B . GFP - Snc1 - PEM protein accumulates in aberrant ER structures in atg1Δ , atg8Δ , and atg11Δ mutant cells . The ER - marker Sec61 was tagged with mCherry in strains from panel A , and the cells were examined by live - cell microscopy . Shown from left to right : DIC , GFP , mCherry , merge , % cells with intracellular GFP - Snc1 - PEM ( number of cells with internal GFP - Snc1 - PEM / number of cells visualized ) , and % cells in which intra - cellular Snc1 - PEM co - localizes with Sec61 .

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B. GFP-Snc1-PEM protein accumulates in aberrant ER structures in atg1Δ, atg8Δ, and atg11Δ mutant cells. The ER-marker Sec61 was tagged with mCherry in strains from panel A, and the cells were examined by live-cell microscopy. Shown from left to right: DIC, GFP, mCherry, merge, % cells with intracellular GFP-Snc1-PEM (number of cells with internal GFP-Snc1-PEM / number of cells visualized), and % cells in which intra-cellular Snc1-PEM co-localizes with Sec61.


C . UPR is induced in atg1Δ , atg8Δ , and atg11Δ mutant cells . Cells from panel A were transformed with a second plasmid that expresses β - gal from a UPR promoter . UPR was determined and expressed as % of the WT response .

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C. UPR is induced in atg1Δ, atg8Δ, and atg11Δ mutant cells. Cells from panel A were transformed with a second plasmid that expresses β-gal from a UPR promoter. UPR was determined and expressed as % of the WT response.


D - E . Unlike deletion of Atg11 , deletion of other known selective Atgs required for the CVT pathway ( Atg19 ) , mitophagy ( Atg32 ) and pexophagy ( Atg36 ) , does not result in increase of GFP - Snc1 - PEM protein level (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D-E. Unlike deletion of Atg11, deletion of other known selective Atgs required for the CVT pathway (Atg19), mitophagy (Atg32) and pexophagy (Atg36), does not result in increase of GFP-Snc1-PEM protein level (D


D - E . Unlike deletion of Atg11 , deletion of other known selective Atgs required for the CVT pathway ( Atg19 ) , mitophagy ( Atg32 ) and pexophagy ( Atg36 ) , does not result in increase of GFP - Snc1 - PEM protein level ( D ) , intra - cellular accumulation of GFP - Snc1 - PEM , ( E ) ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D-E. Unlike deletion of Atg11, deletion of other known selective Atgs required for the CVT pathway (Atg19), mitophagy (Atg32) and pexophagy (Atg36), does not result in increase of GFP-Snc1-PEM protein level (D), intra-cellular accumulation of GFP-Snc1-PEM, (E),


D - E . Unlike deletion of Atg11 , deletion of other known selective Atgs required for the CVT pathway ( Atg19 ) , mitophagy ( Atg32 ) and pexophagy ( Atg36 ) , does not result in increase of GFP - Snc1 - PEM protein level ( D ) , intra - cellular accumulation of GFP - Snc1 - PEM , ( E ) , and induction of the UPR response ( F ) . Wild type ( WT ) , atg19Δ , atg11Δ , atg32Δ , and atg36Δ mutant cells overexpressing GFP - Snc1 - PEM were analyzed as described for panels A - C , respectively . E . Shown from left to right : DIC , GFP , and % cells with intracellular Snc1 - PEM structures . + / - and error bars represent STDEV . Results in this figure represent at least two independent experiments .

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D-E. Unlike deletion of Atg11, deletion of other known selective Atgs required for the CVT pathway (Atg19), mitophagy (Atg32) and pexophagy (Atg36), does not result in increase of GFP-Snc1-PEM protein level (D), intra-cellular accumulation of GFP-Snc1-PEM, (E), and induction of the UPR response (F). Wild type (WT), atg19Δ, atg11Δ, atg32Δ, and atg36Δ mutant cells overexpressing GFP-Snc1-PEM were analyzed as described for panels A-C, respectively. E. Shown from left to right: DIC, GFP, and % cells with intracellular Snc1-PEM structures. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.


A - C . While the level of overexpressed GFP - Snc1 - PEM is increased in atg9 ∆ and atg2 ∆ , but not atg18 ∆ , mutant cells , it does not accumulate in aberrant structures and does not induce UPR . Wild type ( WT ) , atg9 ∆ , atg18 ∆ , and atg2 ∆ mutant cells overexpressing GFP - Snc1 - PEM were analyzed as described for Fig 1A - 1C , respectively . The tested phenotypes : the level of GFP - Snc1 - PEM protein ( A ) ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-C. While the level of overexpressed GFP-Snc1-PEM is increased in atg9∆ and atg2∆, but not atg18∆, mutant cells, it does not accumulate in aberrant structures and does not induce UPR. Wild type (WT), atg9∆, atg18∆, and atg2∆ mutant cells overexpressing GFP-Snc1-PEM were analyzed as described for Fig 1A-1C, respectively. The tested phenotypes: the level of GFP-Snc1-PEM protein (A),


A - C . While the level of overexpressed GFP - Snc1 - PEM is increased in atg9 ∆ and atg2 ∆ , but not atg18 ∆ , mutant cells , it does not accumulate in aberrant structures and does not induce UPR . Wild type ( WT ) , atg9 ∆ , atg18 ∆ , and atg2 ∆ mutant cells overexpressing GFP - Snc1 - PEM were analyzed as described for Fig 1A - 1C , respectively . The tested phenotypes : the level of GFP - Snc1 - PEM protein ( A ) , accumulation of GFP - Snc1 - PEM in aberrant structures ( B )

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A-C. While the level of overexpressed GFP-Snc1-PEM is increased in atg9∆ and atg2∆, but not atg18∆, mutant cells, it does not accumulate in aberrant structures and does not induce UPR. Wild type (WT), atg9∆, atg18∆, and atg2∆ mutant cells overexpressing GFP-Snc1-PEM were analyzed as described for Fig 1A-1C, respectively. The tested phenotypes: the level of GFP-Snc1-PEM protein (A), accumulation of GFP-Snc1-PEM in aberrant structures (B)


A - C . While the level of overexpressed GFP - Snc1 - PEM is increased in atg9 ∆ and atg2 ∆ , but not atg18 ∆ , mutant cells , it does not accumulate in aberrant structures and does not induce UPR . Wild type ( WT ) , atg9 ∆ , atg18 ∆ , and atg2 ∆ mutant cells overexpressing GFP - Snc1 - PEM were analyzed as described for Fig 1A - 1C , respectively . The tested phenotypes : the level of GFP - Snc1 - PEM protein ( A ) , accumulation of GFP - Snc1 - PEM in aberrant structures ( B ) , and induction of the UPR response ( C , atg1 ∆ is shown as a positive control ) .

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A-C. While the level of overexpressed GFP-Snc1-PEM is increased in atg9∆ and atg2∆, but not atg18∆, mutant cells, it does not accumulate in aberrant structures and does not induce UPR. Wild type (WT), atg9∆, atg18∆, and atg2∆ mutant cells overexpressing GFP-Snc1-PEM were analyzed as described for Fig 1A-1C, respectively. The tested phenotypes: the level of GFP-Snc1-PEM protein (A), accumulation of GFP-Snc1-PEM in aberrant structures (B), and induction of the UPR response (C,atg1∆ is shown as a positive control).


D - G . Atg9 is epistatic to Atg11 in macro - ER - phagy . ATG9 was deleted in wild type and atg11 ∆ mutant cells and the effects of overexpression of GFP - Snc1 - PEM were determined in the single and double mutants as described in Fig 1 legend . D . Deletion of ATG9 in wild type ( WT ) or atg11 ∆ mutant cells results in an increase of GFP - Snc1 - PEM protein level similar to the increase in atg11 ∆ mutant cells .

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D-G. Atg9 is epistatic to Atg11 in macro-ER-phagy. ATG9 was deleted in wild type and atg11∆ mutant cells and the effects of overexpression of GFP-Snc1-PEM were determined in the single and double mutants as described in Fig 1 legend. D. Deletion of ATG9 in wild type (WT) or atg11∆ mutant cells results in an increase of GFP-Snc1-PEM protein level similar to the increase in atg11∆ mutant cells.


E . Deletion of ATG9 in wild type or atg11 ∆ mutant cells results in an increase of intracellular GFP - Snc1 - PEM fluorescence . However , only ~ 20 % of the atg9 ∆ single - , and atg9 ∆ atg11 ∆ double - mutant cells accumulate GFP - Snc1 - PEM in aberrant structures , as compared with ~ 75 % of atg11 ∆ mutant cells . Shown from top to bottom : DIC , GFP , % cells with aberrant intracellular GFP - Snc1 - PEM structures , ratio of GFP - Snc1 - PEM fluorescence inside / PM ( 30 cells were analyzed for each strain ) .

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E. Deletion of ATG9 in wild type or atg11∆ mutant cells results in an increase of intracellular GFP-Snc1-PEM fluorescence. However, only ~20% of the atg9∆ single-, and atg9∆ atg11∆ double-mutant cells accumulate GFP-Snc1-PEM in aberrant structures, as compared with ~75% of atg11∆ mutant cells. Shown from top to bottom: DIC, GFP, % cells with aberrant intracellular GFP-Snc1-PEM structures, ratio of GFP-Snc1-PEM fluorescence inside/PM (30 cells were analyzed for each strain).


F . UPR is induced in atg11 ∆ , but not in atg9 ∆ single - and atg9 ∆ atg11 ∆ double - mutant cells overexpressing GFP - Snc1 - PEM .

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F. UPR is induced in atg11∆, but not in atg9∆ single- and atg9∆ atg11∆ double-mutant cells overexpressing GFP-Snc1-PEM.


G . UPR can be induced in atg9 ∆ single - , and atg9 atg11 ∆ double - mutant cells overexpressing GFP - Snc1 - PEM by tunicamycin . + / - and error bars represent STDEV . Results in this figure represent at least two independent experiments .

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G. UPR can be induced in atg9∆ single-, and atg9 atg11∆ double-mutant cells overexpressing GFP-Snc1-PEM by tunicamycin. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.


A - C . ATG9 was deleted in ypt1 - 1 , WT and sec12ts mutant cells and the effect of overexpression of GFP - Snc1 - PEM was determined in single and double mutant cells . Experiments were performed as described for Fig 1A - 1C , respectively . A . Snc1 - PEM is increased ~ 3 fold in atg9 ∆ and sec12ts as compared to ~ 20 fold in ypt1 - 1 mutant cells . Importantly , atg9 ∆ is epstatic to the ypt1 - 1 , but not to the sec12ts , mutation . Left , immuno - blot analysis , increase of the protein level in mutant versus the WT cells is shown under the blot ; right , a bar graph summarizing the quantified

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-C.ATG9 was deleted in ypt1-1, WT and sec12ts mutant cells and the effect of overexpression of GFP-Snc1-PEM was determined in single and double mutant cells. Experiments were performed as described for Fig 1A-1C, respectively. A. Snc1-PEM is increased ~3 fold in atg9∆ and sec12ts as compared to ~20 fold in ypt1-1 mutant cells. Importantly, atg9∆ is epstatic to the ypt1-1, but not to the sec12ts, mutation. Left, immuno-blot analysis, increase of the protein level in mutant versus the WT cells is shown under the blot; right, a bar graph summarizing the quantified data


. Whereas deletion of ATG9 in ypt1 - 1 mutant cells results in three fold lower accumulation of GFP - Snc1 - PEM in aberrant structures ( 85 % to 27 % ) , its deletion in sec12ts mutant cells results in two fold increased accumulation ( ~ 40 % to ~ 80 % ) . Shown from top to bottom : DIC , GFP , and % cells with intracellular Snc1 - PEM structures .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

. Whereas deletion of ATG9 in ypt1-1 mutant cells results in three fold lower accumulation of GFP-Snc1-PEM in aberrant structures (85% to 27%), its deletion in sec12ts mutant cells results in two fold increased accumulation (~40% to ~80%). Shown from top to bottom: DIC, GFP, and % cells with intracellular Snc1-PEM structures.


C . Deletion of ATG9 in ypt1 - 1 mutant cells overexpressing Snc1 - PEM results in ~ 3 . 5 lower UPR ( p - value < 0 . 0005 ) , but a slightly increased UPR in sec12ts mutant cells ( p - value = 0 . 05 ) .

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C. Deletion of ATG9 in ypt1-1 mutant cells overexpressing Snc1-PEM results in ~3.5 lower UPR (p-value<0.0005), but a slightly increased UPR in sec12ts mutant cells (p-value = 0.05).


D . Atg9 is present on aberrant ER structures that accumulate in ypt1 - 1 mutant cells . WT and ypt1 - 1 mutant cells expressing Atg9 - mCherry from the chromosome and GFP - Snc1 - PEM from a 2μ plasmid were analyzed by live - cell microscopy . Whereas in WT cells ( top ) , the GFP - Snc1 - PEM localizes to the cell membrane and Atg9 - mCherry to intracellular puncta , the two proteins co - localize in 100 % of the ypt1 - 1 mutant cells ( bottom ) that accumulate intracellular GFP - Snc1 - PEM structures ( ~ 80 % ) . Shown from left to right : DIC , GFP , mCherry , merge , % cells with intracellular Snc1 - PEM , and % cells with co - localization ( number of cells with observed phenotype / total number of cells analyzed ) .

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D. Atg9 is present on aberrant ER structures that accumulate in ypt1-1 mutant cells. WT and ypt1-1 mutant cells expressing Atg9-mCherry from the chromosome and GFP-Snc1-PEM from a 2μ plasmid were analyzed by live-cell microscopy. Whereas in WT cells (top), the GFP-Snc1-PEM localizes to the cell membrane and Atg9-mCherry to intracellular puncta, the two proteins co-localize in 100% of the ypt1-1 mutant cells (bottom) that accumulate intracellular GFP-Snc1-PEM structures (~80%). Shown from left to right: DIC, GFP, mCherry, merge, % cells with intracellular Snc1-PEM, and % cells with co-localization (number of cells with observed phenotype / total number of cells analyzed).


E . GFP - Snc1 - PEM is delivered to the vacuole for degradation in sec12ts mutant cells . Accumulation of GFP - Snc1 - PEM in vacuoles of sec12ts mutant cells , with and without deletion of the vacuolar protease Pep4 , was determined using the FM4 - 64 dye , which labels the vacuolar membrane . Deletion of PEP4 in sec12ts mutant cells results in an increase percent of cells in which GFP - Snc1 - PEM accumulates inside the vacuole ( from 8 % to 100 % ) . Shown from left to right : DIC , GFP , FM4 - 64 ( vacuolar membrane ) , merge , % cells with aberrant GFP - Snc1 - PEM structures , % cells with GFP - Snc1 - PEM outside vacuole , and % cells with GFP - Snc1 - PEM inside the vacuole . + / - and error bars represent STDEV . Results in this figure represent at least two independent experiments .

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E. GFP-Snc1-PEM is delivered to the vacuole for degradation in sec12ts mutant cells. Accumulation of GFP-Snc1-PEM in vacuoles of sec12ts mutant cells, with and without deletion of the vacuolar protease Pep4, was determined using the FM4-64 dye, which labels the vacuolar membrane. Deletion of PEP4 in sec12ts mutant cells results in an increase percent of cells in which GFP-Snc1-PEM accumulates inside the vacuole (from 8% to 100%). Shown from left to right: DIC, GFP, FM4-64 (vacuolar membrane), merge, % cells with aberrant GFP-Snc1-PEM structures, % cells with GFP-Snc1-PEM outside vacuole, and % cells with GFP-Snc1-PEM inside the vacuole. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.


A . The ypt1 - 1 mutation is epistatic to vps21 ∆ in ER - phagy . yDsRed - Snc1 - PEM was overexpressed in WT , vps21 ∆ , ypt1 - 1 and ypt1 - 1vps21 ∆ double mutant cells that also expressed the autophagosomal marker yEGFP - Atg8 . Cells were analyzed by live - cell microscopy . Shown from left to right : DIC , DsRed , GFP , merge , % cells Atg8 dots , number of Atg8 dots per cell , and % cells in which the Atg8 dots co - localize with Snc1 - PEM . About 50 % of WT and 85 % of vps21 ∆ mutant cells contain ~ 1 dot of Atg8 representing the AP . Importantly , in ~ 70 % of the vps21 ∆ mutant cells Snc1 - PEM co - localizes with the APs , as compared to ~ 4 % in WT cells . In contrast , ~ 90 % ypt1 - 1 and ypt1 - 1vps21 ∆ mutant cells contain three APs per cell , and Snc1 - PEM does not co - localize with them . Arrows point to co - localization ; arrowheads point to either Atg8 dots or GFP - Snc1 - PEM that do not co - localize .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. The ypt1-1 mutation is epistatic to vps21∆ in ER-phagy. yDsRed-Snc1-PEM was overexpressed in WT, vps21∆, ypt1-1 and ypt1-1vps21∆ double mutant cells that also expressed the autophagosomal marker yEGFP-Atg8. Cells were analyzed by live-cell microscopy. Shown from left to right: DIC, DsRed, GFP, merge, % cells Atg8 dots, number of Atg8 dots per cell, and % cells in which the Atg8 dots co-localize with Snc1-PEM. About 50% of WT and 85% of vps21∆ mutant cells contain ~1 dot of Atg8 representing the AP. Importantly, in ~70% of the vps21∆ mutant cells Snc1-PEM co-localizes with the APs, as compared to ~4% in WT cells. In contrast, ~90% ypt1-1 and ypt1-1vps21∆ mutant cells contain three APs per cell, and Snc1-PEM does not co-localize with them. Arrows point to co-localization; arrowheads point to either Atg8 dots or GFP-Snc1-PEM that do not co-localize.


The following effects of overexpression of GFP - Snc1 - PEM in WT and ypt1 - 1 mutant cells , without and with ire1Δ or hac1Δ , were analyzed as described for Fig 1A - 1C , respectively : protein level ( B ) , accumulation of GFP - Snc1 - PEM in aberrant structures ( C ) , and UPR induction ( D ) . B . Deletion of either Ire1 or Hac1 does not affect the level of Snc1 - PEM accumulation in WT or ypt1 - 1 mutant cells .

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The following effects of overexpression of GFP-Snc1-PEM in WT and ypt1-1 mutant cells, without and with ire1Δ or hac1Δ, were analyzed as described for Fig 1A-1C, respectively: protein level (B), accumulation of GFP-Snc1-PEM in aberrant structures (C), and UPR induction (D). B. Deletion of either Ire1 or Hac1 does not affect the level of Snc1-PEM accumulation in WT or ypt1-1 mutant cells.


C . Deletion of either Ire1 or Hac1 does not affect the percent of WT and ypt1 - 1 mutant cells that accumulate aberrant intra - cellular Snc1 - PEM . Shown from top to bottom : DIC , GFP , and % cells with intracellular Snc1 - PEM structures .

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C. Deletion of either Ire1 or Hac1 does not affect the percent of WT and ypt1-1 mutant cells that accumulate aberrant intra-cellular Snc1-PEM. Shown from top to bottom: DIC, GFP, and % cells with intracellular Snc1-PEM structures.


D . Deletion of either Ire1 or Hac1 obliterate UPR in both WT and ypt1 - 1 mutant cells . + / - and error bars represent STDEV . Results in this figure represent at least two independent experiments .

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D. Deletion of either Ire1 or Hac1 obliterate UPR in both WT and ypt1-1 mutant cells. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.


A - C . Overexpressed Snq2 - GFP accumulates in the ER and induces UPR in ypt1 - 1 mutant cells . Snq2 - yEGFP was overexpressed in WT and ypt1 - 1 mutant cells and the following effects were analyzed as described for Fig 1A - 1C , respectively : increase in the level of Snq2 - yEGFP protein ( A )

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A-C. Overexpressed Snq2-GFP accumulates in the ER and induces UPR in ypt1-1 mutant cells. Snq2-yEGFP was overexpressed in WT and ypt1-1 mutant cells and the following effects were analyzed as described for Fig 1A-1C, respectively: increase in the level of Snq2-yEGFP protein (A)


A - C . Overexpressed Snq2 - GFP accumulates in the ER and induces UPR in ypt1 - 1 mutant cells . Snq2 - yEGFP was overexpressed in WT and ypt1 - 1 mutant cells and the following effects were analyzed as described for Fig 1A - 1C , respectively : increase in the level of Snq2 - yEGFP protein ( A ) , accumulation of Snq2 - yEGFP in aberrant ER structures ( B ) ,

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A-C. Overexpressed Snq2-GFP accumulates in the ER and induces UPR in ypt1-1 mutant cells. Snq2-yEGFP was overexpressed in WT and ypt1-1 mutant cells and the following effects were analyzed as described for Fig 1A-1C, respectively: increase in the level of Snq2-yEGFP protein (A), accumulation of Snq2-yEGFP in aberrant ER structures (B),


A - C . Overexpressed Snq2 - GFP accumulates in the ER and induces UPR in ypt1 - 1 mutant cells . Snq2 - yEGFP was overexpressed in WT and ypt1 - 1 mutant cells and the following effects were analyzed as described for Fig 1A - 1C , respectively : increase in the level of Snq2 - yEGFP protein ( A ) , accumulation of Snq2 - yEGFP in aberrant ER structures ( B ) , and induction of the UPR response ( C , GFP - Snc1 - PEM is shown for comparison ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-C. Overexpressed Snq2-GFP accumulates in the ER and induces UPR in ypt1-1 mutant cells. Snq2-yEGFP was overexpressed in WT and ypt1-1 mutant cells and the following effects were analyzed as described for Fig 1A-1C, respectively: increase in the level of Snq2-yEGFP protein (A), accumulation of Snq2-yEGFP in aberrant ER structures (B), and induction of the UPR response (C, GFP-Snc1-PEM is shown for comparison).


D - F . Synergistic effect of co - overexpression of Snq2 - GFP with DsRed - Snc1 - PEM in atg11 ∆ mutant cells . WT ( left ) and atg11 ∆ mutant cells ( right ) were transformed with plasmids for overexpression of Snq2 , Snc1 - PEM or both Snq2 and Snc1 - PEM . D . Immunoblot analysis and quantification . Shown from top to bottom : Snq2 - yEGFP ( using anti - GFP antibodies ) , Ds - Red - Snc1 - PEM ( using anti - Snc1 antibodies ) , G6PDH ( loading control ) , and a bar graph showing fold increase of Snq2 - yEGFP ( green ) and Ds - Red - Snc1 - PEM ( red ) in atg11 ∆ mutant cells over WT .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D-F. Synergistic effect of co-overexpression of Snq2-GFP with DsRed-Snc1-PEM in atg11∆ mutant cells. WT (left) and atg11∆ mutant cells (right) were transformed with plasmids for overexpression of Snq2, Snc1-PEM or both Snq2 and Snc1-PEM. D. Immunoblot analysis and quantification. Shown from top to bottom: Snq2-yEGFP (using anti-GFP antibodies), Ds-Red-Snc1-PEM (using anti-Snc1 antibodies), G6PDH (loading control), and a bar graph showing fold increase of Snq2-yEGFP (green) and Ds-Red-Snc1-PEM (red) in atg11∆ mutant cells over WT.


E . Accumulation of macro - ER - phagy cargos in aberrant intracellular structures using live - cells microscopy . Shown from left to right : DIC , GFP , DsRed and Merge . Shown from top to bottom : Snq2 , Snc1 - PEM , Snq2 + Snc1 - PEM , and % cells with co - localization ( relevant only for co - overexpression ) ( % cells with ER - phagy cargo accumulation in aberrant intracellular structures is shown in S5A Fig ) . F . Fluorescence level of intracellular structures ( ratio over PM ) in atg11 ∆ mutant cells . The bar graph shows Snq2 - yEGFP ( green ) and Ds - Red - Snc1 - PEM ( red ) fluorescence ( 20 cells were analyzed for each strain ) . When co - overexpressed in atg11 ∆ mutant cells , the fluorescence level of either protein accumulating in aberrant structures is ~ 5 fold higher than when overexpressed individually . + / - and error bars represent STDEV . Results in this figure represent at least two independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Accumulation of macro-ER-phagy cargos in aberrant intracellular structures using live-cells microscopy. Shown from left to right: DIC, GFP, DsRed and Merge. Shown from top to bottom: Snq2, Snc1-PEM, Snq2+Snc1-PEM, and % cells with co-localization (relevant only for co-overexpression) (% cells with ER-phagy cargo accumulation in aberrant intracellular structures is shown in S5A Fig). F. Fluorescence level of intracellular structures (ratio over PM) in atg11∆ mutant cells. The bar graph shows Snq2-yEGFP (green) and Ds-Red-Snc1-PEM (red) fluorescence (20 cells were analyzed for each strain). When co-overexpressed in atg11∆ mutant cells, the fluorescence level of either protein accumulating in aberrant structures is ~5 fold higher than when overexpressed individually. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.


A . A diagram showing two groups of ER - resident proteins : those that become macro - ER - phagy cargos ( in green ) , and those that do not ( in blue or grey ) . Like overexpressed Snc1 - PEM or Snq2 , the ER - resident membrane proteins Sec61 ( translocon subunit ) and Hmg1 ( sterol biogenesis ) are transported to the vacuole via macro - ER - phagy . In contrast , the ER - to - Golgi exit regulators Sec12 and Sec13 and the ER lumen chaperone Kar2 are not co - transported to the lysosome through macro - ER - phagy . Four or six different strains were used for this analysis : WT ( YPT1 ) , pep4 ∆ , and pep4 prb1 ∆ , ypt1 - 1 , ypt1 - 1pep4 ∆ , and ypt1 - 1pep4 ∆ prb1 ∆ . In each strain , one ER - resident protein was tagged at its C - terminus ( except for Kar2 ) . ( The pep4 ∆ strains were not used in all experiments because they do not show the full defect ) . The level of ER - resident proteins was determined by immunoblot analysis in cells that either do not ( B - D ) or do overexpress GFP - Snc1 - PEM ( F - H ) . B - C . Sec61 - 3xHA ( B ) and Sec13 - 3x HA ( C ) expressing cells ( 2 independent un - transformed colonies ) were tested by immunoblot analysis ( using anti - HA antibodies ) . Shown from top to bottom : strain genotype , HA - tagged protein , G6PDH ( loading control ) , quantification of HA - tagged protein expressed as average fold of WT ( p - value = 0 . 025 for Sec61 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. A diagram showing two groups of ER-resident proteins: those that become macro-ER-phagy cargos (in green), and those that do not (in blue or grey). Like overexpressed Snc1-PEM or Snq2, the ER-resident membrane proteins Sec61 (translocon subunit) and Hmg1 (sterol biogenesis) are transported to the vacuole via macro-ER-phagy. In contrast, the ER-to-Golgi exit regulators Sec12 and Sec13 and the ER lumen chaperone Kar2 are not co-transported to the lysosome through macro-ER-phagy. Four or six different strains were used for this analysis: WT (YPT1), pep4∆, and pep4 prb1∆, ypt1-1, ypt1-1pep4∆, and ypt1-1pep4∆ prb1∆. In each strain, one ER-resident protein was tagged at its C-terminus (except for Kar2). (The pep4∆ strains were not used in all experiments because they do not show the full defect). The level of ER-resident proteins was determined by immunoblot analysis in cells that either do not (B-D) or do overexpress GFP-Snc1-PEM (F-H). B-C. Sec61-3xHA (B) and Sec13-3x HA (C) expressing cells (2 independent un-transformed colonies) were tested by immunoblot analysis (using anti-HA antibodies). Shown from top to bottom: strain genotype, HA-tagged protein, G6PDH (loading control), quantification of HA-tagged protein expressed as average fold of WT (p-value = 0.025 for Sec61).


D . Summary of immunoblot analyses of ER - resident proteins level in cells that do not overexpress GFP - Snc1 - PEM : WT ( PEP4PRB1 ) vs pep4 ∆ prb1 ∆ ( left ) , ypt1 - 1PEP4PRB1 vs ypt1 - 1pep4 ∆ prb1 ∆ ( right ) . Immunoblots and quantification are shown in Fig 6B - 6C and S6A - S6C Fig . In YPT1 cells , the levels of Sec61 and Hmg1 ( green ) are slightly increased ( 14 and 38 % , respectively ) in strains defective in vacuolar proteolysis ; the levels of Sec12 , Sec13 and Kar2 are not increased ( blue and grey ) . The levels of Sec61 and Hmg1 ( green ) are increased by 2 - 2 . 5 fold in ypt1 - 1 mutant cells when compared to WT cells , regardless if they are defective in vacuolar proteolysis or not . In contrast , the levels of Sec12 , Sec13 and Kar2 are only slightly increased ( blue and grey ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Summary of immunoblot analyses of ER-resident proteins level in cells that do not overexpress GFP-Snc1-PEM: WT (PEP4PRB1) vs pep4∆ prb1∆ (left), ypt1-1PEP4PRB1 vs ypt1-1pep4∆ prb1∆ (right). Immunoblots and quantification are shown in Fig 6B-6C and S6A-S6C Fig. In YPT1 cells, the levels of Sec61 and Hmg1 (green) are slightly increased (14 and 38%, respectively) in strains defective in vacuolar proteolysis; the levels of Sec12, Sec13 and Kar2 are not increased (blue and grey). The levels of Sec61 and Hmg1 (green) are increased by 2-2.5 fold in ypt1-1 mutant cells when compared to WT cells, regardless if they are defective in vacuolar proteolysis or not. In contrast, the levels of Sec12, Sec13 and Kar2 are only slightly increased (blue and grey).


E . The level of the ER - resident protein Sec61 is similar whether GFP - Snc1 - PEM is overexpressed or not . Wild - type cells were transformed with a 2μ plasmid , either empty or for overexpression of GFP - Snc1 - GEM ( 2 independent transformants ) . Shown from top to bottom : plasmid , Sec61 , G6PDH ( loading control ) , GFP - Snc1 - PEM , and quantification of Sec61 expressed as average fold of WT with empty plasmid .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. The level of the ER-resident protein Sec61 is similar whether GFP-Snc1-PEM is overexpressed or not. Wild-type cells were transformed with a 2μ plasmid, either empty or for overexpression of GFP-Snc1-GEM (2 independent transformants). Shown from top to bottom: plasmid, Sec61, G6PDH (loading control), GFP-Snc1-PEM, and quantification of Sec61 expressed as average fold of WT with empty plasmid.


F - G . Sec61 - 3xHA ( F ) and Sec13 - 3xHA ( G ) expressing cells were transformed with a 2μ plasmid for overexpression of GFP - Snc1 - PEM . Cell lysates were tested by immunoblot analysis ( using ant - HA and anti - GFP antibodies ) . Shown from top to bottom : strain genotype , the specific ER - resident protein tested , quantification of the ER - resident protein bands expressed as average fold of WT , GFP - Snc1 - PEM , quantification of the GFP - Snc1 - PEM bands expressed as average fold of WT , and G6PDH ( loading control ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F-G. Sec61-3xHA (F) and Sec13-3xHA (G) expressing cells were transformed with a 2μ plasmid for overexpression of GFP-Snc1-PEM. Cell lysates were tested by immunoblot analysis (using ant-HA and anti-GFP antibodies). Shown from top to bottom: strain genotype, the specific ER-resident protein tested, quantification of the ER-resident protein bands expressed as average fold of WT, GFP-Snc1-PEM, quantification of the GFP-Snc1-PEM bands expressed as average fold of WT, and G6PDH (loading control).


H . Summary of immunoblot analysis of ER - resident proteins level in cells overexpressing GFP - Snc1 - PEM : WT ( PEP4PRB1 ) vs pep4 ∆ prb1 ∆ ( left ) , ypt1 - 1PEP4PRB1 vs ypt1 - 1pep4 ∆ prb1 ∆ ( right ) . Immunoblots and quantification are shown in Fig 6F and 6G and S6F - S6H Fig . In YPT1 cells , like Snc1 - PEM ( black ) , the levels of Hmg1 and Sec61 ( green ) are increased > 10 fold in strains defective in vacuolar proteolysis . In contrast , the levels of Sec12 , Sec13 and Kar2 are not changed ( blue and grey ) . Like Snc1 - PEM ( black ) , the protein levels of Hmg1 and Sec61 ( green ) are increased ~ 15 fold in ypt1 - 1 mutant cells when compared to WT cells , regardless if they are defective in vacuolar proteolysis or not . In contrast , the levels of Sec12 , Sec13 and Kar2 are only slightly increased ( blue and grey ) . + / - and error bars represent STDEV . Results in this figure represent at least two independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Summary of immunoblot analysis of ER-resident proteins level in cells overexpressing GFP-Snc1-PEM: WT (PEP4PRB1) vs pep4∆ prb1∆ (left), ypt1-1PEP4PRB1 vs ypt1-1pep4∆ prb1∆ (right). Immunoblots and quantification are shown in Fig 6F and 6G and S6F-S6H Fig. In YPT1 cells, like Snc1-PEM (black), the levels of Hmg1 and Sec61 (green) are increased >10 fold in strains defective in vacuolar proteolysis. In contrast, the levels of Sec12, Sec13 and Kar2 are not changed (blue and grey). Like Snc1-PEM (black), the protein levels of Hmg1 and Sec61 (green) are increased ~15 fold in ypt1-1 mutant cells when compared to WT cells, regardless if they are defective in vacuolar proteolysis or not. In contrast, the levels of Sec12, Sec13 and Kar2 are only slightly increased (blue and grey). +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.


A . In cells defective in vacuolar proteolysis ( pep4 ∆ prb1 ∆ ) , Hmg1 co - localizes with 70 % of intra - cellular Snc1 - PEM , but Sec13 does not ( at least 36 cells were analyzed for each strain ) . No co - localization was observed for resident ER proteins and overexpressed Snc1 - PEM in WT cells ( PEP4PRB1 ) . Hmg1 ( left ) and Sec13 ( right ) were tagged with mCherry at their C - terminus in PEP4PRB1 ( WT ) and pepb4 ∆ prb1 ∆ mutant cells . Cells were transformed with a 2μ plasmid for overexpression of GFP - Snc1 - PEM and analyzed by live - cell microscopy . Shown from top to bottom : DIC , GFP , mCherry , merge ( yellow ) , % cells with intra - cellular Snc1 - PEM , and % co - localization of the ER protein with intra - cellular Snc1 - PEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. In cells defective in vacuolar proteolysis (pep4∆ prb1∆), Hmg1 co-localizes with 70% of intra-cellular Snc1-PEM, but Sec13 does not (at least 36 cells were analyzed for each strain). No co-localization was observed for resident ER proteins and overexpressed Snc1-PEM in WT cells (PEP4PRB1). Hmg1 (left) and Sec13 (right) were tagged with mCherry at their C-terminus in PEP4PRB1 (WT) and pepb4∆ prb1∆ mutant cells. Cells were transformed with a 2μ plasmid for overexpression of GFP-Snc1-PEM and analyzed by live-cell microscopy. Shown from top to bottom: DIC, GFP, mCherry, merge (yellow), % cells with intra-cellular Snc1-PEM, and % co-localization of the ER protein with intra-cellular Snc1-PEM.


B . The ER - resident protein Hmg1 is delivered to the vacuole with GFP - Snc1 - PEM . The vacuole of PEP4PRB1 ( WT ) and pepb4 ∆ prb1 ∆ mutant cells expressing Hmg1 - mCherry and overexpressing GFP - Snc1 - PEM ( see Panel A , left ) was stained with CMAC - Arg ( blue ) , and the cells were analyzed by live - cell microscopy . In WT cells ( top ) , there is no intracellular Snc1 - PEM and the red - labeled ER is distinguished from the blue vacuole . In contrast , in ~ 75 % pep4 ∆ prb1 ∆ mutant cells ( bottom ) , which are defective in vacuolar proteolysis , Hmg1 ( red ) co - localizes with Snc1 - PEM ( green ) in the vacuole ( blue ) . Shown from left to right : DIC , GFP , mCherry , blue , merge ( white shows merge of the three colors ) , % cells with intracellular Snc1 - PEM , % cells in which Hmg1 with Snc1 - PEM co - localize , and % co - localization of the two proteins with the vacuolar dye ( 38 cells were analyzed for each strain ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The ER-resident protein Hmg1 is delivered to the vacuole with GFP-Snc1-PEM. The vacuole of PEP4PRB1 (WT) and pepb4∆ prb1∆ mutant cells expressing Hmg1-mCherry and overexpressing GFP-Snc1-PEM (see Panel A, left) was stained with CMAC-Arg (blue), and the cells were analyzed by live-cell microscopy. In WT cells (top), there is no intracellular Snc1-PEM and the red-labeled ER is distinguished from the blue vacuole. In contrast, in ~75% pep4∆ prb1∆ mutant cells (bottom), which are defective in vacuolar proteolysis, Hmg1 (red) co-localizes with Snc1-PEM (green) in the vacuole (blue). Shown from left to right: DIC, GFP, mCherry, blue, merge (white shows merge of the three colors), % cells with intracellular Snc1-PEM, % cells in which Hmg1 with Snc1-PEM co-localize, and % co-localization of the two proteins with the vacuolar dye (38 cells were analyzed for each strain).


C . > 80 % of ypt1 - 1 mutant cells , regardless if they are PEP4PRB1 or pep4 ∆ prb1 ∆ , accumulate aberrant GFP - Snc1 - PEM structures . Hmg1 co - localizes with > 70 % of these structures , whereas Sec13 co - localizes with only ~ 15 % of these structures ( 36 - 43 cells were analyzed for each strain ) . The experiment was done as described for Panel A .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. >80% of ypt1-1 mutant cells, regardless if they are PEP4PRB1 or pep4∆ prb1∆, accumulate aberrant GFP-Snc1-PEM structures. Hmg1 co-localizes with >70% of these structures, whereas Sec13 co-localizes with only ~15% of these structures (36-43 cells were analyzed for each strain). The experiment was done as described for Panel A.


D . Summary of microscopy analysis of ER - resident proteins co - localization with GFP - Snc1 - PEM in cells overexpressing GFP - Snc1 - PEM : WT ( PEP4PRB1 ) vs pep4Δ prb1Δ ( left ) , and ypt1 - 1PEP4PRB1 vs ypt1 - 1pep4Δ prb1Δ ( right ) . Microscopy and quantification are shown in Fig 7A and 7C and S7 Fig . In YPT1 cells , whereas > 60 % of Hmg1 and Sec61 ( green ) co - localize with Snc1 - PEM in strains defective in vacuolar proteolysis , Sec12 , Sec13 and Kar2 are not changed significantly ( blue and grey ) . Hmg1 and Sec61 ( green ) co - localize with Snc1 - PEM in > 70 % of ypt1 - 1 and > 80 % of ypt1 - 1pep4Δ prb1Δ mutant cells , whereas Sec13 and Kar2 ( blue and grey ) do so in < 15 % of the cells . The level of Sec12 co - localization with Snc1 - PEM is intermediate , ~ 38 % in ypt1 - 1 and ypt1 - 1pep4Δ prb1Δ . Error bars represent STDEV . Results in panels A and B represent at least two independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Summary of microscopy analysis of ER-resident proteins co-localization with GFP-Snc1-PEM in cells overexpressing GFP-Snc1-PEM: WT (PEP4PRB1) vs pep4Δ prb1Δ (left), and ypt1-1PEP4PRB1 vs ypt1-1pep4Δ prb1Δ (right). Microscopy and quantification are shown in Fig 7A and 7C and S7 Fig. In YPT1 cells, whereas >60% of Hmg1 and Sec61 (green) co-localize with Snc1-PEM in strains defective in vacuolar proteolysis, Sec12, Sec13 and Kar2 are not changed significantly (blue and grey). Hmg1 and Sec61 (green) co-localize with Snc1-PEM in >70% of ypt1-1 and >80% of ypt1-1pep4Δ prb1Δ mutant cells, whereas Sec13 and Kar2 (blue and grey) do so in <15% of the cells. The level of Sec12 co-localization with Snc1-PEM is intermediate, ~38% in ypt1-1 and ypt1-1pep4Δ prb1Δ. Error bars represent STDEV. Results in panels A and B represent at least two independent experiments.


C . Kaplan - Meier Survival plot showing low survival of chd7 mutants after 12 dpf ( N = 5 ) .

O O O O O O O O O O O O O O O O O O O O O O

C. Kaplan-Meier Survival plot showing low survival of chd7 mutants after 12 dpf (N=5).


E . Acetylated tubulin staining in 28 hpf controls ( left ) and mutants ( right ) showing severely affected outbranching of the trigeminal nerve ( Vth cranial nerve ) . Notably , chd7 - / - display reduced branching of the Vth cranial nerve ( arrows ) and axonal arborisation in the tectal area . Graphs showing quantitative analyses of percentage ( n = 5 ) and mean total length of peripheral projections ( n = 6 ) per zebrafish in controls and mutants ( * * * p < 0 . 001 ; * * p < 0 . 005 , Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Acetylated tubulin staining in 28 hpf controls (left) and mutants (right) showing severely affected outbranching of the trigeminal nerve (Vth cranial nerve). Notably, chd7-/- display reduced branching of the Vth cranial nerve (arrows) and axonal arborisation in the tectal area. Graphs showing quantitative analyses of percentage (n=5) and mean total length of peripheral projections (n=6) per zebrafish in controls and mutants (***p<0.001; **p<0.005, Student's t-test).


C - H Total number of GABAergic neurons ( GFP + cells ) in ( C , D ) the optic tectum ( OT ) and cerebellum ( CB ) regions of 5 dpf wild - type and chd7 mutant fish ( n = 16 ; * * * * p < 0 . 0001 ; Student ' s unpaired t - test ) , ( E , F ) the hypothalamus ( hyp ) region ( n = 10 ; * p = 0 . 0182 ; Student ' s t - test ) and ( G , H ) the telencephalon ( tel ) ( n = 7 ; * p = 0 . 0347 ; Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-H Total number of GABAergic neurons (GFP+ cells) in (C, D) the optic tectum (OT) and cerebellum (CB) regions of 5 dpf wild-type and chd7 mutant fish (n=16; ****p<0.0001; Student's unpaired t-test), (E, F) the hypothalamus (hyp) region (n=10; *p=0.0182; Student's t-test) and (G, H) the telencephalon (tel) (n=7; *p=0.0347; Student's t-test).


F - G . Treatment with the ERK signalling inhibitor U0126 ameliorated the number of GABAergic neuron ( n = 10 - 15 ; * * p < 0 . 05 ; * * * * p < 0 . 0001 , one - way ANOVA ) .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F-G. Treatment with the ERK signalling inhibitor U0126 ameliorated the number of GABAergic neuron (n=10-15; **p<0.05; ****p<0.0001, one-way ANOVA).


A . qPCR analysis of PAQR3 expression in a CHD7 - mutation positive patient compared to parental controls set ( N = 4 ; * * * * p < 0 . 0001 ; Student ' s t - test ) . Fold change was calculated according to the 2 ( − ΔΔCt ) method , using HPRT1 and RPS1 as housekeeping genes for normalization . All data were expressed as mean fold change ± SD across replicates , relative to control parents set to 1 ( dotted line ) , N is the number of experimental repeats .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. qPCR analysis of PAQR3 expression in a CHD7-mutation positive patient compared to parental controls set (N=4; ****p<0.0001; Student's t-test). Fold change was calculated according to the 2(−ΔΔCt) method, using HPRT1 and RPS1 as housekeeping genes for normalization. All data were expressed as mean fold change ± SD across replicates, relative to control parents set to 1 (dotted line), N is the number of experimental repeats.


C . ChIP - qPCR assays in LCLs showing decreased occupation of CHD7 on the PAQR3 proximal promoter in a CHD7 - mutation positive patient ( N = 3 ) compared to parental controls ( N = 6 ) ; * p < 0 . 05 ; Student ' s t - test , All data were expressed as mean fold change ± SD , N is the number of experimental repeats .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. ChIP-qPCR assays in LCLs showing decreased occupation of CHD7 on the PAQR3 proximal promoter in a CHD7-mutation positive patient (N=3) compared to parental controls (N=6); *p< 0.05; Student's t-test, All data were expressed as mean fold change ± SD, N is the number of experimental repeats.


A - B . GABAergic neuron defects in wild - type fish treated with ER / Golgi traffic inhibitor BFA ( n = 9 ; * * * * p < 0 . 0001 ; ns , not significant ; One - way ANOVA ) .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

A-B. GABAergic neuron defects in wild-type fish treated with ER/Golgi traffic inhibitor BFA (n=9; ****p<0.0001; ns, not significant; One-way ANOVA).


C - D . Overexpression of paqr3b mRNA improve the number of GABAergic neurons in 3dpf chd7 mutant fish ( n = 7 for chd7 - / - and n = 11 for chd7 - / - + paqr3b mRNA ; * * * * p < 0 . 0001 ; * * p < 0 . 05 ; One - way ANOVA ) . Of note , the rescue experiment was performed at 3 dpf given the transient nature of mRNA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-D. Overexpression of paqr3b mRNA improve the number of GABAergic neurons in 3dpf chd7 mutant fish (n=7 for chd7-/- and n=11 for chd7-/- + paqr3b mRNA; ****p<0.0001; **p<0.05; One-way ANOVA). Of note, the rescue experiment was performed at 3 dpf given the transient nature of mRNA.


B . Lifespan analyses of chd - 7 ( gk290 ) mutants ( n = 339 ; red ) compared to WT ( n = 444 ; black ) . Log - rank test was performed for statistical analyses . * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O 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. Lifespan analyses of chd-7(gk290) mutants (n=339; red) compared to WT (n=444; black). Log-rank test was performed for statistical analyses. ***p < 0.001.


D - F . Example pictures of the GABAergic nervous system cell bodies , commissures , axonal gaps and axonal breaks larger than 50 µm per worm in chd - 7 ( gk290 ) mutants expressing unc - 47p : : mCherry .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D-F. Example pictures of the GABAergic nervous system cell bodies, commissures, axonal gaps and axonal breaks larger than 50 µm per worm in chd-7(gk290) mutants expressing unc-47p::mCherry.


O . Representative images of GABAergic neurons and total number of GABAergic neurons ( GFP + cells ) in the optic tectum ( OT ) and cerebellum ( CB ) regions of wild - type , chd7 mutant and ephedrine - treated fish ( n = 9 ; * * * * p < 0 . 0001 ; One - way ANOVA ) . Treatment with ephedrine ameliorated the number of GABAergic neurons .

O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O 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-CONTROLLED_VAR O O O O O O O

O. Representative images of GABAergic neurons and total number of GABAergic neurons (GFP+ cells) in the optic tectum (OT) and cerebellum (CB) regions of wild-type, chd7 mutant and ephedrine-treated fish (n=9; ****p<0.0001; One-way ANOVA). Treatment with ephedrine ameliorated the number of GABAergic neurons.


( C ) Quantitative real time PCR analysis showing head enrichment of dEsyt ; the X - axis indicates the wild type tissues from which RNA was isolated and Y - axis indicates the dEsyt transcript level expression normalized to the loading control ( RP49 - Ribosomal protein 49 ) , n = 3 replicates ( biological ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Quantitative real time PCR analysis showing head enrichment of dEsyt; the X-axis indicates the wild type tissues from which RNA was isolated and Y-axis indicates the dEsyt transcript level expression normalized to the loading control (RP49- Ribosomal protein 49), n=3 replicates (biological).


( D ) Quantitative real time PCR analysis showing dEsyt transcript level expression in head RNA samples from wild type and sOD . Y - axis indicates the dEsyt transcript level expression normalized to the loading control ( RP49 - Ribosomal protein 49 ) , n = 3 replicates ( biological ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Quantitative real time PCR analysis showing dEsyt transcript level expression in head RNA samples from wild type and sOD. Y-axis indicates the dEsyt transcript level expression normalized to the loading control (RP49- Ribosomal protein 49), n=3 replicates (biological).


( A ) Representative ERG trace from dark reared 0 - 1 day old flies of the indicated genotype stimulated by 2s flash of green light . The duration of the stimulating light is shown . Y - axis shows the amplitude of the ERG response . X - axis shows the duration of the recording .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Representative ERG trace from dark reared 0-1 day old flies of the indicated genotype stimulated by 2s flash of green light. The duration of the stimulating light is shown. Y-axis shows the amplitude of the ERG response. X-axis shows the duration of the recording.


( B ) Graph showing ERG amplitude normalized to body weight . Y - axis shows the ratio of ERG amplitude of individual flies to their body weight . X - axis indicates genotypes . Each data point represents an individual fly tested . Error bar represents s . e . m .

(B) Graph showing ERG amplitude normalized to body weight. Y-axis shows the ratio of ERG amplitude of individual flies to their body weight. X-axis indicates genotypes. Each data point represents an individual fly tested. Error bar represents s.e.m.


( C ) Quantification of the mean fluorescence intensity of the deep pseudopupil formed by the PIP2 probe PH - GFP in the one day old flies of the indicated genotypes ( n = 10 ) .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

(C) Quantification of the mean fluorescence intensity of the deep pseudopupil formed by the PIP2 probe PH-GFP in the one day old flies of the indicated genotypes (n=10).


( D ) Deep pseudopupil imaging of PIP2 levels in the microvillar membrane of photoreceptors . The fluorescence of the PH - GFP probe is depicted . The protocol used is shown with red light illumination periods shown as red bars and flashes of blue light ( a - f ) used for image capture depicted . Representative deep pseudopupil images acquired at specified time points are depicted . Genotypes as indicated .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Deep pseudopupil imaging of PIP2 levels in the microvillar membrane of photoreceptors. The fluorescence of the PH-GFP probe is depicted. The protocol used is shown with red light illumination periods shown as red bars and flashes of blue light (a-f) used for image capture depicted. Representative deep pseudopupil images acquired at specified time points are depicted. Genotypes as indicated.


( E ) Graph quantifying the recovery kinetics of the fluorescent pseudopupil with time , X - axis represents the genotypes , Y - axis represent intensity normalised to the intensity of the first image . ( n = 10 biological replicates ) . Error bars represent s . e . m .

(E) Graph quantifying the recovery kinetics of the fluorescent pseudopupil with time, X-axis represents the genotypes, Y-axis represent intensity normalised to the intensity of the first image. (n=10 biological replicates). Error bars represent s.e.m.


( F ) Quantification of the time course of retinal degeneration . 10 ommatidia from 5 separate flies of each genotype were scored and plotted . Y - axis is the number of intact rhabdomeres / ommatidium . The maximum value possible is 7 . X - axis is the age of the flies post eclosion .

(F) Quantification of the time course of retinal degeneration. 10 ommatidia from 5 separate flies of each genotype were scored and plotted. Y-axis is the number of intact rhabdomeres/ommatidium. The maximum value possible is 7. X-axis is the age of the flies post eclosion.


( G ) Representative optical neutralization images showing rhabdomere integrity of the indicated genotypes . Rearing conditions and the age of the flies are indicated on top . ( * CL - Constant Light )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Representative optical neutralization images showing rhabdomere integrity of the indicated genotypes. Rearing conditions and the age of the flies are indicated on top. (*CL- Constant Light)


( H ) Representative ON images showing the rescue of rhabdomere structural integrity in dEsytKO mutants on expressing the dEsyt : : mCherry transgene . Genotypes of the flies are indicated on the left . Rearing conditions and the age of the flies are indicated on top .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Representative ON images showing the rescue of rhabdomere structural integrity in dEsytKO mutants on expressing the dEsyt::mCherry transgene. Genotypes of the flies are indicated on the left. Rearing conditions and the age of the flies are indicated on top.


( A ) ( i ) Representative optical neutralization ( ON ) images showing rhabdomere structure of the indicated genotypes . Rearing conditions and the age of the flies are indicated on top . ( * CD - Constant Dark ) . ( ii ) TEM images showing a single ommatidium from the photoreceptors of rdgB9 and rdgB9 ; dEsytKO mutants reared in 12 hr L / D cycle ( / ) for 2 days . Scale bar : 1µm ( asterisk indicates degenerated rhabdomere ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) (i) Representative optical neutralization (ON) images showing rhabdomere structure of the indicated genotypes. Rearing conditions and the age of the flies are indicated on top. (*CD- Constant Dark). (ii) TEM images showing a single ommatidium from the photoreceptors of rdgB9 and rdgB9;dEsytKO mutants reared in 12 hr L/D cycle ( / ) for 2 days. Scale bar: 1µm (asterisk indicates degenerated rhabdomere).


( B ) Quantification of the time course taken for retinal degeneration . 10 ommatidia were scored from 5 flies of each genotype and plotted .

O O 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) Quantification of the time course taken for retinal degeneration. 10 ommatidia were scored from 5 flies of each genotype and plotted.


( C ) Representative ERG traces showing the duration of light pulse , X - axis indicates time in msec and Y - axis indicates the average ERG amplitude in mV .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Representative ERG traces showing the duration of light pulse, X-axis indicates time in msec and Y-axis indicates the average ERG amplitude in mV.


( D ) Graph showing ERG amplitude of rdgB9 and rdgB9 ; dEsytKO normalized to body weight . Y - axis shows the ratio of ERG amplitude of individual flies to their body weight . X - axis indicates genotypes . Each data point represents an individual fly tested . Error bar represents s . e . m .

(D) Graph showing ERG amplitude of rdgB9 and rdgB9;dEsytKO normalized to body weight. Y-axis shows the ratio of ERG amplitude of individual flies to their body weight. X-axis indicates genotypes. Each data point represents an individual fly tested. Error bar represents s.e.m.


( E ) Deep pseudopupil imaging of PIP2 levels in the microvillar membrane of photoreceptors . The fluorescence of the PH - GFP probe is depicted . The protocol used is shown with red light illumination periods shown as red bars and flashes of blue light ( a - f ) used for image capture depicted . Representative deep pseudopupil images acquired at specified time points are depicted . Genotypes as indicated .

(E) Deep pseudopupil imaging of PIP2 levels in the microvillar membrane of photoreceptors. The fluorescence of the PH-GFP probe is depicted. The protocol used is shown with red light illumination periods shown as red bars and flashes of blue light (a-f) used for image capture depicted. Representative deep pseudopupil images acquired at specified time points are depicted. Genotypes as indicated.


( F ) Quantification of the mean fluorescence intensity of the PIP2 probe PH - GFP from the deep pseudopupil formed by one day old flies of the indicated genotypes ( n = 10 biological replicates ) .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Quantification of the mean fluorescence intensity of the PIP2 probe PH-GFP from the deep pseudopupil formed by one day old flies of the indicated genotypes (n=10 biological replicates).


( G ) Graph quantifying the recovery kinetics of the fluorescent pseudopupil with time , X - axis represents the genotypes , Y - axis represent intensity normalized to the intensity of the first image . ( n = 10 biological replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Graph quantifying the recovery kinetics of the fluorescent pseudopupil with time, X-axis represents the genotypes, Y-axis represent intensity normalized to the intensity of the first image. (n=10 biological replicates).


( A ) Confocal images showing the localization of endogenous dEsyt protein in photoreceptors of one day old dark reared flies probed with antibody against dEsyt . Scale bar : 5µm . dEsytKO shows no staining when probed with dEsyt antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Confocal images showing the localization of endogenous dEsyt protein in photoreceptors of one day old dark reared flies probed with antibody against dEsyt. Scale bar: 5µm. dEsytKO shows no staining when probed with dEsyt antibody.


( B ) Confocal images showing the localization of exogenously expressed dEsyt : : mCherry protein expressed using the eye specific Rh1 - Gal4 in one day old dark reared flies . Rh1 - Gal4 is shown as a control . A single ommatidium is shown . Scale bar : 5 µm . Phalloidin marks F - actin staining and highlights rhabdomeres R1 - R7 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal images showing the localization of exogenously expressed dEsyt::mCherry protein expressed using the eye specific Rh1-Gal4 in one day old dark reared flies. Rh1-Gal4 is shown as a control. A single ommatidium is shown. Scale bar: 5 µm. Phalloidin marks F-actin staining and highlights rhabdomeres R1-R7.


( C ) Western blot from the head extracts of wild type and dEsytKO probed with the antibody against RDGB . Rearing conditions , age of the flies and genotype is indicated on top of the blot . Tubulin was used as the loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Western blot from the head extracts of wild type and dEsytKO probed with the antibody against RDGB. Rearing conditions, age of the flies and genotype is indicated on top of the blot. Tubulin was used as the loading control.


( D , E ) Confocal images showing the localization of RDGB in wild type and dEsytKO photoreceptors of flies which are ( D ) 1 day old - dark reared and ( E ) 6 days old - exposed to constant illumination . For ( D ) and ( E ) RDGB visualized using an antibody against the endogenous protein . Rhabdomeres are outlined using phalloidin which marks F - actin . Scale bar : 5 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D, E) Confocal images showing the localization of RDGB in wild type and dEsytKO photoreceptors of flies which are (D) 1 day old-dark reared and (E) 6 days old- exposed to constant illumination. For (D) and (E) RDGB visualized using an antibody against the endogenous protein. Rhabdomeres are outlined using phalloidin which marks F-actin. Scale bar: 5 µm.


( A ) TEM images of a single ommatidium from wild type photoreceptors of flies reared in dark ( i ) day 1 ( D1 ) and ( iii ) day 14 ( D14 ) , scale bar : 1µm ( red asterisk indicates R7 photoreceptor ) . Magnified image showing a single photoreceptor from the same ommatidium ( ii ) day 1 ( iv ) day 14 . Scale bar : 200 nm . Origin boxes for magnification shown . Arrow in ( Aiv ) indicates the SMC forming an MCS with the microvillar PM . M - Microvillar plasma membrane , S - Sub microvillar cisternae , MCS - Membrane Contact Site , P - Pigment granules . ( B ) Quantification of the number of MCS per wild type photoreceptor of day 1 and day 14 old flies reared in dark ( from figure 6A ) , Y - axis indicates the number of MCS / photoreceptor . n = 30 photoreceptors from 3 separate flies ( R1 - R6 ) for ( i ) . n = 9 photoreceptors from 3 separate flies ( R7 ) in ( ii ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) TEM images of a single ommatidium from wild type photoreceptors of flies reared in dark (i) day 1 (D1) and (iii) day 14 (D14), scale bar: 1µm (red asterisk indicates R7 photoreceptor). Magnified image showing a single photoreceptor from the same ommatidium (ii) day 1 (iv) day 14. Scale bar: 200 nm. Origin boxes for magnification shown. Arrow in (Aiv) indicates the SMC forming an MCS with the microvillar PM. M- Microvillar plasma membrane, S- Sub microvillar cisternae, MCS- Membrane Contact Site, P- Pigment granules. (B) Quantification of the number of MCS per wild type photoreceptor of day 1 and day 14 old flies reared in dark (from figure 6A), Y-axis indicates the number of MCS/photoreceptor. n=30 photoreceptors from 3 separate flies (R1-R6) for (i). n=9 photoreceptors from 3 separate flies (R7) in (ii).


( C ) TEM images of a single ommatidium from norpAP24 photoreceptors of flies reared in dark ( i ) day 1 and ( iii ) day 14 , scale bar : 1µm ( red asterisk indicates R7 photoreceptor ) . Magnified image showing a single photoreceptor from the same ommatidium ( ii ) day1 ( iv ) day 14 . Scale bar : 200 nm . Arrows indicate the SMC forming an MCS with the microvillar PM . ( D ) Quantification of the number of MCSs per photoreceptor ( from figure 6C ) , X - axis indicates the genotype and age of the flies and Y - axis indicates the number of MCS / photoreceptor ( i ) n = 30 photoreceptors from 3 separate flies ( R1 - R6 ) ( ii ) n = 9 photoreceptors from 3 separate flies ( R7 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) TEM images of a single ommatidium from norpAP24 photoreceptors of flies reared in dark (i) day 1 and (iii) day 14, scale bar: 1µm (red asterisk indicates R7 photoreceptor). Magnified image showing a single photoreceptor from the same ommatidium (ii) day1 (iv) day 14. Scale bar: 200 nm. Arrows indicate the SMC forming an MCS with the microvillar PM. (D) Quantification of the number of MCSs per photoreceptor (from figure 6C), X-axis indicates the genotype and age of the flies and Y-axis indicates the number of MCS/photoreceptor (i) n=30 photoreceptors from 3 separate flies (R1-R6) (ii) n=9 photoreceptors from 3 separate flies (R7)


( E ) TEM images of a single ommatidium from ( i ) wild type and ( iii ) dEsytKO photoreceptors of 0 - 1 day old flies reared in dark , scale bar : 1µm ( red asterisk indicates R7 photoreceptor ) ( ii , iv ) Magnified image showing a single photoreceptor from the ommatidium image shown on the left . Scale bar : 200 nm . Arrows indicate the SMC forming an MCS with the microvillar PM . ( F ) Quantification of the number of MCS per photoreceptor of wild type day 1 v / s dEsytKO day 1 old flies reared in dark , X - axis indicates the genotype and age of the flies and Y - axis indicates the number of MCS / photoreceptor ( i ) n = 30 photoreceptors from 3 separate flies ( R1 - R6 ) ( ii ) n = 9 photoreceptors from 3 separate flies ( R7 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) TEM images of a single ommatidium from (i) wild type and (iii) dEsytKO photoreceptors of 0-1 day old flies reared in dark, scale bar: 1µm (red asterisk indicates R7 photoreceptor) (ii, iv) Magnified image showing a single photoreceptor from the ommatidium image shown on the left. Scale bar: 200 nm. Arrows indicate the SMC forming an MCS with the microvillar PM. (F) Quantification of the number of MCS per photoreceptor of wild type day 1 v/s dEsytKO day 1 old flies reared in dark, X-axis indicates the genotype and age of the flies and Y-axis indicates the number of MCS/photoreceptor (i) n=30 photoreceptors from 3 separate flies (R1-R6) (ii) n=9 photoreceptors from 3 separate flies (R7).


( A ) TEM images of a single ommatidium from photoreceptors of ( i ) dEsytKO - day 1 ( iii ) dEsytKO - day 14 flies reared in dark , scale bar : 1 µm except for ( Ai ) : 2 µm ( red asterisk indicates R7 photoreceptor ) . ( A - ii , iv ) Magnified image showing a single photoreceptor from the ommatidium image shown on the left . Scale bar : 200 nm . Boxes represent the origin boxes for magnification . M - Microvillar plasma membrane , S - Sub microvillar cisternae , MCS - Membrane Contact Site , P - Pigment granules . ( B ) Quantification of the number of MCS per photoreceptor , X - axis indicates the genotype and age of the flies and Y - axis indicates the number of MCS / photoreceptor . n = 9 photoreceptors from 3 separate flies for R7 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) TEM images of a single ommatidium from photoreceptors of (i) dEsytKO- day 1 (iii) dEsytKO- day 14 flies reared in dark, scale bar: 1 µm except for (Ai): 2 µm (red asterisk indicates R7 photoreceptor). (A-ii, iv) Magnified image showing a single photoreceptor from the ommatidium image shown on the left. Scale bar: 200 nm. Boxes represent the origin boxes for magnification. M- Microvillar plasma membrane, S- Sub microvillar cisternae, MCS- Membrane Contact Site, P- Pigment granules. (B) Quantification of the number of MCS per photoreceptor, X-axis indicates the genotype and age of the flies and Y-axis indicates the number of MCS/photoreceptor. n=9 photoreceptors from 3 separate flies for R7.


( C ) TEM images of a single ommatidium from photoreceptors of ( i ) norpAP24 ; dEsytKO - day 1 ( iii ) norpAP24 ; dEsytKO - day 14 flies reared in dark , scale bar : 1 µm ( red asterisk indicates R7 photoreceptor ) . ( C - ii , iv ) Magnified image showing a single photoreceptor from the ommatidium image shown on the left . Scale bar : 200 nm . Arrows indicate the SMC forming an MCS with the microvillar PM . ( D ) Quantification of the number of MCS per photoreceptor , X - axis indicates the genotype and age of the flies and Y - axis indicates the number of MCS / photoreceptor . ( i ) n = 30 photoreceptors from 3 separate flies for R1 - R6 . ( ii ) n = 9 photoreceptors from 3 separate flies for R7 .

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(C) TEM images of a single ommatidium from photoreceptors of (i) norpAP24;dEsytKO- day 1 (iii) norpAP24;dEsytKO- day 14 flies reared in dark, scale bar: 1 µm (red asterisk indicates R7 photoreceptor). (C-ii, iv) Magnified image showing a single photoreceptor from the ommatidium image shown on the left. Scale bar: 200 nm. Arrows indicate the SMC forming an MCS with the microvillar PM. (D) Quantification of the number of MCS per photoreceptor, X-axis indicates the genotype and age of the flies and Y-axis indicates the number of MCS/photoreceptor. (i) n=30 photoreceptors from 3 separate flies for R1-R6. (ii) n=9 photoreceptors from 3 separate flies for R7.


( E ) TEM images of a single ommatidium from photoreceptors of ( i ) rdgB9 - day1 and ( iii ) rdgB9 ; dEsytKO - day 1 flies reared in dark , scale bar : 1 µm ( red asterisk indicates R7 photoreceptor ) . ( E - ii , iv ) Magnified image showing a single photoreceptor from the ommatidium image shown on the left . Scale bar : 200 nm . ( F ) Quantification of the number of MCS per photoreceptor for R1 - R6 in 1 day old dark reared wild type ( from Figure 5B i ) , rdgB9 and rdgB9 ; dEsytKO flies . n = 30 photoreceptors from 3 separate flies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) TEM images of a single ommatidium from photoreceptors of (i) rdgB9-day1 and (iii) rdgB9;dEsytKO - day 1 flies reared in dark, scale bar: 1 µm (red asterisk indicates R7 photoreceptor). (E-ii, iv) Magnified image showing a single photoreceptor from the ommatidium image shown on the left. Scale bar: 200 nm. (F) Quantification of the number of MCS per photoreceptor for R1-R6 in 1 day old dark reared wild type (from Figure 5B i), rdgB9 and rdgB9;dEsytKO flies. n=30 photoreceptors from 3 separate flies.


Time - series measurements of ( B ) E . coli abundance , ( C ) B . subtilis abundance ,

O O O O O O O O O O O O O O O O O O O O O

Time-series measurements of (B) E. coli abundance, (C) B. subtilis abundance,


( H ) Bar plot of measured extracellular pBB275 plasmid concentration at 0 hr and 6 hr in the co - culture composed of E . coli MG1655 - rfp and B . subtilis .

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(H) Bar plot of measured extracellular pBB275 plasmid concentration at 0 hr and 6 hr in the co-culture composed of E. coli MG1655-rfp and B. subtilis.


( B , C ) ( B ) Extracellular pBB275 plasmid concentration or ( C ) extracellular E . coli genomic DNA concentration at 0 or 6 hr in the co - cultures composed of a recA + or recA - E . coli plasmid donor and engineered B . subtilis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C) (B) Extracellular pBB275 plasmid concentration or (C) extracellular E. coli genomic DNA concentration at 0 or 6 hr in the co-cultures composed of a recA+ or recA- E. coli plasmid donor and engineered B. subtilis.


( E ) Extracellular pBB275 or pUC19 plasmid concentration in the E . coli MG1655 monoculture or or co - culture with engineered B . subtilis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Extracellular pBB275 or pUC19 plasmid concentration in the E. coli MG1655 monoculture or or co-culture with engineered B. subtilis.


( F ) HGT efficiencies of pBB275 plasmid ( with or without a f1 ori ) at 6 hr in the co - culture composed of E . coli MG1655 and engineered B . subtilis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) HGT efficiencies of pBB275 plasmid (with or without a f1 ori) at 6 hr in the co-culture composed of E. coli MG1655 and engineered B. subtilis.


( B ) E . coli and B . subtilis abundances in the co - culture in the presence of 0 , 0 . 02 , 0 . 2 or 2 mM IPTG at 6 hr .

O O O O O O O O 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-CONTROLLED_VAR O O O O

(B) E. coli and B. subtilis abundances in the co-culture in the presence of 0, 0.02, 0.2 or 2 mM IPTG at 6 hr.


Time - series measurements of extracellular plasmid or E . coli gDNA concentrations in the co - culture in the presence of ( D ) 0 . 02 mM , ( E ) 0 . 2 mM or ( F ) 2 mM IPTG .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Time-series measurements of extracellular plasmid or E. coli gDNA concentrations in the co-culture in the presence of (D) 0.02 mM, (E) 0.2 mM or (F) 2 mM IPTG.


( H ) Abundance of transformants at 6 hr in the co - culture in the presence of 0 , 0 . 02 , 0 . 2 or 2 mM IPTG .

O O O O O O 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-CONTROLLED_VAR O

(H) Abundance of transformants at 6 hr in the co-culture in the presence of 0, 0.02, 0.2 or 2 mM IPTG.


( C ) Scatter plot of the initial abundance of heat - killed E . coli MG1655 - rfp and extracellular pBB275 plasmid concentration at 0h or 6 hr in the co - culture .

(C) Scatter plot of the initial abundance of heat-killed E. coli MG1655-rfp and extracellular pBB275 plasmid concentration at 0h or 6 hr in the co-culture.


Time - series measurements of ( E ) the abundance of E . coli MG1655 - rfp , ( F ) the abundance of engineered B . subtilis Unpaired t - test was used to determine if the abundances in the presence and absence of chloramphenicol were statistically different . Stars ( * ) indicate p - value of 0 . 0237 and 0 . 0283 for 4 hr and 8 hr , respectively .

O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Time-series measurements of (E) the abundance of E. coli MG1655-rfp, (F) the abundance of engineered B. subtilis Unpaired t-test was used to determine if the abundances in the presence and absence of chloramphenicol were statistically different. Stars (*) indicate p-value of 0.0237 and 0.0283 for 4 hr and 8 hr, respectively.


Time - series measurements of ( G ) extracellular pBB275 plasmid concentration , Unpaired t - test was used to determine if the abundances in the presence and absence of

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Time-series measurements of (G) extracellular pBB275 plasmid concentration, Unpaired t-test was used to determine if the abundances in the presence and absence of chloramphenicol


( C ) Histogram of the number of E . coli plasmid replication origins ColE1 , p15A , CloDF13 or pSC101 identified in Bacillus or non - Bacillus DNA sequences with a sequence length larger than 106 bp ( indicating potential genomic integration ) . ColE1 was found in 6 Bacillus genomes and 43 non - Bacillus genomes . CloDF13 was found in 23 non - Bacillus genomes . p15A and pSC101 were not detected in this NCBI RefSeq dataset .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Histogram of the number of E. coli plasmid replication origins ColE1, p15A, CloDF13 or pSC101 identified in Bacillus or non-Bacillus DNA sequences with a sequence length larger than 106 bp (indicating potential genomic integration). ColE1 was found in 6 Bacillus genomes and 43 non-Bacillus genomes. CloDF13 was found in 23 non-Bacillus genomes. p15A and pSC101 were not detected in this NCBI RefSeq dataset.


C - E . 2a and 2b ESCs maintained in 2i medium were cultured in the absence ( Ctrl ) or presence of OHT and subsequently used for WB ( C )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-E. 2a and 2b ESCs maintained in 2i medium were cultured in the absence (Ctrl) or presence of OHT and subsequently used for WB (C)


C - E . 2a and 2b ESCs maintained in 2i medium were cultured in the absence ( Ctrl ) or presence of OHT and subsequently used for growth curves and RT - qPCR ( D , E ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-E. 2a and 2b ESCs maintained in 2i medium were cultured in the absence (Ctrl) or presence of OHT and subsequently used for growth curves and RT-qPCR (D,E).


F - G . 2a and 2b ESCs were adapted to grow in serum - containing ESC medium , exposed to OHT as indicated and subsequently used for growth curves and RT - qPCR analyses .

F-G. 2a and 2b ESCs were adapted to grow in serum-containing ESC medium, exposed to OHT as indicated and subsequently used for growth curves and RT-qPCR analyses.


H - I . Jmjd2a ( + / - ) ; Jmjd2a ( + / - ) ( H ) or Jmjd2b ( + / - ) ; Jmjd2b ( + / - ) ( I ) mice were inter - crossed and the number of pups alive at time of weaning reported . P values were calculated using a Chi - square test .

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H-I. Jmjd2a(+/-); Jmjd2a(+/-) (H) or Jmjd2b(+/-); Jmjd2b(+/-) (I) mice were inter-crossed and the number of pups alive at time of weaning reported. P values were calculated using a Chi-square test.


A - D . WB and growth curves of 2ac ( A ) , 2abc ( B ) , 2ab ( C ) and 2bc ( D ) ESCs cultured in 2i medium and exposed to OHT as indicated .

A-D. WB and growth curves of 2ac (A), 2abc (B), 2ab (C) and 2bc (D) ESCs cultured in 2i medium and exposed to OHT as indicated.


E . Flow cytometry profiles of 2ac ESCs pulsed with BrdU in 2i medium . The upper table shows the percentages of living cells ( the subG1 fraction is omitted from the analysis ) , which can be classified as being in S ( BrdU + ) , G1 ( 2N DNA content , BrdU - ) or G2 / M phase ( 4N DNA content , BrdU - ) . The percentages of dead cells ( subG1 , events with a < 2N DNA content ) is shown in red .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O

E. Flow cytometry profiles of 2ac ESCs pulsed with BrdU in 2i medium. The upper table shows the percentages of living cells (the subG1 fraction is omitted from the analysis), which can be classified as being in S (BrdU+), G1 (2N DNA content, BrdU-) or G2/M phase (4N DNA content, BrdU-). The percentages of dead cells (subG1, events with a <2N DNA content) is shown in red.


F . RT - qPCR analyses for ESCs cultured in 2i medium .

F. RT-qPCR analyses for ESCs cultured in 2i medium.


G - J . 2ac and 2abc ESCs were adapted to grow in serum - containing ESC medium , exposed to OHT as indicated and subsequently used for growth curves ( G - H ) and RT - qPCR analyses ( I - J ) . The RT - qPCR data are also presented in Appendix Fig S2C - D with a zoom - in on the lower region of the y - axis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G-J. 2ac and 2abc ESCs were adapted to grow in serum-containing ESC medium, exposed to OHT as indicated and subsequently used for growth curves (G-H) and RT-qPCR analyses (I-J). The RT-qPCR data are also presented in Appendix Fig S2C-D with a zoom-in on the lower region of the y-axis.


A - B . 2ac ESCs expressing GFP were cultured in the absence or presence of OHT and subsequently used for morula injections . ( A ) Average volume of the GFP - positive area in chimeric embryos at 24 and 72 hrs after morula injection . Number of embryos used for quantification : Ctrl 24h n = 18 ; + OHT 24h n = 15 ; Ctrl 72h n = 5 ; + OHT 72h n = 5 . Data are presented as mean ± SD . ( B ) Representative images of chimeric embryos . The white bars indicate a length of 32 µm . Relative intensities of the GFP signal are indicated for each image .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-B. 2ac ESCs expressing GFP were cultured in the absence or presence of OHT and subsequently used for morula injections. (A) Average volume of the GFP-positive area in chimeric embryos at 24 and 72 hrs after morula injection. Number of embryos used for quantification: Ctrl 24h n=18; +OHT 24h n=15; Ctrl 72h n=5; +OHT 72h n=5. Data are presented as mean ± SD. (B) Representative images of chimeric embryos. The white bars indicate a length of 32 µm. Relative intensities of the GFP signal are indicated for each image.


C - D . Jmjd2a ( + / - ) ; Jmjd2c ( + / - ) mice were inter - crossed . The number of ( C ) pups alive at weaning or ( D ) embryos recovered at E6 . 5 is presented . P values were calculated using exact binomial test with the most conservative estimate of expected ( - / - ; - / - ) embryos ( 6 . 25 % , assuming no linkage between the Jmjd2a and Jmjd2cloci even though they are located on the same chromosome ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-D. Jmjd2a(+/-); Jmjd2c(+/-) mice were inter-crossed. The number of (C) pups alive at weaning or (D) embryos recovered at E6.5 is presented. P values were calculated using exact binomial test with the most conservative estimate of expected (-/-;-/-) embryos (6.25 %, assuming no linkage between the Jmjd2a and Jmjd2cloci even though they are located on the same chromosome).


A . Examples of ChIP - seq data obtained for Jmjd2a , Jmjd2c ( Pedersen et al , 2014 ) , and H3K4me3 .

O O O O O O O O O O O O O O O O O O O O O O O O

A. Examples of ChIP-seq data obtained for Jmjd2a, Jmjd2c (Pedersen et al, 2014), and H3K4me3.


C . Unsupervised k - means clustering of ChIP - seq tags over all TSSs ( + / - 5kb ) .

O O O O O O O O O O O O O O O O O O O O O O

C. Unsupervised k-means clustering of ChIP-seq tags over all TSSs (+/- 5kb).


D - E . Bar graphs showing the number of Jmjd2a peaks displaying at least 1 bp overlap with ( D ) an H3K4me3 peak . Jmjd2a peaks were classified as TSS - associated ( ' TSS ' ) if overlapping a region of + / - 1kb of a TSS .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D-E. Bar graphs showing the number of Jmjd2a peaks displaying at least 1 bp overlap with (D) an H3K4me3 peak. Jmjd2a peaks were classified as TSS-associated ('TSS') if overlapping a region of +/- 1kb of a TSS.


D - E . Bar graphs showing the number of Jmjd2a peaks displaying at least 1 bp overlap with ( E ) a Jmjd2c peak . Jmjd2a peaks were classified as TSS - associated ( ' TSS ' ) if overlapping a region of + / - 1kb of a TSS .

D-E. Bar graphs showing the number of Jmjd2a peaks displaying at least 1 bp overlap with (E) a Jmjd2c peak. Jmjd2a peaks were classified as TSS-associated ('TSS') if overlapping a region of +/- 1kb of a TSS.


G . ChIP - qPCR validations . Graphs show mean ± SD for 3 technical replicates and are representative of results obtained in at least 2 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. ChIP-qPCR validations. Graphs show mean ± SD for 3 technical replicates and are representative of results obtained in at least 2 independent experiments.


A . Density plots showing average ChIP - seq signals with 95 % confidence intervals of the mean indicated in grey . y - axes show mean Tags per Million ( TPM ) . Data were obtained using the 2ac ESCs line # 4 . TSSs were classified as ' Bound ' if containing binding sites for both Jmjd2a and Jmjd2c within + / - 1kb ( see Fig 4F ) , or ' Not bound ' if neither protein binds within the same region .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Density plots showing average ChIP-seq signals with 95% confidence intervals of the mean indicated in grey. y-axes show mean Tags per Million (TPM). Data were obtained using the 2ac ESCs line #4. TSSs were classified as 'Bound' if containing binding sites for both Jmjd2a and Jmjd2c within +/- 1kb (see Fig 4F), or 'Not bound' if neither protein binds within the same region.


B . Heat map showing H3K9me3 ChIP - seq data for Jmjd2a / c bound TSSs ( + / - 5kb ) sorted according to read number in 2abc # 13 + OHT .

O O O O O O O O O O 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. Heat map showing H3K9me3 ChIP-seq data for Jmjd2a/c bound TSSs (+/- 5kb) sorted according to read number in 2abc #13+OHT.


C . ChIP - qPCR validations for selected Jmjd2a / c targets ( see Fig 4G ) . Graphs show mean ± SD for technical triplicates and are representative of results obtained in at least 2 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. ChIP-qPCR validations for selected Jmjd2a/c targets (see Fig 4G). Graphs show mean ± SD for technical triplicates and are representative of results obtained in at least 2 independent experiments.


A - B . Independent RT - qPCR validations of genes identified as being ( A ) down - regulated or ( B ) up - regulated by expression arrays . Graphs show mean ± SD for technical triplicates and are representative of results obtained with at least 2 different ESCs lines of each genotype in independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-B. Independent RT-qPCR validations of genes identified as being (A) down-regulated or (B) up-regulated by expression arrays. Graphs show mean ± SD for technical triplicates and are representative of results obtained with at least 2 different ESCs lines of each genotype in independent experiments.


C - D . Density plots showing average ChIP - seq signals for ( C ) H3K9me3 or ( D ) H3K36me3 with 95 % confidence intervals of the mean indicated in grey . Plots are shown for genes , which are down - or up - regulated according to the microarray analyses ( FDR < 0 . 05 , absolute fold change log2 ( | FC | ) > 0 . 5 ) for both 2ac and 2abc KO ESCs ( overlaps presented in Appendix Fig S6A ) , or do not show strong expression changes ( log2 ( | FC | ) < 0 . 2 ) . Genes are further classified as " Bound " if containing binding sites for both Jmjd2a and Jmjd2c within + / - 1kb of a TSS .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-D. Density plots showing average ChIP-seq signals for (C) H3K9me3 or (D) H3K36me3 with 95% confidence intervals of the mean indicated in grey. Plots are shown for genes, which are down- or up-regulated according to the microarray analyses (FDR<0.05, absolute fold change log2(|FC|)>0.5) for both 2ac and 2abc KO ESCs (overlaps presented in Appendix Fig S6A), or do not show strong expression changes (log2(|FC|) < 0.2). Genes are further classified as "Bound" if containing binding sites for both Jmjd2a and Jmjd2c within +/- 1kb of a TSS.


E . Heat map presenting array data in the form of log2 ( fold change ) values comparing OHT with control treated ESCs . Genes are shown for which TSS regions ( + / - 1kb ) show the most substantial increase in H3K9me3 levels in OHT treated cells according to the ChIP - seq analyses ( defined in Fig EV4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Heat map presenting array data in the form of log2(fold change) values comparing OHT with control treated ESCs. Genes are shown for which TSS regions (+/- 1kb) show the most substantial increase in H3K9me3 levels in OHT treated cells according to the ChIP-seq analyses (defined in Fig EV4).


A - C . 2ac clones established after transfection with an empty vector ( empty ) or plasmids expressing wild type Jmjd2c ( 2c wt ) or a catalytic mutant ( 2c mut ) were exposed to OHT as indicated and subsequently used for ( A ) WB .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-C. 2ac clones established after transfection with an empty vector (empty) or plasmids expressing wild type Jmjd2c (2c wt) or a catalytic mutant (2c mut) were exposed to OHT as indicated and subsequently used for (A) WB.


A - C . 2ac clones established after transfection with an empty vector ( empty ) or plasmids expressing wild type Jmjd2c ( 2c wt ) or a catalytic mutant ( 2c mut ) were exposed to OHT as indicated and subsequently used for ( B ) RT - qPCR

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-C. 2ac clones established after transfection with an empty vector (empty) or plasmids expressing wild type Jmjd2c (2c wt) or a catalytic mutant (2c mut) were exposed to OHT as indicated and subsequently used for (B) RT-qPCR analyses


A - C . 2ac clones established after transfection with an empty vector ( empty ) or plasmids expressing wild type Jmjd2c ( 2c wt ) or a catalytic mutant ( 2c mut ) were exposed to OHT as indicated and subsequently used for ( C ) serial plating for the generation of growth curves .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-C. 2ac clones established after transfection with an empty vector (empty) or plasmids expressing wild type Jmjd2c (2c wt) or a catalytic mutant (2c mut) were exposed to OHT as indicated and subsequently used for (C) serial plating for the generation of growth curves.


( A ) ANAC032expression in WT plants sprayed with Pst , 6 and 24 hpi , compared to control ( sprayed with 10 mM MgCl2 ( Mock ) ) . Error bars represent means ± SD ( n = 3 ; ' n ' represents independently performed experiments , each including the rosette leaves of at least three plants grown in individual pots ) . ANAC032expression in WT treated with ( B ) SA or MeJA or ( C ) COR for 3 and 6 h compared to non - treated controls . In B and C , means ± SD are given ( n = 3 ; ' n ' represents independently performed experiments , each including at least 20 seedlings ) . FCh , fold change . Asterisks indicate a significant difference from their respective controls ( p < 0 . 01 ; Student ´ s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) ANAC032expression in WT plants sprayed with Pst, 6 and 24 hpi, compared to control (sprayed with 10 mM MgCl2 (Mock)). Error bars represent means ± SD (n = 3; 'n' represents independently performed experiments, each including the rosette leaves of at least three plants grown in individual pots). ANAC032expression in WT treated with (B) SA or MeJA or (C) COR for 3 and 6 h compared to non-treated controls. In B and C, means ± SD are given (n = 3; 'n' represents independently performed experiments, each including at least 20 seedlings). FCh, fold change. Asterisks indicate a significant difference from their respective controls (p < 0.01; Student´s t-test).


( A ) Plants after pressure infiltration with Pst DC3000 , at 4 dpi . The experiment was repeated three times ( each time 9 - 12 plants ) with similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Plants after pressure infiltration with Pst DC3000, at 4 dpi. The experiment was repeated three times (each time 9 - 12 plants) with similar results.


( B ) Pst DC3000 growth in ANAC032 transgenics and WT plants 3 dpi after pressure infiltration . Two independent experiments were performed with three replications per experiment , each replicate consisting of three plants grown in individual pots ( six measurements in total ) . The graph shows data points of the two individual experiments ( I and II ) along with their mean ( Avg ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Pst DC3000 growth in ANAC032 transgenics and WT plants 3 dpi after pressure infiltration. Two independent experiments were performed with three replications per experiment, each replicate consisting of three plants grown in individual pots (six measurements in total). The graph shows data points of the two individual experiments (I and II) along with their mean (Avg).