Plant Physiol. Drug Metab Dispos
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH
QUICK SEARCH:   [advanced]


     

Plant Physiology Preview
Published on January 11, 2008; 10.1104/pp.108.115741

OPEN ACCESS ARTICLE
This Article
Free via Open Access: OA
Right arrow Full Text (Plant Physiology Preview (PDF))
Right arrowOA All Versions of this Article:
146/3/1010    most recent
pp.108.115741v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Goulet, M.-C.
Right arrow Articles by Michaud, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Goulet, M.-C.
Right arrow Articles by Michaud, D.
Agricola
Right arrow Articles by Goulet, M.-C.
Right arrow Articles by Michaud, D.

Received January 1, 2008
Accepted January 8, 2008

Tailoring the Specificity of a Plant Cystatin towards Herbivorous Insect Digestive Cysteine Proteases by Single Mutations at Positively Selected Amino Acid Sites

Marie-Claire Goulet , Cindy Dallaire , Louis-Philippe Vaillancourt , Moustafa Khalf , Amine M. Badri , Andreja Preradov , Marc-Olivier Duceppe , Charles Goulet , Conrad Cloutier , and Dominique Michaud *

Departement de phytologie, Pavillon des Services–INAF; Departement de biologie; Centre de recherche en horticulture, Universite Laval, Quebec (Quebec), Canada G1K 7P4

* Corresponding author; email: Dominique.Michaud{at}plg.ulaval.ca.

Plant cystatins, similar to other defense proteins, include hypervariable, positively selected amino acid sites presumably impacting their biological activity [Kiggundu et al., 2006, Plant J 48: 403–413]. Using 29 single mutants of the eight domain of tomato multicystatin, SlCYS8, we assessed here the potential of site-directed mutagenesis at positively selected amino acid sites to generate cystatin variants with improved inhibitory potency and specificity towards herbivorous insect digestive cysteine proteases. Compared to SlCYS8, several mutants (22 out of 29) exhibited either improved or lowered potency against different model cysteine proteases, strongly suggesting the potential of positively selected amino acids as target sites to modulate the inhibitory specificity of the cystatin towards cysteine proteases of agronomic significance. Accordingly, mutations at positively selected sites strongly influenced the inhibitory potency of SlCYS8 against digestive cysteine proteases of the insect herbivore, Colorado potato beetle (Leptinotarsa decemlineata). In particular, several variants exhibited improved potency against both ‘cystatin-sensitive’ and ‘cystatin-insensitive’ digestive cysteine proteases of this insect. Of these, some variants also showed weaker activity against leaf cysteine proteases of the host plant (potato, Solanum tuberosum), and against a major digestive cysteine protease of the two-spotted stinkbug Perillus bioculatus, an insect predator of Colorado potato beetle showing potential for biological control. Overall, these observations suggest the usefulness of site-directed mutagenesis at positively selected amino acid sites for the engineering of recombinant cystatins with both improved inhibitory potency towards the digestive proteases of target herbivores and weaker potency against non-target cysteine proteases in the host plant or the environment.






HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH
ASPB Publications PLANT PHYSIOLOGY THE PLANT CELL
Copyright © 2008 by the American Society of Plant Biologists