Purification and characterization of a constitutive polygalacturonase associated with the infection process of French bean leaves by Botrytis cinerea

Abstract

The constitutively produced polygalacturonase isoenzyme PG2 was isolated from culture filtrates of Botrytis cinerea, purified to homogeneity, and characterized. The shape of titration curves of PG2 and two other polygalacturonase isoenzymes explained the difficulties found in separating superimposed pectic enzyme activities during the purification procedure. PG2 hydrolyzed sodium polygalacturonate more quickly than pectin. The optimal pH for PG2 activity with polygalacturonate was 4.5 and with pectin, 4.0. PG2 activity was also influenced by the presence of NaCl or CaCl2 in the reaction mixture. Analysis of the breakdown products by paper chromatography and a comparison of the reaction rate by viscosimetry and reducing group assay revealed that PG2 has an endocatalytic mode of action on polygalacturonate. The isoelectric point and the molecular mass of PG2 were estimated to be 9.1 and 23.0 kDa, respectively. Key words: Botrytis cinerea, chromatofocusing, endopolygalacturonase, purification, substrate specificity, titration curve.

Post‐translational modifications of recombinant B. cinerea EPG 6

“…B. cinerea must breach the cell wall during the infection process, and accordingly secretes a great number of enzymes that can degrade cell walls 6. Among these cell‐wall‐degrading enzymes, endopolygalacturonases (EC 3.2.1.15) (EPGs) are in the first array of enzymes secreted.…”

Analysis of Polygalacturonase Enzyme Production in Isolates of Botrytis cinerea Sensitive to Different Fungicides Used in Vineyards

“…It is known that B. cinerea strains isolated from diseased grapes or other plants have the ability to produce PG (Magro et al. 1980; Leone et al. 1990; Johnston and Williamson 1992; Tobias et al.…”

“…It is known that B. cinerea strains isolated from diseased grapes or other plants have the ability to produce PG (Magro et al 1980;Leone et al 1990;Johnston and Williamson 1992;Tobias et al 1995;Rha et al 2001;Cabanne and Doneche 2002) and PNL (Chilosi and Magro 1997) with enzyme forms. The B. cinerea isolates tested in this study differed in PG enzyme activity, isoenzyme expression and banding profiles when they were grown on liquid medium in absence of fungicide.…”

“…Polygalacturonases (PGs) in particular are the first cell wall‐degrading enzymes produced by B. cinerea (Cabanne and Doneche 2002), and PG activity may be composed of many isoenzymes (Magro et al. 1980; Leone et al. 1990; Johnston and Williamson 1992; Tobias et al.…”

“…It is believed that mycelial development within the host, which accounts for symptom development, is linked to pectic enzyme production (Wasfy et al 1978;Reignault et al 2000). Polygalacturonases (PGs) in particular are the first cell wall-degrading enzymes produced by B. cinerea (Cabanne and Doneche 2002), and PG activity may be composed of many isoenzymes (Magro et al 1980;Leone et al 1990;Johnston and Williamson 1992;Tobias et al 1995;Chilosi and Magro 1997;Rha et al 2001;Cabanne and Doneche 2002;Pashkoulov et al 2002). This fungus has been reported to modulate PG-encoding gene expression, providing evidence of a functional role for PG during pathogenesis ten Have et al 2001).…”