Directed proteomics identifies a plant-specific protein rapidly phosphorylated in response to bacterial and fungal elicitors

Abstract

The perception of microbial signal molecules is part of the strategy evolved by plants to survive attacks by potential pathogens. To gain a more complete understanding of the early signaling events involved in these responses, we used radioactive orthophosphate to pulse-label suspension-cultured cells of Arabidopsis in conjunction with two-dimensional gel electrophoresis and mass spectrometry to identify proteins that are phosphorylated rapidly in response to bacterial and fungal elicitors. One of these proteins, AtPhos43, and related proteins in tomato and rice, are phosphorylated within minutes after treatment with flagellin or chitin fragments. By measuring (32)P incorporation into AtPhos43 immunoprecipitated from extracts of elicitor-treated hormone and defense-response mutants, we found that phosphorylation of AtPhos43 after flagellin treatment but not chitin treatment is dependent on FLS2, a receptor-like kinase involved in flagellin perception. Induction by both elicitors is not dependent on salicylic acid or EDS1, a putative lipase involved in defense signaling.

Figure 1.

Phosphoprotein Proteome of flg22-Treated Arabidopsis Cell Culture. (A) Phosphorimage of radiolabeled soluble protein extracted from cells treated for 3 min with water (left) or 10 nM flg22 (right). Proteins that increase in incorporation of ³³P after elicitor treatment are indicated by triangles, and those that decrease are indicated by inverted triangles. An example of a constitutively phosphorylated protein is indicated by horizontal arrows. The phosphoprotein examined in this study, AtPhos43, is labeled. (B) Alignment of the radioactive images of AtPhos43 (top) and its corresponding silver-stained protein (bottom).

Figure 2.

Identification and Sequence of AtPhos43. (A) Spectrum obtained by nanoESI-MS-MS from a single tryptic fragment of the isolated protein. The individual mass peaks represent fragments of the parent ion produced by collision-induced dissociation. m/z, mass-to-charge ratio. (B) Graphic representation of the data obtained from the spectrum in (A). The circles represent the amino acids of the tryptic peptide, with the three amino acids identified in the shaded circles. The total mass of amino acids between the phenylalanine residue and the N terminus of the peptide is represented by m₁. The total mass of amino acids from the glutamine residue to the C terminus is represented by m₃. (C) Peptide sequence tags used to search the Arabidopsis database, and corresponding accession numbers identifying AtPhos43 as the phosphoprotein. (D) Alignment of AtPhos43 (top sequence) with the highly related protein ARP2 (bottom sequence). Residues of ARP2 identical to those of AtPhos43 are indicated by dashes. Gaps in the alignment are indicated by dots. Peptides corresponding to the peptide sequence tags are shown in the shaded boxes. The putative ankyrin domains are shown in the unshaded boxes, and the glycosyl hydrolase family 1 active site motif is underlined.

Figure 3.

AtPhos43 Is Differentially Phosphorylated in Arabidopsis Cell Cultures. (A) Immunoprecipitation of proteins from suspension cell cultures pulse labeled with ³³P after 0 or 4 min of treatment with 10 nM flg22 (flg) or 10 μg/mL chitin fragments (CH). Radioactive image (top) and Coomassie blue stain (bottom) of immunoprecipitated Arabidopsis proteins using preimmune serum (Pre-Imm) or immune serum (Anti-AtPhos43 Ab). Arrows at right indicate the position of AtPhos43. (B) Radioactive image of proteins immunoprecipitated with anti-AtPhos43 antibody from flg22-treated Arabidopsis cultures transformed with a vector expressing AtPhos43-GFP or GFP alone.

Figure 4.

Phos43-Related Proteins Are Phosphorylated in Other Species. Radioactive image of ³²P-labeled proteins immunoprecipitated from tomato or rice suspension cell cultures treated with flg22 or chitin fragments, respectively.

Figure 5.

Phosphoprotein Proteome of flg22-Treated Tomato Cell Culture. (A) Phosphorimage of radiolabeled soluble protein extracted from cells treated for 3 min with water (left) or 10 nM flg22 (right). Proteins that increase in incorporation of ³²P after elicitor treatment are indicated by triangles, and those that decrease are indicated by inverted triangles. An example of a constitutively phosphorylated protein is indicated by horizontal arrows. The location of the Phos43 ortholog is labeled. (B) Confirmation that the spot indicated in (A) is the tomato Phos43 ortholog by 2D separation of immunoprecipitate using anti-AtPhos43 antibody. The top section shows the radioactive image of the silver-stained gel in the bottom section. The spots comigrate to the position indicated in (A).

Figure 6.

AtPhos43 Is Phosphorylated after Treatment of Intact Plants with Microbial Elicitors. Radioactive image of proteins immunoprecipitated by the anti-AtPhos43 antibody from in planta ³²P-labeled Arabidopsis seedlings. Columbia ecotype wild-type (Col WT), fls2-24 mutant, Ws-0 ecotype, eds1-2 mutant, and NahG-expressing seedlings were treated for 10 min with water, 10 μM flg22 (flg), or 100 μg/mL chitin fragments (CH) before immunoprecipitation.