bar : 100 μm . The graph to the right shows the percentage of CD11chi ATMs showing a proximity of ≤ 10 or > 10 μm to blood vessels quantified from 3 biological replicates from 1 experiment , data was expressed as mean ± SD . Statistical significance was determined using an unpaired Student ' s t - test . * * * P < 0 . 001 .

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Figure 6Representative 3D fluorescence imaging of lean and obese (HFD 16 weeks) eWAT samples stained with anti-MHCII (green in lean samples), anti-CD11c (green in obese samples), anti-CD31 (blue), anti-CD169 (pink), and DAPI (white). The two images at the bottom show the images merged for the lean and obese conditions without DAPI. Scale bar: 100 μm (lean) and 70 μm (obese).Higher magnification of specific eWAT areas. Scale bar: 30 μm (lean) and 50 μm (obese). (left panels) Distance of different ATM subsets to blood vessels. Distances (in μm) were calculated using the Imaris software, as described in the Materials and Methods section. The box and whisker plots show the median value and 10-90 percentiles of distance values. The right panels show the distribution of ATM subsets to blood vessels within a certain distance (≤1 μm, 1-10 μm, ≥10 μm). C: CD169+MHCIIlow and CD169+MHChi ATMs in lean eWAT. D: CD169+CD11c- and CD169+CD11c+ ATMs in obese (HFD 16 weeks) eWAT. Four mice from two independent experiments were analyzed (n = 4-5; mean ± SD). Statistical significance was determined using an unpaired Student's t-test. *P < 0.05, ****P < 0.0001, bars with no asterisks showed no significant difference.Model construction based on 3D reconstructed images showing the relative distance of CD11c+ ATMs from traced large blood vessels. Blood vessels larger than 10µm diameter in the image were marked out on the image by tracing (white filaments). CD11c+ ATMs are close (< 10µm) to the traced large blood vessels (red ellipsoids) or far (>10 µm) (grey ellipsoids) from the traced blood vessel. Blood vessels are shown in green. Scale bar: 100 μm. The graph to the right shows the percentage of CD11chi ATMs showing a proximity of ≤10 or >10 μm to blood vessels quantified from 3 biological replicates from 1 experiment, data was expressed as mean ± SD. Statistical significance was determined using an unpaired Student's t-test. ***P < 0.001.


Figure 7Two representative images of eWAT dissected from Evans blue - injected obese ( HFD 16 weeks ) WT and CD169 - DTR mice treated for 7 days with DT . Evans blue dye was extracted from eWAT using formamide and the absorption of the extract was measured at 620 nm ( the absorption values were normalized to the tissue weight in grams ) . A chart showing the normalized mean optical density ± SD ( n = 5 - 7 mice per group ) . Statistical significance was determined using an unpaired Student ' s t - test . * * * P < 0 . 001 . Mmp12 ( H ) and Vegfα ( F ) are highly expressed in MHCIIhi and CD11c + ATMs purified from obese ( HFD 16 weeks ) eWAT . qPCR analysis showed the relative expression in all three ATM subsets ( n = 3 ) , data was expressed as mean ± SD . Statistical significance between the three ATM subpopulations was determined using one - way ANOVA test . * P < 0 . 05 , * * P < 0 . 01 . qPCR analysis showing the relative expression of Vegfα in obese ( HFD 16 weeks ) eWAT of WT ( n = 3 ) or CD169 - DTR mice ( n = 5 ) treated for 7 days with DT , data was expressed as mean ± SD . Statistical significance was determined using an unpaired Student ' s t - test . * * * * P < 0 . 0001 .

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Figure 7Two representative images of eWAT dissected from Evans blue-injected obese (HFD 16 weeks) WT and CD169-DTR mice treated for 7 days with DT. Evans blue dye was extracted from eWAT using formamide and the absorption of the extract was measured at 620 nm (the absorption values were normalized to the tissue weight in grams). A chart showing the normalized mean optical density ± SD (n = 5-7 mice per group). Statistical significance was determined using an unpaired Student's t-test. ***P < 0.001.Mmp12 (H) and Vegfα (F) are highly expressed in MHCIIhi and CD11c+ ATMs purified from obese (HFD 16 weeks) eWAT. qPCR analysis showed the relative expression in all three ATM subsets (n=3), data was expressed as mean ± SD . Statistical significance between the three ATM subpopulations was determined using one-way ANOVA test. *P < 0.05, **P < 0.01. qPCR analysis showing the relative expression of Vegfα in obese (HFD 16 weeks) eWAT of WT (n=3) or CD169-DTR mice (n=5) treated for 7 days with DT, data was expressed as mean ± SD. Statistical significance was determined using an unpaired Student's t-test. ****P < 0.0001.


Figure 8Elastin staining showing more pronounced ECM accumulation in eWAT of CD169 - DTR ( HFD 16 weeks ) when compared with ECM accumulation in the WT obese ( HFD 16 weeks ) control ( DAPI , blue ; elastin , red ; CD31 , green ) . The fourth image in each row shows the three channels merged . Scale bar : 50 μm . Picrosirius red - staining depicts excessive collagen deposits in obese ( HFD 16 weeks ) eWAT of CD169 - DTR mice when compared with collagen deposits in WT controls . Scale bar : 250 μm . Upper panels : brightfield microscopy ; lower panels : fluorescence microscopy . Violin plots showing the pro - inflammatory cytokine profile ( Il - 6 , Tnf - α , and Il - 1β ) of all 23 cell clusters obtained from obese ( HFD 16 weeks ) WT and CD169 - DTR eWAT . Cluster 0 : ASC1 ; Cluster 1 : Stromal cells ; Cluster 2 : VEC1 ; Cluster 3 : VEC2 ; Cluster 4 : NK1 ; Cluster 5 : MAC / Mono1 ; Cluster 6 : ASC2 ; Cluster 7 : B ; Cluster 8 : cDC1 ; Cluster 9 : mono - derived cDC ; Cluster 10 : Th2 / ILC2 / Treg ; Cluster 11 : MAC / Mono3 ; Cluster 12 : Th17 ; Cluster 13 : VEC2 ; Cluster 14 : CD8 ; Cluster 15 : MLCs ; Cluster 16 : CD11b + DC ; Cluster 17 : Neutrophils ; Cluster 18 : cDC2 ; Cluster 19 : cDC ; Cluster 20 : NKT ; Cluster 21 : NK2 ; Cluster 22 : Mast cells / Basophils . ASC : adipocyte stem cell ; VEC : vascular endothelial cell ; MLC : mesothelial - like cell ; NK : natural killer ; MAC : macrophage ; Mono : monocyte ; DC : dendritic cell ; NKT : natural killer T cells .

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Figure 8Elastin staining showing more pronounced ECM accumulation in eWAT of CD169-DTR (HFD 16 weeks) when compared with ECM accumulation in the WT obese (HFD 16 weeks) control (DAPI, blue; elastin, red; CD31, green). The fourth image in each row shows the three channels merged. Scale bar: 50 μm. Picrosirius red-staining depicts excessive collagen deposits in obese (HFD 16 weeks) eWAT of CD169-DTR mice when compared with collagen deposits in WT controls. Scale bar: 250 μm. Upper panels: brightfield microscopy; lower panels: fluorescence microscopy.Violin plots showing the pro-inflammatory cytokine profile (Il-6, Tnf-α, and Il-1β) of all 23 cell clusters obtained from obese (HFD 16 weeks) WT and CD169-DTR eWAT. Cluster 0: ASC1; Cluster 1: Stromal cells; Cluster 2: VEC1; Cluster 3: VEC2; Cluster 4: NK1; Cluster 5: MAC/Mono1; Cluster 6: ASC2; Cluster 7: B; Cluster 8: cDC1; Cluster 9: mono-derived cDC; Cluster 10: Th2/ILC2/Treg; Cluster 11: MAC/Mono3; Cluster 12: Th17; Cluster 13: VEC2; Cluster 14: CD8; Cluster 15: MLCs; Cluster 16: CD11b+ DC; Cluster 17: Neutrophils; Cluster 18: cDC2; Cluster 19: cDC; Cluster 20: NKT; Cluster 21: NK2; Cluster 22: Mast cells/Basophils. ASC: adipocyte stem cell; VEC: vascular endothelial cell; MLC: mesothelial-like cell; NK: natural killer; MAC: macrophage; Mono: monocyte; DC: dendritic cell; NKT: natural killer T cells.


Figure 1 ( a - b ) eIF4A knockdown in either S2 ( a ) or Kc167 ( b ) cells blunts the inactivation of TORC1 upon amino acid removal . Cells were treated with dsRNA targeting GFP as a negative control , or three independent , non - overlapping dsRNAs targeting eIF4A for 5 days and then incubated with complete medium or medium lacking only amino acids for 30 minutes . Representative of 3 biological replicates . ( d ) Impaired inactivation of TORC1 in response to eIF4A knockdown is most apparent upon partial depletion of amino acids , caused by removal of amino acid subsets . After 5 days of knockdown , S2 cells were treated for 30 min with either complete Schneider ' s medium ( + aa ) , Schneider ' s medium lacking all amino acids ( - aa ) or various subsets of amino acids , as indicated ( where EAA " essential amino acids " = H , I , L , K , M , T , W , V ) . Error bars : std dev . n = 3 biological replicates . ( e ) Time course of amino - acid removal reveals that eIF4A knockdown Kc167 cells maintain elevated S6K phosphorylation up to the maximum possible timepoint of 60 minutes when the cells start dying ( see drop in S6K and tubulin levels ) . Representative of two biological replicates . ( f ) eIF4A mutant larvae have impaired TORC1 inactivation upon shifting to food lacking amino acids . Control ( w1118 ) or eIF4A1006 / 1013 1st instar larvae were transferred from standard food to plates containing either standard fly food or PBS / 1 % agarose + 2 % sucrose for 1h prior to lysis and immunoblot analysis . Two biological replicates are shown . Representative of 3 biological replicates .

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Figure 1(a-b) eIF4A knockdown in either S2 (a) or Kc167 (b) cells blunts the inactivation of TORC1 upon amino acid removal. Cells were treated with dsRNA targeting GFP as a negative control, or three independent, non-overlapping dsRNAs targeting eIF4A for 5 days and then incubated with complete medium or medium lacking only amino acids for 30 minutes. Representative of 3 biological replicates.(d) Impaired inactivation of TORC1 in response to eIF4A knockdown is most apparent upon partial depletion of amino acids, caused by removal of amino acid subsets. After 5 days of knockdown, S2 cells were treated for 30 min with either complete Schneider's medium (+aa), Schneider's medium lacking all amino acids (-aa) or various subsets of amino acids, as indicated (where EAA "essential amino acids" = H,I,L,K,M,T,W,V). Error bars: std dev. n=3 biological replicates.(e) Time course of amino-acid removal reveals that eIF4A knockdown Kc167 cells maintain elevated S6K phosphorylation up to the maximum possible timepoint of 60 minutes when the cells start dying (see drop in S6K and tubulin levels). Representative of two biological replicates.(f) eIF4A mutant larvae have impaired TORC1 inactivation upon shifting to food lacking amino acids. Control (w1118) or eIF4A1006/1013 1st instar larvae were transferred from standard food to plates containing either standard fly food or PBS/1% agarose+2%sucrose for 1h prior to lysis and immunoblot analysis. Two biological replicates are shown. Representative of 3 biological replicates.


Figure 2 ( a - a ' ) Although knockdown of eIF4A or eIF3 - S2 equally blunt translation of EGFP from an inducible plasmid ( a ) , only knockdown of eIF4A but not eIF3 - S2 impairs TORC1 inactivation upon amino acid removal ( a ' ) . ( a ) S2 cells , treated with indicated dsRNAs for 5 days , on day 3 transfected with an inducible EGFP plasmid ( pMT - EGFP ) and on day 4 induced for 18h . Non - targeting LacZ dsRNA used as a negative control . ( a ' ) S2 cells treated with indicated dsRNAs for 5 days , then incubated with medium lacking amino acids for indicated time prior to lysis . Representative of two biological replicates . ( b ) Knockdown of eIF4A , but not other translation initiation factors , blunts TORC1 inactivation upon amino acid withdrawal . Kc167 cells treated with indicated dsRNAs for 4 days and then incubated with complete Schneider ' s medium or Schneider ' s medium lacking the indicated amino acids for 30 minutes prior to lysis . ( c ) Inhibition of translation with cycloheximide does not block inactivation of TORC1 upon amino acid withdrawal in Kc167 cells . Kc167 cells treated with either eIF4A dsRNA for 4 days , or cycloheximide ( 50 µg / mL , " CHX " ) for 5 min prior to , as well as during removal of amino acids ( - LIVASTQP ) . CHX treated cells still inactivate TORC1 ( compare lanes 8 to 7 ) whereas eIF4A knockdown cells do not ( lanes 14 vs 13 ) . Quantifications of two biological replicates are shown . CHX and eIF4A samples were normalized to their respective control conditions . ( d ) Knockdown of eIF4A in Kc167 cells does not prevent a drop in intracellular amino acids levels when amino acids are removed from the medium for 30 min . Quantification of total intracellular amino acids , analyzed in a blinded fashion . Levels of individual amino acids shown in Figure S2D . For CHX samples , cycloheximide ( 50µg / mL ) was added 5 minutes prior to , and during treatment with medium containing or lacking amino acids . Statistical significance tested by ANOVA2 using Scheffé ' s multiple comparisons method ( p < 0 . 05 ) . Error bars : std . dev . n = 5 .

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Figure 2(a-a') Although knockdown of eIF4A or eIF3-S2 equally blunt translation of EGFP from an inducible plasmid (a), only knockdown of eIF4A but not eIF3-S2 impairs TORC1 inactivation upon amino acid removal (a'). (a) S2 cells, treated with indicated dsRNAs for 5 days, on day 3 transfected with an inducible EGFP plasmid (pMT-EGFP) and on day 4 induced for 18h. Non-targeting LacZ dsRNA used as a negative control. (a') S2 cells treated with indicated dsRNAs for 5 days, then incubated with medium lacking amino acids for indicated time prior to lysis. Representative of two biological replicates.(b) Knockdown of eIF4A, but not other translation initiation factors, blunts TORC1 inactivation upon amino acid withdrawal. Kc167 cells treated with indicated dsRNAs for 4 days and then incubated with complete Schneider's medium or Schneider's medium lacking the indicated amino acids for 30 minutes prior to lysis.(c) Inhibition of translation with cycloheximide does not block inactivation of TORC1 upon amino acid withdrawal in Kc167 cells. Kc167 cells treated with either eIF4A dsRNA for 4 days, or cycloheximide (50 µg/mL, "CHX") for 5 min prior to, as well as during removal of amino acids (-LIVASTQP). CHX treated cells still inactivate TORC1 (compare lanes 8 to 7) whereas eIF4A knockdown cells do not (lanes 14 vs 13). Quantifications of two biological replicates are shown. CHX and eIF4A samples were normalized to their respective control conditions.(d) Knockdown of eIF4A in Kc167 cells does not prevent a drop in intracellular amino acids levels when amino acids are removed from the medium for 30 min. Quantification of total intracellular amino acids, analyzed in a blinded fashion. Levels of individual amino acids shown in Figure S2D. For CHX samples, cycloheximide (50µg/mL) was added 5 minutes prior to, and during treatment with medium containing or lacking amino acids. Statistical significance tested by ANOVA2 using Scheffé's multiple comparisons method (p<0.05). Error bars: std. dev. n=5.


Figure 3 ( a ) Knockdown of eIF4F components ( eIF - 4E and eIF - 4G ) partially phenocopy the eIF4A knockdown , leading to elevated TORC1 activity upon amino acid removal ( 30 min ) . Knockdown of all other tested translation initiation factors do not cause elevated TORC1 upon amino acid removal ( main Figures 1c , 2a ' and 2b ) . Error bars : std . dev . n = 3 biological replicates . ( b ) Quantification of de novo protein synthesis rates by OPP incorporation reveals that eIF4E and eIF4G knockdowns deplete eIF4F function less efficiently than the eIF4A knockdown , explaining why the effects of eIF4E and eIF4G knockdowns on TORC1 activity ( panel a ) are a bit milder than the eIF4A knockdown . Kc167 cells treated with dsRNA for 4 days then incubated with 20μM Click - it OPP reagent 30min before fixation and fluorescent labeling . Quantification of OPP fluorescence per cell ( nuclear count ) for two independent experiments is displayed ( 3 independent images per condition ) , normalized to the no dsRNA condition . Scale bars : 25µm .

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Figure 3(a) Knockdown of eIF4F components (eIF-4E and eIF-4G) partially phenocopy the eIF4A knockdown, leading to elevated TORC1 activity upon amino acid removal (30 min). Knockdown of all other tested translation initiation factors do not cause elevated TORC1 upon amino acid removal (main Figures 1c, 2a' and 2b). Error bars: std. dev. n=3 biological replicates.(b) Quantification of de novo protein synthesis rates by OPP incorporation reveals that eIF4E and eIF4G knockdowns deplete eIF4F function less efficiently than the eIF4A knockdown, explaining why the effects of eIF4E and eIF4G knockdowns on TORC1 activity (panel a) are a bit milder than the eIF4A knockdown. Kc167 cells treated with dsRNA for 4 days then incubated with 20μM Click-it OPP reagent 30min before fixation and fluorescent labeling. Quantification of OPP fluorescence per cell (nuclear count) for two independent experiments is displayed (3 independent images per condition), normalized to the no dsRNA condition. Scale bars: 25µm.