Potato type I and II serine protease inhibitors are produced by solanaceous plants as a defense mechanism against insects and microbes. Nicotiana alata proteinase inhibitor (NaPI) is a multidomain potato type II inhibitor (pin II) that is produced at high levels in the female reproductive tissues of the ornamental tobacco, Nicotiana alata. The individual inhibitory domains of NaPI target the major classes of digestive enzymes, trypsin and chymotrypsin, in the gut of lepidopteran larval pests. Although consumption of NaPI dramatically reduced the growth and development of a major insect pest, Helicoverpa punctigera, we discovered that surviving larvae had high levels of chymotrypsin activity resistant to inhibition by NaPI. We found a potato type I inhibitor, Solanum tuberosum potato type I inhibitor (StPin1A), was a strong inhibitor of the NaPI-resistant chymotrypsin activity. The combined inhibitory effect of NaPI and StPin1A on H. armigera larval growth in the laboratory was reflected in the increased yield of cotton bolls in field trials of transgenic plants expressing both inhibitors. Better crop protection thus is achieved using combinations of inhibitors in which one class of proteinase inhibitor is used to match the genetic capacity of an insect to adapt to a second class of proteinase inhibitor.chymotrypsin | resistance | Lepidoptera

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A cDNA clone, NA-PI-II, encoding a protein with partial identity to proteinase inhibitor (PI) II of potato and tomato has been isolated from a cDNA library constructed from Nicotiana alata stigma and style mRNA. The cDNA encodes a polypeptide of 397 amino acids with a putative signal peptide of 29 amino acids and six repeated domains, each with a potential reactive site. Domains 1 and 2 have chymotrypsin-specific sites and domains 3, 4, 5, and 6 have sites specific for trypsin. In situ hybridization experiments demonstrated that expression of the gene is restricted to the stigma of both immature and mature pistils. Peptides with inhibitory activity toward chymotrypsin and trypsin have been isolated from stigmas of N. alata. The N-terminal amino acid sequence obtained from this protein preparation corresponds to six regions in the cDNA clone NA-PI-II. The purified PI protein preparation is likely to be composed of a mixture of up to five similar peptides of approximately 6 kD, produced in vivo by proteolytic processing of a 42-kD precursor. The PI may function to protect the reproductive tissue against potential pathogens.

SummaryExpression of the plant defensin NaD1 in transgenic cotton plants increases plant survival, disease tolerance, and yield when grown in soil naturally infested with Fusarium oxysporum and Verticillium dahliae. Importantly, transgenic plants did not exhibit any detrimental agronomic features.