Plant cells recognize chitin fragments for defense signaling through a plasma membrane receptor

Abstract

Chitin is a major component of fungal cell walls and serves as a molecular pattern for the recognition of potential pathogens in the innate immune systems of both plants and animals. In plants, chitin oligosaccharides have been known to induce various defense responses in a wide range of plant cells including both monocots and dicots. To clarify the molecular machinery involved in the perception and transduction of chitin oligosaccharide elicitor, a high-affinity binding protein for this elicitor was isolated from the plasma membrane of suspension-cultured rice cells. Characterization of the purified protein, CEBiP, as well as the cloning of the corresponding gene revealed that CEBiP is actually a glycoprotein consisting of 328 amino acid residues and glycan chains. CEBiP was predicted to have a short membrane spanning domain at the C terminus. Knockdown of CEBiP gene by RNA interference resulted in the suppression of the elicitor-induced oxidative burst as well as the gene responses, showing that CEBiP plays a key role in the perception and transduction of chitin oligosaccharide elicitor in the rice cells. Structural analysis of CEBiP also indicated the presence of two LysM motifs in the extracellular portion of CEBiP. As the LysM motif has been known to exist in the putative Nod-factor receptor kinases involved in the symbiotic signaling between leguminous plants and rhizobial bacteria, the result indicates the involvement of partially homologous plasma membrane proteins both in defense and symbiotic signaling in plant cells.

Fig. 1.

Purification of CEBiP from the plasma membrane of suspension-cultured rice cell. (A) Affinity columns used for purification. Columns 1 and 2, precolumns packed with Sephadex G-75 and glycine-CH-Sepharose 4B, respectively. Column 3, major affinity column of (GlcNAc)₈-APEA-CH-Sepharose 4B. (B) Silver staining of the SDS/PAGE gel. Lanes 1 and 2, proteins bound to precolumn 1 and 2, respectively. Lane 3, proteins eluted from the (GlcNAc)₈-APEA-CH-Sepharose 4B column with elicitor-inactive sugar (cellohexaose and chitosan octasaccharide) solution. Lane 4, proteins finally eluted from the (GlcNAc)₈-APEA-CH-Sepharose 4B column with glycine·HCl buffer. Arrowheads indicate CEBiP and its limited proteolysis product (see Results). (C) Affinity labeling of the proteins eluted from the (GlcNAc)₈-APEA-CH-Sepharose 4B column with glycine·HCl buffer by a [¹²⁵I]APEA-(GlcNAc)₈ derivative, indicating that both 75- and 55-kDa bands retained the binding ability to the elicitor. Unlabeled (GlcNAc)₈ (G, 25 μM) was added to ensure specific binding (+).

Fig. 2.

Structural features of CEBiP. (A) Schematic representation of the genomic DNA (AC099399) (a) and cDNA (b) encoding CEBiP. SP, signal peptide; ORF, open reading frame; NCR, noncoding region; LysM 1/LysM 2, LysM motif; TM, transmembrane region; RNAi, region inserted to the RNAi vector. (B) Amino acid sequence of CEBiP predicted from the cDNA. Solid and dashed underlines indicate the sequences corresponding to the N-terminal and internal peptide sequences obtained from the purified CEBiP, respectively. (C) Alignments of the two CEBiP LysM motifs to the consensus sequences of LysM motifs in the chitinases from Volvox (14), Lotus NFR1 (10), and Lotus NFR5 (11). The amino acid residues conserved in two or more sequences were underlined. Asterisks indicate the conserved residues in all four sequences.

Fig. 3.

Characterization of CEBiP-RNAi cell lines. (A) Western blot analysis of total proteins from the CEBiP-RNAi and nontransformed (NT) cell lines using anti-CEBiP antiserum (first row). The amount of mRNA for CEBiP (second row), GUS (third row), and ubiquitin (fourth row) genes in the CEBiP-RNAi and nontransformed cell lines was analyzed by RT-PCR. The amount of CEBiP mRNA was also determined by quantitative RT-PCR and shown as relative amount to the nontransformant (fifth row). ND, not determined. (B) ROS generation induced by (GlcNAc)₈ elicitor (G, 100 μg/ml) in the CEBiP-RNAi and nontransformed rice cells. One hundred milligrams of the suspension-cultured rice cells were incubated in 1 ml of the medium with (+) or without (−) the elicitor for 30 min at 25°C. (C) Specific suppression of ROS generation induced by chitin oligosaccharide elicitor in the CEBiP-RNAi cell line. Sixty milligrams of the CEBiP-RNAi and nontransformed rice cells were incubated in 1 ml of the medium containing (GlcNAc)₈ (G, 100 ng/ml), lipopolysaccharides (L, 50 μg/ml), chitosan octasaccharide (N, 2 μg/ml), or sterile H₂O (C) for 2 h at 25°C. All of the data were the average of three independent experiments. Error bars indicate standard deviation.

Fig. 4.

Microarray analyses of elicitor-responsive genes in the CEBiP-RNAi and nontransformed (NT) rice cells. Open box, genes responded to the elicitor only in NT; gray box, genes significantly decreased responsiveness to the elicitor (ratio of NT/CEBiP-RNAi >2) in CEBiP-RNAi; black box, genes equally responded to the elicitor in both NT and CEBiP-RNAi; hatched box, genes responded to the elicitor only in CEBiP-RNAi.

Fig. 5.

Decrease of the elicitor binding activity in the plasma membrane preparation from CEBiP-RNAi rice cells. (A) Binding activity of the PM preparation from CEBiP-RNAi and nontransformed (NT) rice cells to the [¹²⁵I]APEA-(GlcNAc)₈ derivative. Twenty micrograms of PM preparation was pretreated with elicitor-active [G, (GlcNAc)₇] or inactive (N, chitosan heptamer) sugars (+) for 30 min before incubation with [¹²⁵I]APEA-(GlcNAc)₈ derivative. All of the data were the average of three independent experiments. Error bars indicate standard deviation. (B) Detection of the elicitor binding protein by affinity labeling with the [¹²⁵I]APEA-(GlcNAc)₈ derivative. Unlabeled (GlcNAc)₈ (G, 25 μM) was added to ensure specific binding (+).

Fig. 6.

Induction of CEBiP expression by chitin oligosaccharide elicitor. Expression of CEBiP induced by (GlcNAc)₇ treatment (1 μg/ml) in the nontransformed rice cells was analyzed by Northern blotting using a cDNA probe.