DOI: 10.5281/zenodo.11048049
title: A pectin acetyl-transferase controls miRNA movement by facilitating plasmodesmata number in Arabidopsis

description:  

The dataset contains all the original raw files for protein and RNA blots, microscopy pictures and additional raw data, sorted by figure and figure panel. NGS data has been deposited on SRA. Additional EXCEL files are provided for qRT-PCR, degree of chlorosis, quantification of aniline blue deposits, quantification of the epidermis cell areas and perimeters  and quantification of CFDA diffusion raw data. The content of each file is:

 

FIGURE 1: 

 

1A: Pictures of pSUC2::amiRSUL and pATML1::amiRSUL rosettes (JPG files), as well as pictures of leaf cross sections of pSUC2::GUS and pATML1::GUS after GUS staining (TIF files).

1B: Northern blots raw data (TIF files) for the detection of amiRSUL and miR165/166 accumulation in pSUC2::amiRSUL and pATML1::amiRSUL backgrounds as compared to the negative WT control.

1C: GraphPad Prism analysis (PRISM file) for the quantification of SUL mRNA accumulation (RT-qPCR) in pSUC2::amiRSUL and pATML1::amiRSUL backgrounds as compared to the WT control.

1D: Pictures of pCA1::P19WT and pCA1::P19WG rosettes in the pATML1::amiRSUL background, as compared to the WT and the pATML1::amiRSUL plants (JPG files), and picture of leaf cross section of pCA1::GUS after GUS staining (TIF files).

1E: Northern blots and Western blots raw data (TIF files) for the detection of amiRSUL in P19-immunoprecipitates in the pATML1::amiRSUL background leaves without or with pCA1::P19WT and pCA1::P19WG transgenes.

1F: GraphPad Prism analysis (PRISM file) for the quantification of amiRSUL accumulation (RT-qPCR) in total, lower epidermis, mesophyll or vascular-Meselect-enriched fractions, in the pATML1::amiRSULbackground.

1G: Northern blots raw data (TIF files) for the detection of amiRSUL in scions or rootstocks of various grafting combinations: WT/WT, pSUC2::amiRSUL/pSUC2::amiRSUL, pSUC2::amiRSUL/WT, pATML1::amiRSUL/pATML1::amiRSUL, pATML1::amiRSUL/WT.

 

 

FIGURE 2: 

 

2B: Pictures of PMR5 or pmr5-2 rosettes in the pATML1::amiRSUL background (JPG files).

2C: Pictures of PMR5 or pmr5-2 rosettes in the pATML1::amiRSUL background (JPG files) and GraphPad Prism analysis (PRISM file) of the degree of leaf chlorosis of the corresponding plants as compared to WT.

2D: GraphPad Prism analysis (PRISM file) for the quantification of SUL mRNA accumulation (RT-qPCR) in PMR5 or pmr5-2 rosette leaves in the pATML1::amiRSUL background.

2E: Northern blots and Western blots raw data (TIF files) for the detection of amiRSUL and endogenous miRNAs in AGO1-immunoprecipitates in the PMR5 or pmr5-2 rosette leaves in the pATML1::amiRSULbackground.

2F: GraphPad Prism analysis (PRISM file) for the quantification of SUL mRNA accumulation (RT-qPCR) in total or Meselect-enriched epidermis fraction from PMR5 or pmr5-2 rosettes in the pATML1::amiRSULbackground.

2G: GraphPad Prism analysis (PRISM file) for the quantification of PHB mRNA accumulation (RT-qPCR) in total or Meselect-enriched epidermis fraction from PMR5 or pmr5-2 rosettes in the pATML1::amiRSULbackground.

2H: GraphPad Prism analysis (PRISM file) for the quantification of ARF10, NAC1 and MYB65 mRNA accumulation (RT-qPCR) in PMR5 or pmr5-2 rosette leaves in the pATML1::amiRSUL background.

2I: Pictures of PMR5 or pmr5-2 rosettes in the pATML1::amiRSUL background without or with the pCA1::P19WT transgene (JPG files).

2J: Northern blots and Western blots raw data (TIF or JPG files) for the detection of amiRSUL and endogenous miRNAs in P19-immunoprecipitates in PMR5 or pmr5-2 rosettes in the pATML1::amiRSULbackground the with pCA1::P19WT transgene.

2K: Pictures of PMR5 or pmr5-2 rosettes in the pSUC2::amiRSUL or pSUC2::SUL-LUS backgrounds (JPG files).

 

 

FIGURE 3: 

 

3A: Confocal pictures (LSM and JPG files) and analysis (EXCEL file) of the GFP signal detected in the young seedling leaves of pATML1::GFPER and pATML1::GFPsol in the PMR5 or pmr5-2 backgrounds.

3B: Pictures (JPG files) and analysis (EXCEL and PRISM files) of the primary infection foci observed on WT or pmr5-1 rosettes 5 days post-infection by TuMV-GFP.

3C: Western blot raw data (TIF files) for the detection of TuMV-GFP seven days after inoculation of the virus in the WT or pmr5-1 backgrounds.

3D: Northern blot raw data (TIF files) for the detection of amiRSUL and miR159 in scions or rootstocks of various grafting combinations: WT/WT, pSUC2::amiRSUL/WT, pSUC2::amiRSUL/pmr5-1.

3E: GraphPad Prism analysis (PRISM file) for the quantification of SUL mRNA accumulation (RT-qPCR) in roots of various grafting combinations: WT/WT, pSUC2::amiRSUL/WT, WT/pmr5-1 and pSUC2::amiRSUL/pmr5-1.

3F: Confocal pictures (LSM and JPG files) of WT, pmr5-1 and hst-1 roots after Fuchsin staining.

3G: Confocal pictures (LSM and JPG files) of pSUC2::GFPsol roots in the WT or pmr5-1 backgrounds.

3H: Confocal pictures (LSM files) and GraphPad Prism analysis (PRISM file) of aniline blue deposites detected on PMR5 or pmr5-2 rosette leaves in the pATML1::amiRSUL background.

3I: Confocal pictures (LSM and JPG files) and analysis (EXCEL and PRISM files) of PDLP3:YFP fluorescing spots in the WT or pmr5-1 backgrounds. Measures of cell wall length (PRISM file).

3J: Scanning electron microscopy images (TIF and JPG files) of shoot apical meristems of WT or pmr5-1seedlings.

3K: Analysis (EXCEL and PRISM files) of the number of periclinal and anticlinal plasmodesmata observed in the shoot apical meristem of WT or pmr5-1 seedlings.

3L: Confocal pictures (LSM files) and analysis (PRISM file) of the CF diffusion in the PMR5 or pmr5-2rosette leaves in the pATML1::amiRSUL background.

3M: Northern blot raw data (TIF files) for the detection of amiRSUL and miR159 in scions or rootstocks of various grafting combinations: WT/WT, pSUC2::amiRSUL/WT, pSUC2::amiRSUL(hst-1)/WT and pSUC2::amiRSUL(pmr5-2)/WT. Assembly file (TIF file) and mounting (Adobe Illustrator file).

 

 

FIGURE 4: 

 

4A: Pictures (JPG files) of pmr5-2 rosettes in the pATML1::amiRSUL background complemented with a WT or point mutated versions (S142A, D379A or H382A) of pPMR5::PMR5:eGFP.

4B: Pictures (JPG files) of PMR5 and pmr5-2 rosettes in the pATML1::amiRSUL background complemented or not with a WT or point mutated versions (S142A, D379A or H382A) of pPMR5::PMR5:eGFP and GraphPad Prism analysis (PRISM file) of the degree of leaf chlorosis of the corresponding plants.

4C: Pictures (JPG files) of PMR6 or pmr6-3 rosettes in the pATML1::amiRSUL background and in the PMR5 or pmr5-2 backgrounds.

4D: GraphPad Prism analyses (PRISM files) for the quantification of SUL or PHB mRNA accumulation (RT-qPCR) in pATML1::amiRSUL rosettes in the PMR5, pmr5-2 and pmr6-3 backgrounds.

4E: Confocal pictures (LSM files) and GraphPad Prism analysis (PRISM file) of aniline blue deposites detected on WT or pmr6-3 rosette leaves. GraphPad Prism analysis (PRISM file) of the cell wall length.

4F: Confocal pictures (LSM files) and analysis (PRISM file) of the CF diffusion in the WT or pmr6-3rosette leaves.

4G: Pictures (JPG files) and degree of leaf chlorosis analysis (PRISM file) of WT, pmr5-2, pmr6-3 andpmr5-2 pmr6-3  rosettes in the pATML1::amiRSUL background.

4H: Pictures (JPG files) of WT, tbr-1 and rwa2-3 rosettes in the pATML1::amiRSUL background and in the PMR5 or pmr5-2 backgrounds.

4I: GraphPad Prism analyses (PRISM file) for the quantification of SUL mRNA accumulation (RT-qPCR) in pATML1::amiRSUL rosettes in the WT, tbr-1 and rwa2-3 backgrounds.

4J: Confocal pictures (LSM files) and GraphPad Prism analysis (PRISM file) of aniline blue deposites detected on WT, tbr-1 and rwa2-3 rosette leaves. GraphPad Prism analysis (PRISM file) of the cell wall length.

4K: Confocal pictures (LSM files) and analysis (PRISM file) of the CF diffusion in WT, tbr-1 and rwa2-3rosette leaves.

4M-N: Confocal pictures (PPT file) of the LM19 and LM20 signal intensities obtained by immunolocalization on WT or pmr5-1 cotyledon epidermal monolayers and negative controls. Analyses (EXCEL and PRISM files) for the quantification of the LM19 and LM20 signal intensities obtained by immunolocalization on WT, pmr5-1 or pmr5-2 cotyledon epidermal monolayers. Immunolocalization protocole (DOCX file) and presentation of representative pictures with negative controls (PPTX file).

4O-P-Q: Analyses (EXCEL and PRISM files) for the quantification of the elastic moduli of the epidermal cell wall and the cellulose microfibril diameters of WT or pmr5-1 cotyledon epidermal monolayer. Atomic force microscopy pictures (JPG files) of cellulose microfibrils of WT or pmr5-1 cotyledon epidermal monolayer.

4R: Confocal pictures (LSM files) and GraphPad Prism analysis (PRISM file) of aniline blue deposites detected on WT and galt9-3 rosette leaves. GraphPad Prism analysis (PRISM file) of the cell wall length.

4S: Confocal pictures (LSM files) and analysis (PRISM file) of the CF diffusion in WT and galt9-3rosette leaves.

 

 

SUPPLEMENTARY FIGURE 1:

 

S1A: Pictures (JPG files) of WT, pSUC2::amiRSUL and pSUC2::SUL-LUS rosettes.

S1C: Pictures (JPG files) of T1 transformants of pATML1::amiRSUL(pri-319) rosettes.

S1D: Northern blot raw data (TIF files) for the detection of amiRSUL and miR165-166 in T1 transformants of pATML1::amiRSUL(pri-319) rosette leaves.

S1E: Pictures (JPG files) of T1 transformants of pATML1::amiRSUL(pri-390) rosettes.

S1F: Northern blot raw data (TIF files) for the detection of amiRSUL and miR165-166 in T1 transformants of pATML1::amiRSUL(pri-390) rosette leaves.

S1G: Pictures (JPG files) of WT, dcl1-11, ago1-42, hyl1-2 and dcl2-1 dcl3-1 dcl4-2 rosettes without or with the pATML1::amiRSUL transgene.

 

 

SUPPLEMENTARY FIGURE 2:

 

S2A: Pictures (TIF files) of leaf cross sections of WT and pCA1::GUS after GUS staining.

S2B: Pictures (JPG files) of pCA1::P19WT and pCA1::P19WG individual T1 plants obtained in the pATML1::amiRSUL background.

S2C: Pictures (JPG files) and degree of leaf chlorosis analysis (PRISM file) of pCA1::P19WT and pCA1::P19WG rosettes in the pATML1::amiRSUL background, as compared to the WT and the pATML1::amiRSUL plants.

S2D: GraphPad Prism analysis (PRISM file) for the quantification of ATML1 and CA1 mRNAaccumulation (RT-qPCR) in total, lower epidermis, mesophyll or vascular-Meselect-enriched fractions, in the pATML1::amiRSUL background.

 

 

SUPPLEMENTARY FIGURE 3:

 

S3A: Pictures (JPG files) of rosettes used for the allelism test, to validate pmr5-2 as the causal mutations for the reduction of the amiRSUL-dependent chlorosis.

S3B: Pictures (JPG files) of pATML1::amiRSUL rosettes in the WT, pmr5-1 and pmr5-2 backgrounds.

S3C: Pictures (JPG files) of WT, pmr5-1 and pmr5-2 rosettes, without the pATML1::amiRSUL transgene.

S3D: Analyses (EXCEL and PRISM files) of the WT, pmr5-1 and pmr5-2 rosette areas.

S3E: Pictures (JPG files) of PMR5 or pmr5-1 rosettes without or with pATML1::amiRSUL transgenes.

S3F: Northern blot raw data (PSD files) for the detection of amiRSUL in PMR5 or pmr5-1 rosettes without or with pATML1::amiRSUL transgenes.

S3G: Pictures (JPG files) of T1 transformants of pPMR5::PMR5:eGFP in the pATML1::amiRSUL(pmr5-2)background, as compared to the pATML1::amiRSUL and pATML1::amiRSUL(pmr5-2) rosettes. Confocal pictures (LSM and JPG files) of the pPMR5::PMR5:GFP signal in epidermis, mesohyll and vasculature.

S3H: Picture (LSM file) obtained by confocal microscopy of the co-infiltration of pPMR5::PMR5:GFPconstruct together with the p35S::mCherryER in Nicotiana bentamiana leaf.

 

 

SUPPLEMENTARY FIGURE 4:

 

S4A: Northern blot raw data (TIF files) for the detection of amiRSUL and miR159 in PMR5 or pmr5-1rosette leaves in the pATML1::amiRSUL background.

S4B: Analyses (EXCEL and PRISM files) of the mature miRNA average normalized read count obtain by sRNA sequencing from 2 biological replicates of 3 week-old rosettes in the pmr5-2 or in the pmr5-1backgrounds (both without the pATML1::amiRSUL transgene) as compared to the WT control.

S4C: GraphPad Prism analysis (PRISM file) for the quantification of ATML1 mRNA accumulation (RT-qPCR) in total or Meselect-enriched epidermis fraction from PMR5 or pmr5-2 rosettes in the pATML1::amiRSUL background.

S4D: Pictures (JPG files) of PMR5 or pmr5-1 seeds containing the 35S::CHS-RNAi (CHS-RNAi)transgene or not (NT)and analysis (PRISM file) for the quantification of CHS mRNA accumulation (RT-qPCR) in the corresponding genotypes (from rosette leaf tissues).

S4E: GraphPad Prism analysis (PRISM file) for the quantification of AGO1, DCL1, SPL10, TIR1, CSD2,CIP4, SCL6-IV and GRF2 mRNA accumulation (RT-qPCR) in PMR5 or pmr5-2 rosette leaves in the pATML1::amiRSUL background.

S4G: Pictures (JPG files) and degree of leaf chlorosis analysis (PRISM file) of pmr5-1, pSUC2::amiRSULand pSUC2::amiRSUL(pmr5-1) rosettes.

 

 

SUPPLEMENTARY FIGURE 5:

 

S5A: Pictures (PSD or JPG files) of the fluorescent signal of pSUC2::tmGFP9 and pSUC2::GFPsolseedlings in the WT or pmr5-1 backgrounds.

S5B: Confocal pictures (LSM and JPG files) of pATML1::GFPER and pATML1::GFPsol young seedling leaves in the PMR5 or pmr5-2 backgrounds.

S5C: Analysis (EXCEL and PRISM files) of the integrated relative mean GFP intensities from the pATML1::GFPER and pATML1::GFPsol young seedling leaves in the PMR5 or pmr5-2 backgrounds.

 

 

SUPPLEMENTARY FIGURE 6:

 

S6A: Analysis of the number of TuMV-GFP infection foci on WT, pmr5-1 and pmr5-2 rosette leaves (EXCEL file).

S6B: Western blot raw data (TIF files) for the detection of TuMV-GFP signals in infected and not infected WT or pmr5-2 rosettes. Mounting of the results (AI file).

S6C: Western blot raw data (TIF files) for the detection of PMR5-GFP in shoots or roots of pmr5-1 and pPMR5::PMR5:GFP(pmr5-1).

S6D: Confocal pictures (LSM and JPG files) of pSUC2::GFPsol roots in the WT or pmr5-1 backgrounds (extra to Fig3G).

S6E: GraphPad Prism analysis (PRISM file) of aniline blue deposites detected on PMR5 (WT) or pmr5-1rosette leaves. GraphPad Prism analysis (PRISM file) of the cell wall length.

S6F: Confocal pictures (LSM and JPG files) of the fluorescent signal observed on the adaxial side (up) or the abaxial side (down) of Arabidopsis rosette leaves, 5 minutes after CFDA drop deposit (5 µL).

 

 

SUPPLEMENTARY FIGURE 7:

 

S7A: Western blot raw data (TIF and PDF files) for the detection of PMR5-GFP in pmr5-2 rosettes in the pATML1::amiRSUL background complemented with a WT or point mutated versions (S142A, D379A or H382A) of pPMR5::PMR5:eGFP.

S7B: Pictures (JPG files) of WT or pmr6-3 rosettes in the pSUC2::amiRSUL background.

S7C: GraphPad Prism analysis (PRISM file) for the quantification of SUL mRNA accumulation (RT-qPCR) in WT or pmr6-3 rosette leaves in the pSUC2::amiRSUL background.

S7D: Pictures (JPG files) of WT or pmr6-3 rosettes in the pSUC2::SUL-LUS background.

S7E: GraphPad Prism analysis (PRISM file) for the quantification of SUL mRNA accumulation (RT-qPCR) in WT or pmr6-3 rosette leaves in the pSUC2::SUL-LUS background.

S7F: GraphPad Prism analyses (PRISM file) for the quantification of PHB, ARF10, ARF17 and MYB65mRNA accumulation (qRT-PCR) in WT or pmr6-3 rosettes.

S7G: Pictures (JPG files) and degree of leaf chlorosis analysis (PRISM file) of WT, tbr-1 and rwa2-3rosettes in the pATML1::amiRSUL or pATML1::amiRSUL(pmr5-2) backgrounds. Genotyping results are also indicated (DOC file) for the tbr-1 mutant in the pATML1::amiRSUL(pmr5-2) background.

S7H: Confocal pictures (PPT file) of the LM19 and LM20 signal intensities obtained by immunolocalization on WT or pmr5-2 cotyledon epidermal monolayers and negative controls. Immunolocalization protocole (DOCX file).

S7I: GraphPad Prism analyses (PRISM file) for the quantification of GALT9 mRNA accumulation (qRT-PCR) in WT or galt9-3 rosette leaves.