Abstract
M.Sc.
Plants have evolved a complex array of biochemical pathways that enable them to recognise and respond to signals from the environment. At present, little is known about the signal transduction pathways that are activated during a plant's response to attack by a pathogen, although this knowledge is central to our understanding of disease susceptibily and resistance. A common form of plant resistance is the restriction of pathogen proliferation to a small zone surrounding the site of infection. In many cases, this restriction is accompanied by localized death of host tissues, known as the hypersensitive response. In addition to local defense responses, many plants respond to infection by activating defenses in uninfected parts of the plant. As a result, the entire plant is more resistant to a secondary infection. This systemic acquired resistance can persist for several weeks or more and often confers crossresistance to unrelated pathogens.
Fungal polygalacturonases (PGs) catalyze the fragmentation and the solubilisation of the
homogalacturonan in the plant cell wall. These enzymes might have important functions
during plant colonization by a fungus. PGs have also been shown to activate plant defense responses, likely because they generate oligogalacturonides with elicitor activity from the plant cell wall. A polygalacturonase inhibiting protein (PGIP), found in the plant cell wall of many plants, forms a specific complex with fungal PGs and favours the accumulation of elicitor-active oligogalacturonides in vitro.
An agarose diffusion assay was used to screen the extracts from Verticillium dahliae for PG activity and ensuing inhibition by purified cotton PGIP. Quantitative determination of
differences in polygalacturonase activity in the extracts were performed using a reducing
sugar assay. There may be more than one isoform of PG present since the
polygalacturonases produced by fungi are likely to be to a mixture of exo- and endo-PGs.
Polygalacturonase was therefore isolated from 18-day-old culture filtrates of V. dahliae. The enzyme was partially purified by means of ammonium sulphate precipitation and gel
chromatography. The band responsible for PG activity was identified and characterized,
having a molecular weight of approximately 28-31 kDa, and a pl of 5.1 - 5.9. Kinetic studies indicate a Km of 0.33% and V,„,,of 0.85 pmoles reducing units / min.
A commercial preparation of endo-PG from Aspergillus niger was used as a control. This
endo-PG had a molecular weight of 68 kDa and a pl point of 3.6 and 5.1, suggesting there
were at least two isoforms of endo-PG present. Kinetic studies indicate a K m of 0.33% and V,,„ of 1.07 gmoles reducing units / min.