Differential expression of the potato proteinase inhibitor II promoter in transgenic tobacco

We studied temporal and spatial expression patterns of the potato proteinase inhibitor II (PI-II) promoter, using transgenic tobacco (Nkotiana tabacum L cv. Xanthi) plants that carried a fusion between the PI-II promoter and the chloramphenicol acetyltransferase (cat) gene. Pl-ll promoter activity was low when plants were young, but increased as plants grew. In 8-week-old plants, old leaves showed higher activity than young leaves. At flowering stage (ca. 15 weeks), the overall promoter activity was reduced to a lower level except in the petals. Compared with stems or petioles at the flowering stage, the roots and floral organs showed minimal activity for the Pl-ll promoter. We used several environmental stimuli to examine the induction of the Pl-ll promoter in different organs. Promoter induction was effected by wounding or methyl jasmonate in stems, petioles, sepals, and leaves. The induction was highest in leaves, as was sucrose-enhanced wound induction. These results suggest that the Pl-ll gene is temporally and spatially regulated. We also established a transient assay system in tobacco BY2 suspension cells to elucidate the upstream regulatory region of the Pl-ll promoter. A field strength of 0.75 kV/cm and 400 μF capacitance were optimal electroporation conditions for our transient assay.     

Sugar response element enhances wound response of potato proteinase inhibitor II promoter in transgenic tobacco

The promoter region of the potato proteinase inhibitor II (PI-II) gene was studied to identifycis-acting regulatory sequences involved in sugar response using transgenic tobacco plants. The 5 control region covering an 892 nucleotide sequence upstream from the cap site and a 32 nucleotide untranslated region of the PI-II promoter was able to activate a reporter chloramphenicol acetyltransferase (cat) gene by wounding or by incubating in a sugar-free medium. This wound response was further enhanced by sugar. Hexoses, disaccharides, and some trisaccharides were strong inducers whereas pentoses, deoxy sugars, sugar acids, TCA cycle intermediates, amino acids, and other carbohydrates had little effect on the promoter activity. Deletion of the sequence between-892 and-573 abolished the wound response but not the sugar response. An additional 5 deletion to-453 removed the sugar inducibility. Locations of thecis-acting regulatory elements were further elucidated by 3 deletion analysis. Deletion of the downstream region from-520 did not affect the wound of sugar response of the promoter. However, 3 deletion mutant-574 was unable to respond to sugar but did respond weakly to wounding. Further deletion to-624 abolished both responses. Therefore, it can be concluded that a wound response element is located in between-624 and-574 and that the response is further enhanced by a sugar response element located in the sequence between-573 and-520.     

First report on a potato I family chymotrypsin inhibitor from the seeds of a Cucurbitaceous plant, Momordica cochinchinensis

A 7514-Da chymotrypsin inhibitor was isolated from the seed extract of Momordica cochinchinensis (Family Cucurbitaceae) by chromatography on chymotrypsin-Sepharose 4B and subsequently by C18 reversed-phase HPLC. This inhibitor, named MCoCl, possessed remarkable thermostability and was stable from pH 2 to 12. MCoCl also inhibited subtilisin, but had at least 50-fold lower inhibitory activity towards trypsin and elastase. Amino acid sequencing of a peptide fragment of MCoCl revealed a sequence of 23 amino acids. Comparison of this sequence and the molecular mass with those of other protease inhibitors suggests that MCoCl belongs to the potato I inhibitor family.     

Selective loss of cysteine residues and disulphide bonds in a potato proteinase inhibitor II family

Disulphide bonds between cysteine residues in proteins play a key role in protein folding, stability, and function. Loss of a disulphide bond is often associated with functional differentiation of the protein. The evolution of disulphide bonds is still actively debated; analysis of naturally occurring variants can promote understanding of the protein evolutionary process. One of the disulphide bond-containing protein families is the potato proteinase inhibitor II (PI-II, or Pin2, for short) superfamily, which is found in most solanaceous plants and participates in plant development, stress response, and defence. Each PI-II domain contains eight cysteine residues (8C), and two similar PI-II domains form a functional protein that has eight disulphide bonds and two non-identical reaction centres. It is still unclear which patterns and processes affect cysteine residue loss in PI-II. Through cDNA sequencing and data mining, we found six natural variants missing cysteine residues involved in one or two disulphide bonds at the first reaction centre. We named these variants Pi7C and Pi6C for the proteins missing one or two pairs of cysteine residues, respectively. This PI-II-7C/6C family was found exclusively in potato. The missing cysteine residues were in bonding pairs but distant from one another at the nucleotide/protein sequence level. The non-synonymous/synonymous substitution (Ka/Ks) ratio analysis suggested a positive evolutionary gene selection for Pi6C and various Pi7C. The selective deletion of the first reaction centre cysteine residues that are structure-level-paired but sequence-level-distant in PI-II illustrates the flexibility of PI-II domains and suggests the functionality of their transient gene versions during evolution.     

Purification and characterization from tobacco (Nicotiana tabacum) leaves of six small, wound-inducible, proteinase isoinhibitors of the potato inhibitor II family.

Six small molecular mass, wound-inducible trypsin and chymotrypsin inhibitor proteins from tobacco (Nicotiana tabacum) leaves were isolated to homogeneity. The isoinhibitors, cumulatively called tobacco trypsin inhibitor (TTI), have molecular masses of approximately 5500 to 5800 D, calculated from gel filtration analysis and amino acid content. The amino acid sequence of the entire 53 residues of one isoinhibitor, TTI-1, and the sequence of 36 amino acid residues from the N terminus of a second isoinhibitor, TTI-5, were determined. The two isoinhibitors differ only at residue 11, which is threonine in TTI-1 and lysine in TTI-5. The isoinhibitors are members of the potato inhibitor II family and show considerable identity with the small molecular mass members of this family, which include the eggplant inhibitor, two small molecular mass trypsin and chymotrypsin inhibitors from potatoes, and an inhibitor from pistils of the ornamental plant Nicotiana alata. Antibodies produced against the isoinhibitors in rabbits were used in radial immunoassays to quantify both the systemic wound inducibility of TTI in tobacco leaves and its constitutive levels in flowers.     

Accumulation of a chymotrypsin inhibitor in transgenic tobacco can affect the growth of insect pests

A member of the potato proteinase inhibitor II (PPI II) gene family that encodes for a chymotrypsin iso-inhibitor has been introduced into tobacco (Nicotiana tabacum) usingAgrobacterium tumefaciens-mediated T-DNA transfer. Analysis of the primary transgenic plants (designated R₀) confirmed that the introduced gene is being expressed and the inhibitor accumulates as an intact and fully functional protein. For insect feeding trials, progeny from the self-fertilization of R₀ plants (designated R₁) were used. Leaf tissue, either from transgenic or from control (non-transgenic) plants, was fed to larvae ofChrysodeixis eriosoma (Lepidoptera: Noctuidae, green looper),Spodoptera litura (F.) (Lepidoptera: Noctuidae) andThysanoplusia orichalcea (F.) (Lepidoptera: Noctuidae) and insect weight gain (increase in fresh weight) measured. Consistently,C. eriosoma larvae fed leaf tissue from transgenic plants expressing thePPI II gene grew slower than insects fed leaf tissue from non-transgenic plants or transgenic plants with no detectablePPI II protein accumulation. However, larvae of bothS. litura andT. orichalcea consistently demonstrated similar or faster growth when fed leaf tissue from transgenic plants compared with those fed non-transgenic plants. In agreement with the feeding trials, the chymotrypsin iso-inhibitor extracted from transgenic tobacco effectively retarded chymotrypsin-like activity measured inC. eriosoma digestive tract extracts, but not in extracts fromS. litura. We conclude, therefore, that for certain insects the use of chymotrypsin inhibitors should now be evaluated as an effective strategy to provide field resistance against insect pests in transgenic plants, but further, that a single proteinase inhibitor gene may not be universally effective against a range of insect pests. The significance of these observations is discussed with respect to the inclusion of chymotrypsin inhibitors in the composite of insect pest resistance factors that have been proposed for introduction into crop plants.     

Structure and induction pattern of a novel proteinase inhibitor class II gene of tobacco

A cDNA and a corresponding genomic clone encoding a protein with partial identity to type II proteinase inhibitors from potato, tomato and Nicotiana alata, were isolated from tobacco libraries. The protein of 197 amino acids contains a putative signal peptide of 24 residues and three homologous domains, each with a different reactive site. The tobacco PI-II gene is not expressed in leaves of healthy plants, but is locally induced in leaves subjected to different types of stress (TMV infection, wounding, UV irradiation) and upon ethephon treatment. As opposed to the analogous PI-II genes of potato and tomato, the tobacco gene is not systemically induced by wounding or pathogenic infection. A far-upstream region in the PI-II promoter, containing various direct and indirect repeats, shares considerable sequence similarity to a similar region in the stress-inducible Cu/Zn-superoxide dismutase gene of N. plumbaginifolia.     

Both wound-inducible and tuber-specific expression are mediated by the promoter of a single member of the potato proteinase inhibitor II gene family

A chimeric gene consisting of 1.3 kb of the 5' regulatory region of a member of the potato proteinase inhibitor II gene family, the coding region of the bacterial β-glucuronidase (GUS) gene and 260 bp of the proteinase inhibitor II 3'-untranslated region containing the poly(A) addition site was introduced into potato and tobacco by Agrobacterium tumefaciens mediated transformation. Analysis of transgenic plants demonstrates systemic, wound-inducible expression of this gene in stem and leaves of potato and tobacco. Constitutive expression was found in stolons and tubers of non-wounded potato plants. Histochemical experiments based on the enzymatic activity of the GUS protein indicate an association of the proteinase inhibitor II promoter activity with vascular tissue in wounded as well as in systemically induced non-wounded leaves, petioles, potato stems and in developing tubers. These data prove that one single member of the proteinase inhibitor II gene family contains cis-active elements, which are able to respond to both developmental and environmental signals. Furthermore they support the hypothesis of an inducing signal (previously called proteinase inhibitor inducing factor), which is released at the wound site and subsequently transported to non-wounded parts of the plant via the vascular system from where it is released to the surrounding tissue.     

Inhibition of Colorado potato beetle larvae by a locust proteinase inhibitor peptide expressed in potato

The cDNA for a 73-mer peptide containing two locust serine proteinase inhibitors was cloned, fused to the constitutive CaMV35S promoter and introduced into potato by Agrobacterium-mediated transformation. From 23 independent transgenic lines, three with high mRNA level and proteinase inhibitory activity were propagated in vitro and transferred to pots. The peptide from the leaves was identified by its N-terminal sequence and by K_i values against chymotrypsin and trypsin. Colorado potato beetle larvae reared on transgenic plants grew slightly but significantly more slowly than those on control plants. This supports the notion that expression of multifunctional proteinase inhibitors of insect origin might be a good strategy to improve insect resistance in plants.     

LTCI, a novel chymotrypsin inhibitor of the potato I family from the earthworm Lumbricus terrestris. Purification, cDNA cloning, and expression

A novel chymotrypsin inhibitor of the potato I protease inhibitor family from the earthworm Lumbricus terrestris was purified. The inhibitor, named LTCI, was isolated by methanol extraction, affinity chromatography on immobilized methylchymotrypsin, and ion exchange chromatography followed by RP–HPLC. The 7076 Da inhibitor consists of a single polypeptide chain of 64-amino-acid residues without disulfide bridges. LTCI is the first of the potato I protease inhibitors with Tyr in position P1 of the reactive site. cDNA analysis revealed that LTCI is produced as a 86-amino-acid precursor with a 22-amino-acid secretory signal peptide. RT–PCR analysis demonstrates that LTCI mRNA is expressed in body wall, intestine, and coelomocytes. The recombinant LTCI was produced in Escherichia coli as a fusion protein with intein and chitin binding domain using IMPACT™–CN system.     

Phage display of a double-headed proteinase inhibitor: Analysis of the binding domains of potato proteinase inhibitor II

Potato proteinase inhibitor II (PI2) is a serine proteinase inhibitor composed of two domains that are thought to bind independently to proteinases. To determine the activities of each domain separately, various inactive and active domain combinations were constructed by substituting amino acid residues in the active domains by alanines. These derivatives were expressed as soluble protein inEscherichia coli and exposed on M13 phage as fusions to gene 3 in a phagemid system for monovalent phage display. Inactivation of both active domains by Ala residues reduced binding of phage to trypsin and chymotrypsin by 95%. Ten times more phage were bound to proteinases by domain II compared to domain I, while a point mutation (Leu⁵ Arg) altered the binding specificity of domain I of PI2 phage from chymotrypsin to trypsin. The mutants were used to show that functional PI2 phage mixed with nonfunctional PI2 phage could be enriched 323 000-fold after three rounds of panning. Thus, these results open up the possibility to use phage display for the selection of engineered PI2 derivatives with improved binding characteristics towards digestive proteinases of plants pests.The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number L37519 (p303.51).     

Functional analysis of the 3'' control region of the potato wound-inducible proteinase inhibitor II gene.

Proteinase inhibitor genes are expressed strongly in specific plant tissues under both developmental and environmental regulation. We have studied the role of the 3' control region of the potato proteinase inhibitor II gene (PI-II) that is inducible in leaves in response to herbivore attacks or other severe wounding. Comparison of the terminator from the PI-II gene with two different terminators from the 6b and 7 genes, driven by a common PI-II promoter-cat fusion molecule, indicated that the PI-II terminator provided the most efficient expression of cat. The PI-II terminator also caused a significantly elevated cat gene expression driven by the cauliflower mosaic virus 35S promoter. The increase in the level of expression is probably not due to the presence of an enhancer element in the PI-II terminator region, but to cis-acting elements involved in mRNA processing or stability. Both transient and stable transformation analyses of the deletion mutants in the 3'-flanking sequence indicated that about a 100-base pair DNA fragment surrounding the polyadenylation site is essential for the efficient gene expression. This region seems to consist of several regulatory elements, including the conserved sequence, CGTGTCTT, which is located 9 bases downstream from the polyadenylation site. The elements appear to contribute to the increased stability of mRNAs containing the PI-II terminator.     

Molecular interactions between an insect predator and its herbivore prey on transgenic potato expressing a cysteine proteinase inhibitor from rice

Transgenic plants expressing resistance to herbivorous insects may represent a safe and sustainable pest control alternative if they do not interfere with the natural enemies of target pests. Here we examined interactions between oryzacystatin I (OCI), a proteinase inhibitor from rice genetically engineered into potato (Solanum tuberosum cv. Kennebec, line K52) to increase resistance to insect herbivory, and the insect predator Perillus bioculatus. This stinkbug is a relatively specialized predator of caterpillars and leaf-beetle larvae, and may also include plant sap in its predominantly carnivorous diet. One of its preferred prey is Colorado potato beetle (Leptinotarsa decemlineata), a major target of insect resistance development for potato field crops. Gelatin/sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed that a major fraction of proteinase (gelatinase) activity in P. bioculatus extracts is OCI-sensitive. Among five gelatinolytic bands detected, the slowest-moving one (proteinase I) was inhibited strongly by purified OCI expressed in Escherichia coli or by OCI-transgenic potato extracts, while three other proteinases were partly sensitive to these treatments. There was also evidence of slight inhibition of proteinase I by untransformed potato foliage, suggesting the presence of a natural inhibitor related to OCI at low level in potato foliage. Interestingly, only about 50% of the maximum potential activity of proteinase I was recovered in extracts of P. bioculatus feeding on L. decemlineata larval prey on a diet of OCI-potato foliage, indicating that the predator was sensitive to OCI in the midgut of its prey. However, P. bioculatus on OCI-prey survived, grew and developed normally, indicating ability to compensate prey-mediated exposure to the OCI inhibitor. Confinement of P. bioculatus to potato foliage provided no evidence that potato plant-derived nutrition is a viable alternative to predation, restriction to potato foliage in fact being inferior to free water for short-term survival of nonfeeding first-instar larvae. These results support the view that OCI, an effective inhibitor of a substantial fraction of digestive enzymatic potential in P. bioculatus, should not interfere with its predation potential when expressed in potato plants fed to its prey at a maximum level of approximately 0.8% of total soluble proteins in mature foliage.     

Adaptation of Helicoverpa armigera (Lepidoptera: Noctuidae) to a proteinase inhibitor expressed in transgenic tobacco

A giant taro proteinase inhibitor (GTPI) cDNA was expressed in transgenic tobacco using three different gene constructs. The highest expression level obtained was ca. 0.3% of total soluble protein when the cDNA was driven by the Arabidopsis rbcS ats1 promoter. Repeated feeding trials with Helicoverpa armigera larvae fed on clonally derived T₀ and T₁ plants expressing GTPI demonstrated that, relative to those fed on control plants, some growth inhibition (22–40%) occurs, but there was no increase in larval mortality. Proteinase activities of larvae fed on GTPI-expressing tobacco or GTPI-containing diet were examined to monitor the spectrum of digestive proteinases in the midgut. Total proteinase activity was reduced by 13%, but GTPI-insensitive proteinase activity was increased by up to 17%. Trypsin was inhibited by 58%, but chymotrypsin and elastase were increased by 26% and 16% respectively. These results point to an adaptive mechanism in this insect that elevates the levels of other classes of proteinases to compensate for the trypsin activity inhibited by dietary proteinase inhibitors.