ABSTRACT
Novel forms of Pin-II type proteinase inhibitor (PIs) cDNAs (CanPIs) having three or four inhibitory repeat domains (IRD) were isolated from the developing green fruits of Capsicum annuum. Deduced amino acid (aa) sequences of the CanPIs showed up to 15% sequence divergence among each other or reported inhibitors (CanPI-1AF039398, CanPI-2AF221097). Amino acid sequence analysis of these CanPIs revealed that three IRD PIs have trypsin inhibitory sites, while four IRD CanPIs have both trypsin and chymotrypsin inhibitory sites. Four CanPIs, two having three IRD (CanPI-3AY986465 and CanPI-5DQ005912) and two having four IRD (CanPI-7DQ005913 and CanPI-9DQ005915), were cloned in Pichia pastoris to express recombinant CanPIs. Recombinant CanPIs inhibited 90% of bovine trypsin (TI), while chymotrypsin inhibition (CI) varied with the number of chymotrypsin inhibitory sites in the CanPIs. Recombinant inhibitors inhibited over 70% of the gut proteinase activity of Helicoverpa armigera. H. armigera larvae fed on recombinant CanPIs individually incorporated into artificial diet, showed 35% mortality; in addition, weight gain in H. armigera larvae and pupae was severely reduced compared to controls. Of the four CanPIs, CanPI-7, which has two sites for TI and CI, was the only one to have a consistently antagonistic effect on H. armigera growth and development. We conclude that among the four recombinant PIs tested, CanPIs containing diverse IRDs are best suited for developing insect-resistant transgenic plants.
Subject(s)
Capsicum , Moths/growth & development , Peptide Hydrolases/pharmacology , Plant Proteins/chemistry , Protease Inhibitors/pharmacology , Amino Acid Sequence , Animals , Chymotrypsin/antagonists & inhibitors , Chymotrypsin/metabolism , Cloning, Molecular , Codon, Terminator , DNA, Complementary , Escherichia coli/genetics , Fruit/chemistry , Fruit/metabolism , Larva/drug effects , Larva/metabolism , Molecular Sequence Data , Peptide Hydrolases/chemistry , Peptide Hydrolases/genetics , Pichia/genetics , Plant Proteins/genetics , Plant Proteins/pharmacology , Protease Inhibitors/analysis , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , Protein Sorting Signals , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacology , Sequence Homology, Amino Acid , Transformation, Genetic , Trypsin/metabolism , Trypsin Inhibitors/chemistry , Trypsin Inhibitors/metabolism , Trypsin Inhibitors/pharmacologyABSTRACT
Tomato (Lycopersicon esculentum, Mill; cultivar- Dhanashree) proteinase inhibitors (PIs) were tested for their trypsin inhibitory (TI) and Helicoverpa armigera gut proteinases inhibitory (HGPI) activity in different organs of the tomato plants. Analysis of TI and HGPI distribution in various parts of the plant showed that flowers accumulated about 300 and 1000 times higher levels of TI while 700 and 400 times higher levels of HGPI as compared to those in leaves and fruits, respectively. Field observation that H. armigera larvae infest leaves and fruits but not the flowers could be at least partially attributed to the protective role-played by the higher levels of PIs in the flower tissue. Tomato PIs inhibited about 50-80% HGP activity of H. armigera larvae feeding on various host plants including tomato, of larvae exposed to non-host plant PIs and of various larval instars. Tomato PIs were found to be highly stable to insect proteinases wherein incubation of inhibitor with HGP even for 3h at optimum conditions did not affect inhibitory activity. Bioassay using H. armigera larvae fed on artificial diet containing tomato PIs revealed adverse effect on larval growth, pupae development, adult formation and fecundity.
Subject(s)
Flowers/metabolism , Food Preferences , Fruit/metabolism , Moths/physiology , Plant Leaves/metabolism , Protease Inhibitors/metabolism , Solanum lycopersicum/metabolism , Animals , Cattle , Chymotrypsin/antagonists & inhibitors , Chymotrypsin/metabolism , Flowers/chemistry , Fruit/chemistry , Solanum lycopersicum/anatomy & histology , Solanum lycopersicum/drug effects , Solanum lycopersicum/parasitology , Moths/growth & development , Plant Leaves/chemistry , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Trypsin/metabolism , Trypsin Inhibitors/chemistry , Trypsin Inhibitors/metabolism , Trypsin Inhibitors/pharmacologyABSTRACT
Two serine proteinases from the midgut of Helicoverpa armigera have been partially purified and characterized. One proteinase, HGP-1, was capable of hydrolyzing a synthetic substrate of elastase and was inhibited by elastatinal. The second proteinase, HGP-2, was inhibited by a trypsin inhibitor. Molecular weights of HGP-1 and HGP-2 were approximately 26.0 and 29.0kDa, respectively. Both the proteinases exhibited alkaline pH optima in the range of 10-11. Furthermore, interaction of HGP-1 and HGP-2 with proteinase inhibitors (PIs) from host and non-host plants was studied. HGP-1 was not only insensitive to a PI from chickpea (host) but was also able to degrade it. The same PI from chickpea was able to inhibit over 50% activity of HGP-2. On the contrary, PIs from potato (non-host) showed strong inhibition of both, HGP-1 and HGP-2 and also demonstrated protection of chickpea seed proteins from digestion by both the HGPs. These results could provide important clues in designing strategies for sustainable use of plant PIs in developing insect-tolerant transgenic plants.
Subject(s)
Cicer/chemistry , Moths/enzymology , Protease Inhibitors/pharmacology , Serine Endopeptidases/metabolism , Animals , Cicer/metabolism , Feeding Behavior , Gastrointestinal Tract/enzymology , Larva/enzymology , Plant Proteins/metabolism , Protease Inhibitors/metabolism , Substrate SpecificityABSTRACT
Relative quantification of reported gut proteinase cDNAs from Helicoverpa armigera larvae fed on various host plants (cotton, chickpea, pigeonpea, tomato and okra), non-host plant PIs (winged bean, bitter gourd, ground nut, and capsicum) and during larval development has been carried out using semi-quantitative RT-PCR. Five trypsin-like and three chymotrypsin-like proteinases were categorized as insensitive or sensitive to most of the proteinase inhibitors (PIs) and insensitive/sensitive to specific PIs based on their expression analysis. These results were supported by amino acid sequence analysis, where diverged amino acids were observed in the regions, which are reported to be involved in typical trypsin-trypsin inhibitor interactions and critical for proteinase inhibitor resistance. Among exopeptidases (five aminopeptidase and three carboxypeptidase), HaAmi4 and HaAmi5 of aminopeptidase and HaCar1 of carboxypeptidase exhibited considerable differential expression. Elastase and cathepsin B-like proteinases were expressed at negligible levels. The proteases identified in the study would be ideal candidates for further interactions studies with protease inhibitors to understand the structural reasons of protease inhibitor insensitivity.
Subject(s)
Digestive System/enzymology , Gene Expression Regulation, Enzymologic , Moths/enzymology , Peptide Hydrolases/genetics , Animals , Base Sequence , DNA Primers , Larva/enzymology , Moths/growth & development , Peptide Hydrolases/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Two proteinase inhibitors (PIs), CapA1 and CapA2, were purified from Capsicum annum Linn. Var. Phule Jyoti leaves and assessed for their in vitro and in vivo activity against Helicoverpa armigera gut proteinases (HGPs). Both the inhibitors exhibited molecular weights of about 12 kDa with inhibitory activity against bovine trypsin and chymotrypsin indicating presence of probable two-inhibitor repeats of PIN II family. CapA1 and CapA2 inhibited 60-80% HGP (azocaseinolytic) activity of fourth instar larvae feeding on various host plants while 45-65% inhibition of HGP activity of various instars (II to VI) larvae reared on artificial diet. The partial purification of HGP isoforms, their characterization with synthetic inhibitors and inhibition by C. annum PIs revealed that most of the trypsin-like activity (68-91%) of HGPs was sensitive to C. annum PIs while 39-85% chymotrypsin-like activity of HGPs was insensitive to these inhibitors. The feeding of C. annum leaf extracts and two purified PIs in various doses to H. armigera larvae for two successive generations through artificial diet demonstrated their potential in inhibiting larval growth and development, delay in pupation period and dramatic reduction in fecundity and fertility. This is the first report-demonstrating efficacy of C. annum PIs against insect gut proteinases as well as larval growth and development of H. armigera.
