Serge Yelle

Selective inhibition of Colorado potato beetle cathepsin H by oryzacystatins I and II

The use of oryzacystatins I and II, two cysteine proteinase inhibitors naturally produced in rice grains, represents an attractive way for the control of Coleoptera insect pests. The present study was done to analyze the inhibitory effect of recombinant oryzacystatins produced in Escherichia coli as fusion proteins against digestive proteinases of the major pest Colorado potato beetle (Leptinotarsa decemlineata Say). Both inhibitors had a significant effect on total proteolytic activity, but maximal inhibitions ranged from 20 to 80% for pHs varying from 5.0 to 7.0, respectively. This pH‐dependent efficiency of plant cystatins was due to the selective inactivation of potato beetle cathepsin H, as demonstrated by the use of inhibitors with different specificities against cathepsins B and H. These results demonstrate the importance of having an adequate knowledge of insect proteinases specifically recognized by the inhibitors to be used in pest control strategies.

Synthesis of active oryzacystatin I in transgenic potato plants

SummaryTransformation of potato (Solanum tuberosum L.) with cysteine proteinase inhibitor (PI) genes represents a potential way of controlling the major insect pest Colorado potato beetle (CPB; Leptinotarsa decemlineata Say). The present study describes the Agrobacterium-mediated transformation of potato (cv. Kennebec) with an oryzacystatin I (OCI) cDNA clone linked to a CaMV 35S promoter. The transgenic plants accumulated active OCI in potato leaves, as demonstrated by the papain-inhibitory activity of transgenic plant leaf extracts. In addition to their anti-papain activity, the extracts also caused a partial but significant inhibition of CPB digestive proteinases, similar to that observed with pure inhibitors. Recombinant OCI did not alter the activity of the major potato leaf endogenous proteinases, which seemed to be of the serine-type. Therefore we suggest that the OCI cDNA can be used for the production of CPB-resistant transgenic potato plants without interfering with endogenous proteinases of these plants.

Complete nucleotide sequence of the maize (Zea mays L.) sucrose synthase 2 cDNA.

Suc synthase (UDP-G~C:D-FIW 2-glucosyl-transferase, EC 2.4.1.13) is an enzyme that catalyzes the reversible conversion of UDP and Suc to UDP-Glc and Fru. In maize (Zea mays L.), two isozymes of Suc synthase, SSl and SS2, have been described previously (Chourey, 1981). The two proteins are biochemically similar and are immunologically crossreactive. However, the two isoforms are readily separable by nondenaturing (Chourey et al., 1988) and SDS electrophoretic analysis, and a difference of 6 kD is detectable (NguyenQuoc et al., 1990). In addition, the cellular distribution of the two molecules varies as well (Chen and Chourey, 1989; Heinlein and Starlinger, 1989). SS1 is present most abundantly in the developing kemels, where it accounts for as much as 10% of the total protein, whereas SS2 can reach up to 3% in heterotrophic leaves (Nguyen-Quoc et al., 1990). Genetic (Chourey, 1981), biochemical (Chourey, 1981; Echt and Chourey, 1985), and molecular analysis have demonstrated that SS1 and SS2 isozymes are encoded by separate genes, designated Shl and Susl. The Shl locus has been cloned and its entire DNA sequence determined (Werr et al., 1985). It contains 16 exons and yields a mature mRNA of 2746 bp that encodes a peptide of 802 amino acids. The Susl gene has also been cloned and mapped to chromosome 9, approximately 40 map units from the Shl locus (McCarty et al., 1986; Gupta et al., 1988). As a further step toward understanding the differential regulation of the expression of the two SS genes, we are working on the second Suc synthase gene, Susl. In this paper, we report the complete sequence of the SS2 cDNA corresponding to its mRNA sequence (Table I). The complete sequence was determined by sequencing a cDNA clone containing an incomplete coding sequence of SS2 as described previously (Gupta et al., 1988). The missing 5’ end sequence was completed by using the 5’ AmpliFINDER RACE Kit (Clontech) and poly(A)+-rich RNA isolated from young leaves of maize sh bz-m4 mutant, which cames a complete deletion of the Shl locus. The combination of the two partia1 clones yields a sequence of 2914 nucleotides in length with an open reading frame encoding a putative peptide of 763 amino acids. Compared

Genetic and Molecular Genetic Regulation of Soluble and Insoluble Carbohydrate Composition in Tomato

Fruit quality is determined by the amount and composition of soluble and insoluble solids. In tomato fruit the soluble solids are comprised primarily of mono and disaccharides, whereas the insoluble solids are comprised primarily of cell wall polysaccharides. To assess the feasibility of improving tomato fruit quality by manipulating monogenic traits, genetic and molecular genetic manipulation of soluble and insoluble carbohydrate composition has been carried out. A trait reversing the monosaccharide to disaccharide ratio of soluble carbohydrate in tomato fruit has been transferred from Lycopersicon chmielewskii to Lycopersicon esculentum and shown to be governed by a single recessive gene. Biochemical characterization of this trait has led to the tentative cloning of a gene that may control this trait, thus providing the means to genetically engineer one aspect of soluble sugar composition. Insoluble polysaccharides, including pectins and hemicelluloses, are degraded during tomato fruit ripening. To manipulate the extent and timing of this change in cell wall polymer structure, we have cloned genes encoding pectin and hemicellulose-degrading enzymes and are using these genes to modify their expression in transgenic plants. Together, these experiments are providing the basis to assess the feasibility of achieving incremental enhancement of complex traits that control components of fruit quality by identifying specific biochemical targets for genetic manipulation.

Regeneration of Transgenic Plants from Electroporated Protoplasts of Asparagus officinalis L

An effective method for consistent regeneration of transgenic asparagus (Asparagus officinalis L) plants from electroporated protoplasts is described. Transgenic plants containing β-glucuronidase (GUS) and neomycin-phosphotransferase (NPT II) genes were obtained by electroporating callus-derived protoplasts of Asparagus officinalis L. Embryogenic callus tissue and plants from four kanamycin resistant lines expressed P-glucuronidase activity, as revealed by histological staining. The amplification of genomic DNA by polymerase chain reaction revealed the presence of both GUS and NPT II genes in transformed callus tissue and plants. Southern hybridization confirmed the integration of these genes into the asparagus genome.