Genomics and Proteomics Analyses Revealed Novel Candidate Pesticidal Proteins in a Lepidopteran-Toxic Bacillus thuringiensis Strain

Abstract

Discovery and identification of novel insecticidal proteins in Bacillus thuringiensis (Bt) strains are of crucial importance for efficient biological control of pests and better management of insect resistance. In this study, the Bt strain KhF, toxic for Plodia interpunctella and Grapholita molesta larvae, underwent genomics and proteomics analyses to achieve a better understanding of the bases of its pathogenicity. The whole-genome sequencing results revealed that the KhF strain contained nine coding sequences with homologies to Bt insecticidal genes. The lepidopteran toxic mixture of spores and crystals of this Bt strain was subjected to liquid chromatography and tandem mass spectrometry (LC-MS/MS) to assess the protein composition. The results of the proteomic analyses, combined with the toxin gene sequences, revealed that two of the main components of the crystals were two new candidate pesticidal proteins, named KhFA and KhFB. These proteins showed a similarity lower than 36% to the other known Bt toxins. The phylogenetic analysis showed that the KhFA and KhFB grouped with the newly denominated Xpp and Mpp (former ETX/Mtx) pesticidal protein groups, respectively. Altogether, this study has led to the discovery of two novel candidate pesticidal toxins in the lepidopteran toxic KhF strain.

Isolation, Genomic, and Proteomic Characterization of a Novel Neotropical Strain of Bacillus thuringiensis with Mosquitocidal Activities

“…Metalloproteinases have been described as virulence factors involved in Bt pathogenesis, increasing the toxicity of pesticidal proteins [52]. The immune inhibitor A (InhA) has been identified as the main metalloproteinase associated with pesticidal proteins in sporecrystal mixtures of Bt strains [25,66]. Interestingly, in this study, the high protein homology to Metallophosphoesterase (Mppe) was the most abundant protease in Bt TOD651.…”

“…Thus, proteomic analysis of the pesticidal proteins that make up the parasporal crystal is essential to understand the toxicity of novel and commercial Bt strains [19]. In this context, the combination of genomic and proteomic analyses is a powerful tool for the accurate identification of pesticidal proteins and virulence factors of Bt strains [23][24][25], and estimations of the abundance of such proteins can be achieved in purified parasporal crystals and spore-crystal mixtures [25,26].…”

Abundance, distribution, and expression of nematicidal crystal protein genes in Bacillus thuringiensis strains from diverse habitats

“…The protein identification of the solubilized crystals was performed as described by Khorramnejad et al ( 2020 ). In short, the Bt strains were grown in liquid CCY medium for 2 days to allow sporulation.…”

“…The sample was then centrifuged at 24,000 × g for 15 min at 4 °C, and the supernatant was used for protein detection. The detection of the protein content was performed by LC–MS/MS at the proteomics facility of the SCIE (Servei Central de Suport a la Investigació Experimental) at the University of Valencia as described elsewhere (Khorramnejad et al 2020 ), after trypsin digestion using a Mass Spectrometter nanoESI qQTOF (6600plus TripleTOF, ABSCIEX). The Protein Pilot v 5.0 (ABSciex, Madrid, Spain) default parameters were used to generate the peptides list, and the protein identification was obtained by the Paragon algorithm (Shilov et al 2007 ), using two different databases: the SwissProt database (versión 200,601) and a customized database kindly provided by Dr. Joaquin Gomis-Cebolla (Polytechnic University of Valencia, Spain) that groups all the Cry, Cyt, Mtx-like, Bin-like, Sip, and Vip proteins available in public databases and patents.…”

Development of a new broad-spectrum microencapsulation-based spray drying formulation of Bacillus thuringiensis subsp. kurstaki IMBL-B9 for the control of moths

“…Bt-based bioinsecticides have been available commercially since the late 1920s, however, their use is limited due to their narrow host spectrum, a short shelf life, and the development of pest resistance (Chandrasena et al, 2018). To address these challenges, researchers have collected Bt samples from various locations globally to isolate novel strains with broad host spectra and high insecticidal activity (Nair et al, 2018;Cao et al, 2020;Khorramnejad et al, 2020;Yin et al, 2020). Despite these efforts, there remains a shortage of Bt strains with both high toxicity and an expended host range, thus highlighting the need for novel insecticidal proteins which address these shortcomings.…”