A deletion mutant ndv200 of the Bacillus thuringiensis vip3BR insecticidal toxin gene is a prospective candidate for the next generation of genetically modified crop plants resistant to lepidopteran insect damage

Abstract

Ectopic expression of a deletion mutant ( ndv200 ) of Bacillus thuringiensis vip3BR gene in tobacco plant provided almost complete protection against major crop pests cotton boll worm ( Helicoverpa armigera ), black cut worm ( Agrotis ipsilon ) and cotton leaf worm ( Spodoptera littoralis ). Whereas vip3BR transgenic tobacco plant failed to protect themselves from these insects and showed resistance towards cotton leaf worm only. An analogous form of the Bacillus thuringiensis vip3Aa insecticidal toxin gene, named vip3BR, was identified and characterized, and exhibited similar attributes to the well-known Vip3Aa toxin. Vip3BR possessed broad-spectrum lepidopteran-specific insecticidal properties effective against most major crop pests of the Indian subcontinent. A Vip3BR toxin protein N-terminal deletion mutant, Ndv200, showed increased insecticidal potency relative to the native toxin, which conferred efficacy against four major crop pests, including cotton boll worm (Helicoverpa armigera), black cut worm (Agrotis ipsilon), cotton leaf worm (Spodoptera littoralis), and rice yellow stem borer (Scirpophaga incertulas). Ligand blot analysis indicated the Ndv200 toxin recognized the same larval midgut receptors as the native Vip3BR toxin, but differed from receptors recognized by Cry1A toxins. In the present study, we tested the prospect of the vip3BR and ndv200 toxin gene as candidate in development of insect-resistant genetically engineered crop plants by generating transgenic tobacco plant. The study revealed that the ndv200 mutant of vip3BR insecticidal toxin gene is a strong and prospective candidate for the next generation of genetically modified crop plants resistant to lepidopteran insects.

Critical amino acids for the insecticidal activity of Vip3Af from Bacillus thuringiensis: Inference on structural aspects

“… 50 suggested a putative cleavage site between residue Thr 10 and Arg 11 in Vip3Aa based on the S score prediction. Removal of N-terminal 12 amino acids (in Arg 12 ) was shown in Vip3Ab1 28 and we have here shown that the 20 kDa N-terminal fragment of Vip3Af1 is also cleaved after Arg 11 .…”

“…4 ). The 20 kDa fragment, obtained after trypsin treatment of the Vip3Af1(WT) (and present in patterns “a”, “d”, and “e”), yielded the sequence ALPSF, the first amino acid corresponding to Ala 12 . The N-terminal sequence of the 62 kDa was DXXPA, which only matched with the query sequence on DSSPA (D = Asp 199 ).…”

“…The largest fragment from the trypsin treatment, the 62–66 kDa polypeptide, was formerly considered to be the core active toxin 1 , 3 – 5 , 8 , 12 , 33 , 37 , 43 – 49 . However, it has been recently shown that the 20 kDa fragment is required for toxicity, presumably by protecting the 62–66 kDa polypeptide from protease degradation 28 .…”

Bacterial Vegetative Insecticidal Proteins (Vip) from Entomopathogenic Bacteria

“…A 3-year study on the field performance of VipCot expressing just the Vip3Aa protein indicated that the plants were highly efficacious against H. armigera early in the season but that efficacy declined as the season progressed although not so drastically as Cry1Ac in Bollgard or Ingard cotton (117). In 2015, the first modified Vip3A protein, with improved toxicity, was introduced into tobacco, conferring almost total protection against H. armigera, A. ipsilon, and S. littoralis (118).…”

Insights into the Structure of the Vip3Aa Insecticidal Protein by Protease Digestion Analysis

“…While Li et al [28] found that deletion of the N-terminal first 189 amino acids abolished the insecticidal activity of a chimeric Vip3AcAa protein, Gayen et al [35] found that a Vip3Aa deletion mutant lacking the first 200 amino acids of the protein not only did not abolish the activity, but it slightly enhanced it against the three insect species tested, and that the expression of this deleted protein in tobacco plants provided even higher plant protection against several feeding insects than the expression of the wild type protein [36]. It is possible that the discrepancy between these two studies is due to the method used for the expression and purification of the deletion mutants, if not to the different Vip3A proteins used.…”