Genetic Engineering Approach for Next-Generation of Bt-Based Agents: Comparison
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Please note this is a comparison between Version 2 by Jason Zhu and Version 1 by Igor Maksimov.

Bacillus thuringiensis Berliner (Bt) and B. cereus sensu stricto Frankland and Frankland are closely related species of aerobic, spore-forming bacteria included in the B. cereus sensu lato group. This group is one of the most studied, but it remains also the most mysterious species of bacteria. Despite more than a century of research on the features of these ubiquitous bacteria, there are a lot of questionable issues related to their taxonomy, resistance to external influences, endophytic existence, their place in multidimensional relationships in the ecosystem, and many others.

  • endophytes
  • insecticide
  • nematicide
  • Cry
  • Vip
  • Bt-crops

1. Introduction

Now, genetic modifications of Bt serve the purpose of dissolving two main problems of Bt-preparations, such as increase in bacteria tolerance to the impact of environmental factors and improving its insecticidal effects against different pests. The strategies of genetic engineering approaches to constructing strains with the required properties are as follows: (1) the up-regulation of the key enzyme gene involved in the target compound biosynthesis; (2) relieving the inhibition and/or repression of the key enzyme; and (3) the interruption of the pathways for synthesizing by-products [165][1]. The development of next-generation artificially improved Bt strains or strains heterologically producing Bt-toxins involves a broad spectrum of DNA reorganization, such as site-directed mutagenesis (SDM), the suppression and overexpression of genes, including RNA interference (RNAi) [166,167][2][3] Initially, RNAi was proposed as a suggestive strategy for the inhibition of viral infection. It is a post-transcriptional gene regulation mechanism characteristic of (possibly) all eukaryotes, including insect pests [106,107,108][4][5][6]. The mechanism is triggered by double-stranded RNA (dsRNA) precursors that are processed into short-interfering RNA (siRNA) duplexes, which then realize the recognition and repression of complementary dsRNAs, such as mRNAs or viral genomic RNAs [168][7].

3.1. Improvement of Insecticidal Properties of Bacterial Strains

2. Improvement of Insecticidal Properties of Bacterial Strains

Currently, genetic engineering approaches make it possible to transfer/supplement/modify genes encoding insectotoxin to other Bt strains or strains of another bacterial species using homologous recombination [169][8]. Current information on Cry- and non-Cry genes, which were used for the recombination of a broad spectrum of bacterial strains is assumed in [106,170][4][9]. An important tool for the recombination of Bt strains is site-specific recombination (SSR), which is useful for engineering strains with original combinations of Cry toxins genes with improved insecticidal activity [166,167,171][2][3][10]. It seems interesting to create endophytic Bt or other bacterial species whose populations in the internal tissues of plants would be safe from the environment and have greater activity against pests. These investigations originated in the last decade of the 20th century when the Bt gene encoding Cry1Aa was expressed in root-associated P. fluorescens [172][11]. Thus, the introduction of the cry1Ia gene into the endophytic strain B. subtilis 26D does not lead to the loss of the endophytic status of B. subtilis 26DCryChS line and gives impetus to its insecticidal and aphicidal activity in vitro and in planta [29,30][12][13]. Endophytic Burkholderia pyrrocinia JKSH007 heterologically expressing the Btcry218 gene showed an effectiveness against Bombyx mori L. [173][14]. The ability of Pantoea agglomerans 33.1:pJTT expressing cry1Ac7 to inhabit Saccharum officinarum L. tissues was confirmed by re-isolation from the plant’s rhizosphere, roots and shoots. Thus, the introduction of an exogenous gene did not affect the plant–host interaction but increased the mortality of Diatraea saccharalis Fabricius, 1794 fed on inoculated stems [174][15]. The transfer of “useful” insectotoxin genes from other economically important Bt strains to endophytic bacteria, as well as the maintenance of their consortiums, should contribute to the creation of new-generation biological agents based on them. At the same time, modern technologies for editing microbial genomes based on the CRISPRCas9 platform [168,175][7][16] can be proposed to disable the α-exotoxin and β-exotoxin synthesis of Bt.

3.2. Approaches to the Development of UV-Tolerant Bt

3. Approaches to the Development of UV-Tolerant Bt

The problem of the UV-irradiation susceptibility of Bt seriously restricts its effective use. The exogenous addition of UV protective agents, such as rhodamine B or methyl green, can protect spores from the light [176][17]. Subsequently, for the same purpose, latex particles, ethanol, and olive oil have been used to encapsulate Bt in colloidosomes [177,178][18][19].
Homologous recombination technology was used for the insertion of the yhfS gene encoding acetyl-CoA acyltransferase in Bt LLP29 R-yhfS. The loss of the yhfS gene in the knockout strain Bt LLP29 Δ-yhfS led to the reduction in antioxidant ability and reduced UV resistance of the mutant [179][20]. The cell-surface exposure of chitinase Chi9602ΔSP was developed on the basis of Bt BMB171 using two repeat N-terminal regions of autolysin (Mbgn)2 as the anchoring motif. After continuous culturing for 120 h, the line of Bt expressing chitinase Chi9602ΔSP showed narrow pH tolerance and obviously enhanced UV radiation resistance capacity in addition to a high inhibitory effect towards phytopathogenic fungi, F. oxysporum FB012 and Botryosphaeria berengeriana FB016 [180][21]. CRISPR/Cas9 systems have been used to knock out the homogentisate-1,2-dioxygenase (hmgA) gene and obtain a melanin-producing mutant Bt HD-1-1 hmgA. The anti-UV test shows that melanin arranges protection to both Bt cells and Cry toxin crystals. After UV-irradiation the strain Bt HD-1-1 hmgA still had an 80% insecticidal activity against H. armigera, while the wild line only had about 20% [8][22].
Cry genes have been expressed in P. fluorescens and Anabaena sp. to increase the damage to crystals from UV light [181[23][24],182], as well as in E. coli; B. megaterium [183][25]; B. subtilis [83][26]; Clavibacter xyli Davis et al. 1984; Herbaspirillum seropedicae Baldani et al. 1986; R. leguminosarum [170][9]; Beauveria bassiana (Bals.-Criv.) Vuill., 1912 [184][27]; etc. The recombinant strain P. fluorescens is the base of biopesticide “CellCapTM” (Mycogen Corp.; Indianapolis, IN, USA), contains encapsulated Cry toxins [185][28]. The increase in the amount of Bt-plants can be partially attributed to the means of the protection of insectotoxins from UV rays [72][29].

3.3. Bt Crops Prospects

4. Bt Crops Prospects

Since 1996, genetically engineered Bt crops have been planted in the fields, which led to a “gene revolution” in agricultural production [186,187][30][31]. By the early 21th century, Bt-potato, Bt-cotton, Bt-maize, Bt-eggplant, etc. were actively distributed worldwide, which allowed for a significant reduction in the amount of chemical insecticides used in a number of countries [96,188][32][33]. However, this approach lead to a fairly rapid spread of resistant pest populations [80,189,190][34][35][36]. To overcome insect resistance, it is possible to introduce Bt crops containing more than two genes encoding insecticidal proteins [189,190][35][36]. The Bollgard cotton variety bearing Cry1Ac gene decreased the viability of pink bollworm Pectinophora gossypiella (Saunders, 1844) and corn earworm Helicoverpa zea Boddie, 1850. Plants of the Bollgard II variety, expressing two Bt endotoxins, expand the spectrum of protective features against lepidopteran pests [191][37]. Bt-cotton with cassettes of protective genes (1Ac/Cry2Ab/Vip3A), (Cry1Ab/Cry2Ac/Vip3Aa19) or (Cry1Ac/Cry1F/Vip3A) was cultivated in the 2016–2017 season on more than 90% of the arable lands of Australia [192][38].
Currently, the creation of plants containing not only Cry or Vip genes but also containing other gene sequences is of interest in order to increase the effectiveness of biological plant protection against pests. Recently, the US EPA approved a transgenic corn, SmartStaxPRO, expressing the Cry3Bb1 protein, and a dsRNA complementing the RNA of the vacuolar protein DvSnf7 of Diabrotica virgifera LeConte, 1858 [193,194][39][40]. A vector containing information about dsRNA targeting the acid methyltransferase gene of the juvenile hormone biosynthesis of H. armigera was inserted into the genome of Bt-cotton and impaired the resistance of pest compared to plants expressing only insectotoxic proteins [195][41]. The RNAi-mediated knockdown of H. armigera acetylcholinesterase, the ecdysone receptor, and v-ATPase-A genes by producing dsRNAs homologous to genetic targets in potato plants led to mortality and abnormal development in the larva of this insect (recorded ten days post feeding) [167][3].
Apparently, the application of single Bt genes to modify plant genomes will be gradually replaced by multiple Bt toxin genes or Bt with other nucleotide sequences.

3.4. Bt as a Means of dsRNAs Deliverance

5. Bt as a Means of dsRNAs Deliverance

The important problem of interference methods is dsRNA degradation by nucleases in the gut lumen and tissues of insects. Retaining dsRNA molecules in the gut or hemocoel of pest insects is the key aspect of an effective dsRNA delivery [166][2]. Thus, dsRNase catalyzing the specific cleavage of dsRNA has been found in the saliva of Lygus lineolaris Palisot de Beauvois, 1818 [196][42]; pea aphid Acyrthosiphon pisum Harris, 1776 [197][43]; Schistocerca gregaria Forskal, 1775 [198][44]; H. armigera [167][3]; etc. A dsRNA cassette targeting the multiple genes of H. armigera revealed more rapid cleavage in midgut juice compared to the hemolymph [167][3], and for this reason, the Bt-mediated appearance of pores in digestion membranes, can improve the efficacy of dsRNAs along with dsRNAs targeting dsRNases [199][45]. The stability of mRNA provided by, for example, the Shine–Dalgarno sequence (GAAAG-GAGG), is a promising factor of the high-level expression of Cry genes in Bt [167,200,201][3][46][47]. The binding of the 30S ribosomal subunit to this sequence might prevent mRNA cleavage by RNAses of pest. The use of a sporulation-dependent promoter of Cry genes of Bt for the transcription of the target dsRNA sequence, leads to the fact that the dsRNA will be spontaneously produced during the sporulation phase [201,202][47][48]. It has been demonstrated that the incorporation of plasmid pBtdsSBV-VP1, which carries out dsRNA complemental to the VP1 sequence of the sacbrood virus (SBV) in Bt 4Q7 and the subsequent appliance of exogenous total RNA, leads to a decrease in SVB severance in Apis cerana (Fabricius, 1793) families [202][48]. Then, the plasmid pBtdsSBV-VP1 was inserted into the Bt NT0423, which expresses Cry1 protein, resulting in SBV replication being repressed in A. cerana bees as well as the viability of the A. cerana parasite Galleria mellonella L. [200][46]. These results demonstrated that dsRNA-expressing Bt products could be efficiently exploited for the control of both viral diseases and insect pests simultaneously.
And furthermore, it is possible to enhance the toxicity of Cry toxins using dsRNA cassettes. Thus, Bt strains 8010AKi and BMB171AKi expressing the dsRNA of the arginine kinase gene (PxAK) of P. xylostella, flanking two ends with the promoter Pro3α, effectively decreased PxAK expression in ones treated with the composition with wild Cry-producing Bt 8010 and caused a higher lewel of mortality of the pest [203][49]. Separately, E. coli HT115 dsINT expressing the dsRNA of integrin β1 subunit gene (SeINT) cause a less than 50% mortality rate against Spodoptera exigua Hubner, 1808 larvae, and E. coli expressing Cry1Ca led to a maximal 58% mortality rate of the pest. When S. exigua larvae were treated with the Cry1Ca-expressing bacteria (E. coli or Bt subsp. aizawai from commercial Xentari insecticide) after treatment with E. coli HT115 dsINT, the insecticidal activity of the Cry1Ca was significantly enhanced up to about 80% [201][47]. The nuclease gene HaREase characteristic for Lepidoptera is up-regulated by dsRNA and affects RNAi in H. armigera. When this gene was knocked out using the CRISPR/Cas9 system, the midgut epithelium structure was not affected in the ΔHaREase mutant, but when larvae were fed an artificial diet with sublethal doses (2.5 or 4 μg/g) of Cry1Ac, the growth rate of the ΔHaREase line was repressed significantly [204][50]. The insecticidal activity of the Bt-based biopesticide Xentari™ (Valent BioSciences) against larvae of S. littoralis was significantly enhanced by pre-treatment with dsRNA-Bac targeted against the Sl 102 gene, which is responsible for insect cell aggregation and encapsulation to protect against Bt infection [205][51]. Likewise, the efficacy of biological preparations based on live Bt cells was enhanced when used together with dsRNA-Bac specific to sequences of the P. xylostella Pxf gene [206][52], which caused insect resistance to Cry1Ac toxin. The RNAi-mediated suppression of the Cat L-like gene encoding the lysosomal cathepsin L-like cysteine protease of Bombyx mori led to an increase in larvae mortality under the influence of Bt subsp. kurstaki strain ABTS-351 (Dipel®, Valent BioSciences, Libertyville, IL, USA) [207][53]. It is probably that the increase in insect resistance to biocontrol agents based on Bt strains producing toxins and the use of this bacterium as a platform for the expression of dsRNA can help in pest control using the Cry + RNAi strategy [205,206,207][51][52][53].

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