An ABA-serotonin module regulates root suberization and salinity tolerance.

Suberin in roots acts as a physical barrier preventing water/mineral losses. In Arabidopsis, root suberization is regulated by abscisic acid (ABA) and ethylene in response to nutrient stresses. ABA also mediates coordination between microbiota and root endodermis in mineral nutrient homeostasis. However, it is not known whether this regulatory system is common to plants in general, and whether there are other key molecule(s) involved. We show that serotonin acts downstream of ABA in regulating suberization in rice and Arabidopsis and negatively regulates suberization in rice roots in response to salinity. We show that ABA represses transcription of the key gene (OsT5H) in serotonin biosynthesis, thus promoting root suberization in rice. Conversely, overexpression of OsT5H or supplementation with exogenous serotonin represses suberization and reduces tolerance to salt stress. These results identify an ABA-serotonin regulatory module controlling root suberization in rice and Arabidopsis, which is likely to represent a general mechanism as ABA and serotonin are ubiquitous in plants. These findings are of significant importance to breeding novel crop varieties that are resilient to abiotic stresses and developing strategies for production of suberin-rich roots to sequestrate more CO2 , helping to mitigate the effects of climate change.

pCAT vectors overcome inefficient electroporation of Cupriavidus necator H16.

Cupriavidus necator H16 is a chemolithoautotroph with a range of industrial biotechnological applications. Advanced metabolic engineering in the bacterium, however, is impeded by low transformation efficiency, making it difficult to introduce and screen new genetic functions rapidly. This study systematically characterized the broad host range plasmids pBHR1, pBBR1MCS-2 and pKT230 used frequently for C. necator engineering. Kanamycin resistance cassette (KanR) and a truncated sequence of the replication origin (Rep) are contributing factors to C. necator low electroporation transformation efficiency. Consequently, a series of modular minimal plasmids, named pCAT, were constructed. pCAT vectors transform C. necator H16 with a > 3000-fold higher efficiency (up to 107 CFU/µg DNA) compared to control plasmids. Further, pCAT vectors are highly stable, expressing reporter proteins over several days of serial cultivation in the absence of selection pressure. Finally, they can be assembled rapidly from PCR or synthesized DNA fragments, and restriction-ligation reactions can be efficiently electroporated directly into C. necator, circumventing the requirement to use Escherichia coli for plasmid maintenance or propagation. This study demonstrates that an understanding of the behaviour of the constituent parts of plasmids in a host is key to efficient propagation of genetic information, while offering new methods for engineering a bacterium with desirable industrial biotechnological features.

Virus-induced plant volatiles mediate the olfactory behaviour of its insect vectors.

Plant viruses can manipulate their hosts to release odours that are attractive or repellent to their insect vectors. However, the volatile organic compounds (VOCs), either individually or as mixtures, which play a key role in the olfactory behaviour of insect vectors remains largely unknown. Our study focused on green rice leafhoppers (GRLHs) vectoring rice dwarf virus (RDV) revealed that RDV infection significantly induced the emission of (E)-ß-caryophyllene and 2-heptanol by rice plants, which influenced the olfactory behaviour of both non-viruliferous and viruliferous GRLHs. (E)-ß-caryophyllene attracted non-viruliferous GRLHs to settle on RDV-infected plants, but neither attracted nor repelled viruliferous GRLHs. In contrast, 2-heptanol repelled viruliferous GRLHs to settle on RDV-infected plants, but neither repelled nor attracted non-viruliferous GRLHs. Suppression of (E)-ß-caryophyllene synthase OsCAS via CRISPR-Cas9 to generate oscas-1 plants enabled us to confirm the important role played by (E)-ß-caryophyllene in modulating the virus-vector-host plant interaction. These novel results reveal the role of these virus-induced VOCs in modulating the behaviour of its GRLH insect vector and may facilitate the design of new strategies for disease control through manipulation of plant volatile emissions.

Volatile Organic Compounds as Insect Repellents and Plant Elicitors: an Integrated Pest Management (IPM) Strategy for Glasshouse Whitefly (Trialeurodes vaporariorum).

The glasshouse whitefly (Trialeurodes vaporariorum Westwood) is a polyphagous arthropod pest that is of particular detriment to glasshouse grown tomato (Solanum lycopersicum) across temperate regions of the world. Control of whiteflies with synthetic pesticides has resulted in the evolution of resistant genotypes and a reduction in natural enemies, thus highlighting the need for environmentally sound control strategies. Volatile organic compounds (VOCs) offer an environmentally benign alternative to synthetic chemical sprays and this study explored the use of VOCs as insect repellents and plant defence elicitors to control whiteflies on tomato in a commercial glasshouse setting. Limonene in the form of a volatile dispenser system was found to successfully repel whitefly from the target crop and increased fruit yield by 32% during a heavy whitefly infestation. Analysis of tomato herbivore induced plant volatiles (HIPVs) led us to select methyl salicylate (MeSA) as the plant elicitor and application of MeSA to un-infested tomato plants was found to successfully reduce whitefly population development and increase yield by 11%, although this difference was marginally statistically significant. Combination of these two methods was also effective but whitefly abundance in combined plots was similar to the standalone limonene treatment across the course of the experiment. All of the VOC based control methods we used had a negative impact on whitefly performance, with more pronounced effects during the first few weeks of infestation. In subsequent laboratory experiments, we found elevated peroxidase (POD) activity and a significant increase in TPX1 and PR1 transcripts in MeSA treated plants. This led us to deduce that MeSA immediately induced plant defences, rather than priming them. We did however see evidence for residual priming, as plants treated with MeSA and infested with whiteflies produced significantly higher levels of POD activity than whitefly infestation alone. Despite the fact that our treatments failed to synergise, our methods can be optimised further, and the effectiveness of the standalone treatments is promising for future studies. In particular, our repellent limonene dispensers were extremely effective at deterring whiteflies and offer a low economic cost and easy to implement whitefly control option. The methods we have used here could be incorporated into current integrated pest management (IPM) systems, a sustainable approach to pest control which will be central to our efforts to manage whitefly populations under glass in the future.

Septoria Leaf Blotch and Reduced Nitrogen Availability Alter WRKY Transcription Factor Expression in a Codependent Manner.

A major cause of yield loss in wheat worldwide is the fungal pathogen Zymoseptoria tritici, a hemibiotrophic fungus which causes Septoria leaf blotch, the most destructive wheat disease in Europe. Resistance in commercial wheat varieties is poor, however, a link between reduced nitrogen availability and increased Septoria tolerance has been observed. We have shown that Septoria load is not affected by nitrogen, whilst the fungus is in its first, symptomless stage of growth. This suggests that a link between nitrogen and Septoria is only present during the necrotrophic phase of Septoria infection. Quantitative real-time PCR data demonstrated that WRKYs, a superfamily of plant-specific transcription factors, are differentially expressed in response to both reduced nitrogen and Septoria. WRKY39 was downregulated over 30-fold in response to necrotrophic stage Septoria, whilst changes in the expression of WRKY68a during the late biotrophic phase were dependent on the concentration of nitrogen under which wheat is grown. WRKY68a may therefore mediate a link between nitrogen and Septoria. The potential remains to identify key regulators in the link between nitrogen and Septoria, and as such, elucidate molecular markers for wheat breeding, or targets for molecular-based breeding approaches.

Applying Statistical Design of Experiments To Understanding the Effect of Growth Medium Components on Cupriavidus necator H16 Growth.

Cupriavidus necator H16 is gaining significant attention as a microbial chassis for range of biotechnological applications. While the bacterium is a major producer of bioplastics, its lithoautotrophic and versatile metabolic capabilities make the bacterium a promising microbial chassis for biofuels and chemicals using renewable resources. It remains necessary to develop appropriate experimental resources to permit controlled bioengineering and system optimization of this microbe. In this study, we employed statistical design of experiments to gain understanding of the impact of components of defined media on C. necator growth and built a model that can predict the bacterium's cell density based on medium components. This highlighted medium components, and interaction between components, having the most effect on growth: fructose, amino acids, trace elements, CaCl2, and Na2HPO4 contributed significantly to growth (t values of <-1.65 or >1.65); copper and histidine were found to interact and must be balanced for robust growth. Our model was experimentally validated and found to correlate well (r2 = 0.85). Model validation at large culture scales showed correlations between our model-predicted growth ranks and experimentally determined ranks at 100 ml in shake flasks (ρ = 0.87) and 1 liter in a bioreactor (ρ = 0.90). Our approach provides valuable and quantifiable insights on the impact of medium components on cell growth and can be applied to model other C. necator responses that are crucial for its deployment as a microbial chassis. This approach can be extended to other nonmodel microbes of medical and industrial biotechnological importance.IMPORTANCE Chemically defined media (CDM) for cultivation of C. necator vary in components and compositions. This lack of consensus makes it difficult to optimize new processes for the bacterium. This study employed statistical design of experiments (DOE) to understand how basic components of defined media affect C. necator growth. Our growth model predicts that C. necator can be cultivated to high cell density with components held at low concentrations, arguing that CDM for large-scale cultivation of the bacterium for industrial purposes will be economically competitive. Although existing CDM for the bacterium are without amino acids, addition of a few amino acids to growth medium shortened lag phase of growth. The interactions highlighted by our growth model show how factors can interact with each other during a process to positively or negatively affect process output. This approach is efficient, relying on few well-structured experimental runs to gain maximum information on a biological process, growth.

Induction of defense in cereals by 4-fluorophenoxyacetic acid suppresses insect pest populations and increases crop yields in the field.

Synthetic chemical elicitors, so called plant strengtheners, can protect plants from pests and pathogens. Most plant strengtheners act by modifying defense signaling pathways, and little is known about other mechanisms by which they may increase plant resistance. Moreover, whether plant strengtheners that enhance insect resistance actually enhance crop yields is often unclear. Here, we uncover how a mechanism by which 4-fluorophenoxyacetic acid (4-FPA) protects cereals from piercing-sucking insects and thereby increases rice yield in the field. Four-FPA does not stimulate hormonal signaling, but modulates the production of peroxidases, H2O2, and flavonoids and directly triggers the formation of flavonoid polymers. The increased deposition of phenolic polymers in rice parenchyma cells of 4-FPA-treated plants is associated with a decreased capacity of the white-backed planthopper (WBPH) Sogatella furcifera to reach the plant phloem. We demonstrate that application of 4-PFA in the field enhances rice yield by reducing the abundance of, and damage caused by, insect pests. We demonstrate that 4-FPA also increases the resistance of other major cereals such as wheat and barley to piercing-sucking insect pests. This study unravels a mode of action by which plant strengtheners can suppress herbivores and increase crop yield. We postulate that this represents a conserved defense mechanism of plants against piercing-sucking insect pests, at least in cereals.

The Impact of Environmental Stress on Bt Crop Performance.

Bt crops have been grown commercially for more than two decades. They have proven remarkably effective in the control of target insect pests. However, Bt crops can become less effective under various forms of environmental stress. Most studies in this area have considered the effect of environmental stress on Bt insecticidal protein levels or target pest mortality, but not both, resulting in a lack of mechanistic analysis. In this review, we critically examine previous research addressing the impact of environmental stress on the effectiveness of Bt crops. We find that the body of research data is not sufficiently robust to allow the reliable prediction of the performance of Bt crops under extreme climatic conditions.

Evolution of Asian Corn Borer Resistance to Bt Toxins Used Singly or in Pairs.

Transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) have revolutionized pest control, but the benefits of this approach have been reduced by the evolution of resistance in pests. The widely adopted 'pyramid strategy' for delaying resistance entails transgenic crops producing two or more distinct toxins that kill the same pest. The limited experimental evidence supporting this strategy comes primarily from a model system under ideal conditions. Here we tested the pyramid strategy under nearly worst-case conditions, including some cross-resistance between the toxins in the pyramid. In a laboratory selection experiment with an artificial diet, we used Bt toxins Cry1Ab, Cry1F, and Cry1Ie singly or in pairs against Ostrinia furnacalis, one of the most destructive pests of corn in Asia. Under the conditions evaluated, pairs of toxins did not consistently delay the evolution of resistance relative to single toxins.

A proteomic-based approach to study underlying molecular responses of the small intestine of Wistar rats to genetically modified corn (MON810).

A genetically modified (GM) commercial corn variety, MON810, resistant to European corn borer, has been shown to be non-toxic to mammals in a number of rodent feeding studies carried out in accordance with OECD Guidelines. Insect resistance results from expression of the Cry1Ab gene encoding an insecticidal Bt protein that causes lysis and cell death in susceptible insect larvae by binding to midgut epithelial cells, which is a key determinant of Cry toxin species specificity. Whilst whole animal studies are still recognised as the 'gold standard' for safety assessment, they only provide indirect evidence for changes at the cellular/organ/tissue level. In contrast, omics-based technologies enable mechanistic understanding of toxicological or nutritional events at the cellular/receptor level. To address this important knowledge-gap and to gain insights into the underlying molecular responses in rat to MON810, differential gene expression in the epithelial cells of the small intestine of rats fed formulated diets containing MON810, its near isogenic line, two conventional corn varieties, and a commercial (Purina™) corn-based control diet were investigated using comparative proteomic profiling. Pairwise and five-way comparisons showed that the majority of proteins that were differentially expressed in the small intestine epithelial cells in response to consumption of the different diets in both 7-day and 28-day studies were related to lipid and carbohydrate metabolism and protein biosynthesis. Irrespective of the diet, a limited number of stress-related proteins were shown to be differentially expressed. However these stress-related proteins differed between diets. No adverse clinical or behavioural effects, or biomarkers of adverse health, were observed in rats fed GM corn compared to the other corn diets. These findings suggest that MON810 has negligible effects on the small intestine of rats at the cellular level compared with the well-documented toxicity observed in susceptible insects.

Companion planting with French marigolds protects tomato plants from glasshouse whiteflies through the emission of airborne limonene.

Horticulturalists and gardeners in temperate regions often claim that planting marigolds next to tomato plants protects the tomatoes from the glasshouse whitefly (Trialeurodes vaporariorum Westwood). If shown to hold true, this technique could be used in larger-scale tomato production, protecting the crop and helping to introduce greater plant diversity into these agro-ecosystems. Here we present two large-scale glasshouse trials corresponding to the two main ways growers are likely to use marigolds to control whiteflies. In the first, marigolds are grown next to tomato throughout the growing period and we quantify whitefly population growth from the seedling stage over a 48 day infestation period. Here we show that association with marigolds significantly slows whitefly population development. Introducing additional whitefly-attractive 'pull' plants around the perimeter of plots has little effect, but reducing the proportion of marigolds and introducing other non-hosts of whiteflies (basil, nasturtium and Chinese cabbage) also reduces whitefly populations on tomato. The second experiment assesses the efficacy of marigolds when used as an 'emergency' measure. Here we allow whitefly populations to build to a high density on unprotected tomatoes then introduce marigolds and assess whitefly population over a further period. Following laboratory work showing limonene to be a major chemical component of French marigolds and a negative behaviour response of whiteflies to this compound, limonene dispensers are added as an additional treatment to this experiment. "Emergency" marigold companion planting yielded minimal reductions in whitefly performance, but the use of limonene dispensers was more effective. Our work indicates that companion planting short vine tomatoes with French marigolds throughout the growing season will slow development of whitefly populations. Introducing marigolds to unprotected tomatoes after significant whitefly build-up will be less effective. The use of limonene dispensers placed near to tomato plants also shows promise. It is argued that this work supports the possibility of the development of a mixture of tomato companion plants that infer 'associational resistance' against many major invertebrate pests of tomato. Such a mixture, if comprising edible or ornamental plants, would be economically viable, would reduce the need for additional chemical and biological control, and, if used outdoors, would generate plant-diverse agro-ecosystems that are better able to harbour invertebrate wildlife.

OXI1 kinase plays a key role in resistance of Arabidopsis towards aphids (Myzus persicae).

Plants have co-evolved with a diverse array of pathogens and insect herbivores and so have evolved an extensive repertoire of constitutive and induced defence mechanisms activated through complex signalling pathways. OXI1 kinase is required for activation of mitogen-activated protein kinases (MAPKs) and is an essential part of the signal transduction pathway linking oxidative burst signals to diverse downstream responses. Furthermore, changes in the levels of OXI1 appear to be crucial for appropriate signalling. Callose deposition also plays a key role in defence. Here we demonstrate, for the first time, that OXI1 plays an important role in defence against aphids. The Arabidopsis mutant, oxi1-2, showed significant resistance both in terms of population build-up (p < 0.001) and the rate of build-up (p < 0.001). Arabidopsis mutants for ß-1,3-glucanase, gns2 and gns3, showed partial aphid resistance, significantly delaying developmental rate, taking two-fold longer to reach adulthood. Whilst ß-1,3-glucanase genes GNS1, GNS2, GNS3 and GNS5 were not induced in oxi1-2 in response to aphid feeding, GNS2 was expressed to high levels in the corresponding WT (Col-0) in response to aphid feeding. Callose synthase GSL5 was up-regulated in oxi1-2 in response to aphids. The results suggest that resistance in oxi1-2 mutants is through induction of callose deposition via MAPKs resulting in ROS induction as an early response. Furthermore, the results suggest that the ß-1,3-glucanase genes, especially GNS2, play an important role in host plant susceptibility to aphids. Better understanding of signalling cascades underpinning tolerance to biotic stress will help inform future breeding programmes for enhancing crop resilience.

Resistance of rice to insect pests mediated by suppression of serotonin biosynthesis.

Rice is one of the world's most important foods, but its production suffers from insect pests, causing losses of billions of dollars, and extensive use of environmentally damaging pesticides for their control1,2. However, the molecular mechanisms of insect resistance remain elusive. Although a few resistance genes for planthopper have been cloned, no rice germplasm is resistant to stem borers. Here, we report that biosynthesis of serotonin, a neurotransmitter in mammals3, is induced by insect infestation in rice, and its suppression confers resistance to planthoppers and stem borers, the two most destructive pests of rice2. Serotonin and salicylic acid derive from chorismate4. In rice, the cytochrome P450 gene CYP71A1 encodes tryptamine 5-hydroxylase, which catalyses conversion of tryptamine to serotonin5. In susceptible wild-type rice, planthopper feeding induces biosynthesis of serotonin and salicylic acid, whereas in mutants with an inactivated CYP71A1 gene, no serotonin is produced, salicylic acid levels are higher and plants are more insect resistant. The addition of serotonin to the resistant rice mutant and other brown planthopper-resistant genotypes results in a loss of insect resistance. Similarly, serotonin supplementation in artificial diet enhances the performance of both insects. These insights demonstrate that regulation of serotonin biosynthesis plays an important role in defence, and may prove valuable for breeding insect-resistant cultivars of rice and other cereal crops.

RNAi-mediated knockdown of the voltage gated sodium ion channel TcNav causes mortality in Tribolium castaneum.

The voltage-gated sodium ion channel (VGSC) belongs to the largest superfamily of ion channels. Since VGSCs play key roles in physiological processes they are major targets for effective insecticides. RNA interference (RNAi) is widely used to analyse gene function, but recently, it has shown potential to contribute to novel strategies for selectively controlling agricultural insect pests. The current study evaluates the delivery of dsRNA targeted to the sodium ion channel paralytic A (TcNav) gene in Tribolium castaneum as a viable means of controlling this insect pest. Delivery of TcNav dsRNA caused severe developmental arrest with larval mortalities up to 73% post injection of dsRNA. Injected larvae showed significant (p < 0.05) knockdown in gene expression between 30-60%. Expression was also significantly (p < 0.05) reduced in pupae following injection causing 30% and 42% knockdown for early and late pupal stages, respectively. Oral delivery of dsRNA caused dose-dependant mortalities of between 19 and 51.34%; this was accompanied by significant (p < 0.05) knockdown in gene expression following 3 days of continuous feeding. The majority of larvae injected with, or fed, dsRNA died during the final larval stage prior to pupation. This work provides evidence of a viable RNAi-based strategy for insect control.

Novel resistance mechanisms of a wild tomato against the glasshouse whitefly.

The glasshouse whitefly, Trialeurodes vaporariorum, is an important pest of many crop plants including tomato, Solanum lycopersicum. Many wild tomato species exhibit a higher resistance to whiteflies. Therefore, locating the source of this enhanced resistance and breeding it into commercial tomato species is an important strategy to reduce the impact of pests on crops. Here, we assessed the pest resistance of Lycopersicon pimpinellifolium by comparing oviposition and feeding data from T. vaporariorum on this wild tomato species with data collected from a susceptible commercial tomato, S. lycopersicum var. 'Elegance'. The location of resistance factors was examined by use of electrical penetration graph (EPG) studies on these tomato species. Results show that whiteflies preferentially settled on the commercial tomato more often in 80 % of the replicates when given free choice between the two tomato species and laid significantly fewer eggs on L. pimpinellifolium. Whiteflies exhibited a shorter duration of the second feeding bout, reduced pathway phase probing, longer salivation in the phloem and more non-probing activities in the early stages of the EPG on the wild tomato species compared to the commercial tomato. These findings evidence that a dual mode of resistance is present in this wild tomato against T. vaporariorum: a post-penetration, pre-phloem resistance mechanism and a phloem-located factor, which to the best of our knowledge is the first time that evidence for this has been presented. These findings can be used to inform future breeding strategies to increase the resistance of commercial tomato varieties against this important pest.

Proteomic analysis shows that stress response proteins are significantly up-regulated in resistant diploid wheat (Triticum monococcum) in response to attack by the grain aphid (Sitobion avenae).

The grain aphid Sitobion avenae (F.) is a major pest of wheat, acting as a virus vector as well as causing direct plant damage. Commonly grown wheat varieties in the UK have only limited resistance to this pest. The present study was carried out to investigate the potential of a diploid wheat line (ACC20 PGR1755), reported as exhibiting resistance to S. avenae, to serve as a source of resistance genes. The diploid wheat line was confirmed as partially resistant, substantially reducing the fecundity, longevity and growth rate of the aphid. Proteomic analysis showed that approximately 200 protein spots were reproducibly detected in leaf extracts from both the resistant line and a comparable susceptible line (ACC5 PGR1735) using two-dimensional gel electrophoresis and image comparison software. Twenty-four spots were significantly up-regulated (>2-fold) in the resistant line after 24 h of aphid feeding (13 and 11 involved in local and systemic responses, respectively). Approximately 50 % of all differentially expressed protein spots were identified by a combination of database searching with MS and MS/MS data, revealing that the majority of proteins up-regulated by aphid infestation were involved in metabolic processes (including photosynthesis) and transcriptional regulation. However, in the resistant line only, several stress response proteins (including NBS-LRR-like proteins) and oxidative stress response proteins were identified as up-regulated in response to aphid feeding, as well as proteins involved in DNA synthesis/replication/repair. This study indicates that the resistant diploid line ACC20 PGR1755 may provide a valuable resource in breeding wheat for resistance to aphids.

Transgenic plants expressing ω-ACTX-Hv1a and snowdrop lectin (GNA) fusion protein show enhanced resistance to aphids.

Recombinant fusion proteins containing arthropod toxins have been developed as a new class of biopesticides. The recombinant fusion protein Hv1a/GNA, containing the spider venom toxin ω-ACTX-Hv1a linked to snowdrop lectin (GNA) was shown to reduce survival of the peach-potato aphid Myzus persicae when delivered in artificial diet, with survival <10% after 8 days exposure to fusion protein at 1 mg/ml. Although the fusion protein was rapidly degraded by proteases in the insect, Hv1a/GNA oral toxicity to M. persicae was significantly greater than GNA alone. A construct encoding the fusion protein, including the GNA leader sequence, under control of the constitutive CaMV 35S promoter was transformed into Arabidopsis; the resulting plants contained intact fusion protein in leaf tissues at an estimated level of 25.6 ± 4.1 ng/mg FW. Transgenic Arabidopsis expressing Hv1a/GNA induced up to 40% mortality of M. persicae after 7 days exposure in detached leaf bioassays, demonstrating that transgenic plants can deliver fusion proteins to aphids. Grain aphids (Sitobion avenae) were more susceptible than M. persicae to the Hv1a/GNA fusion protein in artificial diet bioassays (LC50 = 0.73 mg/ml after 2 days against LC50 = 1.81 mg/ml for M. persicae), as they were not able to hydrolyze the fusion protein as readily as M. persicae. Expression of this fusion protein in suitable host plants for the grain aphid is likely to confer higher levels of resistance than that shown with the M. persicae/Arabidopsis model system.

Downregulation and mutation of a Cadherin gene associated with Cry1Ac resistance in the Asian Corn Borer, Ostrinia furnacalis (Guenée).

Development of resistance in target pests is a major threat to long-term use of transgenic crops expressing Bacillus thuringiensis (Bt) Cry toxins. To manage and/or delay the evolution of resistance in target insects through the implementation of effective strategies, it is essential to understand the basis of resistance. One of the most important mechanisms of insect resistance to Bt crops is the alteration of the interactions between Cry toxins and their receptors in the midgut. A Cry1Ac-selected strain of Asian corn borer (ACB), Ostrinia furnacalis, a key pest of maize in China, evolved three mutant alleles of a cadherin-like protein (OfCAD) (MPR-r1, MPR-r2 and MPR-r3), which mapped within the toxin-binding region (TBR). Each of the three mutant alleles possessed two or three amino acid substitutions in this region, especially Thr1457→Ser. In highly resistant larvae (ACB-Ac200), MPR-r2 had a 26-amino acid residue deletion in the TBR, which resulted in reduced binding of Cry1Ac compared to the MPR from the susceptible strain, suggesting that the number of amino acid deletions influences the level of resistance. Furthermore, downregulation of OfCAD gene (ofcad) transcription was observed in the Cry1Ac resistant strain, ACB-Ac24, suggesting that Cry1Ac resistance in ACB is associated with the downregulation of the transcript levels of the cadherin-like protein gene. The OfCAD identified from ACB exhibited a high degree of similarity to other members of the cadherin super-family in lepidopteran species.