Identification and Evaluations of Novel Insecticidal Proteins from Plants of the Class Polypodiopsida for Crop Protection against Key Lepidopteran Pests.

Various lepidopteran insects are responsible for major crop losses worldwide. Although crop plant varieties developed to express Bacillus thuringiensis (Bt) proteins are effective at controlling damage from key lepidopteran pests, some insect populations have evolved to be insensitive to certain Bt proteins. Here, we report the discovery of a family of homologous proteins, two of which we have designated IPD083Aa and IPD083Cb, which are from Adiantum spp. Both proteins share no known peptide domains, sequence motifs, or signatures with other proteins. Transgenic soybean or corn plants expressing either IPD083Aa or IPD083Cb, respectively, show protection from feeding damage by several key pests under field conditions. The results from comparative studies with major Bt proteins currently deployed in transgenic crops indicate that the IPD083 proteins function by binding to different target sites. These results indicate that IPD083Aa and IPD083Cb can serve as alternatives to traditional Bt-based insect control traits with potential to counter insect resistance to Bt proteins.

Molecular characterization of the insecticidal activity of double-stranded RNA targeting the smooth septate junction of western corn rootworm (Diabrotica virgifera virgifera).

The western corn rootworm (WCR, Diabrotica virgifera virgifera) gene, dvssj1, is a putative homolog of the Drosophila melanogaster gene, snakeskin (ssk). This gene encodes a membrane protein associated with the smooth septate junction (SSJ) which is required for the proper barrier function of the epithelial lining of insect intestines. Disruption of DVSSJ integrity by RNAi technique has been shown previously to be an effective approach for corn rootworm control, by apparent suppression of production of DVSSJ1 protein leading to growth inhibition and mortality. To understand the mechanism that leads to the death of WCR larvae by dvssj1 double-stranded RNA, we examined the molecular characteristics associated with SSJ functions during larval development. Dvssj1 dsRNA diet feeding results in dose-dependent suppression of mRNA and protein; this impairs SSJ formation and barrier function of the midgut and results in larval mortality. These findings suggest that the malfunctioning of the SSJ complex in midgut triggered by dvssj1 silencing is the principal cause of WCR death. This study also illustrates that dvssj1 is a midgut-specific gene in WCR and its functions are consistent with biological functions described for ssk.

Susceptible and mCry3A resistant corn rootworm larvae killed by a non-hemolytic Bacillus thuringiensis Cyt1Aa mutant.

The western corn rootworm (WCR) Diabrotica virgifera virgifera causes substantial damage in corn. Genetically modified (GM) plants expressing some Bacillus thuringiensis (Bt) insecticidal Cry proteins efficiently controlled this pest. However, changes in WCR susceptibility to these Bt traits have evolved and identification of insecticidal proteins with different modes of action against WCR is necessary. We show here for the first time that Cyt1Aa from Bt exhibits toxicity against WCR besides to the dipteran Aedes aegypti larvae. Cyt1Aa is a pore-forming toxin that shows no cross-resistance with mosquitocidal Cry toxins. We characterized different mutations in helix α-A from Cyt1Aa. Two mutants (A61C and A59C) exhibited reduced or absent hemolytic activity but retained toxicity to A. aegypti larvae, suggesting that insecticidal and hemolytic activities of Cyt1Aa are independent activities. These mutants were still able to form oligomers in synthetic lipid vesicles and to synergize Cry11Aa toxicity. Remarkably, mutant A61C showed a five-fold increase insecticidal activity against mosquito and almost 11-fold higher activity against WCR. Cyt1Aa A61C mutant was as potent in killing WCR that were selected for resistance to mCry3A as it was against unselected WCR indicating that this toxin could be a useful resistance management option in the control of WCR.

Enhancement of Bacillus thuringiensis Cry1Ab and Cry1Fa Toxicity to Spodoptera frugiperda by Domain III Mutations Indicates There Are Two Limiting Steps in Toxicity as Defined by Receptor Binding and Protein Stability.

Bacillus thuringiensis Cry1Ab and Cry1Fa toxins are environmentally safe insecticides that control important insect pests. Spodoptera frugiperda is an important maize pest that shows low susceptibility to Cry1A toxins, in contrast to Cry1Fa, which is highly active against this pest and is used in transgenic maize for S. frugiperda control. The ß16 region from domain III of Cry1Ab has been shown to be involved in interactions with receptors such as alkaline phosphatase (ALP) or aminopeptidase (APN) in different lepidopteran insects. Alanine-scanning mutagenesis of amino acids of Cry1Ab ß16 (509STLRVN514) revealed that certain ß16 mutations, such as N514A, resulted in increased toxicity of Cry1Ab for S. frugiperda without affecting the toxicity for other lepidopteran larvae, such as Manduca sexta larvae. Exhaustive mutagenesis of N514 was performed, showing that the Cry1Ab N514F, N514H, N514K, N514L, N514Q, and N514S mutations increased the toxicity toward S. frugiperda A corresponding mutation was constructed in Cry1Fa (N507A). Toxicity assays of wild-type and mutant toxins (Cry1Ab, Cry1AbN514A, Cry1AbN514F, Cry1Fa, and Cry1FaN507A) against four S. frugiperda populations from Mexico and one from Brazil revealed that Cry1AbN514A and Cry1FaN507A consistently showed 3- to 18-fold increased toxicity against four of five S. frugiperda populations. In contrast, Cry1AbN514F showed increased toxicity in only two of the S. frugiperda populations analyzed. The mutants Cry1AbN514A and Cry1AbN514F showed greater stability to midgut protease treatment. In addition, binding analysis of the Cry1Ab mutants showed that the increased toxicity correlated with increased binding to brush border membrane vesicles and increased binding affinity for S. frugiperda ALP, APN, and cadherin receptors.IMPORTANCESpodoptera frugiperda is the main maize pest in South and North America and also is an invasive pest in different African countries. However, it is poorly controlled by Bacillus thuringiensis Cry1A toxins expressed in transgenic crops, which effectively control other lepidopteran pests. In contrast, maize expressing Cry1Fa is effective in the control of S. frugiperda, although its effectiveness is being lost due to resistance evolution. Some of the Cry1Ab domain III mutants characterized here show enhanced toxicity for S. frugiperda without loss of toxicity to Manduca sexta Thus, these Cry1Ab mutants could provide useful engineered toxins that, along with other Cry toxins, would be useful for developing transgenic maize expressing stacked proteins for the effective control of S. frugiperda and other lepidopteran pests in the field.

A selective insecticidal protein from Pseudomonas mosselii for corn rootworm control.

The coleopteran insect western corn rootworm (WCR, Diabrotica virgifera virgifera) is an economically important pest in North America and Europe. Transgenic corn plants producing Bacillus thuringiensis (Bt) insecticidal proteins have been useful against this devastating pest, but evolution of resistance has reduced their efficacy. Here, we report the discovery of a novel insecticidal protein, PIP-47Aa, from an isolate of Pseudomonas mosselii. PIP-47Aa sequence shows no shared motifs, domains or signatures with other known proteins. Recombinant PIP-47Aa kills WCR, two other corn rootworm pests (Diabrotica barberi and Diabrotica undecimpunctata howardi) and two other beetle species (Diabrotica speciosa and Phyllotreta cruciferae), but it was not toxic to the spotted lady beetle (Coleomegilla maculata) or seven species of Lepidoptera and Hemiptera. Transgenic corn plants expressing PIP-47Aa show significant protection from root damage by WCR. PIP-47Aa kills a WCR strain resistant to mCry3A and does not share rootworm midgut binding sites with mCry3A or AfIP-1A/1B from Alcaligenes that acts like Cry34Ab1/Cry35Ab1. Our results indicate that PIP-47Aa is a novel insecticidal protein for controlling the corn rootworm pests.

Control of Western Corn Rootworm (Diabrotica virgifera virgifera) Reproduction through Plant-Mediated RNA Interference.

RNA interference (RNAi) in transgenic maize has recently emerged as an alternative mode of action for western corn rootworm (Diabrotica virgifera virgifera) control which can be combined with protein-based rootworm control options for improved root protection and resistance management. Currently, transgenic RNAi-based control has focused on suppression of genes that when silenced lead to larval mortality. We investigated control of western corn rootworm reproduction through RNAi by targeting two reproductive genes, dvvgr and dvbol, with the goal of reducing insect fecundity as a new tool for pest management. The results demonstrated that exposure of adult beetles, as well as larvae to dvvgr or dvbol dsRNA in artificial diet, caused reduction of fecundity. Furthermore, western corn rootworm beetles that emerged from larval feeding on transgenic maize roots expressing dvbol dsRNA also showed significant fecundity reduction. This is the first report of reduction of insect reproductive fitness through plant-mediated RNAi, demonstrating the feasibility of reproductive RNAi as a management tool for western corn rootworm.

An Enterotoxin-Like Binary Protein from Pseudomonas protegens with Potent Nematicidal Activity.

Soil microbes are a major food source for free-living soil nematodes. It is known that certain soil bacteria have evolved systems to combat predation. We identified the nematode-antagonistic Pseudomonas protegens strain 15G2 from screening of microbes. Through protein purification we identified a binary protein, designated Pp-ANP, which is responsible for the nematicidal activity. This binary protein inhibits Caenorhabditis elegans growth and development by arresting larvae at the L1 stage and killing older-staged worms. The two subunits, Pp-ANP1a and Pp-ANP2a, are active when reconstituted from separate expression in Escherichia coli The binary toxin also shows strong nematicidal activity against three other free-living nematodes (Pristionchus pacificus, Panagrellus redivivus, and Acrobeloides sp.), but we did not find any activity against insects and fungi under test conditions, indicating specificity for nematodes. Pp-ANP1a has no significant identity to any known proteins, while Pp-ANP2a shows ∼30% identity to E. coli heat-labile enterotoxin (LT) subunit A and cholera toxin (CT) subunit A. Protein modeling indicates that Pp-ANP2a is structurally similar to CT/LT and likely acts as an ADP-ribosyltransferase. Despite the similarity, Pp-ANP shows several characteristics distinct from CT/LT toxins. Our results indicate that Pp-ANP is a new enterotoxin-like binary toxin with potent and specific activity to nematodes. The potency and specificity of Pp-ANP suggest applications in controlling parasitic nematodes and open an avenue for further research on its mechanism of action and role in bacterium-nematode interaction.IMPORTANCE This study reports the discovery of a new enterotoxin-like binary protein, Pp-ANP, from a Pseudomonas protegens strain. Pp-ANP shows strong nematicidal activity against Caenorhabditis elegans larvae and older-staged worms. It also shows strong activity on other free-living nematodes (Pristionchus pacificus, Panagrellus redivivus, and Acrobeloides sp.). The two subunits, Pp-ANP1a and Pp-ANP2a, can be expressed separately and reconstituted to form the active complex. Pp-ANP shows some distinct characteristics compared with other toxins, including Escherichia coli enterotoxin and cholera toxin. The present study indicates that Pp-ANP is a novel binary toxin and that it has potential applications in controlling parasitic nematodes and in studying toxin-host interaction.

An Alcaligenes strain emulates Bacillus thuringiensis producing a binary protein that kills corn rootworm through a mechanism similar to Cry34Ab1/Cry35Ab1.

Crops expressing Bacillus thuringiensis (Bt)-derived insecticidal protein genes have been commercially available for over 15 years and are providing significant value to growers. However, there remains the need for alternative insecticidal actives due to emerging insect resistance to certain Bt proteins. A screen of bacterial strains led to the discovery of a two-component insecticidal protein named AfIP-1A/1B from an Alcaligenes faecalis strain. This protein shows selectivity against coleopteran insects including western corn rootworm (WCR). Transgenic maize plants expressing AfIP-1A/1B demonstrate strong protection from rootworm injury. Surprisingly, although little sequence similarity exists to known insecticidal proteins, efficacy tests using WCR populations resistant to two different Cry proteins show that AfIP-1A/1B and mCry3A differ in their mode of action while AfIP-1A/1B and the binary Cry34Ab1/Cry35Ab1 protein share a similar mode. These findings are supported by results of competitive binding assays and the similarity of the x-ray structure of AfIP-1A to Cry34Ab1. Our work indicates that insecticidal proteins obtained from a non-Bt bacterial source can be useful for developing genetically modified crops and can function similarly to familiar proteins from Bt.

A selective insecticidal protein from Pseudomonas for controlling corn rootworms.

The coleopteran insect western corn rootworm (WCR) (Diabrotica virgifera virgifera LeConte) is a devastating crop pest in North America and Europe. Although crop plants that produce Bacillus thuringiensis (Bt) proteins can limit insect infestation, some insect populations have evolved resistance to Bt proteins. Here we describe an insecticidal protein, designated IPD072Aa, that is isolated from Pseudomonas chlororaphis. Transgenic corn plants expressing IPD072Aa show protection from WCR insect injury under field conditions. IPD072Aa leaves several lepidopteran and hemipteran insect species unaffected but is effective in killing WCR larvae that are resistant to Bt proteins produced by currently available transgenic corn. IPD072Aa can be used to protect corn crops against WCRs.

Discovery of midgut genes for the RNA interference control of corn rootworm.

RNA interference (RNAi) is a promising new technology for corn rootworm control. This paper presents the discovery of new gene targets - dvssj1 and dvssj2, in western corn rootworm (WCR). Dvssj1 and dvssj2 are orthologs of the Drosophila genes snakeskin (ssk) and mesh, respectively. These genes encode membrane proteins associated with smooth septate junctions (SSJ) which are required for intestinal barrier function. Based on bioinformatics analysis, dvssj1 appears to be an arthropod-specific gene. Diet based insect feeding assays using double-stranded RNA (dsRNA) targeting dvssj1 and dvssj2 demonstrate targeted mRNA suppression, larval growth inhibition, and mortality. In RNAi treated WCR, injury to the midgut was manifested by "blebbing" of the midgut epithelium into the gut lumen. Ultrastructural examination of midgut epithelial cells revealed apoptosis and regenerative activities. Transgenic plants expressing dsRNA targeting dvssj1 show insecticidal activity and significant plant protection from WCR damage. The data indicate that dvssj1 and dvssj2 are effective gene targets for the control of WCR using RNAi technology, by apparent suppression of production of their respective smooth septate junction membrane proteins located within the intestinal lining, leading to growth inhibition and mortality.

Sequencing, de novo assembly and annotation of a pink bollworm larval midgut transcriptome.

BACKGROUND: The pink bollworm Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) is one of the world's most important pests of cotton. Insecticide sprays and transgenic cotton producing toxins of the bacterium Bacillus thuringiensis (Bt) are currently used to manage this pest. Bt toxins kill susceptible insects by specifically binding to and destroying midgut cells, but they are not toxic to most other organisms. Pink bollworm is useful as a model for understanding insect responses to Bt toxins, yet advances in understanding at the molecular level have been limited because basic genomic information is lacking for this cosmopolitan pest. Here, we have sequenced, de novo assembled and annotated a comprehensive larval midgut transcriptome from a susceptible strain of pink bollworm. FINDINGS: A de novo transcriptome assembly for the midgut of P. gossypiella was generated containing 46,458 transcripts (average length of 770 bp) derived from 39,874 unigenes. The size of the transcriptome is similar to published midgut transcriptomes of other Lepidoptera and includes up to 91 % annotated contigs. The dataset is publicly available in NCBI and GigaDB as a resource for researchers. CONCLUSIONS: Foundational knowledge of protein-coding genes from the pink bollworm midgut is critical for understanding how this important insect pest functions. The transcriptome data presented here represent the first large-scale molecular resource for this species, and may be used for deciphering relevant midgut proteins critical for xenobiotic detoxification, nutrient digestion and allocation, as well as for the discovery of protein receptors important for Bt intoxication.

A pigeonpea gene confers resistance to Asian soybean rust in soybean.

Asian soybean rust (ASR), caused by the fungus Phakopsora pachyrhizi, is one of the most economically important crop diseases, but is only treatable with fungicides, which are becoming less effective owing to the emergence of fungicide resistance. There are no commercial soybean cultivars with durable resistance to P. pachyrhizi, and although soybean resistance loci have been mapped, no resistance genes have been cloned. We report the cloning of a P. pachyrhizi resistance gene CcRpp1 (Cajanus cajan Resistance against Phakopsora pachyrhizi 1) from pigeonpea (Cajanus cajan) and show that CcRpp1 confers full resistance to P. pachyrhizi in soybean. Our findings show that legume species related to soybean such as pigeonpea, cowpea, common bean and others could provide a valuable and diverse pool of resistance traits for crop improvement.

Eighteen New Candidate Effectors of the Phytonematode Heterodera glycines Produced Specifically in the Secretory Esophageal Gland Cells During Parasitism.

Heterodera glycines, the soybean cyst nematode, is the number one pathogen of soybean (Glycine max). This nematode infects soybean roots and forms an elaborate feeding site in the vascular cylinder. H. glycines produces an arsenal of effector proteins in the secretory esophageal gland cells. More than 60 H. glycines candidate effectors were identified in previous gland-cell-mining projects. However, it is likely that additional candidate effectors remained unidentified. With the goal of identifying remaining H. glycines candidate effectors, we constructed and sequenced a large gland cell cDNA library resulting in 11,814 expressed sequence tags. After bioinformatic filtering for candidate effectors using a number of criteria, in situ hybridizations were performed in H. glycines whole-mount specimens to identify candidate effectors whose mRNA exclusively accumulated in the esophageal gland cells, which is a hallmark of many nematode effectors. This approach resulted in the identification of 18 new H. glycines esophageal gland-cell-specific candidate effectors. Of these candidate effectors, 11 sequences were pioneers without similarities to known proteins while 7 sequences had similarities to functionally annotated proteins in databases. These putative homologies provided the bases for the development of hypotheses about potential functions in the parasitism process.

Agricultural input traits: past, present and future.

For thousands of years farm practices have evolved as new innovations have become available. Farmers want more value per unit of land, clean fields, and high yields with less input. Plants with incorporated pest resistance and herbicide resistance help meet these needs through increased yield, reduced chemical use, and reduced soil impacts. Although researchers have developed useful traits for a wide variety of plant species, only a few traits are available commercially; however, global adoption of these traits has and continues to increase rapidly. Availability of future traits will be dependent on input not only from researchers, but from governments, interest groups, processors, distributors and ultimately consumers, in addition to the farmers that drive demand for transgenic seed.