Assay for proteinase inhibition in Tripsacum-Zea diploperennis X maize hybrids resistant to Western corn rootworm --Eubanks, M, Cook, C A defense response of plants to insect herbivory is production of proteinase inhibitors (PIs) with insecticidal activity. Zhao et al. (Plant Physiol. 111:1299-1306, 1996) demonstated that cysteine proteinase inhibitors have inhibitory activity against gut cysteine proteinases in third instar larvae of Western corn rootworm, Diabrotica virgifera LeConte (Coleoptera). Eastern gamagrass, Tripsacum dactyloides L., is resistant to Western corn rootworm (T. Branson, Ann. Entomol. Soc. Amer. 64:861-863, 1972), and resistance from Tripsacum has been transferred to corn using diploid perennial teosinte, Zea diploperennis Iltis, Doebley and Guzmán, as a bridging species (M. W. Eubanks MNL 71: 1999; Amer. J. Bot. (suppl.):84:116, 1997; MNL 70:22-23, 1996; MNL 68:40-41, 1994). In an insect bioassay for selection of rootworm resistant plants, three week old plants were infested with 70 newly hatched first instar diapausing Western corn rootworm larvae per plant. In order to measure the papain inhibitory activity and investigate whether there was a correlation between proteinase inhibition and resistance to corn rootworm, leaf samples were collected from the plants three weeks after infestation just prior to harvesting them for scoring the roots according to the Iowa 1-6 root rating scale.

Bioassay #2 Twelve lines were tested in NSF Phase II insect bioassay #2 conducted at the Duke University Phytotron. The lines included corn inbred W64A, five breeding lines A, C, (97-3 X C), E, and (97-1 X 97-3), and five hybrid lines in a Tripsacum cytoplasm TC64, (TC64 X TC), (TC64 X 97-1), (TC64 X A), and (TC64 X 97-5). Root ratings for all lines are summarized below in Table 1. A root rating of 1 or 2 indicates strong resistance to corn rooworm herbivory.

Table 1. Summary of Root Ratings in Insect Bioassay #2
 
     
Root Ratings
Line Treated Controls 1 2 3 4 5 6
A 50 17 3 4 21 22 0 0
C 15 4 2 5 4 4 0 0
97-3 X C 18 6 3 2 9 4 0 0
E 32 8 2 4 17 9 0 0
E reciprocal 24 7 2 7 7 8 0 0
97-1 X 97-3 29 10 2 4 14 9 0 0
97-3 X 97-1 23 2 2 3 10 8 0 0
TC64 17 10 1 1 8 6 1 0
TC64 X TC 13 5 1 3 5 4 0 0
TC64 X 97-1 25 10 1 4 5 13 0 2
TC64 X A 14 6 0 0 3 5 3 3
TC64 X 97-5 14 6 0 1 3 8 0 2
W64A 3 3 0 0 3 0 0 0

Papain Assay - A modification of the technique in Koiwa et al. (Plant J. 14:371-379, 1998) was used to determine papain inhibitory activity in the corn leaves. Four disks of leaf material yielding ~0.1 g/sample or ~7 cm2/sample were taken from each plant three weeks after infestation with corn rootworm larvae. Leaf disks were frozen at -80 C, then ground to a fine powder using liquid nitrogen in 1.5 ml microcentrifuge tubes. To extract the inhibitor, 1 ml of a solution of 50mM phosphate buffer pH 7.2, with 150 mM NaCl and 2 mM EDTA:2Na, was added to the leaf powder, approximately 10 ml per gram leaf tissue (fresh weight). This was vortexed for 10 s, then centrifuged at 12 000 g for 15 min. To preactivate the papain, 1 volume of 20µg/ml papain in 25mM NaPi pH 7.0, 20 mM 2-mercaptoethanol was incubated for 10 min at 40 C, combined with 2 volumes of 0.25M NaPi pH 6.0, 2.5mM EDTA:2Na, and then kept on ice. Next 0.1 ml of the inhibitor solution and 0.3 ml of the preactivated papain solution were combined and incubated for 5 min at 40 C. The reaction was started by adding 0.2 ml of 2.3mM (final concentration) Na-benzoyl-DL- arginine-b-naphthylamide (BANA). This was incubated for 10 min at 40C, then 1 ml of 2% HCl in ethanol was added to stop the reaction, and finally 1 ml of 0.06% p-dimethylaminocinnamaldehyde in ethanol was added to develop color. The samples were held for at least 30 min at room temperature for full color development. The absorbance of the final mixture was read in a spectrophotometer (Perkin-Elmer Lambda 3B) at 540 nm. The inhibitory activity was expressed as the percentage decrease in absorbance relative to the reaction with no inhibitor.

Statistical Analysis - Although there was considerable range in the inhibitory activity of individual plants for each line tested (see Table 2 below), correlation analysis revealed no relationship between proteinase inhibition and root rating. It is thus concluded that the resistance mechanism conferred via Tripsacum does not involve proteinase inhibition insecticidal activity like the Bt endotoxin that has been engineered into transgenic corn for resistance to European corn borer and is being developed for resistance to corn rootworm.

Table 2. Mean and Coefficient of Variation for Proteinase Inhibition Per Line Tested.
 
Line N* Min Max Mean CV
A 53 31.78 75.73 52.97 19.9
C 19 27.51 62.65 41.99 26.4
97-3 X C 23 33.13 60.94 46.09 16.3
E 40 35.24 73.90 50.70 17.1
E reciprocal 32 26.36 67.65 49.11 24.3
97-1 X 97-3 41 28.52 70.45 50.33 20.4
97-3 X 97-1 29 25.63 73.64 54.26 20.2
TC64 40 25.90 77.24 50.32 26.1
TC64 X TC 18 36.38 75.71 52.19 23.6
TC64 X 97-1 36 27.67 73.00 53.72 23.4
TC64 X A 20 25.35 68.43 45.68 26.4
TC64 X 97-5 20 39.15 74.73 56.51 18.4
W64A 6 46.83 73.21 59.84 17.4

*N is the number of treated and control plants sampled for the papain assay.
 
 
 
 


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