Polygalacturonase inhibiting proteins from Allium porrum L. and their role in plant tissue against fungal endo- polygalacturonases

Five endo-polygalacturonases (PGs), three produced in culture filtrate by Fusarium moniliforme, Sclerotium cepivorum and Botrytis aclada, respectively, and two (one acidic and one basic isoform) obtained from Sclerotinia sclerotiorum soybean infected hypocotyls, were purified in order to characterize the activity of polygalacturonase inhibitor(s) (PGIP(s)) from leek stalk tissue (Allium porrum L.). 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 from S. sclerotiorum, followed by the acidic endo-PG of S. sclerotiorum and the endo-PG from B. aclada. In contrast, no inhibition of endo-PGs from S. cepivorum and F. moniliforme was 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. moniliforme and S. cepivorum showed the maximum activity in uronide releasing. The endo-PGs of S. sclerotiorum (acidic PG) and B. aclada were active only when high levels were used while the basic endo-PG of S. sclerotiorum was not active in combustion with any level of PGIP. These results indicate that a close relationship exists between PGIP activity in vitro and the ability of PGIP to protect leek tissue from endo-PG degradation