Purification and characterization of two basic β-1,3-glucanases induced in Colletotrichum lindemuthianum-infected bean seedlings

Daugrois, Jean Heinrich; Lafitte, Claude; Barthe, Jean-Paul; Faucher, Catherine; Touzé, A.; Esquerré-Tugayé, Marie-Thérèse

doi:10.1016/0003-9861(92)90017-q

Cited by 10 publications

(6 citation statements)

References 25 publications

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“…In this respect, these enzymes are similar to other chitinases and fl-l,3-glucanases isolated from pea pods (Mauch et al 1988a) and bean (Boller et al 1983;Daugrois et al 1992). An endo cleaving mechanism appears to be more suitable for effectively inhibiting the growth of an invading fungal pathogen, because glucan and chitin oligosaccharides liberated from the fungal cell wall could serve as additional elicitors to further induce or enhance defence reactions .…”

Section: Discussionmentioning

confidence: 87%

Purification, characterization and differential hormonal regulation of a ?-1,3-glucanase and two chitinases from chickpea (Cicer arietinum L.)

Vogelsang

Barz

1993

Planta

163

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Chickpea (Cicer arietinum L.) cell-suspension cultures were used to isolate one beta-1,3-glucanase (EC 3.2.1.29) and two chitinases (EC 3.2.1.14). The beta-1,3-glucanase (M(r) = 36 kDa) and one of the chitinases (M(r) = 32 kDa) belong to class I hydrolases with basic isoelectric points (10.5 and 8.5, respectively) and were located intracellularly. The basic chitinase (BC) was also found in the culture medium. The second chitinase (M(r) = 28 kDa), with an acidic isoelectric point of 5.7, showed homology to N-terminal sequences of class III chitinases and represented the main protein accumulating in the culture medium. Polyclonal antibodies raised against the basic beta-1,3-glucanase (BG) and the acidic chitinase (AC) were shown to be monospecific. The anti-AC antiserum failed to recognize the BC on immune blots, confirming the structural diversity between class I and class III chitinases. Neither chitinase exhibited lysozyme activity. All hydrolases were endo in action on appropriate substrates. The BC inhibited the hyphal growth of several test fungi, whereas the AC failed to show any inhibitory activity. Expression of BG activity appeared to be regulated by auxin in the cell culture and in the intact plant. In contrast, the expression of neither chitinase was apparently influenced by auxin, indicating a differential hormonal regulation of beta-1,3-glucanase and chitinase activities in chickpea. After elicitation of cell cultures or infection of chickpea plants with Ascochyta rabiei, both system were found to have hydrolase patterns which were qualitatively and quantitatively comparable.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Discussionmentioning

confidence: 87%

Purification, characterization and differential hormonal regulation of a ?-1,3-glucanase and two chitinases from chickpea (Cicer arietinum L.)

Vogelsang

Barz

1993

Planta

163

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“…Fragments were ligated in pGEM-T (Promega, Madison, Wis., USA) and ampli®ed in the Escherichia coli strain XL1 blue according to standard procedures (Sambrook et al 1989). Nucleotide sequencing of the inserts (Sanger et al 1977) con®rmed that they corresponded to the above-mentioned chitinase and b-1,3-glucanase clones, as well as to the b-1,3-glucanase isoform which was previously isolated from infected bean seedlings and partly sequenced (Daugrois et al 1992). Radioactive labelling of the probes was performed with [a 32 P]dCTP by random priming as described by Feinberg and Vogelstein (1983).…”

Section: Methodsmentioning

confidence: 99%

Differential elicitation of defense responses by pectic fragments in bean seedlings

Boudart

Lafitte

Barthe

et al. 1998

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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 signi®cant activity or only a weak elicitor eect on the defence of susceptible or resistant bean seedlings. The dierential elicitor eect 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. La®tte et al., 1993, Mol PlantMicrob Interact 6: 628±634) to elicit defence responses in a cultivar-speci®c manner.

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“…␤-1,3-glucanase activity was assessed by measuring the reducing sugars released from 1% (w/v) laminarin (Sigma) by incubation of 5 mg of laminarin with 25 L of the dialyzed protein extract in 50 mm acetate buffer (pH 5.2) at 50°C for 30 min, according to a previously published procedure (Daugrois et al, 1992). The amount of reducing sugars was estimated by the Somogyi (1952) procedure.…”

Section: ␤-13-glucanase Assaymentioning

confidence: 99%

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

et al. 2003

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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...

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Purification and characterization of two basic β-1,3-glucanases induced in Colletotrichum lindemuthianum-infected bean seedlings

Cited by 10 publications

References 25 publications

Purification, characterization and differential hormonal regulation of a ?-1,3-glucanase and two chitinases from chickpea (Cicer arietinum L.)

Purification, characterization and differential hormonal regulation of a ?-1,3-glucanase and two chitinases from chickpea (Cicer arietinum L.)

Differential elicitation of defense responses by pectic fragments in bean seedlings

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

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