A COMPLEX OF GENES INVOLVED IN ADAPTATION OF Leptinotarsa decemlineata LARVAE TO INDUCED POTATO DEFENSE

Abstract

The Colorado potato beetle (Leptinotarsa decemlineata) is the most important pest of potato in many areas of the world. One of the main reasons for its success lies in the ability of its larvae to counteract plant defense compounds. Larvae adapt to protease inhibitors (PIs) produced in potato leaves through substitution of inhibitor-sensitive digestive cysteine proteases with inhibitor-insensitive cysteine proteases. To get a broader insight into the basis of larval adaptation to plant defenses, we created a "suppression subtractive hybridisation" library using cDNA from the gut of L. decemlineata larvae fed methyl jasmonate-induced or uninduced potato leaves. Four hundred clones, randomly selected from the library, were screened for their relevance to adaptation with DNA microarray hybridizations. Selected enzyme systems of beetle digestion were further inspected for changes in gene expression using quantitative PCR and enzyme activity measurements. We identified two new groups of digestive cysteine proteases, intestains D and intestains E. Intestains D represent a group of structurally distinct digestive cysteine proteases, of which the tested members are strongly upregulated in response to induced plant defenses. Moreover, we found that other digestive enzymes also participate in adaptation, namely, cellulases, serine proteases, and an endopolygalacturonase. In addition, juvenile hormone binding protein-like (JHBP-like) genes were upregulated. All studied genes were expressed specifically in larval guts. In contrast to earlier studies that reported experiments based on PI-enriched artificial diets, our results increase understanding of insect adaptation under natural conditions.

Clitocypin, a fungal cysteine protease inhibitor, exerts its insecticidal effect on Colorado potato beetle larvae by inhibiting their digestive cysteine proteases

“…Digestive cysteine proteases are accompanied by digestive serine and aspartic proteases in CPB larval guts [9,41,42], therefore their inhibition by clitocypin was also assessed, since they complement cysteine proteases during adaptation to dietary cysteine protease inhibitors. Clitocypin did not inhibit the chymotrypsin-like serine protease activity, measured with substrate Boc-Gly-Arg-Arg-MCA at pH 8.8 in the presence of E-64 (Fig.…”

“…Profiles of gene expression in CPB larvae fed with transgenic potato leaves were monitored by isolating RNA from their midguts at day 8 of the second feeding trial, as described [9]. Six biological samples were obtained for each group by pooling midguts from two or three larval specimens from the same group for each sample.…”

“…CPB larvae utilize predominantly cysteine proteases, intestains, for protein digestion. In response to dietary protease inhibitors they overexpress these cysteine proteases, express insensitive cysteine proteases, proteases of other catalytic classes and proteases that inactivate ingested protease inhibitors [5][6][7][8][9][10].…”

Transcriptional Analysis of The Adaptive Digestive System of The Migratory Locust in Response to Plant Defensive Protease Inhibitors

“…Detailed analyses of differentially expressed transcripts showed a notable upregulation of several transcripts encoding hexamerin-like proteins, which are known to be juvenile hormone binding proteins (JHBPs)19. Interestingly, one other study performed in the Colorado potato beetle, Leptinotarsa decemlineata , also demonstrated an induced expression of several JHBPs after feeding on potato plants with induced defences16. These findings could suggest a more general role for JHBPs or JH in the regulation of the digestive process in insects.…”

“…Like in other insects, the underlying regulatory mechanisms of this adaptation are however still unknown. Up to now, only few studies have tried to identify broader transcriptional changes associated with this phenomenon81415161718. However, these studies mostly investigated long-term changes after dietary PI uptake and did not focus on the initial driving forces regulating the response.…”