Insect Responses to Dietary Serine PIs

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Despite successes in enhancing the resistance of transgenic plants towards insects by the expression of foreign PIs, other examples of this technology have given disappointing results, with little or no protection observed. For example Jongsma et al50 produced transgenic tobacco plants expressing the chymotrypsin/trypsin-specific potato inhibitor II (PI-II) constitutively. In contrast to results reported above in Section 2.4, the growth of Spodoptera exigua larvae fed with detached leaves of plants expressing PI-II was not affected. These results, with other observations, enabled the authors to show that the insect could respond to the

Serine Proteinase

Fig. 2.4. Protection of strawbery plants expressing the trypsin inhibitor from cowpea (CpTI) against larvae ofvine weevil (Otiorhyncus sulcatus). The plant on the left is a transgenic strawbery expressing CpTI; the plant on the right is a control. Both plants were exposed to vine weevil larvae in a glasshouse trial. (Photo courtesy of Julie Graham, Scottish Crop Research Institute, Invergowrie, Dundee, Scotland.)

Fig. 2.4. Protection of strawbery plants expressing the trypsin inhibitor from cowpea (CpTI) against larvae ofvine weevil (Otiorhyncus sulcatus). The plant on the left is a transgenic strawbery expressing CpTI; the plant on the right is a control. Both plants were exposed to vine weevil larvae in a glasshouse trial. (Photo courtesy of Julie Graham, Scottish Crop Research Institute, Invergowrie, Dundee, Scotland.)

presence of PIs in its diet by inducing novel proteinase activities which are insensitive to inhibition. In the example quoted, larvae exposed to plants constitutively expressing PI-II exhibited a change in the nature of their protease enzymes. They contained lower levels of proteinase activity sensitive to inhibition by PI-II than larvae raised on control plants (by 4-fold), but had higher levels of proteinase activity which was insensitive to inhibition by PI-II (by 2.5-fold). The insects reared on transgenic plants thus contained protease activity which had a greatly reduced sensitivity to inhibition by the transgene product, explaining why no antimetabolic effect was observed. The authors also observed induction of proteinase activity insensitive to the endogenous wound-induced proteinase inhibitors of tobacco, and suggested that this was a general mechanism through which insects were able to overcome the accumulation of PIs caused by the wounding response in plants. Similar induction of inhibitor-insensitive proteinase activity was also observed by Broadway51 in two other lepidopteran larvae as a result of chronic ingestion of cabbage PIs in artificial diet.

Results recently reported by our group52 have clarified the molecular mechanisms involved in the response of lepidopteran larvae to PIs. The trypsin-like activity in Helicoverpa armigera larvae has been shown to be sensitive to inhibition by soybean Kunitz trypsin inhibitor (SKTI) in vitro, but preliminary assays had suggested that transgenic plants expressing this inhibitor showed little or no antimetabolic effects. Whereas insects fed on control artificial diet for 7 days contained trypsin-like activity which could be inhibited almost totally (94%) by SKTI, insects fed on diet containing SKTI showed adaptation and contained trypsin-like activity which was relatively insensitive to inhibition by SKTI (25% inhibition). The insects were shown to contain relatively large gene families encoding serine proteinases by Southern blot analysis of genomic DNA, and by direct sequencing of cDNA clones; at least 28 genes encoding trypsin- and chymotrypsin-like proteins were identified. Differences in sequences were observed when different "trypsins" and "chymot-rypsins" were compared. Whereas the catalyti-cally active residues were conserved, as were residues in the substrate binding pocket which determined the specificity of cleavage, residues in the areas of contact between the proteinases and protein PIs were found to be variable. This variability was suggested to lead to differing interactions between different trypsins or chymotrypsins and PIs. The expression of different genes was differentially affected by exposure of the insects to SKTI. Chymotrypsin-encoding mRNAs generally increased in level whereas some trypsin-encoding mRNAs decreased, while others increased or showed little change. We suggested that the presence of families of trypsin and chymotrypsin genes encoding proteinases with different sensitivities to PIs allows the insect to adapt to the presence of PIs in its diet. Thus, exposure to an inhibitor causes the synthesis of "sensitive" proteinases to be repressed, while the synthesis of "insensitive" proteinases is up-regulated. Time course experiments showed that the response of mRNA levels to dietary inhibitor is relatively rapid, and occurs over a timescale of hours (Fig. 2.5). This response is significantly faster than the wound-induced accumulation of PIs in plants (timescale approx. 48 h), and thus would be able to protect the insect from the plant's response to wounding, in agreement with the observations ofJongsma et al50 Although these experiments have provided a basis to explain the poor performance of some transgenic crops expressing PIs when attacked by lepidopteran pests, the data leave many questions unanswered. H. armigera is a highly polyphagous pest, which must be able to adapt to a wide range of foodstuffs containing different PIs, and thus may be atypical of insects generally in possessing the capacity to induce a range of proteases with differing sensitivities to inhibitors. However, large families of genes encoding serine proteinases have been reported in dipteran insects, and other Lepidoptera like Manduca sexta also have more than one gene encoding these enzymes, since several cDNA clones have been isolated, or multiple isoforms of the enzyme are present. In contrast the spruce budworm, Choristoneura fumiferana has been reported to contain only a single trypsin-encoding gene by Southern blotting of genomic DNA.53 The extent to which different PIs cause different changes in gene expression, and thus give rise to different mixtures of dietary proteinases is unknown. The response may be an "all-or-nothing" switch, or it may be finely tuned to adjust the complement of dietary proteinases to the composition of the diet. Finally, the mechanism by which the insect "senses" the presence of PIs and adjusts gene expression accordingly remains unknown for phytophagous insects. Results obtained with blood-feeding insects such as mosquito and blackfly (reviewed in Lehane et al54) show a strong induction of activity of trypsin-encoding genes by a blood meal, and also suggest that the activity of some proteinase genes (the so-called "late" trypsin genes) is controlled by the products of others ("early" trypsin genes). In this scheme, peptides produced by the action of the "early" trypsin enzymes (and other proteases) act as signals to induce the expression of the "late" trypsin genes.55 Interestingly, in the mosquito, addition of a PI, SKTI, to the diet blocks the activation of "late" trypsin genes, providing another example of inhibitors affecting insect proteinase gene expression. The relevance of these results to lepidopteran larvae, which feed continuously, is difficult to assess, but it seems likely that similar control mechanisms are present and allow the expression of proteinase genes to be controlled by dietary components.

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