Georges Boudart

Cell wall proteins: a new insight through proteomics

Cell wall proteins are essential constituents of plant cell walls; they are involved in modifications of cell wall components, wall structure, signaling and interactions with plasma membrane proteins at the cell surface. The application of proteomic approaches to the cell wall compartment raises important questions: are there technical problems specific to cell wall proteomics? What kinds of proteins can be found in Arabidopsis walls? Are some of them unexpected? What sort of post-translational modifications have been characterized in cell wall proteins to date? The purpose of this review is to discuss the experimental results obtained to date using proteomics, as well as some of the new questions challenging future research.

Differential elicitation of defense responses by pectic fragments in bean seedlings

Abstract. The cell walls of two near-isogenic lines of bean (Phaseolus vulgaris L.) seedlings, susceptible or resistant to the bean anthracnose pathogen Colletotrichum lindemuthianum, were digested with the pure endopolygalacturonase (endoPG; EC 3.2.1.15) isolated from the fungus. The solubilized pectic fragments were separated according to their charge and size. Analysis of their uronic acid contents showed that their elution patterns were quite dissimilar, depending on whether they originated from the resistant or the susceptible host plant. Their sugar compositions revealed that neutral sugars were more abundant in the fragments released from the resistant plant than from the susceptible one, while the reverse was true for acidic residues. The fragments solubilized from the resistant plant induced an increase of pathogenesis-related (PR) proteins when challenged on resistant or susceptible bean seedlings, both at the transcript and enzyme-activity levels. On the other hand, pectic fragments released from susceptible bean cell walls exhibited either no significant activity or only a weak elicitor effect on the defence of susceptible or resistant bean seedlings. The differential elicitor effect observed between pectic fragments was inversely correlated to their acidity. Thus, endoPG-released pectic fragments from bean cell walls exhibited the same ability as the endoPG itself (C. Lafitte et al., 1993, Mol Plant-Microb Interact 6: 628–634) to elicit defence responses in a cultivar-specific manner.

Elicitor Activity of a Fungal Endopolygalacturonase in Tobacco Requires a Functional Catalytic Site and Cell Wall Localization

CLPG1, an endopolygalacturonase (endoPG) gene of Colletotrichum lindemuthianum, was transferred to tobacco (Nicotiana tabacum) leaves by using the Agrobacterium tumefaciens transient delivery system. The following four constructs were prepared:CLPG1, with or without its signal peptide (SP; PG1, PG1ΔSP); CLPG1 with the tobaccoexpansin1 SP instead of its own SP (Exp::PG1ΔSP); and a mutated version of the latter on two amino acids potentially involved in the catalytic site of CLPG1 (D202N/D203N). Chlorotic and necrotic lesions appeared 5 to 7 d postinfiltration, exclusively in response to CLPG1 fused to the expansin SP. The lesions were correlated to the production of an active enzyme. Necrosis-inducing activity, as well as endoPG activity, were completely abolished by site-directed mutagenesis. Ultrastructural immunocytolocalization experiments indicated that the expansin SP addressed CLPG1 to the cell wall. Staining of parenchyma cells revealed the progressive degradation of pectic material in junction zones and middle lamella as a function of time after infiltration, ultimately leading to cell separation. A 30% decrease in the GalUA content of the cell walls was simultaneously recorded, thereby confirming the hydrolytic effect of CLPG1 on pectic polysaccharides, in planta. The elicitor activity of CLPG1 was further illustrated by the induction of defense responses comprising active oxygen species and β-1,3-glucanase activity, before leaf necrosis. Altogether, the data demonstrate that an appropriate SP and a functional catalytic site are required for the proper expression and elicitor activity of the fungal endoPG CLPG1 in tobacco.

Isolation of Plant Cell Wall Proteins

The quality of a proteomic analysis of a cell compartment strongly depends on the reliability of the isolation procedure for the cell compartment of interest. Plant cell walls possess specific drawbacks: (1) the lack of a surrounding membrane may result in the loss of cell wall proteins (CWP) during the isolation procedure; (2) polysaccharide networks of cellulose, hemicelluloses, and pectins form potential traps for contaminants such as intracellular proteins; (3) the presence of proteins interacting in many different ways with the polysaccharide matrix require different procedures to elute them from the cell wall. Three categories of CWP are distinguished: labile proteins that have little or no interactions with cell wall components, weakly bound proteins extractable with salts, and strongly bound proteins. Two alternative protocols are decribed for cell wall proteomics: (1) nondestructive techniques allowing the extraction of labile or weakly bound CWP without damaging the plasma membrane; (2) destructive techniques to isolate cell walls from which weakly or strongly bound CWP can be extracted. These protocols give very low levels of contamination by intracellular proteins. Their application should lead to a realistic view of the cell wall proteome at least for labile and weakly bound CWP extractable by salts.

Cell Wall Proteome

In this chapter, we will focus on the contribution of proteomics to the identification and determination of the structure and function of CWPs as well as discussing new perspectives in this area. The great variety of proteins found in the plant cell wall is described. Some families, such as glycoside hydrolases, proteases, lectins, and inhibitors of cell wall modifying enzymes, are discussed in detail. Examples of the use of proteomic techniques to elucidate the structure of various cell wall proteins, especially with post-translational modifications such as Nglycosylations, proline hydroxylation and O-glycosylations, addition of GPI anchors, and phosphorylation, are given. Finally, the emerging understanding of the functions of cell wall proteins is discussed, as well as proposals for future research.

Elicitation of β-1,3-Glucanase in Bean Seedlings by Endopolygalacturonase of Colletotrichum Lindemuthianum

Pectic oligosaccharides derived from plant cell walls have been shown to elicit defence responses in plants. They are released by hydrolytic enzymes secreted by pathogenic microorganisms, and act as endogenous elicitors. In this study, we investigated the effect of the pure endopolygalacturonase (endoPG ) prepared from the culture filtrate of Colletotrichum lindemuthianum race β on defence induction in Phaseolus vulgaris. The effects of race β endoPG treatment and of race β infection on the induction of β-1,3-glucanase were compared.