Cry1Ac toxin‐binding proteins from Helicoverpa armigera brush border membrane vesicles were identified by an improved pull‐down method that involves coupling Cry1Ac to CNBr agarose combined with liquid chromatography–tandem mass spectrometry (LC‐MS/MS). According to the LC‐MS/MS results, Cry1Ac toxin could bind to six classes of aminopeptidase‐N, alkaline phosphatase, cadherin‐like protein, ATP‐binding cassette transporter subfamily C protein (ABCC2), actin, ATPase, polycalin, and some other proteins not previously characterized as Cry toxin‐binding molecules such as dipeptidyl peptidase or carboxyl/choline esterase and some serine proteases. This is the first report that suggests the direct binding of Cry1Ac toxin to ABCC2 in H. armigera.
The bacterium produces several insecticidal proteins, such as the crystal proteins (Cry) and the vegetative insecticidal proteins (Vip). In this work, we report that a specific interaction between two toxins creates insecticidal synergism and unravel the molecular basis of this interaction. When applied together, the three-domain Cry toxin Cry9Aa and the Vip Vip3Aa exhibited high insecticidal activity against an important insect pest, the Asiatic rice borer (). We found that these two proteins bind specifically to brush border membrane vesicles of and that they do not share binding sites because no binding competition was observed between them. Binding assays revealed that the Cry9Aa and Vip3Aa proteins interacted with high affinity. We mapped their specific interacting regions by analyzing binding of Cry9Aa to overlapping fragments of Vip3Aa and by analyzing binding of Vip3Aa to individual domains of Cry9Aa. Binding to peptide arrays helped narrow the binding sites to domain II loop-3 of Cry9Aa and toTKKMKTL in Vip3Aa. Site-directed mutagenesis confirmed that these binding regions participate in binding that directly correlates with the synergism between the two proteins. In summary, we show that the Cry9Aa and Vip3Aa toxins display potent synergy based on a specific interaction between them. Our results further our understanding of the complex synergistic activities among toxins and are highly relevant to the development of toxin combinations for effective insect control and for delaying development of insect resistance.
BackgroundFor a long time, the Christie-Atkinson-Munch-Peterson (CAMP) test has been a standard test for the identification of Streptococcus agalactiae, and a positive result for S.agalactiae has been considered sensitive enough.MethodsTo confirm whether a positive CAMP test is a requirement for the identification of S.agalactiae, five suspected CAMP-negative S.agalactiae isolates from two hospitals, confirmed as Gram-positive and catalase-negative streptococci, were verified by the CAMP test in three batches of plates from two manufacturers and identified by the Phoenix system, MALDI-TOF MS, the PCR assay and the 16S rDNA gene sequencing.ResultsAll five suspected strains were S.agalactiae, four of which were CAMP-negative and one of which was not S.agalactiae by the PCR assay.ConclusionsA positive CAMP test was lacking sensitivity for the identification of S.agalactiae, and the question of whether the cfb gene is worthy of targeting should be further studied.
Bacillus thuringiensis is a well-known entomopathogenic bacterium that produces vegetative insecticidal proteins (Vips, including Vip1, Vip2, Vip3, and Vip4) during the vegetative phase. Here, we purified Vip1 and Vip2 from B. thuringiensis and characterized the insecticidal effects of these protoxins. Bioassay results showed that a 1:1 mixture of Vip1Ad and Vip2Ag, purified by ion-affinity chromatography independently, exhibited insecticidal activity against Holotrichia parallela larvae, with a 50% lethal concentration value of 2.33 μg/g soil. The brush border membrane (BBM) in the midgut of H. parallela larvae was destroyed after feeding the Vip1Ad and Vip2Ag mixture. Vacuolization of the cytoplasm and slight destruction of BBM were detected with Vip2Ag alone, but not with Vip1Ad alone. Notably, Vip1Ad bound to BBM vesicles (BBMVs) strongly, whereas Vip2Ag showed weak binding; however, binding of Vip2Ag to BBMV was increased when Vip1Ad was added. Ligand blotting showed that Vip2Ag did not bind to Vip1Ad but bound to Vip1Ad-t (Vip1Ad was activated by trypsin), suggesting the activation of Vip1Ad was important for their binary toxicity. Thus, our findings suggested that Vip1Ad may facilitate the binding of Vip2Ag to BBMVs, providing a basis for studies of the insecticidal mechanisms of Vip1Ad and Vip2Ag.
Vip3Aa was first identified as a protein secreted during the vegetative growth phase of Bacillus thuringiensis (Bt) bacteria and which shows high insecticidal toxicity against lepidopteran insect pests (Estruch et al., 1996). Bt strains formulated as bio-insecticides only had low amounts of Vip3Aa secreted to the medium. Here, we report that Vip3Aa proteins produced by three different Bt strains, including an industrial strain, were indeed not secreted to the culture solution when grown in sporulation medium, but were retained in the mother cell compartment. In order to further investigate the Vip3Aa secretion and location, we grew the strains in rich medium. We found that in rich medium, a fraction of Vip3Aa was secreted, suggesting that Vip3Aa secretion is nutrient-dependent. Regardless of the growth conditions, we found that Vip3Aa retained in cell pellets exhibited high toxicity against Spodoptera frugiperda larvae. Hence, we speculate that the accumulation of Vip3Aa protein in the mother cell compartment under sporulation conditions could still be used as an efficient strategy for industrial production in commercial Bt strains.
Spinosyns, including spinosad and spinetoram, act on the insect central nervous system, gradually paralyzing or destroying the target insect. Spinosad resistance is associated with loss‐of‐function mutations in the nicotinic acetylcholine receptor (nAChR) α6 subunit in a number of agricultural pests. Using gene editing, nAChR α6 has been verified as a target for spinosyns in five insect species. Recently, a point mutation (G275E) in exon 9 of nAChR α6 was identified in spinosad‐resistant strains of Thrips palmi and Tuta absoluta. To date, no in vivo functional evidence has been obtained to support that this mutation is involved in spinosyn resistance in lepidopteran pests. In this study, the G275E mutation was introduced into the nAChR of Spodoptera exigua using clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR‐associated protein 9 (Cas9) gene‐editing technology. Reverse transcriptase‐polymerase chain reaction and sequencing confirmed that this mutation was present in exon 9 of the nAChR transcripts in the edited 275E strain. The results of bioassays showed that the 275E strain was highly resistant to spinosad (230‐fold) and spinetoram (792‐fold) compared to the unedited background strain, directly confirming that the G275E mutation of the nAChR α6 subunit confers high levels of spinosyn resistance in S. exigua. Inheritance analysis showed that the resistance trait is autosomal and incompletely recessive. This study employs a reverse genetics approach to validate the functional role played by the G275E mutation in nAChR α6 of S. exigua in spinosyns resistance and provides another example of the use of CRISPR/Cas9 gene‐editing technology to confirm the role played by candidate target site mutations in insecticide resistance.
To improve the novel Bacillus thuringiensis insecticidal gene cry2Ah1 toxicity, two mutants cry2Ah1-vp (V354VP) and cry2Ah1-sp (V354SP) were performed. SWISS-MODEL analysis showed two mutants had a longer loop located between β-4 and β-5 of domain II, resulting in higher binding affinity with brush border membrane vesicles (BBMV) of Helicoverpa armigera comparing with Cry2Ah1. The cry2Ah1, cry2Ah1-vp, and cry2Ah1-sp were optimized codon usage according to plant codon bias, and named mcry2Ah1, mcry2Ah1-vp, and mcry2Ah1-sp. They were transformed into tobacco via Agrobacterium-mediated transformation and a total of 4, 8, and 24 transgenic tobacco plants were obtained, respectively. The molecular detection showed the exogenous gene was integrated into tobacco genome, and successfully expressed at the transcript and translation levels. Cry2Ah1 protein in transgenic tobacco plants varied from 4.41 to 40.28 μg g−1 fresh weight. Insect bioassays indicated that all transgenic tobacco plants were highly toxic to both susceptible and Cry1Ac-resistant cotton bollworm larvae, and the insect resistance efficiency to Cry1Ac-resistant cotton bollworm was highest in mcry2Ah1-sp transgenic tobacco plants. The results demonstrated that cry2Ah1 was a useful Bt insecticidal gene to susceptible and Cry1Ac-resistant cotton bollworm and had potential application for insect biocontrol and as a candidate for pyramid strategy in Bt crops.
Herein, cationic latex particles (CL) of different particle sizes were introduced as a cross-linking center to enhance the mechanical properties of the hydrophobically-associated hydrogels (P(AAm-co-HMA)-CL).
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