Polygalacturonase inhibiting proteins fromAllium porrumL. and their role in plant tissue against fungal endo-polygalacturonases

Abstract

Five endo-polygalacturonases (PGs), three produced in culture filtrate byFusarium moniliforme, Sclerotium cepivorumandBotrytis aclada,respectively, and two (one acidic and one basic isoform) obtained fromSclerotinia sclerotiorumsoybean infected hypocotyls, were purified in order to characterize the activity of polygalacturonase inhibitor(s) (PGIP(s)) from leek stalk tissue (Allium porrumL.). Three apparently different PGIPs (PGIP-I, PGIP-II and PGIP-III) were purified from the leek tissue. The two more abundant PGIPs (PGIP-I and PGIP-III), although possessing similar pIs of about 6.5, differed in chromatographic behaviour, their molecular mass (39 and 42 kDa, respectively), and specific activity when assayed with the fungal endo-PGs. In addition, PGIP-I was solubilized from tissue homogenate with a low-salt buffer whilst PGIP-III needed a high-salt buffer for extraction (behaving as an ionically wall-bound protein). PGIP-II had very similar properties to PGIP-I, but was extracted using the high-salt buffer. The purified PGIPs and the crude leek extract showed similar inhibition activity patterns against the five fungal endo-PGs. The maximum inhibition activity was observed against the basic endo-PG fromS. sclerotiorum,followed by the acidic endo-PG ofS. sclerotiorumand the endo-PG fromB. aclada.In contrast, no inhibition of endo-PGs fromS. cepivorumandF. moniliformewas observed. Four different concentrations of the five fungal endo-PGs were incubated separately with slices of leek stalk, and the galacturonides released in the incubation mixture were measured. At every level used the endo-PGs ofF. moniliformeandS. cepivorumshowed the maximum activity in uronide releasing. The endo-PGs ofS. sclerotiorum(acidic PG) andB. acladawere active only when high levels were used while the basic endo-PG ofS. sclerotiorumwas not active in combustion with any level of PGIP. These results indicate that a close relationship exists between PGIP activityin vitroand the ability of PGIP to protect leek tissue from endo-PG degradation.