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Pectate lyase from Fusarium solani f. sp. pisi: purification, characterization, in vitro translation of the mRNA, and
M S Crawford1, P E Kolattukudy
1Institute of Biological Chemistry, Washington State University, Pullman 99164.
Abstract:
Since indirect experimental evidence suggested that penetration of Fusarium solani f. sp. pisi into its host (Pisum sativum) involved pectin-degrading enzymes (W. Köller, C. R. Allan, and P. E. Kolattukudy (1982) Physiol, Plant Pathol. 20, 47-60), direct tests were made for the production of such degradative enzymes by this pathogen. When the organism was grown on pectin, a pectate lyase (EC 4.2.2.2) was released into the media. This lyase was purified to apparent homogeneity from the culture filtrate by a two-step process involving passage through DEAE-Sephacel followed by hydrophobic interaction chromatography on octyl-Sepharose. The enzyme cleaved polygalacturonate chains in an endo fashion. The molecular mass of the mature extracellular form of this enzyme was estimated to be 26 kDa. The isoelectric point of the enzyme was 8.3 and the optimum pH for activity was 9.4. Calcium was required for activity and evidence is presented that calcium probably interacts with the substrate rather than the enzyme. When antibodies prepared against this enzyme were used for Western blot analysis of the extracellular culture fluid, a single band was observed at 26 kDa. Following in vitro translation of poly(A)+ RNA, a 29-kDa precursor polypeptide was precipitated by the antibodies. Antibodies inhibited both the catalytic activity of the enzyme and the ability of the fungus to infect pea stems, strongly suggesting that this lyase is involved in pathogenesis.
Insights
Fusarium solani f. sp. pisi produces a pectate lyase enzyme essential for infecting pea plants. This enzyme degrades pectin, facilitating pathogen penetration and disease development.
Area of Science:
- Plant Pathology
- Enzymology
- Molecular Biology
Background:
- Indirect evidence suggested pectin-degrading enzymes facilitate Fusarium solani f. sp. pisi infection of pea plants.
- Direct investigation was needed to confirm enzyme production and its role in pathogenesis.
Purpose of the Study:
- To directly test for pectin-degrading enzyme production by Fusarium solani f. sp. pisi.
- To purify and characterize the enzyme involved in pea stem infection.
- To confirm the enzyme's role in pathogenesis.
Main Methods:
- Culturing the fungus on pectin to induce enzyme release.
- Purification of pectate lyase using DEAE-Sephacel and hydrophobic interaction chromatography.
- Enzyme activity assays, molecular mass estimation, isoelectric focusing, and pH optimum determination.
- Antibody production and Western blot analysis for protein identification.
- In vitro translation and antibody precipitation to study the precursor polypeptide.
Main Results:
- A pectate lyase (EC 4.2.2.2) was isolated and purified from the culture filtrate.
- The extracellular enzyme has a molecular mass of 26 kDa, an isoelectric point of 8.3, and an optimal pH of 9.4.
- Calcium is required for activity, likely interacting with the substrate.
- Antibodies against the lyase inhibited both enzyme activity and fungal infection of pea stems.
Conclusions:
- The purified pectate lyase is directly involved in the pathogenesis of Fusarium solani f. sp. pisi on pea plants.
- The enzyme's ability to degrade pectin is crucial for the fungus to penetrate its host.
- This study provides direct evidence linking a specific enzyme to plant disease development.