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The ability of plants to respond to changes in the environment is crucial to their survival and reproductive success. The impact of increasing the atmospheric CO2 concentration (a[CO2]), mediated by behavioral and developmental responses of stomata, on crop performance remains a concern under all climate change scenarios, with potential impacts on future food security. To identify possible beneficial traits that could be exploited for future breeding, phenotypic variation in morphological traits including stomatal size and density, as well as physiological responses and, critically, the effect of growth [CO2] on these traits, was assessed in six wheat relative accessions (including Aegilops tauschii, Triticum turgidum ssp. Dicoccoides, and T. turgidum ssp. dicoccon) and five elite bread wheat T. aestivum cultivars. Exploiting a range of different species and ploidy, we identified key differences in photosynthetic capacity between elite hexaploid wheat and wheat relatives. We also report differences in the speed of stomatal responses which were found to be faster in wheat relatives than in elite cultivars, a trait that could be useful for enhanced photosynthetic carbon gain and water use efficiency. Furthermore, these traits do not all appear to be influenced by elevated [CO2], and determining the underlying genetics will be critical for future breeding programmes.

Although stomata are typically found in greater numbers on the abaxial surface, wheat flag leaves have greater densities on the adaxial surface. We determine the impact of this less common stomatal patterning on gaseous fluxes using a novel chamber that simultaneously measures both leaf surfaces. Using a combination of differential illuminations and CO

The wheat flag leaf is the main contributor of photosynthetic assimilates to developing grains. Understanding how canopy architecture strategies affect source strength and yield will aid improved crop design. We used an eight-founder population to investigate the genetic architecture of flag leaf area, length, width and angle in European wheat. For the strongest genetic locus identified, we subsequently created a near-isogenic line (NIL) pair for more detailed investigation across seven test environments. Genetic control of traits investigated was highly polygenic, with colocalisation of replicated quantitative trait loci (QTL) for one or more traits identifying 24 loci. For QTL QFll.niab-5A.1 (FLL5A), development of a NIL pair found the FLL5A+ allele commonly conferred a c. 7% increase in flag and second leaf length and a more erect leaf angle, resulting in higher flag and/or second leaf area. Increased FLL5A-mediated flag leaf length was associated with: (1) longer pavement cells and (2) larger stomata at lower density, with a trend for decreased maximum stomatal conductance (Gsmax ) per unit leaf area. For FLL5A, cell size rather than number predominantly determined leaf length. The observed trade-offs between leaf size and stomatal morphology highlight the need for future studies to consider these traits at the whole-leaf level. ispartof: NEW PHYTOLOGIST vol:237 issue:5 pages:1558-1573 ispartof: location:England status: published

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.

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-β, TaRca2-α, and TaRca2-β. Furthermore, we compared wheat Rca with one of the two Rca isoforms from rice (Oryza sativa; OsRca-β) 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 ± 0.3°C, the temperature at which Rubisco activation velocity by Rca was halved. By contrast, TaRca1-β was more thermotolerant, with a thermal midpoint of 42°C, matching that of rice OsRca-β. Mutations of the TaRca2-β isoform based on sequence alignment of the thermostable TaRca1-β from wheat, OsRca-β 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 decades.

The mechanistic basis of tolerance to heat stress was investigated in Oryza sativa and two wild rice species, Oryza meridionalis and Oryza australiensis. The wild relatives are endemic to the hot, arid Australian savannah. Leaf elongation rates and gas exchange were measured during short periods of supra-optimal heat, revealing species differences. The Rubisco activase (RCA) gene from each species was sequenced. Using expressed recombinant RCA and leaf-extracted RCA, the kinetic properties of the two isoforms were studied under high temperatures. Leaf elongation was undiminished at 45°C in O. australiensis. The net photosynthetic rate was almost 50% slower in O. sativa at 45°C than at 28°C, while in O. australiensis it was unaffected. Oryza meridionalis exhibited intermediate heat tolerance. Based on previous reports that RCA is heat-labile, the Rubisco activation state was measured. It correlated positively with leaf elongation rates across all three species and four periods of exposure to 45°C. Sequence analysis revealed numerous polymorphisms in the RCA amino acid sequence from O. australiensis. The O. australiensis RCA enzyme was thermally stable up to 42°C, contrasting with RCA from O. sativa, which was inhibited at 36°C. We attribute heat tolerance in the wild species to thermal stability of RCA, enabling Rubisco to remain active.

Stomata are the primary gatekeepers for CO2 uptake for photosynthesis and water loss via transpiration and therefore play a central role in crop performance. Although stomatal conductance (gs) and assimilation rate (A) are often highly correlated, studies have demonstrated an uncoupling between A and gs that can result in sub-optimal physiological processes in dynamic light environments. Wheat (Triticum aestivum L.) is exposed to changes in irradiance due to leaf self-shading, moving clouds and shifting sun angle to which both A and gs respond. However, stomatal responses are generally an order of magnitude slower than photosynthetic responses, leading to non-synchronized A and gs responses that impact CO2 uptake and water use efficiency (iWUE). Here we phenotyped a panel of eight wheat cultivars (estimated to capture 80% of the single nucleotide polymorphism variation in North–West European bread wheat) for differences in the speed of stomatal responses (to changes in light intensity) and photosynthetic performance at different stages of development. The impact of water stress and elevated [CO2] on stomatal kinetics was also examined in a selected cultivar. Significant genotypic variation was reported for the time constant for stomatal opening (Ki, P = 0.038) and the time to reach 95% steady state A (P = 0.045). Slow gs opening responses limited A by ∼10% and slow closure reduced iWUE, with these impacts found to be greatest in cultivars Soissons, Alchemy and Xi19. A decrease in stomatal rapidity (and thus an increase in the limitation of photosynthesis) (P < 0.001) was found during the post-anthesis stage compared to the early booting stage. Reduced water availability triggered stomatal closure and asymmetric stomatal opening and closing responses, while elevated atmospheric [CO2] conditions reduced the time for stomatal opening during a low to high light transition, thus suggesting a major environmental effect on dynamic stomatal kinetics. We discuss these findings in terms of exploiting various traits to develop ideotypes for specific environments, and suggest that intraspecific variation in the rapidity of stomatal responses could provide a potential unexploited breeding target to optimize the physiological responses of wheat to dynamic field conditions.

Stomata are the primary gatekeepers for CO

Night temperature (NT) increases faster than day temperature with global warming and this may have strong implications for crop yield. One of the assumptions is the increase in night respiration and thus the increase in carbon losses over time. Some rice cultivars (indica, aus and japonica types) were grown in the field in the Philippines and in heated greenhouses in Montpellier, France, to quantify the response of respiration to increased NT and its implication at crop level. Increased NT by 3.8 and 5.4 °C over the whole crop cycle in two greenhouse experiments and by 1.9 °C in the field from 33 days after transplanting up to maturity using a free-air-controlled enhancement increased night respiration rate significantly. Overall, the cost in CH 2 O of night respiration over crop duration was 17 to 20% (field) and 8 to 18% (greenhouse) of the potential shoot dry matter (including that lost by respiration). Additional respiratory burden due to increased NT was only 1 to 7% of the potential shoot dry matter at maturity, and was not associated with significant loss in additional dry matter. The absence of any dry matter reduction with increased NT within indica cultivars questions the assumptions of recent papers.

The cotton leaf worm Spodoptera littoralis is known for causing serious damages to various crops. In this study, the susceptibility/tolerance of this larvae to four Vip3A (Vip3Aa, Vip3Ae, Vip3Ad and Vip3Af) toxins was investigated. UnlikeVip3Ad which showed no activity to S. littoralis, Vip3Aa, Vip3Ae and Vip3Af exhibited high toxicity to this larva with LC50 of 228.42 ng/cm2, 65.71 ng/cm2, and 388.90 ng/cm2, respectively. Activation of the 90 kDa Vip3A proteins by S. littoralis larvae juice generated four major bands of sizes 62, 45, 33 and 22 kDa. Binding experiments between biotinylated Vip3A toxins and the brush border membrane vesicles (BBMV) revealed two binding proteins of 55 and 100 kDa with Vip3Aa. Vip3Ae and Vip3Af recognized one single putative receptor of 65 kDa, whereas Vip3Ad did not bind to S. littoralis BBMV. In histopathological observations, Vip3Aa, Vip3Ae and Vip3Af toxins showed approximately similar damages on S. littoralis midgut including rupture and disintegration of epithelial layer and cellular vacuolization. These findings showed that Vip3Aa, Vip3Ae and Vip3Af might be useful for controlling S. littoralis.

The pore-forming domain of Bacillus thuringiensis insecticidal Cry toxins is formed of seven amphipathic α-helices. Because pore formation is thought to involve conformational changes within this domain, the possible role of its interhelical loops in this crucial step was investigated with Cry9Ca double mutants, which all share the previously characterized R164A mutation, using a combination of homology modeling, bioassays and electrophysiological measurements. The mutations either introduced, neutralized or reversed an electrical charge carried by a single residue of one of the domain I loops. The ability of the 28 Cry9Ca double mutants to depolarize the apical membrane of freshly isolated Manduca sexta larval midguts was tested in the presence of either midgut juice or a cocktail of protease inhibitors because these conditions had been shown earlier to greatly enhance pore formation by Cry9Ca and its R164A single-site mutant. Most mutants retained toxicity toward neonate larvae and a pore-forming ability in the electrophysiological assay, which were comparable to those of their parental toxin. In contrast, mutants F130D, L186D and V189D were very poorly toxic and practically inactive in vitro. On the other hand, mutant E129A depolarized the midgut membrane efficiently despite a considerably reduced toxicity, and mutant Q192E displayed a reduced depolarizing ability while conserving a near wild-type toxicity. These results suggest that the conditions found in the insect midgut, including high ionic strength, contribute to minimizing the influence of surface charges on the ability of Cry9Ca and probably other B. thuringiensis toxins to form pores within their target membrane.

The pore-forming ability of the Bacillus thuringiensis toxin Cry9Ca, its two single-site mutants R164A and R164K, and the 55-kDa fragment resulting from its proteolytic cleavage at R164 was evaluated under a variety of experimental conditions using an electrophysiological assay. All four toxin preparations depolarized the apical membrane of freshly isolated third-instar Manduca sexta midguts bathing in a solution containing 122 mM KCl at pH 10.5, but the 55-kDa fragment was considerably more active than Cry9Ca and its mutants. The activity of the latter toxins was greatly enhanced, however, when the experiments were conducted in the presence of fifth-instar M. sexta midgut juice. This effect was also observed after midgut juice proteins had been denatured by heating at 95 degrees C or after inorganic ions and small molecules had been removed from the midgut juice by extensive dialysis. A similar stimulation of toxin activity was also observed when the experiments were carried out in the presence of the lipids extracted from an equivalent volume of midgut juice. Depolarization of the cell membrane was also greatly enhanced, in the absence of midgut juice, by the addition of a cocktail of water-soluble protease inhibitors. These results indicate that, depending on the cleavage site and on the experimental conditions used, further proteolysis of the activated Cry9Ca toxin can either stimulate or be detrimental to its activity and that M. sexta midgut juice probably contains protease inhibitors that could play a major role in the activity of B. thuringiensis toxins in the insect midgut.

Global warming causes night temperature (NT) to increase faster than day temperature in the tropics. According to crop growth models, respiration incurs a loss of 40-60% of photosynthate. The thermal sensitivity of night respiration (R(n)) will thus reduce biomass. Instantaneous and acclimated effects of NT on R(n) of leaves and seedlings of two rice cultivars having a variable level of carbohydrates, induced by exposure to different light intensity on the previous day, were investigated. Experiments were conducted in a greenhouse and growth chambers, with R(n) measured on the youngest fully expanded leaves or whole seedlings. Dry weight-based R(n) was 2.6-fold greater for seedlings than for leaves. Leaf R(n) was linearly related to starch (positive intercept) and soluble sugar concentration (zero intercept). Increased NT caused higher R(n) at a given carbohydrate concentration. The change of R(n) at NT increasing from 21 °C to 31 °C was 2.4-fold for the instantaneous response but 1.2- to 1.7-fold after acclimation. The maintenance component of R(n) (R(m)'), estimated by assimilate starvation, averaged 28% in seedlings and 34% in leaves, with no significant thermal effect on this ratio. The acclimated effect of increased NT on R(m)' across experiments was 1.5-fold for a 10 °C increase in NT. No cultivar differences were observed in R(n) or R(m)' responses. The results suggest that the commonly used Q10=2 rule overestimates thermal response of respiration, and R(n) largely depends on assimilate resources.

Binding studies using (125)I-Cry9Ca and biotinylated-Cry1Ba proteins showed the occurrence of independent binding sites for these proteins in Ostrinia nubilalis. Our results, along with previously available binding data, indicate that combinations of Cry1A or Cry1Fa proteins with Cry1Ba and/or Cry9Ca could be a good strategy for the resistance management of O. nubilalis.

We report the purification, cloning and characterization of an aminopeptidase N from the midgut epithelium of Manduca sexta that binds Cry1Ab5, an insecticidal crystal protein [ICP] from Bacillus thuringiensis. Sequence information derived from this M. sexta aminopeptidase N was used for the cloning of an aminopeptidase N from the midgut brush-border membrane of Plutella xylostella, an insect species of which some populations acquired resistance against Cry1Ab5. Affinity chromatography on a Cry1Ab5 matrix was used to isolate a 120-kDa glycoprotein from the larval midgut of the lepidopteran M. sexta. On ligand blots the purified 120-kDa protein discriminates between the lepidopteran-specific Cry1Ab5 and the coleopteran-specific Cry3A delta-endotoxin. Internal amino acid sequences from the 120-kDa protein were used for the design of degenerate oligonucleotides. From a nested PCR with M. sexta midgut cDNA as template, a DNA fragment was obtained which shows similarity to prokaryotic and eukaryotic aminopeptidase N genes. This PCR fragment was used to screen cDNA libraries of larval midguts from M. sexta and P. xylostella. From the M. sexta midgut cDNA library a 2973-bp nucleotide sequence was cloned. The ORF of the sequence encodes a 942-residue aminopeptidase N (M. sexta Apn2) containing two hydrophobic regions. The NH2-terminal hydrophobic region corresponds to a secretory signal sequence and the COOH-terminal hydrophobic region is typical of glycosylphosphatidylinositol (glycosyl-PtdIns)-anchored proteins. Low-stringency hybridization of the P. xylostella midgut cDNA library with M. sexta apn2 probes enabled the isolation of a 3118-bp sequence with an ORF encoding a 946-residue preproprotein. This aminopeptidase N (P. xylostella Apn1) displays 61% amino acid identity to M. sexta Apn2 and contains a COOH-terminal signal peptide for glycosyl-PtdIns anchor addition. Both M. sexta Apn2 and P. xylostella Apn1 contain four Cys residues, which are highly conserved among eukaryotic aminopeptidase N molecules. Treatment of Sf9 cells expressing the P. xylostella apn1 gene with PtdIns-specific phospholipase C demonstrated that P. xylostella Apn1 is attached to the insect cell membrane by a glycosyl-PtdIns anchor.

First generation of insect-protected transgenic corn (Bt-corn) was based on the expression of Cry1Ab or Cry1Fa proteins. Currently, the trend is the combination of two or more genes expressing proteins that bind to different targets. In addition to broadening the spectrum of action, this strategy helps to delay the evolution of resistance in exposed insect populations. One of such examples is the combination of Cry1A.105 with Cry1Fa and Cry2Ab to control O. nubilalis and S. frugiperda. Cry1A.105 is a chimeric protein with domains I and II and the C-terminal half of the protein from Cry1Ac, and domain III almost identical to Cry1Fa. The aim of the present study was to determine whether the chimeric Cry1A.105 has shared binding sites either with Cry1A proteins, with Cry1Fa, or with both, in O. nubilalis and in S. frugiperda. Brush-border membrane vesicles (BBMV) from last instar larval midguts were used in competition binding assays with (125)I-labeled Cry1A.105, Cry1Ab, and Cry1Fa, and unlabeled Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, Cry2Ab and Cry2Ae. The results showed that Cry1A.105, Cry1Ab, Cry1Ac and Cry1Fa competed with high affinity for the same binding sites in both insect species. However, Cry2Ab and Cry2Ae did not compete for the binding sites of Cry1 proteins. Therefore, according to our results, the development of cross-resistance among Cry1Ab/Ac, Cry1A.105, and Cry1Fa proteins is possible in these two insect species if the alteration of shared binding sites occurs. Conversely, cross-resistance between these proteins and Cry2A proteins is very unlikely in such case.

Vip3Aa, Vip3Ad, Vip3Ae, and Vip3Af proteins from Bacillus thuringiensis were tested for their toxicity against Spodoptera frugiperda and Agrotis ipsilon. Vip3Ad was non-toxic to the two species. Vip3Ae and Vip3Af were significantly more toxic than Vip3Aa against S. frugiperda, both as protoxins and as toxins. Against A. ipsilon, Vip3Ae protoxin was more toxic than Vip3Aa and Vip3Af protoxins. Purification by metal-chelate affinity chromatography significantly affected Vip3Ae toxicity against the two insect species.

Cry1Fa insecticidal protein was successfully radiolabeled with 125 I-Na. Specific binding to brush border membrane vesicles was shown for the lepidopteran species Ostrinia nubilalis , Spodoptera frugiperda , Spodoptera exigua , Helicoverpa armigera , Heliothis virescens , and Plutella xylostella . Homologous competition assays were performed to obtain equilibrium binding parameters ( K d [dissociation constant] and R t [concentration of binding sites]) for these six insect 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 field. silcac@uv.es; ferrej@uv.es

Of 188 Australian Bacillus thuringiensis strains screened for genes encoding soluble insecticidal proteins by polymerase chain reaction/restriction-length fragment polymorphism (RFLP) analysis, 87% showed the presence of such genes. Although 135 isolates (72%) produced an RFLP pattern identical to that expected for vip3A genes, 29 isolates possessed a novel vip-like gene. The novel vip-like gene was cloned from B. thuringiensis isolate C81, and sequence analysis demonstrated that it was 94% identical to the vip3Ba1 gene. The new gene was designated vip3Bb2. Cell-free supernatants from both the B. thuringiensis strain C81 and from Escherichia coli expressing the Vip3Bb2 protein were toxic for the cotton bollworm, Helicoverpa armigera.

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 K d (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 R t (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.

Economically important crops, such as maize and cotton, have been transformed with genes encoding insecticidal proteins from Bacillus thuringiensis (Bt) to confer them protection against the most important insect pests. Of the 114 million hectares globally planted with GM crops in 2007, over one third are insect-resistant Bt crops, and the area keeps increasing every year. The potential for insects to evolve resistance to GM insecticidal plants is considered to be one of the main threats to this technology, since resistance to Bt sprayable products has been demonstrated. Insect resistance management plans for this new class of pesticides are encouraged and became mandatory in the USA. Of the several strategies considered, a high dose of the insecticidal protein along with an adjacent refuge plot of non-Bt plants has been chosen as the most effective. Second generation Bt cotton combines two insecticidal proteins with unique target sites. Such “pyramided” Bt crops hold great promise and, in combination with the high dose/refuge strategy, will likely confer maximum protection to the Bt crop technology against insect resistance. So far, no case of resistance evolution to Bt crops has been reported.

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A novel vip3 -related gene was identified in Bacillus thuringiensis . This novel gene is 2,406 bp long and codes for a 91-kDa protein (801 amino acids). This novel protein exhibits between 61 and 62% similarity with Vip3A proteins and is designated Vip3Ba1. Vip3Ba1 has several specific features. Differences between Vip3Ba1 and the Vip3A proteins are spread throughout the sequence but are more frequent in the C-terminal part from amino acid 456 onward. The regions containing the two proteolytic processing sites, which are highly conserved among the Vip3A toxins, are markedly different in Vip3Ba1. The pattern DCCEE (Asp Cys Cys Glu Glu) is repeated four times between position 463 and 483 in Vip3Ba1, generating the sequence 463-DCCEEDCCEEDCCEEDCCEE-483. This sequence, which is rich in negatively charged amino acids, also contains 73% of the cysteines present in Vip3Ba1. This repeated sequence is not present in Vip3A proteins. The Vip3Ba1protein was produced in Escherichia coli and tested against Ostrinia nubilalis and Plutella xylostella , and it generated significant growth delays but had no larvicidal effect, indicating that its host range might be different than that of Vip3A proteins.

Bacillus thuringiensis isolates from different ecological regions and sources of China were analyzed to study the distribution and diversity of cry genes and to detect the presence of novel cry genes. Strains containing cry1-type genes were the most abundant and represent 237 of the 310 B. thuringiensis isolates (76.5%). About 70 and 15.5% of the isolates contained a cry2 gene or cry9 gene, respectively, while 10.0% of the strains did not contain a cry1, cry2, or cry9 gene. Among the cry1 containing isolates, cry1A (67.7%), cry1I (60.6%), cry1C (43.9%), and cry1D (39.4%) genes were the most abundant. Forty-three different cry1 gene profiles were detected in this collection. Several cry1 genes were associated at a high frequency, such as the cry1C-cry1D and cry1A-cry1I gene combination. The cry1A and cry2 amplicons were digested with selected restriction enzymes to examine sequence diversity. Based on this RFLP analysis, one novel cry1A-type gene was observed.

▪ Abstract Bacillus thuringiensis (Bt) is a valuable source of insecticidal proteins for use in conventional sprayable formulations and in transgenic crops, and it is the most promising alternative to synthetic insecticides. However, evolution of resistance in insect populations is a serious threat to this technology. So far, only one insect species has evolved significant levels of resistance in the field, but laboratory selection experiments have shown the high potential of other species to evolve resistance against Bt. We have reviewed the current knowledge on the biochemical mechanisms and genetics of resistance to Bt products and insecticidal crystal proteins. The understanding of the biochemical and genetic basis of resistance to Bt can help design appropriate management tactics to delay or reduce the evolution of resistance in insect populations.

Little information is available on the systemic effects of Bacillus thuringiensis toxins in the hemocoel of insects. In order to test whether B. thuringiensis -activated toxins elicit a toxic response in the hemocoel, we measured the effect of intrahemocoelic injections of several Cry1 toxins on the food intake, growth, and survival of Lymantria dispar (Lepidoptera) and Neobellieria bullata (Diptera) larvae. Injection of Cry1C was highly toxic to the Lymantria larvae and resulted in the complete inhibition of food intake, growth arrest, and death in a dose-dependent manner. Cry1Aa and Cry1Ab (5 μg/0.2 g [fresh weight] [g fresh wt]) also affected growth and food intake but were less toxic than Cry1C (0.5 μg/0.2 g fresh wt). Cry1E and Cry1Ac (5 μg/0.2 g fresh wt) had no toxic effect upon injection. Cry1C was also highly toxic to N. bullata larvae upon injection. Injection of 5 μg/0.2 g fresh wt resulted in rapid paralysis, followed by hemocytic melanization and death. Lower concentrations delayed pupariation or gave rise to malformation of the puparium. Finally, Cry1C was toxic to brain cells of Lymantria in vitro. The addition of Cry1C (20 μg/ml) to primary cultures of Lymantria brain cells resulted in rapid lysis of the cultured neurons.

Several insect species have developed resistance to insecticidal crystal proteins from Bacillus thuringiensis, either through laboratory selection, or under field conditions. In this chapter we review the current knowledge on the biochemical and genetic mechanisms of resistance to B. thuringiensis. This knowledge will be important in the design of appropriate tactics to manage the development of resistance in insect populations.

A little more than 10 years ago, it was demonstrated that genes, from any organism, could be introduced into the plant genome and be expressed by plant cells, conferring new properties on these transgenic plants. Crops are threatened by a vast array of weeds, pests and diseases and annually we use some 2 5 million tons of pesticides, worth some US$ 30 billion. Crop protection through plant engineering will reduce the dependency on pesticides and will in certain cases provide a more effective protection. Crops such as corn, cotton, soybeans and rape seed, engineered so that they are tolerant to non-selective, environmentally friendly herbicides, are expected on the market in 1996.

Current knowledge of biochemical mechanisms of insect resistance to Bacillus thuringiensis is reviewed. Available information on resistance inheritance and on patterns of cross-resistance is included. Modification of the binding sites for B. thuringiensis insecticidal crystal proteins has been found in different populations of three insect species. This resistance mechanism seems to be inherited as a single recessive or partially recessive major gene, and the resistance levels reached are high. Altered proteolytic processing of B. thuringiensis crystal proteins has been suggested to be involved in one case of resistance. From the available data it seems that binding site modification is the most significant resistance mechanism under field conditions.

We have investigated the protein composition and the insecticidal spectrum of crystals of 29 Bacillus thuringiensis strains active against lepidopteran larvae. All crystals contained proteins of 130 to 140 kilodaltons (kDa) which could be grouped into three types by the molecular weight of the protoxin and the trypsin-activated core fragment. Proteins of the three types showed a characteristic insecticidal spectrum when tested against five lepidopteran species. Type A crystal proteins were protoxins of 130 or 133 kDa, which were processed into 60-kDa toxins by trypsin. Several genes encoding crystal proteins of this type have been cloned and sequenced earlier. They are highly conserved in the N-terminal half of the toxic fragment and were previously classified in three subtypes (the 4.5-, 5.3-, and 6.6-kilobase subtypes) based on the restriction map of their genes. The present study shows that different proteins of these three subtypes were equally toxic against Manduca sexta and Pieris brassicae and had no detectable activity against Spodoptera littoralis . However, the 4.5-, 5.3-, and 6.6-kilobase subtypes differed in their toxicity against Heliothis virescens and Mamestra brassicae . Type B crystal proteins consisted of 140-kDa protoxins with a 55-kDa tryptic core fragment. These were only active against one of the five insect species tested ( P. brassicae ). The protoxin and the trypsin-activated toxin of type C were 135- and 63-kDa proteins, respectively. Proteins of this type were associated with high toxicity against S. littoralis and M. brassicae . A panel of 35 monoclonal antibodies was used to compare the structural characteristics of crystal proteins of the three different types and subtypes. Each type of protein could be associated with a typical epitope structure, indicating an unambiguous correlation between antigenic structure and insect specificity.

To study the molecular basis of differences in the insecticidal spectrum of Bacillus thuringienesis delta-endotoxins, we have performed binding studies with three delta-endotoxins on membrane preparations from larval insect mid-gut. Conditions for a standard binding assay were established through a detailed study of the binding of 125I-labeled Bt2 toxin, a recombinant B. thuringiensis delta-endotoxin, to brush border membrane vesicles of Manduca sexta. The toxins tested (Bt2, Bt3 and Bt73 toxins) are about equally toxic to M. sexta but differ in their toxicity against Heliothis virescens. Equilibrium binding studies revealed saturable, high-affinity binding sites on brush border membrane vesicles of M. sexta and H. virescens. While the affinity of the three toxins was not significantly different on H. virescens vesicles, marked differences in binding site concentration were measured which reflected the differences in in vivo toxicity. Competition experiments revealed heterogeneity in binding sites. For H. virescens, a three-site model was proposed. In M. sexta, one population of binding sites is shared by all three toxins, while another is only recognized by Bt3 toxin. Several other toxins, non-toxic or much less toxic to M. sexta than Bt2 toxin, did not or only marginally displace binding of 125I-labeled Bt2 toxin in this insect. No saturable binding of this toxin was observed to membrane preparations from tissues of several non-susceptible organisms. Together, these data provide new evidence that binding to a specific receptor on the membrane of gut epithelial cells is an important determinant with respect to differences in insecticidal spectrum of B. thuringiensis insecticidal crystal proteins.

The toxicity and pore-forming ability of the Bacillus thuringiensis Cry9Ca insecticidal toxin, its single-site mutants, R164A and R164K, and the 55-kDa fragment resulting from its proteolytic cleavage at residue 164 were investigated using Manduca sexta neonate larvae and fifth-instar larval midgut brush border membrane vesicles, respectively. Neither the mutations nor the proteolytic cleavage altered Cry9Ca toxicity. Compared with Cry1Ac, Cry9Ca and its mutants formed large poorly selective pores in the vesicles. Pore formation was highly dependent on pH, however, especially for wild-type Cry9Ca and both mutants. Increasing pH from 6.5 to 10.5 resulted in an irregular step-wise decrease in membrane permeabilization that was not related to a change in the ionic selectivity of the pores. Pore formation was much slower with Cry9Ca and its derivatives, including the 55-kDa fragment, than with Cry1Ac and its rate was not influenced by the presence of protease inhibitors or a reducing agent.