Genetic markers for western corn rootworm resistance to Bt toxin.

Western corn rootworm (WCR) is a major maize (Zea mays L.) pest leading to annual economic losses of more than 1 billion dollars in the United States. Transgenic maize expressing insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) are widely used for the management of WCR. However, cultivation of Bt-expressing maize places intense selection pressure on pest populations to evolve resistance. Instances of resistance to Bt toxins have been reported in WCR. Developing genetic markers for resistance will help in characterizing the extent of existing issues, predicting where future field failures may occur, improving insect resistance management strategies, and in designing and sustainably implementing forthcoming WCR control products. Here, we discover and validate genetic markers in WCR that are associated with resistance to the Cry3Bb1 Bt toxin. A field-derived WCR population known to be resistant to the Cry3Bb1 Bt toxin was used to generate a genetic map and to identify a genomic region associated with Cry3Bb1 resistance. Our results indicate that resistance is inherited in a nearly recessive manner and associated with a single autosomal linkage group. Markers tightly linked with resistance were validated using WCR populations collected from Cry3Bb1 maize fields showing significant WCR damage from across the US Corn Belt. Two markers were found to be correlated with both diet (R2 = 0.14) and plant (R2 = 0.23) bioassays for resistance. These results will assist in assessing resistance risk for different WCR populations, and can be used to improve insect resistance management strategies.

Western corn rootworm (Diabrotica virgifera virgifera) transcriptome assembly and genomic analysis of population structure.

BACKGROUND: Western corn rootworm (WCR) is one of the most significant insect pests of maize in North America. WCR has dramatically increased its range in the last century, invading key maize production areas in the US and abroad. In addition, this species has a history of evolving traits that allow it to escape various control options. Improved genetic and genomic resources are crucial tools for understanding population history and the genetic basis of trait evolution. Here we produce and analyze a transcriptome assembly for WCR. We also perform whole genome population resequencing, and combine these resources to better understand the evolutionary history of WCR. RESULTS: The WCR transcriptome assembly presented here contains approximately 16,000 unigenes, many of which have high similarity to other insect species. Among these unigenes we found several gene families that have been implicated in insecticide resistance in other species. We also identified over 500,000 unigene based SNPs among 26 WCR populations. We used these SNPs to scan for outliers among the candidate genes, and to understand how population processes have shaped genetic variation in this species. CONCLUSIONS: This study highlights the utility of transcriptomic and genomic resources as foundational tools for dealing with highly adaptive pest species. Using these tools we identified candidate gene families for insecticide resistance and reveal aspects of WCR population history in light of the species' recent range expansion.

Reconciliation of sequence data and updated annotation of the genome of Agrobacterium tumefaciens C58, and distribution of a linear chromosome in the genus Agrobacterium.

Two groups independently sequenced the Agrobacterium tumefaciens C58 genome in 2001. We report here consolidation of these sequences, updated annotation, and additional analysis of the evolutionary history of the linear chromosome, which is apparently limited to the biovar I group of Agrobacterium.

Expression of the Arabidopsis thaliana BBX32 gene in soybean increases grain yield.

Crop yield is a highly complex quantitative trait. Historically, successful breeding for improved grain yield has led to crop plants with improved source capacity, altered plant architecture, and increased resistance to abiotic and biotic stresses. To date, transgenic approaches towards improving crop grain yield have primarily focused on protecting plants from herbicide, insects, or disease. In contrast, we have focused on identifying genes that, when expressed in soybean, improve the intrinsic ability of the plant to yield more. Through the large scale screening of candidate genes in transgenic soybean, we identified an Arabidopsis thaliana B-box domain gene (AtBBX32) that significantly increases soybean grain yield year after year in multiple transgenic events in multi-location field trials. In order to understand the underlying physiological changes that are associated with increased yield in transgenic soybean, we examined phenotypic differences in two AtBBX32-expressing lines and found increases in plant height and node, flower, pod, and seed number. We propose that these phenotypic changes are likely the result of changes in the timing of reproductive development in transgenic soybean that lead to the increased duration of the pod and seed development period. Consistent with the role of BBX32 in A. thaliana in regulating light signaling, we show that the constitutive expression of AtBBX32 in soybean alters the abundance of a subset of gene transcripts in the early morning hours. In particular, AtBBX32 alters transcript levels of the soybean clock genes GmTOC1 and LHY-CCA1-like2 (GmLCL2). We propose that through the expression of AtBBX32 and modulation of the abundance of circadian clock genes during the transition from dark to light, the timing of critical phases of reproductive development are altered. These findings demonstrate a specific role for AtBBX32 in modulating soybean development, and demonstrate the validity of expressing single genes in crops to deliver increased agricultural productivity.

Phenotypic variation and host interactions of Xenorhabdus bovienii SS-2004, the entomopathogenic symbiont of Steinernema jollieti nematodes.

Xenorhabdus bovienii (SS-2004) bacteria reside in the intestine of the infective-juvenile (IJ) stage of the entomopathogenic nematode, Steinernema jollieti. The recent sequencing of the X. bovienii genome facilitates its use as a model to understand host - symbiont interactions. To provide a biological foundation for such studies, we characterized X. bovienii in vitro and host interaction phenotypes. Within the nematode host X. bovienii was contained within a membrane bound envelope that also enclosed the nematode-derived intravesicular structure. Steinernema jollieti nematodes cultivated on mixed lawns of X. bovienii expressing green or DsRed fluorescent proteins were predominantly colonized by one or the other strain, suggesting the colonizing population is founded by a few cells. Xenorhabdus bovienii exhibits phenotypic variation between orange-pigmented primary form and cream-pigmented secondary form. Each form can colonize IJ nematodes when cultured in vitro on agar. However, IJs did not develop or emerge from Galleria mellonella insects infected with secondary form. Unlike primary-form infected insects that were soft and flexible, secondary-form infected insects retained a rigid exoskeleton structure. Xenorhabdus bovienii primary and secondary form isolates are virulent towards Manduca sexta and several other insects. However, primary form stocks present attenuated virulence, suggesting that X. bovienii, like Xenorhabdus nematophila may undergo virulence modulation.

Metabolically engineered soybean seed with enhanced threonine levels: biochemical characterization and seed-specific expression of lysine-insensitive variants of aspartate kinases from the enteric bacterium Xenorhabdus bovienii.

Threonine (Thr) is one of a few limiting essential amino acids (EAAs) in the animal feed industry, and its level in feed rations can impact production of important meat sources, such as swine and poultry. Threonine as well as EAAs lysine (Lys) and methionine (Met) are all synthesized via the aspartate family pathway. Here, we report a successful strategy to produce high free threonine soybean seed via identification of a feedback-resistant aspartate kinase (AK) enzyme that can be over-expressed in developing soybean seed. Towards this goal, we have purified and biochemically characterized AK from the enteric bacterium Xenorhabdus bovienii (Xb). Site-directed mutagenesis of XbAK identified two key regulatory residues Glu-257 and Thr-359 involved in lysine inhibition. Three feedback-resistant alleles, XbAK_T359I, XbAK_E257K and XbAK_E257K/T359I, have been generated. This study is the first to kinetically characterize the XbAK enzyme and provide biochemical and transgenic evidence that Glu-257 near the catalytic site is a critical residue for the allosteric regulation of AK. Furthermore, seed-specific expression of the feedback-resistant XbAK_T359I or XbAK_E257K allele results in increases of free Thr levels of up to 100-fold in R(1) soybean seed when compared to wild-type. Expression of feedback-sensitive wild-type AK did not substantially impact seed Thr content. In addition to high Thr, transgenic seed also showed substantial increases in other major free amino acid (FAA) levels, resulting in an up to 3.5-fold increase in the total FAA content. The transgenic seed was normal in appearance and germinated well under greenhouse conditions.

Genome sequence of Azotobacter vinelandii, an obligate aerobe specialized to support diverse anaerobic metabolic processes.

Azotobacter vinelandii is a soil bacterium related to the Pseudomonas genus that fixes nitrogen under aerobic conditions while simultaneously protecting nitrogenase from oxygen damage. In response to carbon availability, this organism undergoes a simple differentiation process to form cysts that are resistant to drought and other physical and chemical agents. Here we report the complete genome sequence of A. vinelandii DJ, which has a single circular genome of 5,365,318 bp. In order to reconcile an obligate aerobic lifestyle with exquisitely oxygen-sensitive processes, A. vinelandii is specialized in terms of its complement of respiratory proteins. It is able to produce alginate, a polymer that further protects the organism from excess exogenous oxygen, and it has multiple duplications of alginate modification genes, which may alter alginate composition in response to oxygen availability. The genome analysis identified the chromosomal locations of the genes coding for the three known oxygen-sensitive nitrogenases, as well as genes coding for other oxygen-sensitive enzymes, such as carbon monoxide dehydrogenase and formate dehydrogenase. These findings offer new prospects for the wider application of A. vinelandii as a host for the production and characterization of oxygen-sensitive proteins.

Genome sequences of three agrobacterium biovars help elucidate the evolution of multichromosome genomes in bacteria.

The family Rhizobiaceae contains plant-associated bacteria with critical roles in ecology and agriculture. Within this family, many Rhizobium and Sinorhizobium strains are nitrogen-fixing plant mutualists, while many strains designated as Agrobacterium are plant pathogens. These contrasting lifestyles are primarily dependent on the transmissible plasmids each strain harbors. Members of the Rhizobiaceae also have diverse genome architectures that include single chromosomes, multiple chromosomes, and plasmids of various sizes. Agrobacterium strains have been divided into three biovars, based on physiological and biochemical properties. The genome of a biovar I strain, A. tumefaciens C58, has been previously sequenced. In this study, the genomes of the biovar II strain A. radiobacter K84, a commercially available biological control strain that inhibits certain pathogenic agrobacteria, and the biovar III strain A. vitis S4, a narrow-host-range strain that infects grapes and invokes a hypersensitive response on nonhost plants, were fully sequenced and annotated. Comparison with other sequenced members of the Alphaproteobacteria provides new data on the evolution of multipartite bacterial genomes. Primary chromosomes show extensive conservation of both gene content and order. In contrast, secondary chromosomes share smaller percentages of genes, and conserved gene order is restricted to short blocks. We propose that secondary chromosomes originated from an ancestral plasmid to which genes have been transferred from a progenitor primary chromosome. Similar patterns are observed in select Beta- and Gammaproteobacteria species. Together, these results define the evolution of chromosome architecture and gene content among the Rhizobiaceae and support a generalized mechanism for second-chromosome formation among bacteria.

Optical mapping as a routine tool for bacterial genome sequence finishing.

BACKGROUND: In sequencing the genomes of two Xenorhabdus species, we encountered a large number of sequence repeats and assembly anomalies that stalled finishing efforts. This included a stretch of about 12 Kb that is over 99.9% identical between the plasmid and chromosome of X. nematophila. RESULTS: Whole genome restriction maps of the sequenced strains were produced through optical mapping technology. These maps allowed rapid resolution of sequence assembly problems, permitted closing of the genome, and allowed correction of a large inversion in a genome assembly that we had considered finished. CONCLUSION: Our experience suggests that routine use of optical mapping in bacterial genome sequence finishing is warranted. When combined with data produced through 454 sequencing, an optical map can rapidly and inexpensively generate an ordered and oriented set of contigs to produce a nearly complete genome sequence assembly.

Predicting prokaryotic ecological niches using genome sequence analysis.

Automated DNA sequencing technology is so rapid that analysis has become the rate-limiting step. Hundreds of prokaryotic genome sequences are publicly available, with new genomes uploaded at the rate of approximately 20 per month. As a result, this growing body of genome sequences will include microorganisms not previously identified, isolated, or observed. We hypothesize that evolutionary pressure exerted by an ecological niche selects for a similar genetic repertoire in those prokaryotes that occupy the same niche, and that this is due to both vertical and horizontal transmission. To test this, we have developed a novel method to classify prokaryotes, by calculating their Pfam protein domain distributions and clustering them with all other sequenced prokaryotic species. Clusters of organisms are visualized in two dimensions as 'mountains' on a topological map. When compared to a phylogenetic map constructed using 16S rRNA, this map more accurately clusters prokaryotes according to functional and environmental attributes. We demonstrate the ability of this map, which we term a "niche map", to cluster according to ecological niche both quantitatively and qualitatively, and propose that this method be used to associate uncharacterized prokaryotes with their ecological niche as a means of predicting their functional role directly from their genome sequence.

Functional genome annotation through phylogenomic mapping.

Accurate determination of functional interactions among proteins at the genome level remains a challenge for genomic research. Here we introduce a genome-scale approach to functional protein annotation--phylogenomic mapping--that requires only sequence data, can be applied equally well to both finished and unfinished genomes, and can be extended beyond single genomes to annotate multiple genomes simultaneously. We have developed and applied it to more than 200 sequenced bacterial genomes. Proteins with similar evolutionary histories were grouped together, placed on a three dimensional map and visualized as a topographical landscape. The resulting phylogenomic maps display thousands of proteins clustered in mountains on the basis of coinheritance, a strong indicator of shared function. In addition to systematic computational validation, we have experimentally confirmed the ability of phylogenomic maps to predict both mutant phenotype and gene function in the delta proteobacterium Myxococcus xanthus.

Genomic analyses of bacterial respiratory and cytochrome c assembly systems: Bordetella as a model for the system II cytochrome c biogenesis pathway.

An analysis of thirty-three genomes of selected bacteria for the presence of specific respiratory pathways and cytochrome c biogenesis systems has led to observations on respiration and biogenesis. A table summarizing these results is presented. The data suggested that Bordetella pertussis would be an excellent genetic model to study the System II cytochrome c biogenesis pathway. These observations are discussed and the results of genetic studies on System II biogenesis in B. pertussis are presented as a case for the power of comparative genomics. System II is present in organisms as diverse as Helicobacter, Neisseria, Porphyromonas, mycobacteria, cyanobacteria, and plants (chloroplasts), indicating this pathway's prominence and that horizontal transfer of system II (and/or System I) must have occurred on multiple occasions.