Nitrogen source,concentration, and NH<Subscript>4</Subscript><Superscript>+</Superscript>:NO<Subscript>3</Subscript><Superscript>−</Superscript> ratio influence shoot regeneration and hyperhydricity in tissue cultured <Emphasis Type="Italic">Aloe polyphylla</Emphasis>

We report on the transformation and expression in sugar beet (Beta vulgaris) hairy roots of a Nicotiana alata NaPI gene encoding a serine proteinase inhibitor (PI) that has been shown to effectively reduce the population of a number of insect pests. Using in-gel analysis, two PI protein activities were detected at approximately 24- and/or 28-kDa in hairy roots generated via Agrobacterium rhizogenes-mediated gene transfer. Immunoblot analysis revealed the presence of the expected ~40 kDa precursor, and in some transformants, a ~20 kDa processing intermediate and the mature 6-kDa PIs. In general, processing of the precursor in the clonal lines was reduced or not detected. The reduced efficiency of post-translational processing of the N. alata PI precursor may be attributed to modification and/or altered folding of the recombinant protein or distinct post-translational machinery functioning in sugar beet hairy roots and Nicotiana. Disclaimer: Mention and/or use of a commercial or proprietary product to the exclusion of others does not constitute endorsement by the USDA.     

Expression of a plant cystatin confers partial resistance to Globodera,full resistance is achieved by pyramiding a cystatin with natural resistance

Constructs based on a cysteine proteinase inhibitor (cystatin) from sunflower and a protein engineered variant of a rice cystatin (Oc-ID86) conferred similar levels of resistance on potato plants to Globodera spp as chicken egg white cystatin (CEWC) under the control of CaMV35S. The level of resistance on challenge by 6.5 viable eggs Globodera spp g^–1 soil in a small scale field trial was similar to that provided by the natural partial resistance of cv. Sante. PCR of an internal transcribed spacer region of the ribosomal cistron of the nematode genome established that the challenging population was a mixture of G. pallida and G. rostochiensis with no evidence of a differential effect of the transgenic resistance on the reproductive success of the two species. Transformation of Sante and the South American cultivar Maria Huanca with CaMV35S/CEWC raised the status of both cultivars from partial to full resistance in this study. The results establish the potential of plant cystatins and demonstrate an additive effect of expressing CEWC with natural resistance genes.     

The serine acetyltransferase gene family in Arabidopsis thaliana and the regulation of its expression by cadmium

Expression of the serine acetyltransferase (SAT) gene family from Arabidopsis thaliana was investigated in response to treatment with the heavy metal cadmium (Cd). A fourth member of the SAT gene family, Sat-106, was also cloned and the complete SAT gene family from A. thaliana is discussed. Northern analysis of the gene family revealed tissue-specific expression patterns for each isogene. A. thaliana plants grown under 50 M CdCl₂ for a 24 h time course were also used for northern analysis. Expression of all SAT genes was increased to some extent by Cd treatment. Sat-5 expression showed particularly high levels of induction in the leaves of treated plants and was chosen for study by in situ hybridisation. Sat-5 expression was induced in the root and stem cortex and the leaf lamella and trichomes in response to heavy metal stress. SAT and its product O-acetylserine have previously been shown to be implicated in the control of sulphate reduction and cysteine biosynthesis in plants. These results suggest that specific SAT isoforms have a role in increasing cysteine production under conditions of heavy-metal stress when increased biosynthesis of glutathione and phytochelatins is required for detoxification purposes.     

Inducible expression of a fusion gene encoding two proteinase inhibitors leads to insect and pathogen resistance in transgenic rice

Plant proteinase inhibitors (PIs) are considered as candidates for increased insect resistance in transgenic plants. Insect adaptation to PI ingestion might, however, compromise the benefits received by transgenic expression of PIs. In this study, the maize proteinase inhibitor (MPI), an inhibitor of insect serine proteinases, and the potato carboxypeptidase inhibitor (PCI) were fused into a single open reading frame and introduced into rice plants. The two PIs were linked using either the processing site of the Bacillus thuringiensis Cry1B precursor protein or the 2A sequence from the foot‐and‐mouth disease virus (FMDV). Expression of each fusion gene was driven by the wound‐ and pathogen‐inducible mpi promoter. The mpi‐pci fusion gene was stably inherited for at least three generations with no penalty on plant phenotype. An important reduction in larval weight of Chilo suppressalis fed on mpi‐pci rice, compared with larvae fed on wild‐type plants, was observed. Expression of the mpi‐pci fusion gene confers resistance to C. suppressalis (striped stem borer), one of the most important insect pest of rice. The mpi‐pci expression systems described may represent a suitable strategy for insect pest control, better than strategies based on the use of single PI genes, by preventing insect adaptive responses. The rice plants expressing the mpi‐pci fusion gene also showed enhanced resistance to infection by the fungus Magnaporthe oryzae, the causal agent of the rice blast disease. Our results illustrate the usefulness of the inducible expression of the mpi‐pci fusion gene for dual resistance against insects and pathogens in rice plants.     

Assessment of Nematode Resistance in Wheat Transgenic Plants Expressing Potato Proteinase Inhibitor (<Emphasis Type="Italic">PIN2</Emphasis>) Gene

Serine proteinase inhibitors (IP’s) are proteins found naturally in a wide range of plants with a significant role in the natural defense system of plants against herbivores. The question addressed in the present study involves assessing the ability of the serine proteinase inhibitor in combating nematode infestation. The present study involves engineering a plant serine proteinase inhibitor (pin2) gene into T. durum PDW215 by Agrobacterium-mediated transformation to combat cereal cyst nematode (Heterodera avenae) infestation. Putative T₀ transformants were screened and positive segregating lines analysed further for the study of the stable integration, expression and segregation of the genes. PCR, Southern analysis along with bar gene expression studies corroborate the stable integration pattern of the respective genes. The transformation efficiency is 3%, while the frequency of escapes was 35.71%. χ² analysis reveals the stable integration and segregation of the genes in both the T₁ and T₂ progeny lines. The PIN2 systemic expression confers satisfactory nematode resistance. The correlation analysis suggests that at p < 0.05 level of significance the relative proteinase inhibitor (PI) values show a direct positive correlation vis-à-vis plant height, plant seed weight and also the seed number.     

The isolation and characterisation of the tapetum-specific Arabidopsis thaliana A9 gene

The Brassica napus cDNA clone A9 and the corresponding Arabidopsis thaliana gene have been sequenced. The B. napus cDNA and the A. thaliana gene encode proteins that are 73% identical and are predicted to be 10.3 kDa and 11.6 kDa in size respectively. Fusions of an RNase gene and the reporter gene -glucuronidase to the A. thaliana A9 promoter demonstrated that in tobacco the A9 promoter is active solely in tapetal cells. Promoter activity is first detectable in anthers prior to sporogenous cell meiosis and ceases during microspore premitotic interphase.The deduced A9 protein sequence has a pattern of cysteine residues that is present in a superfamily of seed plant proteins which contains seed storage proteins and several protease and -amylase inhibitors.     

Effective transgenic resistance to Globodera pallida in potato field trials

A cysteine proteinase inhibitor expressed in potato plants provides the first demonstration that transgenic resistance to nematodes such as the potato cyst nematode Globodera pallida can be effective under field conditions. The highest level of resistance obtained in the field for one of the four transformed lines of the normally fully susceptible Solanum tuberosum tuberosum cv. Désirée was 70±9%. The partially resistant cv. Sante that is currently of commercial use in the UK showed a resistance of 85±3%. In containment experiments a change in G. pallida population to one known to be virulent against cv. Sante caused a significant loss of its resistance from 80±4% to only 51±6%. In contrast, the resistance of transgenic Désirée was similar for challenge by the avirulent and virulent populations with values of 68±6% and 71±4%, respectively. Constitutive expression of the cystatin had no detrimental effect on either number or weight of tubers and the weight of the haulm for 3 of the 4 lines in the field. The results establish that transgenic field resistance against G. pallida can be achieved. Clearly food and environmental safety must be assured before commercialisation can be contemplated. However, a prima facie case can be made that the technology is benign and can reduce the use of environmentally hazardous nematicides.     

A Kunitz trypsin inhibitor from chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) that exerts anti-metabolic effect on podborer (<Emphasis Type="Italic">Helicoverpa armigera</Emphasis>) larvae

Chickpea (Cicer arietinum L.) seeds contain Bowman–Birk proteinase inhibitors, which are ineffective against the digestive proteinases of larvae of the insect pest Helicoverpa armigera. We have identified and purified a low expressing proteinase inhibitor (PI), distinct from the Bowman–Birk Inhibitors and active against H. armigera gut proteinases (HGP), from chickpea seeds. N-terminal sequencing of this HGP inhibitor revealed a sequence similar to reported pea (Pisum sativum) and chickpea -l-fucosidases and also homologous to legume Kunitz inhibitors. The identity was confirmed by matrix assisted laser desorption ionization – time of flight analysis of tryptic peptides and isolation of DNA sequence coding for the mature protein. Available sequence data showed that this protein forms a distinct phylogenetic cluster with Kunitz inhibitors from Glycine max, Medicago truncatula, P. sativum and Canavalia lineata. The isolated coding sequence was cloned into a yeast expression vector and produced as a recombinant protein in Pichia pastoris. -l-fucosidase activity was not detectable in purified or recombinant protein, by solution assays. The recombinant protein did not inhibit chymotrypsin or subtilisin activity but did exhibit stoichiometric inhibition of trypsin, comparable to soybean Kunitz trypsin inhibitor. The recombinant protein exhibited higher inhibition of total HGP activity as compared to soybean kunitz inhibitor, even though it preferentially inhibited HGP-trypsins. H. armigera larvae fed on inhibitor-incorporated artificial diet showed significant reduction in average larval weight after 18 days of feeding demonstrating potent antimetabolic activity. The over-expression of this gene in chickpea could act as an endogenous source of resistance to H. armigera.     

Three unrelated protease inhibitors enhance accumulation of pharmaceutical recombinant proteins in Nicotiana benthamiana

Agroinfiltrated Nicotiana benthamiana is a flexible and scalable platform for recombinant protein (RP) production, but its great potential is hampered by plant proteases that degrade RPs. Here, we tested 29 candidate protease inhibitors (PIs) in agroinfiltrated N. benthamiana leaves for enhancing accumulation of three unrelated RPs: glycoenzyme α‐Galactosidase; glycohormone erythropoietin (EPO); and IgG antibody VRC01. Of the previously described PIs enhancing RP accumulation, we found only cystatin SlCYS8 to be effective. We identified three additional new, unrelated PIs that enhance RP accumulation: N. benthamiana NbPR4, NbPot1 and human HsTIMP, which have been reported to inhibit cysteine, serine and metalloproteases, respectively. Remarkably, accumulation of all three RPs is enhanced by each PI similarly, suggesting that the mechanism of degradation of unrelated RPs follows a common pathway. Inhibitory functions HsTIMP and SlCYS8 are required to enhance RP accumulation, suggesting that their target proteases may degrade RPs. Different PIs additively enhance RP accumulation, but the effect of each PI is dose‐dependent. Activity‐based protein profiling (ABPP) revealed that the activities of papain‐like Cys proteases (PLCPs), Ser hydrolases (SHs) or vacuolar processing enzymes (VPEs) in leaves are unaffected upon expression of the new PIs, whereas SlCYS8 expression specifically suppresses PLCP activity only. Quantitative proteomics indicates that the three new PIs affect agroinfiltrated tissues similarly and that they all increase immune responses. NbPR4, NbPot1 and HsTIMP can be used to study plant proteases and improve RP accumulation in molecular farming.     

Transformation of white poplar (Populus alba L.) with a novel Arabidopsis thaliana cysteine proteinase inhibitor and analysis of insect pest resistance

Transgenic white poplar (Populus alba L.) plants expressing a novel Arabidopsis thaliana cysteine proteinase inhibitor (Atcys) gene have been produced using Agrobacterium tumefaciens-mediated gene transfer. Internodal stem segments of cv. Villafranca were co-cultivated with the EHA105 pBI-Atcys A. tumefaciens strain. Sixteen putative transgenic plant lines were regenerated from different calli with a transformation efficiency of 11%. The integration and expression of the cysteine proteinase inhibitor (Atcys) gene into the plant genome was confirmed by Southern and northern blot analyses. Papain inhibitory activity was detected in poplar transgenic tissues by means of a specific in vitro assay. Such activity was sufficient to inhibit most of the digestive proteinase activity of chrysomelid beetle (Chrysomela populi L.) and confer resistance to C. populi larvae on selected transgenic plants. A close correspondence between the inhibition of papain and resistance to poplar leaf beetle was observed in all tested transgenic lines. Our results indicate that Atcys could be succesfully employed in breeding programmes aimed at the selection of new poplar genotypes resistant to major insect pests.     

Molecular cloning and expression of a proteinase gene from Lactococcus lactis subsp. cremoris H2 and construction of a new lactococcal vector pFX1

The 6.5 kb HindIII DNA fragment of the Lactococcus lactis subsp. cremoris H2 plasmid pDI21 was cloned into Escherichia coli POP13 with NM1149, and also directly into Lactococcus lactis subsp. lactis 4125 using a newly-constructed broad host-range vector pFX1. Proteinase was experessed in both transformed organisms. The proteinase resembles a PI type since it preferentially degraded -casein. The restriction map of the 6.5 kb proteinase gene fragment has minor differences from those of published plamid proteinase genes. High-efficiency electroporation with pFX1 provides a direct approach for gene cloning in lactococci.Abbreviations cfu colony forming units - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulphonic acid] Dedicated to Prof. Dr. G. Drews on the occasion of his 65th birthday     

Evaluation of Perennial Glycine Species for Response to Meloidogyne Incognita,Rotylenchulus Reniformis,and Pratylenchus Penetrans

Root-knot (Meloidogyne incognita (Kofoid & White) Chitwood), reniform (Rotylenchulus reniformis Lindford & Oliveira), and lesion nematodes (Pratylenchus penetrans (Cobb) Filipjev & Schuurmans Stekhoven) are plant-parasitic nematodes that feed on soybean (Glycine max (L.) Merr.) roots, limiting seed production. The availability of resistance in soybeans to these nematodes is limited. However, new sources of resistance can be discovered in wild relatives of agronomic crops. Perennial Glycine species, wild relatives to soybean, are a source of valuable genetic resources with the potential to improve disease resistance in soybean. To determine if these perennials have resistance against nematodes, 18 accessions of 10 perennial Glycine species were evaluated for their response to M. incognita and R. reniformis, and eight accessions of six perennial Glycine species were evaluated for their response to P. penetrans. Pot experiments were conducted for M. incognita and R. reniformis in a growth chamber and in vitro experiments were conducted for P. penetrans. We found both shared and distinct interactions along the resistance-susceptible continuum in response to the three plant-parasitic nematode species. Ten and 15 accessions were classified as resistant to M. incognita based on eggs per gram of root and gall index, respectively. Among them, G. tomentella plant introductions (PIs) 446983 and 339655 had a significantly lower gall index than the resistant soybean check cv. Forrest. Of three R. reniformis resistant accessions identified in this study, G. tomentella PI 441001 showed significantly greater resistance to R. reniformis than the resistant check cv. Forrest based on nematodes per gram of root. In contrast, no resistance to P. penetrans was recorded in any perennial Glycine species.     

A study of integrative transformation in Schizosaccharomyces pombe

Summary Organ-specific and constitutive expression of the Arabidosis HSP18.2 gene under normal growth conditions (22° C) was observed in transgenic A. thaliana, which carried a fusion gene composed of the promoter region of HSP18.2, one of the genes for low molecular weight heat-shock proteins in Arabidopsis, and the gene for -glucuronidase (GUS) from Escherichia coli. In order to clarify the organ-specific nature of promoter expression, various mutations that affect flower morphology were introduced into the transgenic Arabidopsis line, AHS9. The results show that the pattern of expression observed in sepals, filaments, and styles is regulated in a structure-dependent manner, and suggest that the HSP18.2 gene might have an important role in the process of differentiation of flower buds, as do several other stress-related genes.     

Molecular cloning,recombinant gene expression,and antifungal activity of cystatin from taro (<Emphasis Type="Italic">Colocasia esculenta</Emphasis> cv. Kaosiung no. 1)

A cDNA clone, designated CeCPI, encoding a novel phytocystatin was isolated from taro corms (Colocasia esculenta) using both degenerated primers/RT-PCR amplification and 5-/3-RACE extension. The full-length cDNA gene is 1,008 bp in size, encodes 206 amino acid residues, with a deduced molecular weight of 29 kDa. It contains a conserved reactive site motif Gln-Val-Val-Ser-Gly of cysteine protease inhibitors, and another consensus ARFAV sequence for phytocystatin. Sequence analysis revealed that CeCPI is phylogenetically closely related to Eudicots rather than to Monocots, despite taro belonging to Monocot. Recombinant GST–CeCPI fusion protein was overexpressed in Escherichia coli and its inhibitory activity against papain was identified on gelatin/SDS-PAGE. These results confirmed that recombinant CeCPI protein exhibited strong cysteine protease inhibitory activity. Investigation of its antifungal activity clearly revealed a toxic effect on the mycelium growth of phytopathogenic fungi, such as Sclerotium rolfsii Sacc. etc., at a concentration of 80 g recombinant CeCPI/ ml. Moreover, mycelium growth was completely inhibited and the sclerotia lysed at a concentration of 150–200 g/ml. Further studies have demonstrated that recombinant CeCPI is capable of acting against the endogenous cysteine proteinase in the fungal mycelium.     

Proteinases and amylases of larval midgut of Zabrotes subfasciatus reared on cowpea (Vigna unguiculata) seeds

Proteinase and amylase activities in larval midguts of the bruchid beetle Zabrotes subfasciatus (Boh.) (Coleoptera: Bruchidae) reared on cowpea (Vigna unguiculata (L.) Walp.) seeds were investigated. We could detect and isolate a proteolytic activity with a pH optimum of 5.5 (on azo-casein as substrate) which was activated by thiol reagents and inhibited by several compounds reactive against-SH groups. None of the plant protein inhibitors of serine proteinases utilized were effective inhibitors of this activity. This activity has characteristics of a cysteine class proteinase. We could also detect and isolate a proteolytic activity with a pH optimum of 3.5 (on hemoglobin as substrate) which was not influenced by activators or inhibitors of cysteine, serine, or metalloproteinases. This activity was totally inhibited by pepstatin, a specific inhibitor of aspartic proteinases. We conclude that this activity is due to an aspartic class proteinase. We found also that the aspartic class proteolytic activity is higher than the cysteine class proteinase activity in the midguts of Z. subfasciatus. This seems to be contrary to what is found in Callosobruchus maculatus (F.) larvae midguts. An amylolytic activity with the charateristics of an -amylase was also detected and isolated.

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Résumé Les activités protéinase et amylase ont été étudiées sur l'intestin moyen de larves de Zabrotes subfasciatus Boh. (Coléo, Bruchidae), élevées sur graines de Vigna unguiculata Walp. Nous avons pu déceler et isoler une activité protéolytique optimale à pH 5,5 (sur substrat d'azo-caséine) activée par des réactifs thiol et inhibée par plusieurs composés réagissant aux groupements SH. Aucun inhibiteur végétal des sérine-protéases utilisé n'a inibé efficacement cette activité qui présente les caractéristiques des protéines de la famille des cystéines. Nous avons pu déceler et isoler aussi une activité protéolytique optimale au pH 3,5 (sur hémoglobine comme substrat) qui n'était pas modifiée par les activateurs ou les inhibiteurs de cystéine, de sérine ou de métalloprotéinases. Cette activité était totalement inhibée par la pepstatine, inhibiteur spécifique des protéinases aspartiques. Nous en concluons que l'activité est due à une protéinase de la famille aspartique. Nous avons trouvé aussi que l'activité protéolytique de la famille aspartique était supérieure à l'activité protéinase de la famille cystéine dans l'intestin moyen de Z. subfasciatus. Ceci semble l'inverse de ce qui a été observé dans l'intestin moyen des larves de Callosobruchus maculatus F. (C.P. Silva & al, in litt.). Une activité amylolytique ayant les caractéristiques d'une -amylase a aussi été décelée.

     

Protein digestion in cereal aphids (Sitobion avenae) as a target for plant defence by endogenous proteinase inhibitors

Gut extracts from cereal aphids (Sitobion avenae) showed significant levels of proteolytic activity, which was inhibited by reagents specific for cysteine proteases and chymotrypsin-like proteases. Gut tissue contained cDNAs encoding cathepsin B-like cysteine proteinases, similar to those identified in the closely related pea aphid (Acyrthosiphon pisum). Analysis of honeydew (liquid excreta) from cereal aphids fed on diet containing ovalbumin showed that digestion of ingested proteins occurred in vivo. Protein could partially substitute for free amino acids in diet, although it could not support complete development. Recombinant wheat proteinase inhibitors (PIs) fed in diet were antimetabolic to cereal aphids, even when normal levels of free amino acids were present. PIs inhibited proteolysis by aphid gut extracts in vitro, and digestion of protein fed to aphids in vivo. Wheat subtilisin/chymotrypsin inhibitor, which was found to inhibit serine and cysteine proteinases, was more effective in both inhibitory and antimetabolic activity than wheat cystatin, which inhibited cysteine proteases only. Digestion of ingested protein is unlikely to contribute significantly to nutritional requirements when aphids are feeding on phloem, and the antimetabolic activity of dietary proteinase inhibitors is suggested to result from effects on proteinases involved in degradation of endogenous proteins.     

Isolation and characterization of a cysteine protease from the latex of Araujia hortorum fruits

A new protease (araujiain h l) was purified to mass spectroscopy homogeneity from the latex of Araujia hortorum Fourn. (Asclepiadaceae) fruits by ultracentrifugation and ion exchange chromatography. The enzyme has a molecular mass of 24,031 (mass spectrometry) and an isoelectric point higher than 9.3. The optimum pH range for casein hydrolysis was 8.0–9.5. The enzyme showed remarkable caseinolytic activity at high temperatures, although its thermal stability decayed rapidly. The proteinase was activated by thiol compounds and inhibited by common thiol-blocking reagents, particularly E-64 and HgCl₂, suggesting the enzyme belongs to the cysteine protease family. The concentration of active sites as determined by titration with E-64 was 3.3 M. When assayed on N--CBZ-amino acid-p-nitrophenyl esters, the enzyme showed higher preference for the glutamine derivative, followed by those of alanine, asparagine, glycine, and leucine, in decreasing order. Partial homology (36–48%) with other plant cysteine proteinases was observed in an internal fragment obtained by Protease V8 treatment.     

Purification and properties of a coagulant thrombin-like enzyme from the venom of Bothrops leucurus

A thrombin-like enzyme from Bothrops leucurus venom, named leucurobin (leuc), was purified by gel filtration, affinity and ion exchange chromatographies. Physicochemical studies indicated that the purified enzyme is a 35 kDa monomeric glycoprotein on SDS-PAGE under reducing conditions, which decreased to 29 kDa after deglycosylation with N-glycosidase F (PNGase F). The amino acid sequence of leuc was determined by automated sequencing of the intact native protein and peptides produced by digestion of the S-pyridyl-ethylated protein with trypsin. The protein sequence exhibits significant similarities with other serine proteases reported from snake venoms, and contains two potential sites of N-linked glycosylation. The proteinase split off fibrinopeptide A (FPA) rapidly from human fibrinogen; however, only negligible traces of fibrinopeptide B (FPB) were observed. In addition, the enzyme released the N-terminal peptide (Mr = 4572) containing the first 42 residues from the Bβ-chain. Leuc could neither activate factor XIII nor release kinins from heat-treated bovine plasma. Its specific clotting activity was equivalent to 198 NIH thrombin U/mg on human fibrinogen. Kinetic properties of leuc were determined using representative chromogenic substrates. The enzyme evoked the gyroxin syndrome when injected into the tail veins of mice at levels of 0.143 μg/g mouse. The inhibitory effects of PMSF and benzamidine on the amidolytic activity suggest that leuc is a serine proteinase, and inhibition by β-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Antibothropic serum, SBTI and EDTA had little or no effect on its amidolytic activity. However, the clotting effect of the enzyme was strongly inhibited by antibothropic serum. A Dixon plot showed that the hydrolysis of Bz-l-Arg-pNA by leuc was competitively inhibited by benzamidine (K_i = 1.61 ± 0.25 mM).