High affinity RGD‐binding sites at the plasma membrane ofArabidopsis thalianalinks the cell wall

Abstract

SummaryThe heptapeptide Tyr-Gly-Arg-Gly-Asp-Ser-Pro containing the sequence Arg-Gly-Asp (RGD -the essential structure recognised by animal cells in substrate adhesion molecules) was tested on epidermal cells of onion and cultured cells of Arabidopsis upon plasmolysis. Dramatic changes were observed on both types of cells following treatment: on onion cells, Hechtian strands linking the cell wall to the membrane were lost, while Arabidopsis cells changed from concave to convex plasmolysis. A control heptapeptide Tyr-Gly-Asp-Gly-Arg-Ser-Pro had no effect on the shape of plasmolysed cells. Protoplasts isolated from Arabidopsis cells agglutinate in the presence of ProNectinF, a genetically engineered protein of 72 kDa containing 13 RGD sequences: several protoplasts may adhere to a single molecule of ProNectinF. The addition of the RGDheptapeptide disrupted the adhesion between the protoplasts. Purified plasma membrane from Arabidopsis cells exhibits specific binding sites for the iodinated RGDheptapeptide. The binding is saturable, reversible, and two types of high affinity sites (K d1 ≈ 1 nM, and K d2 ≈ 40 nM) can be discerned. Competitive inhibition by several structurally related peptides and proteins noted the specific requirement for the RGD sequence. Thus, the RGD-binding activity of Arabidopsis fulfils the adhesion features of integrins, i.e. peptide specificity, subcellular location, and involvement in plasma membrane-cell wall attachments.

High affinity recognition of a Phytophthora protein by Arabidopsis via an RGD motif

“…The synthesis and iodination of three peptides, YGRGDSP, YGRGESP and YGDGRSP, was performed as previously described [7].…”

“…The binding of radiolabelled YGRGDSP heptapeptide to purified plasma membrane vesicles was performed as previously described [7]. The photolabelling of plasma membrane vesicles with the mono-iodinated N-(4-azidosalicylyl) AGRGDSP heptapeptide was carried out in 50 mM BTP (bis-tris propane) (pH 7.0, HCl) containing 0.25 M sucrose as follows.…”

“…To examine whether IPI-O resembles RGD peptides in disrupting cell wall -plasma membrane attachments [7] we plasmolysed undifferentiated cells from a suspension culture of A. thaliana and observed the plasma membrane -cell wall attachments by microscopy, in the absence or presence of recombinant MBP-IPI-O proteins. Plasmolysis was induced in the absence of MBP-IPI-O with the addition of 0.5 M CaCl 2 .…”

“…Integrins are also chosen by pathogens as targets for binding, and the RGD motif is present in surface proteins of several pathogens that attach to mammalian cells [5]. In plants, synthetic peptides containing the RGD motif can disrupt the adhesion between plasma membrane and cell wall [6][7] and can interfere with physiological processes thereby affecting, e.g., development [8][9][10][11], mechano-perception [12] and the interaction with microorganisms [13][14][15]. In particular, expression of cell wall associated defence responses triggered by fungal pathogens is dependent on adhesion between the plasma membrane and the plant cell wall [15].…”

“…Previously, we demonstrated that Arabidopsis thaliana contains plasma membrane proteins that can bind peptides containing the RGD motif [7]. We hypothesised that the high affinity RGD-binding sites at the plasma membrane may be the plants' counterparts of integrins and to further identify these membrane components we developed a photoaffinity method to cross-link an RGD-containing peptide to its potential targets on purified plasma membrane.…”