Date of Award

1980

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Abstract

Proteinase inhibitor activity was measured in the upper leaves of tomato plants, Lycopersicon esculentum Mill., 48 hr after wounding single lower leaves. Tomato selection 33 (TS 33) previously selected for its resistance to tomato race O of Phytophthora infestans (Mont.) deBary was used in this study. Spectrophotometric determination of total trypsin inhibitor activity (TIA) and chymotrypsin inhibitor activity (CTIA) in crude extracts and after affinity chromatography, showed that significantly higher levels of inhibitory activity were present in wounded plants than in unwounded controls. Seven inhibitor peaks designated A through G were resolved by isoelectric focusing of affinity column eluates on a 3.5 to 10.0 pH gradient, from both wounded and control plants. Two additional peaks with CTIA and TIA, designated A(,1) and C(,1) were resolved by isoelectric focusing on narrower pH gradients (pH 7.0-10.0). All inhibitor peaks from wounded plants contained more CTIA and TIA than unwounded controls. Agar double diffusion immunological assays showed that inhibitors from peaks A, A(,1), B, C(,1), and C with isoelectric points of 9.5, 8.9, 8.3, 8.2, and 8.0, respectively, were serologically related to Inhibitor I. A, C(,1), and C also reacted strongly with Inhibitor II antiserum, showing that complete resolution of Inhibitors I and II by isoelectric focusing was not accomplished. Proteinase inhibitors in peaks, E, F, and G apparently have not been described in past studies based on three criteria: (1) they have differing pI's (6.5, 5.9, and 4.7, respectively, (2) they are better trypsin than chymotrypsin inhibitors, and (3) they are serologically unrelated to Inhibitors I and II. In a similar study inhibitor levels were measured and compared in tomato selections TS 33, incompatible, or TS 19, compatible, to Phytophthora infestans, race O. The initial or endogenous level of inhibitors was greater in uninoculated plants of TS 33 than in TS 19. Generally, CTIA and TIA declined in both tomato selections after inoculation when measured in crude extracts or in eluates from affinity chromatographic columns. Profiles of isoelectric focused peaks revealed the same inhibitor peaks in all host-fungus combinations that were present in wounded plants. A comparison of inhibitory activity in the electro-focused proteins from inoculated and uninoculated tomato plants showed that there was a decline in CTIA and TIA in most peaks following infection. These observations suggest that the decrease in inhibitory activity is a general effect of the pathogen on the tomato plants rather than a qualitative shift to production of different inhibitors. The results from this study failed to corroborate a previous report from this laboratory suggesting that an overall increase in proteinase inhibitor activity was associated with the incompatible response of TS 33 inoculated with race O of P. infestans.

Pages

67

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