Journal: Plant Direct

Article Title: Untargeted Proteomics Identifies Plant Substrates of the Bacterial‐Derived ADP‐Ribosyltransferase AvrRpm1

doi: 10.1002/pld3.70115

Figure Lengend Snippet: ADP‐ribosylated substrates of AvrRpm1 but not HopF2 can be detected by immunoblots, enriched by the engineered Af1521 Macro domain, and identified by untargeted proteomics. (A) Detection of AvrRpm1 substrates by an α‐ADPr immunoblot. Protein extracts from Dex‐treated Col‐0 wild type and the indicated transgenic lines were separated by SDS‐PAGE, electroblotted onto PVDF membrane and probed with α‐HA and α‐mono‐ADPr antibodies. CBB = Coomassie brilliant blue–stained membrane as loading control. (B) Enrichment of ADP‐ribosylated proteins from the indicated Arabidopsis lines by the engineered Af1521 Macro domain and detection in an α‐pan‐ADPr immunoblot. Approximately 15% of the ADPr‐enriched fraction was loaded onto the gel. “sol. extract”= soluble protein extract. The membrane was stained with Amido black as loading control. This experiment was repeated twice with similar results. (C) Depletion of potentially competing nucleotide analogs from plant extracts prior to the Af1521 affinity purification facilitates enrichment of ADP‐ribosylated proteins. “Col‐0” and “AvrRpm1‐HA #18” indicate the respective protein extracts before ADPr‐enrichment. “ADPr‐enriched” = AvrRpm1 sample, direct pulldown with the Af1521 Macro domain. “G‐25‐>ADPr‐enriched” = AvrRpm1 sample, preclearing by G‐25 Sepharose followed by pulldown with the Af1521 Macro domain. ADP‐ribosylated proteins were detected as in (B). (D) HCD MS 2 spectrum of the ADP‐ribosylated peptide FGDWDENNPSSADGYTHIFNK from the RIN4 C‐NOI identified by untargeted proteomics ( z = 3, mass = 2954.09 Da). Unmodified fragment ions and fragment ions with a neutral loss of AMP are indicated in the sequence logos. The horizontal line designates the possible sequence window for ADP‐ribosylation. The ADPr marker ions are shown in beige. Asterisks indicate b‐ions with a neutral loss of AMP (Δ347.06 Da).

Article Snippet: Proteins were detected using α‐HA 3F10 antibody (Merck, 11867423001, 1:4000), α‐Flag M2 antibody (Merck, F3165, 1:1000), α‐pan‐ADPr‐binding reagent (Merck, MABE1016, 1:4000), α‐mono‐ADPr AbD33204 (Bio‐Rad, 1:2000), or α‐ADPr E6F6A (Cell Signaling, #83732, 1:5000) in 2.5% nonfat dry milk in TBST overnight at 4°C.

Techniques: Western Blot, Transgenic Assay, SDS Page, Membrane, Staining, Control, Affinity Purification, Sequencing, Marker

Journal: Plant Direct

Article Title: Untargeted Proteomics Identifies Plant Substrates of the Bacterial‐Derived ADP‐Ribosyltransferase AvrRpm1

doi: 10.1002/pld3.70115

Figure Lengend Snippet: AvrRpm1 ADP‐ribosylates Arabidopsis MAMI and PLDGAMMA3. (A) HCD MS 2 spectrum of the ADP‐ribosylated MAMI peptide VVGEGLVIDEWKER ( z = 3, mass = 2168.92 Da). The ADPr marker ions are shown in beige. Unmodified y‐ions and y‐ions with a neutral loss of AMP are indicated above the sequence logo. The horizontal line designates the possible sequence window for ADP‐ribosylation. Asterisks indicate ions with a neutral loss of AMP (Δ347.06 Da). (B) Detection of ADP‐ribosylated StrepII‐HA‐MAMI transiently coexpressed with AvrRpm1‐Flag in Nicotiana benthamiana . Protein extracts were separated by SDS‐PAGE, electroblotted onto PVDF membrane and probed with α‐HA, α‐pan‐ADPr, or α‐Flag antibodies. The membrane was stained with Amido black as loading control. “19K” indicates a sample from leaves expressing only the silencing suppressor 19K. The results are representative of three independent experiments. Images of the complete membranes are shown in Figure . (C) ADP‐ribosylated peptide AYHPVYNETMSMGGGSSNEFGQWLDK ( z = 3, mass = 3519.31 Da) from PLDGAMMA3‐HA‐StrepII coexpressed with AvrRpm1‐Flag in N. benthamiana . Unmodified y‐ions and y‐ions with a neutral loss of AMP are indicated above the sequence logo. The horizontal line designates the possible sequence window for ADP‐ribosylation. Asterisks indicate ions with a neutral loss of AMP (Δ347.06 Da).

Article Snippet: Proteins were detected using α‐HA 3F10 antibody (Merck, 11867423001, 1:4000), α‐Flag M2 antibody (Merck, F3165, 1:1000), α‐pan‐ADPr‐binding reagent (Merck, MABE1016, 1:4000), α‐mono‐ADPr AbD33204 (Bio‐Rad, 1:2000), or α‐ADPr E6F6A (Cell Signaling, #83732, 1:5000) in 2.5% nonfat dry milk in TBST overnight at 4°C.

Techniques: Marker, Sequencing, SDS Page, Membrane, Staining, Control, Expressing