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

Abstract

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, PG1⌬SP); CLPG1 with the tobacco expansin1 SP instead of its own SP (Exp::PG1⌬SP); 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 ␤-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.Endopolygalacturonases (endoPGs) are a class of pectinases that participate in the degradation of plant cell walls by catalyzing the hydrolysis of the homogalacturonan domain of pectic polysaccharides, a linear chain of ␣-1,4-linked galacturonosyl residues. Depending on the source of enzyme, the degradation proceeds either by a strict endo-mode or by an endo-/exo-mode of cleavage (Cook et al., 1999). The released pectic fragments are mostly composed of linear oligogalacturonides (OGAs). The extent of degradation varies according to the level of methylesterification of the ␣-d,1-4-linked GalUA residues that compose the linear homogalacturonan chain because endoPGs only cleave unesterified rows of GalUA. Degradation can also be controlled by the presence of polygalacturonase-inhibiting proteins (PGIPs), a class of Leu-rich repeat proteins found in the cell walls of many plants.The OGAs released by cleavage of homogalacturonan were the first oligosaccharins, that is biologically active oligosaccharides (Darvill et al., 1992), that were isolated from commercial pectin and plant cell walls. OGA-induced responses have been reviewed extensively (Côté and Hahn, 1994; Ridley et al., 2001). The likelihood that such fragments elicit defense mechanisms when produced in planta is based on their effects when they are externally supplied to plant cells and tissues. Thus, early responses such as plasma membrane depolarization, ion fluxes, and c...

Transgenic Expression of a Fungal endo-Polygalacturonase Increases Plant Resistance to Pathogens and Reduces Auxin Sensitivity

“…Enhanced expression of basal and inducible defense responses and increased resistance to pathogens were previously observed in plants expressing pectin-degrading enzymes. Basal levels of polyphenoloxidase activity and induction of Phe ammonia lyase upon wounding are enhanced in transgenic potato tubers expressing a bacterial pectate lyase (Wegener, 2002), and accumulation of b-1,3-glucanase and H 2 O 2 is induced in tobacco leaves by the transient expression of a PG from Colletotrichum lindemuthianum (Boudart et al, 2003). Moreover, H 2 O 2 accumulates in tomato plants that have increased PG activity upon antisense expression of the regulatory subunit of a wound-inducible endogenous PG (PGbS); these plants exhibit constitutive expression of wound-inducible genes and enhanced resistance to insect attack (OrozcoCardenas and Ryan, 2003).…”

Characterization of the dry bean polygalacturonase-inhibiting protein (PGIP) gene family during Sclerotinia sclerotiorum (Sclerotiniaceae) infection

“…The capacity of PGs to elicit defense responses unrelated to their enzymatic activity has been recently proposed for B. cinerea-grapevine interaction (Poinssot et al, 2003). However, the enzymatic activity of PG from Colletrotrichum lindemuthianum has been shown to be essential for activating protective responses in tobacco (Boudart et al, 2003).…”

Cellulose Binding Domains of a Phytophthora Cell Wall Protein Are Novel Pathogen-Associated Molecular Patterns

“…Ultrathin sections (90 nm thickness) were cut with a diamond knife on an UltraCut E ultramicrotome (Reichert-Leica) and collected on gold grids. Immunogold labeling experiments were performed as described by Boudart et al (2003) using polyclonal antibodies directed against the protein part of CBEL (dilution, 1:50) and goat antirabbit antibodies coupled to colloidal gold (Sigma-Aldrich) at a dilution of 1:400. After contrasting with 5% uranyl acetate, the sections were observed on an H600 electron microscope (Hitachi) at 75 kV.…”