Journal
INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY
Volume 39, Issue 1, Pages 55-61Publisher
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.ibmb.2008.09.012
Keywords
Protease inhibitor; Mustard trypsin inhibitor MTI2; Plutella xylostelin; Arabidopsis thaliana; Trypsin
Categories
Funding
- International Agricultural Centre (The Netherlands)
- Key Laboratory of Vegetable Genetics and Physiology, Ministry of Agriculture (China)
- National Basic Research and Development Program of China [2006CB102003]
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The mustard trypsin inhibitor family has so far only been described among cruciferous species which represent the host plants for the specialist diamondback moth (DBM), Plutella xylostella. The performance of a Dutch and Chinese strain of DBM was assessed on transgenic Arabidopsis expressing Mustard Trypsin Inhibitor 2 MTI2 at a level of 84 mu g/g fresh weight equivalent to 72 mu M. No significant differences in larval mortality or development were found relative to the control. Trypsin activity in gut extracts from larvae feeding on either control or transgenic plants were titrated with MTI2 and SKTI (Soybean Kunitz Trypsin Inhibitor) to assess the basis of the insensitivity to MTI2. The specific trypsin activity per gut of larvae reared on MTI2 plants was not significantly higher compared to the control, and ca. 80% of trypsin activity could be inhibited by both inhibitors in both treatments, suggesting no specific induction of PI-insensitive activity in response to MTI2 in the diet. On the basis of the apparent equilibrium dissociation constant of Plutella trypsins for MTI2 (80 nM), the gut trypsin concentration (4.8 mu M), and the MTI2 concentration in the leaves (12 mu M) it was calculated that 99% of the gut trypsin activity sensitive to MTI2 should be inhibited in vivo, unless MTI2 was degraded. Indeed, we found that a pre-incubation of MTI2 and SKTI with gut proteases for 3 h resulted in complete loss of inhibitory activity of MTI2, but not of SKTI, at the concentration ratios found in planta. This process was enzymatic as it was inactivated by heat. Gut extracts of larvae reared on control or MTI2 leaves were equally well capable of this degradation indicating that the inactivating enzymes are constitutively expressed. In conclusion, it appears that the insensitivity of the diamondback moth to MTI2 can be sufficiently explained by the specific degradation of MTI2, thereby protecting itself against this protease inhibitor which is part of the defense of cruciferous plant species. (C) 2008 Elsevier Ltd. All rights reserved.
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