Protease inhibitors are a promising complement to Bt toxins for the development of insect-resistant transgenic crops, but their limited specificity against proteolytic enzymes and the ubiquity of protease-dependent processes in living organisms raise questions about their eventual non-target effects in agroecosystems. After a brief overview of the main factors driving the impacts of insect-resistant transgenic crops on non-target organisms, the possible effects of protease inhibitors are discussed from a multitrophic perspective, taking into account not only the target herbivore proteases but also the proteases of other organisms found along the trophic chain, including the plant itself. Major progress has been achieved in recent years towards the design of highly potent broad-spectrum inhibitors and the field deployment of protease inhibitor-expressing transgenic plants resistant to major herbivore pests. A thorough assessment of the current literature suggests that, whereas the non-specific inhibitory effects of recombinant protease inhibitors in plant food webs could often be negligible and their 'unintended' pleiotropic effects in planta of potential agronomic value, the innocuity of these proteins might always remain an issue to be assessed empirically, on a case-by-case basis.

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Recombinant protease inhibitors for herbivore pest control: a multitrophic perspective

Plant serine protease inhibitors are defense proteins crafted by nature for inhibiting serine proteases. Use of eco-friendly, sustainable and effective protein molecules which could halt or slow down metabolism of nutrients in pest would be a pragmatic approach in insect pest management of crops. The host-pest complexes that we observe in nature are evolutionary dynamic and inter-depend on other defense mechanisms and interactions of other pests or more generally speaking symbionts with the same host. Insects have co-evolved and adapted simultaneously, which makes it necessary to investigate serine protease inhibitors in non-host plants. Such novel serine protease inhibitors are versatile candidates with vast potential to overcome the host inhibitor-insensitive proteases. In a nutshell exploring and crafting plant serine proteinase inhibitors (PIs) for controlling pests effectively must go on. Non-host PI seems to be a better choice for coevolved insensitive proteases. Transgenic plants expressing wound inducible chimaeric PIs may be an outstanding approach to check wide spectrum of gut proteinases and overcome the phenomenon of resistance development. Thus, this article focuses on an entire array of plant serine protease inhibitors that have been explored in the past decade, their mode of action and biological implications as well as applications.

Serine protease inhibitors in plants: nature’s arsenal crafted for insect predators

The serine protease inhibitors (SPIs) are widely distributed in living organisms like bacteria, fungi, plants, and humans. The main function of SPIs as protease enzymes is to regulate the proteolytic activity. In plants, most of the studies of SPIs have been focused on their physiological role. The initial studies carried out in plants showed that SPIs participate in the regulation of endogenous proteolytic processes, as the regulation of proteases in seeds. Besides, it was observed that SPIs also participate in the regulation of cell death during plant development and senescence. On the other hand, plant SPIs have an important role in plant defense against pests and phytopathogenic microorganisms. In the last 20 years, several transgenic plants over-expressing SPIs have been produced and tested in order to achieve the increase of the resistance against pathogenic insects. Finally, in molecular farming, SPIs have been employed to minimize the proteolysis of recombinant proteins expressed in plants. The present review discusses the potential biotechnological applications of plant SPIs in the agriculture field.

Plant Serine Protease Inhibitors: Biotechnology Application in Agriculture and Molecular Farming

Buckwheat is a kind cereal mainly grown in cold plateau and mountainous districts. The seeds and food production made from buckwheat demonstrated great nutritional value and protective effects towards various kinds of disease. In this review, the antibacterial, trypsin inhibiting, antitumor, hypocholesterol, hypotensive and antidiabetic effects of buckwheat proteins and their enzyme hydrolysates were summarized and discussed. Many naturally occurring peptides isolated from buckwheat seeds are certified to be multiple functional compounds, such as buckwheat antimicrobial peptides, trypsin inhibitors, antitumor proteins, hypotensive peptides and antioxidant peptides. Besides its trypsin inhibiting activity upon proteases, buckwheat trypsin inhibitors also revealed antimicrobial activity towards fungi, Gram-positive and Gram-negative bacteria and antitumor activity against various kinds of cancer cells. The antitumor effects and the trypsin inhibiting activity are related with the special active site of the peptide molecules, while the hypolipidemic property and the hypotensive activity are most probably associating with the unique amino acids composition of buckwheat proteins, for the reason that the hydrolyzed small peptides still possess the relevant activity. Buckwheat peptides show prospective application in function food area and traditional medicine research. And structure–activity relationship of peptides attracts much more interests in recent years.

Advance on the benefits of bioactive peptides from buckwheat

This review considers the main groups of hydrolytic enzymes associated with plant pathogens, as well as proteinaceous inhibitors of these enzymes, acting as the components of plant defense system. The role of hydrolases is described in the development of a pathological process in plant tissues. Significance of hydrolase inhibitors in the development of plant resistance to pathogens is analyzed. It is proposed that specific interactions in the “host plant–pathogen” system, involving hydrolytic enzymes and their proteinaceous inhibitors, depend on the nutritional specialization of fungi.

Hydrolytic enzymes and their proteinaceous inhibitors in regulation of plant–pathogen interactions

The possibility to use agrobacterial transformation of leaf discs to produce resistance to bacterial infections in tobacco and potato plants by introduction of a single gene encoding the serine proteinase inhibitor BWI-1a (ISP) from buckwheat seeds is shown. All studied PCR-positive transgenic plants exhibited antibacterial activity in biotests. It was shown that the presence of just a single gene of serine proteinase inhibitor provides sufficient protection at least against two bacterial phytopathogens, Pseudomonas syringae pv. tomato and Clavibacter michiganensis sbsp. michiganensis. The biotest including tobacco plant infection by the white wings butterfly in the green house has also demonstrated the existence of protective effect in transgenic tobacco plants. Significant genotypic variations in the protection efficiency were found between members of different genera of the same family (potato and tobacco) as well as between different lines of the same species. Northern blot analysis of four transgenic potato lines and three tobacco lines transformed by a vector plasmid containing the ISP gene of serine proteinases BWI-1a from buckwheat seeds has shown the presence of the expected size mRNA transcript.

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Use of buckwheat seed protease inhibitor gene for improvement of tobacco and potato plant resistance to biotic stress.

In this paper, the results of long-term screening of independently derived transgenic tobacco plants carrying the synthetic BWI-1a gene of serine proteases inhibitor from buckwheat are presented. For several years periodic spot checks of persistence and expression of the heterologous protective genes in vegetatively cloned collections and seeds of transgenic plants were conducted. The persistence of expression of the target gene after ten years of passage of plants in aseptic culture without selective pressure in their seed progeny for at least three generations and derived callus was shown. Extracts of tissues of all the variants of transgenic plants inhibited the growth of phytopathogenic bacteria and the germination of spores of fungi. The degree of the suppression of a pathogen in this case was hardly reduced. In the second seed generation, the number of defective seeds increased and there was a sharp decline of germination ability of the seeds even in nonselective conditions.

Molecular genetic analysis of collection of transgenic tobacco plants with buckwheat serine proteases inhibitor gene during long-term subculture.

In order to study the possibility of creating new plant forms resistant to phytopathogens, a collection of transgenic plants of model tobacco culture with new different plant protective genes was obtained by the agrobacterial transformation method. First an addition of a collection with serine proteinase inhibitor BWI-1a (ISP) from buckwheat with fragments of a spidroin gene as putable enhancer by vector constructions different designs was done. Secondly, transgenic plants with an antimicrobial peptide from sinthetic wheat Triticum kiharae and with defensine from Stellaria media. Comparative study of physiological characteristics of transgenic plants in biotests in vivo (with isolated leaves) and in vitro (with well biotests) was carried out. Regardless of the design of the vector construction, the target genes were expressed to a different extent in the tissues of all transgenic plants and their seed and vegetative progenies and gave their tissues antibacterial activity, indicating the synthesis of the functional protein. The introduction to the tobacco tissues of the heterologic plant protective genes of different nature that plants use in different defense mechanisms led to a similar increase in antibacterial activity of the transgenic tobacco tissues.

V. G. Bogush

Papers

Use of buckwheat seed protease inhibitor gene for improvement of tobacco and potato plant resistance to biotic stress.

Molecular genetic analysis of collection of transgenic tobacco plants with buckwheat serine proteases inhibitor gene during long-term subculture.

Comparative Analysis of Transgenic Tobacco Plants with Different Heterologic Plant Defensive Genes