Effects of mti-2

Overexpressed in transgenic plants, protease inhibitors showed insecticidal effects against several insect taxa. We transformed potato internodes with the mustard trypsin inhibitor mti-2 gene. Among the 35 independent transgenic potato lines obtained via Agrobacterium tumefasciens transformation, four (DM6, DM7, DM11, and DM19) were selected for their high level of MTI-2 (at least to 30% of trypsin activity inhibition). Feeding assays were carried out to evaluate their effects on the green-peach aphid, Myzus persicae (Sternorrhyncha: Aphididae). Prereproductive period, nymphal mortality, adult fecundity, and doubling time of M. persicae populations were monitored on nontransformed potato plants (NT) and the four selected DM lines. Compared to NT plants, DM19 did not induce any effect on M. persicae. In contrast, DM7 and DM11 increased nymphal survival by approximately 20%. DM6 and DM11 lines slightly enhanced M. persicae daily fecundity and intrinsic rate of natural increase, leading to a reduction of the doubling time of the populations by 1 day. DM6 did not impact nymphal mortality, whereas with the DM11 almost all the nymphs survived. Potato plants transformed with the mti-2 gene variably affected the life history of M. persicae but did not show any insecticidal effect on the aphid

Uses of phage display in agriculture: A review of food-related protein-protein interactions discovered by biopanning over diverse baits

This review highlights discoveries made using phage display that impact the use of agricultural products. The contribution phage display made to our fundamental understanding of how various protective molecules serve to safeguard plants and seeds from herbivores and microbes is discussed. The utility of phage display for directed evolution of enzymes with enhanced capacities to degrade the complex polymers of the cell wall into molecules useful for biofuel production is surveyed. Food allergies are often directed against components of seeds; this review emphasizes how phage display has been employed to determine the seed component(s) contributing most to the allergenic reaction and how it has played a central role in novel approaches to mitigate patient response. Finally, an overview of the use of phage display in identifying the mature seed proteome protection and repair mechanisms is provided. The identification of specific classes of proteins preferentially bound by such protection and repair proteins leads to hypotheses concerning the importance of safeguarding the translational apparatus from damage during seed quiescence and environmental perturbations during germination. These examples, it is hoped, will spur the use of phage display in future plant science examining protein-ligand interactions

Purification of a trypsin inhibitor from Cocculus hirsutus and identification of its biological activity

Proteinase inhibitors play a significant role in plant defense against insect pests and phytopathogens by inhibiting their proteases. A thermotolerant monomeric trypsin inhibitor with molecular weight ∼18kD was purified from Cocculus hirsutus (ChTI) using trypsin sepharose affinity column. Western blot analysis using ChTI IgY revealed its presence in vegetative parts and seeds. The second and third instar larvae of H. armigera fed with ChTI (5000TIU/ml) resulted in 84.59 and 58.71% reduction in mean larval weight respectively. An increase in the larval growth period was observed in ChTI fed larvae at all instars and inhibitor fed larvae could not complete their life cycle. ChTI caused 74 and 59.53% inhibition of bovine trypsin and Helicoverpa gut proteases respectively. ChTI exhibited strain specificity and inhibited growth and development of plant fungal pathogens. Bioassay studies on yeast strains indicated that ΔYNK and MNN1 are more sensitive to ChTI. The results suggest that phosphodiester linkage in cell wall components is likely to be the key determinants for binding of ChTI. Taken together, these studies indicate that ChTI is a potential candidate for development of transgenic plants against foliar diseases and insect pests

Plant improvement for insect resistance: Testing of the candidate organism Beauveria bassiana, transgenic tobacco expressing protease inhibitors, and rapid screen of insect resistance genes in an agroinfiltration transient expression system

This study focused on three aspects of plant improvement for insect resistance including: testing of candidate organisms for their production of insecticidal proteins, testing of transgenic plants expressing insect resistance genes, and testing novel systems for the evaluation of insect resistance genes. In the initial part of this study, the candidate fungus Beauveria bassiana was tested for its production of insecticidal proteins through a series of insect bioassays containing fungal protein extracts. These extracts were shown to be orally toxic to Plutella xylostella (diamondback moth) and Spodoptera frugiperda (fall armyworm). Assays involving protease treatments significantly decreased mortality indicating the presence of a protein based oral toxin. The following research tested transgenic tobacco plants expressing proteinase inhibitors from Brassica oleracea (cabbage) and Manduca sexta (tobacco hornworm) on the insect pests Helicoverpa zea (corn earworm) and Heliothis virescens (tobacco budworm). Insects fed transgenic tobacco were able to adapt to the recombinant proteinase inhibitors to varying degrees and resulted in no major impacts on insect growth and development. The last part of this study tested a novel insect resistance gene screening system. Agroinfiltrated tobacco transiently co-expressing genes encoding GFP with either a known insecticidal protein (Bt Cry1Ac) or a candidate gene (Brassica oleracea proteinase inhibitor, BoPI) were fed to larval H. zea. Insects fed the known insecticidal protein experienced high mortality. Insects fed tobacco expressing GFP and BoPI showed significant decreases in growth compared to those fed GFP only tissue. Insects feeding on GFP only tissue showed unexpected increases in growth and development compared to insects fed control tissue. Agroinfiltration coupled with an insect bioassay constitutes an efficient system for the evaluation of candidate insect resistance genes

Isolation of a serine Kunitz trypsin inhibitor from leaves of Terminalia arjuna

A serine Kunitz protease inhibitor was isolated from the semi-mature leaves of Terminalia arjuna, a host plant for Antheraea mylitta, using ammonium sulphate fractionation, gel permeation chromatography and trypsin–sepharose affinity chromatography. A 29-fold purification of T. arjuna Trypsin Inhibitor (TaTI) with a yield recovery of 3.2% was achieved. The purified protease inhibitor (TaTI) was resolved into a single protein band corresponding to molecular weight of 19.0 kDa on 12% SDS–PAGE under non-reducing conditions, whereas an additional band of 21.5 kDa was observed when the same fraction was resolved on SDS–PAGE under reducing conditions in the presence of 2-mercaptoethanol. TaTI inhibited both trypsin and chymotrypsin, but showed higher affinity for trypsin compared to chymotrypsin. However, it is more effective on bovine trypsin than midgut trypsin of tasar silkworm. TaTI retains its activity over a wide range of temperatures (0–100°C) and pH (2.0–8.0), with pH optimum of 8.0. These observations indicate that TaTI is not only specific to tasar silkworm but also to bovine serine proteases. Hence it can be considered as a generalist protease inhibitor

In Vitro Antibacterial Activity of Cysteine Protease Inhibitor from Kiwifruit (Actinidia deliciosa)

The need for replacing traditional pesticides with alternative agents for the management of agricultural pathogens is rising worldwide. In this study, a cysteine proteinase inhibitor (CPI), 11 kDa in size, was purified from green kiwifruit to homogeneity. We examined the growth inhibition of three plant pathogenic Gram-negative bacterial strains by kiwi CPI and attempted to elucidate the potential mechanism of the growth inhibition. CPI influenced the growth of phytopathogenic bacteria Agrobacterium tumefaciens (76.2 % growth inhibition using 15 mu M CPI), Burkholderia cepacia (75.6 % growth inhibition) and, to a lesser extent, Erwinia carotovora (44.4 % growth inhibition) by inhibiting proteinases that are excreted by these bacteria. Identification and characterization of natural plant defense molecules is the first step toward creation of improved methods for pest control based on naturally occurring molecules

A genome walking strategy for the identification of eukaryotic nucleotide sequences adjacent to known regions

Determination of nucleotide sequences adjacent to a known region is a recurring need in many genome scale studies. Various methods have been developed based on PCR techniques in order to fulfill th..

Purification and partial characterisation of a protease inhibitor from Mimosa diplotricha

Search for inhibitors to insect proteases is one of many strategies to control pests. Previous work has demonstrated successful purification of effective inhibitors from plant origin. Thus, the current study attempted to purify protease inhibitors from locally available medicinal plants. The study demonstrated that the ethanolic extracts of Mimosa diplotricha leaves caused a significant 80% reduction in bovine trypsin activity. The inhibitory property of the proteinaceous nature of the extract was reconfirmed through qualitative analysis using the detection of trypsin inhibitors on the SDS-PAGE technique. The ammonium precipitated trypsin inhibitor was purified using Hi-Trap G25 and resolved into a single band with a molecular weight of approximately 20.8 kDa. By using the Dixon plot the competitive inhibitor has a Ki value of 2.16 × 10-4 mM. The purified protein inhibited the protease extract of Chrysomya megacephala at IC50 of 28 μg/mL. The results highlighted the presence of trypsin inhibitor in Mimosa diplotricha and its potential as a pest control agent

Identification and characterisation of tomato torrado virus, a new plant picorna-like virus from tomato

A new virus was isolated from tomato plants from the Murcia region in Spain which showed symptoms of ‘torrado disease’ very distinct necrotic, almost burn-like symptoms on leaves of infected plants. The virus particles are isometric with a diameter of approximately 28 nm. The viral genome consists of two (+)ssRNA molecules of 7793 (RNA1) and 5389 nts (RNA2). RNA1 contains one open reading frame (ORF) encoding a predicted polyprotein of 241 kDa that shows conserved regions with motifs typical for a protease-cofactor, a helicase, a protease and an RNA-dependent RNA polymerase. RNA2 contains two, partially overlapping ORFs potentially encoding proteins of 20 and 134 kDa. These viral RNAs are encapsidated by three proteins with estimated sizes of 35, 26 and 23 kDa. Direct protein sequencing mapped these coat proteins to ORF2 on RNA2. Phylogenetic analyses of nucleotide and derived amino acid sequences showed that the virus is related to but distinct from viruses belonging to the genera Sequivirus, Sadwavirus and Cheravirus. This new virus, for which the name tomato torrado virus is proposed, most likely represents a member of a new plant virus genus