Proteinase inhibitors from Nicotiana alata enhance plant resistance to insect pests

The ornamental tobacco (Nicotiana alata) produces one 6-kDa chymotrypsin inhibitor and four 6-kDa trypsin inhibitors from a single 40.3-kDa precursor protein. Three different approaches have been used to assess the potential of these proteinase inhibitors (PIs) in insect control. The first was an in-vitro approach in which all five inhibitors, the single chymotrypsin inhibitor or three of the four trypsin inhibitors were tested for their ability to inhibit gut protease activity in insects from four orders. The second approach was to incorporate the N. alata PIs in the artificial diet of the native budworm (Helicoverpa punctigera) and the black field cricket (Teleogryllus commodus). H. punctigera larvae and T. commodus nymphs had a significant (P<0.01) reduction in growth after ingestion of the PI and were more lethargic than insects on the control diet. Several of the H. punctigera larvae also failed to complete moulting at the third or fourth instar. The third approach was to express the N. alata PIs in transgenic tobacco under the control of the 35S CaMV promoter. When H. punctigera larvae were fed tobacco leaves expressing the N. alata PIs at 0.2% soluble protein, significant (P<0.01) differences in mortality and/or growth rate were observed.