Specific binding of Bacillus thuringiensis Cry2A insecticidal proteins to a common site in the midgut of Helicoverpa species

For a long time, it has been assumed that the mode of action of Cry2A toxins was unique and different from that of other three-domain Cry toxins due to their apparent nonspecific and unsaturable binding to an unlimited number of receptors. However, based on the homology of the tertiary structure among three-domain Cry toxins, similar modes of action for all of them are expected. To confirm this hypothesis, binding assays were carried out with 125 I-labeled Cry2Ab. Saturation assays showed that Cry2Ab binds in a specific and saturable manner to brush border membrane vesicles (BBMVs) of Helicoverpa armigera. Homologous-competition assays with 125 I-Cry2Ab demonstrated that this toxin binds with high affinity to binding sites in H. armigera and Helicoverpa zea midgut. Heterologous-competition assays showed a common binding site for three toxins belonging to the Cry2A family (Cry2Aa, Cry2Ab, and Cry2Ae), which is not shared by Cry1Ac. Estimation of Kd (dissociation constant) values revealed that Cry2Ab had around 35-fold less affinity than Cry1Ac for BBMV binding sites in both insect species. Only minor differences were found regarding Rt (concentration of binding sites) values. This study questions previous interpretations from other authors performing binding assays with Cry2A toxins and establishes the basis for the mode of action of Cry2A toxins

A single point mutation in the C-terminal extension of wheat Rubisco activase dramatically reduces ADP inhibition via enhanced ATP binding affinity

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (Rca) is a AAA+ enzyme that uses ATP to remove inhibitors from the active site of Rubisco, the central carboxylation enzyme of photosynthesis. Rca α and β isoforms exist in most higher plant species, with the α isoform being identical to the β form but having an additional 25-45 amino acids at the Rca C terminus, known as the C-terminal extension (CTE). Rca is inhibited by ADP, and the extent of ADP sensitivity of the Rca complex can be modulated by the CTE of the α isoform, particularly in relation to a disulfide bond structure that is specifically reduced by the redox-regulatory enzyme thioredoxin-f. Here, we introduced single point mutations of Lys-428 in the CTE of Rca-α from wheat (Triticum aestivum) (TaRca2-α). Substitution of Lys-428 with Arg dramatically altered ADP inhibition, independently of thioredoxin-f regulation. We determined that the reduction in ADP inhibition in the K428R variant is not due to a change in ADP affinity, as the apparent constant for ADP binding was not altered by the K428R substitution. Rather, we observed that the K428R substitution strongly increased ATP substrate affinity and ATP-dependent catalytic velocity. These results suggest that the Lys-428 residue is involved in interacting with the γ-phosphate of ATP. Considering that nucleotide-dependent Rca activity regulates Rubisco and thus photosynthesis during fluctuating irradiance, the K428R substitution could potentially provide a mechanism for boosting the performance of wheat grown in the dynamic light environments of the field.Supported by Marie Skłodowska-Curie Individual Fellowship 706115 Heat Wheat and currently by Australian Research Council Grant CE14010000

Binding Site Alteration Is Responsible for Field-Isolated Resistance to Bacillus thuringiensis Cry2A Insecticidal Proteins in Two Helicoverpa Species

Background Evolution of resistance by target pests is the main threat to the long-term efficacy of crops expressing Bacillus thuringiensis (Bt) insecticidal proteins. Cry2 proteins play a pivotal role in current Bt spray formulations and transgenic crops and they complement Cry1A proteins because of their different mode of action. Their presence is critical in the control of those lepidopteran species, such as Helicoverpa spp., which are not highly susceptible to Cry1A proteins. In Australia, a transgenic variety of cotton expressing Cry1Ac and Cry2Ab (Bollgard II) comprises at least 80% of the total cotton area. Prior to the widespread adoption of Bollgard II, the frequency of alleles conferring resistance to Cry2Ab in field populations of Helicoverpa armigera and Helicoverpa punctigera was significantly higher than anticipated. Colonies established from survivors of F2 screens against Cry2Ab are highly resistant to this toxin, but susceptible to Cry1Ac. Methodology/Principal Findings Bioassays performed with surface-treated artificial diet on neonates of H. armigera and H. punctigera showed that Cry2Ab resistant insects were cross-resistant to Cry2Ae while susceptible to Cry1Ab. Binding analyses with 125I-labeled Cry2Ab were performed with brush border membrane vesicles from midguts of Cry2Ab susceptible and resistant insects. The results of the binding analyses correlated with bioassay data and demonstrated that resistant insects exhibited greatly reduced binding of Cry2Ab toxin to midgut receptors, whereas no change in 125I-labeled-Cry1Ac binding was detected. As previously demonstrated for H. armigera, Cry2Ab binding sites in H. punctigera were shown to be shared by Cry2Ae, which explains why an alteration of the shared binding site would lead to cross-resistance between the two Cry2A toxins. Conclusion/Significance This is the first time that a mechanism of resistance to the Cry2 class of insecticidal proteins has been reported. Because we found the same mechanism of resistance in multiple strains representing several field populations, we conclude that target site alteration is the most likely means that field populations evolve resistance to Cry2 proteins in Helicoverpa spp. Our work also confirms the presence in the insect midgut of specific binding sites for this class of proteins. Characterizing the Cry2 receptors and their mutations that enable resistance could lead to the development of molecular tools to monitor resistance in the [email protected]; [email protected]

A Conserved Sequence from Heat-Adapted Species Improves Rubisco Activase Thermostability in Wheat

The central enzyme of photosynthesis, Rubisco, is regulated by Rubisco activase (Rca). Photosynthesis is impaired during heat stress, and this limitation is often attributed to the heat-labile nature of Rca. We characterized gene expression and protein thermostability for the three Rca isoforms present in wheat (Triticum aestivum), namely TaRca1-b, TaRca2-a, and TaRca2-b. Furthermore, we compared wheat Rca with one of the two Rca isoforms from rice (Oryza sativa; OsRca-b) and Rca from other species adapted to warm environments. The TaRca1 gene was induced, whereas TaRca2 was suppressed by heat stress. The TaRca2 isoforms were sensitive to heat degradation, with thermal midpoints of 35°C 6 0.3°C, the temperature at which Rubisco activation velocity by Rca was halved. By contrast, TaRca1-b was more thermotolerant, with a thermal midpoint of 42°C, matching that of rice OsRca-b. Mutations of the TaRca2-b isoform based on sequence alignment of the thermostable TaRca1- b from wheat, OsRca-b from rice, and a consensus sequence representing Rca from warm-adapted species enabled the identification of 11 amino acid substitutions that improved its thermostability by greater than 7°C without a reduction in catalytic velocity at a standard 25°C. Protein structure modeling and mutational analysis suggested that the thermostability of these mutational variants arises from monomeric and not oligomeric thermal stabilization. These results provide a mechanism for improving the heat stress tolerance of photosynthesis in wheat and potentially other species, which is a desirable outcome considering the likelihood that crops will face more frequent heat stress conditions over the coming decadesThis work was supported by a Marie Skłodowska‐Curie Individual Fellowship from the European Commission (706115 Heat Wheat) and by the Department of Industry, Innovation, Science, Research, and Tertiary Education, Australian Government/Australian Research Council (CE140100008) to A.P.

Competition binding experiments with <i>H. punctigera</i> BBMV.

<p>Binding of ¹²⁵I-Cry2Ab (A) and ¹²⁵I-Cry1Ac (B) to BBMVs from <i>H. punctigera</i> at increasing concentrations of unlabeled competitor: Cry2Ab (•), Cry2Ae (○), and Cry1Ac (□).</p

Binding of ¹²⁵I-Cry2Ab proteins to BBMV from <i>Helicoverpa spp.</i> revealed by autoradiography.

<p>¹²⁵I-Cry2Ab was incubated with BBMV in the absence or the presence of an excess of competitor, and the pellet obtained after centrifuging the reaction mixture was subjected to SDS-PAGE and exposed to an X-ray film for 10 days. (A) ¹²⁵I-Cry2Ab binding to <i>H. armigera</i>: lane 1, a sample of ¹²⁵I-Cry2Ab protein used in the binding assays; lane 2, ¹²⁵I-Cry2Ab incubated with BBMV in the absence of competitor; lane 3, homologous competition (excess of unlabeled Cry2Ab); lane 4, ¹²⁵I-Cry2Ab incubated with BBMV from SP15-resistant insects. (B) ¹²⁵I-Cry2Ab binding to <i>H. punctigera</i>: lane 1, ¹²⁵I-Cry2Ab incubated with BBMV in the absence of competitor; lane 2, homologous competition; lane 3, ¹²⁵I-Cry2Ab incubated with BBMV from Hp4-13-resistant insects; lane 4, a sample of the ¹²⁵I-Cry2Ab protein used in the binding assays.</p

Binding parameters in <i>H. punctigera</i>^a.

a<p>Mean ± SEM.</p>b<p>Values are expressed in picomoles per milligram of BBMV protein.</p

Bioassays with Cry2Ab resistant and susceptible <i>H. armigera</i> and <i>H. punctigera.</i>

a<p>Values are in µg/cm².</p>b<p>Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009975#pone.0009975-Mahon2" target="_blank">[26]</a>.</p>c<p>Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009975#pone.0009975-Bird1" target="_blank">[28]</a>.</p>d<p>LC₅₀ and slopes could not be calculated as there was little or no mortality at the maximum concentration tested (0.25 mg/cm²).</p>e<p>Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009975#pone.0009975-Downes2" target="_blank">[27]</a>.</p

Binding of ¹²⁵I-Cry proteins to BBMV from <i>H. punctigera</i>.

<p>Binding of iodinated Cry proteins to <i>H. punctigera</i> at increasing concentrations of BBMV from the susceptible LHP strain (•) and the resistant Hp4-13 strain (□). Non-specific binding was determined by adding an excess of unlabeled protein to the reaction. Specific binding was calculated by subtracting the non-specific binding from the total binding. (A) Specific binding of ¹²⁵I-Cry2Ab. (B) Specific binding of ¹²⁵I-Cry1Ac. Data points in figure A represent the means of two replicates.</p