Targeted insertion of large DNA sequences by homology-directed repair or non-homologous end joining in engineered tobacco BY-2 cells using designed zinc finger nucleases.

Targeted integration of recombinant DNA fragments into plant genomes by DNA double-strand break (DSB) repair mechanisms has become a powerful tool for precision engineering of crops. However, many targeting platforms require the screening of many transgenic events to identify a low number of targeted events among many more random insertion events. We developed an engineered transgene integration platform (ETIP) that uses incomplete marker genes at the insertion site to enable rapid phenotypic screening and recovery of targeted events upon functional reconstitution of the marker genes. The two marker genes, encoding neomycin phosphotransferase II (nptII) and Discosoma sp. red fluorescent protein (DsRed) enable event selection on kanamycin-containing selective medium and subsequent screening for red fluorescent clones. The ETIP design allows targeted integration of donor DNA molecules either by homology-directed repair (HDR) or non-homologous end joining (NHEJ)-mediated mechanisms. Targeted donor DNA integration is facilitated by zinc finger nucleases (ZFN). The ETIP cassette was introduced into Nicotiana tabacum BY-2 suspension cells to generate target cell lines containing a single copy locus of the transgene construct. The utility of the ETIP platform has been demonstrated by targeting DNA constructs containing up to 25-kb payload. The success rate for clean targeted DNA integration was up to 21% for HDR and up to 41% for NHEJ based on the total number of calli analyzed by next-generation sequencing (NGS). The rapid generation of targeted events with large DNA constructs expands the utility of the nuclease-mediated gene addition platform both for academia and the commercial sector.

Development of an activation tagging system for maize.

Activation Tagging, distributing transcriptional enhancers throughout the genome to induce transcription of nearby genes, is a powerful tool for discovering the function of genes in plants. We have developed a transposable element system to distribute a novel activation tagging element throughout the genome of maize. The transposon system is built from the Enhancer/Suppressor (En/Spm) transposon system and uses an engineered seed color marker to show when the transposon excises. Both somatic and germinal excision events can be detected by the seed color. The activation tagging element is in a Spm-derived non-autonomous transposon and contains four copies of the Sugarcane Bacilliform Virus-enhancer (SCBV-enhancer) and the AAD1 selectable marker. We have demonstrated that the transposon can give rise to germinal excision events that can re-integrate into non-linked genomic locations. The transposon has remained active for three generations and events displaying high rates of germinal excision in the T2 generation have been identified. This system can generate large numbers of activation tagged maize lines that can be screened for agriculturally relevant phenotypes.

Zinc finger nuclease-mediated targeting of multiple transgenes to an endogenous soybean genomic locus via non-homologous end joining.

Emerging genome editing technologies hold great promise for the improvement of agricultural crops. Several related genome editing methods currently in development utilize engineered, sequence-specific endonucleases to generate DNA double strand breaks (DSBs) at user-specified genomic loci. These DSBs subsequently result in small insertions/deletions (indels), base substitutions or incorporation of exogenous donor sequences at the target site, depending on the application. Targeted mutagenesis in soybean (Glycine max) via non-homologous end joining (NHEJ)-mediated repair of such DSBs has been previously demonstrated with multiple nucleases, as has homology-directed repair (HDR)-mediated integration of a single transgene into target endogenous soybean loci using CRISPR/Cas9. Here we report targeted integration of multiple transgenes into a single soybean locus using a zinc finger nuclease (ZFN). First, we demonstrate targeted integration of biolistically delivered DNA via either HDR or NHEJ to the FATTY ACID DESATURASE 2-1a (FAD2-1a) locus of embryogenic cells in tissue culture. We then describe ZFN- and NHEJ-mediated, targeted integration of two different multigene donors to the FAD2-1a locus of immature embryos. The largest donor delivered was 16.2 kb, carried four transgenes, and was successfully transmitted to T1 progeny of mature targeted plants obtained via somatic embryogenesis. The insertions in most plants with a targeted, 7.1 kb, NHEJ-integrated donor were perfect or near-perfect, demonstrating that NHEJ is a viable alternative to HDR for gene targeting in soybean. Taken together, these results show that ZFNs can be used to generate fertile transgenic soybean plants with NHEJ-mediated targeted insertions of multigene donors at an endogenous genomic locus.

The cotton centromere contains a Ty3-gypsy-like LTR retroelement.

The centromere is a repeat-rich structure essential for chromosome segregation; with the long-term aim of understanding centromere structure and function, we set out to identify cotton centromere sequences. To isolate centromere-associated sequences from cotton, (Gossypium hirsutum) we surveyed tandem and dispersed repetitive DNA in the genus. Centromere-associated elements in other plants include tandem repeats and, in some cases, centromere-specific retroelements. Examination of cotton genomic survey sequences for tandem repeats yielded sequences that did not localize to the centromere. However, among the repetitive sequences we also identified a gypsy-like LTR retrotransposon (Centromere Retroelement Gossypium, CRG) that localizes to the centromere region of all chromosomes in domestic upland cotton, Gossypium hirsutum, the major commercially grown cotton. The location of the functional centromere was confirmed by immunostaining with antiserum to the centromere-specific histone CENH3, which co-localizes with CRG hybridization on metaphase mitotic chromosomes. G. hirsutum is an allotetraploid composed of A and D genomes and CRG is also present in the centromere regions of other AD cotton species. Furthermore, FISH and genomic dot blot hybridization revealed that CRG is found in D-genome diploid cotton species, but not in A-genome diploid species, indicating that this retroelement may have invaded the A-genome centromeres during allopolyploid formation and amplified during evolutionary history. CRG is also found in other diploid Gossypium species, including B and E2 genome species, but not in the C, E1, F, and G genome species tested. Isolation of this centromere-specific retrotransposon from Gossypium provides a probe for further understanding of centromere structure, and a tool for future engineering of centromere mini-chromosomes in this important crop species.

The genome of woodland strawberry (Fragaria vesca).

The woodland strawberry, Fragaria vesca (2n = 2x = 14), is a versatile experimental plant system. This diminutive herbaceous perennial has a small genome (240 Mb), is amenable to genetic transformation and shares substantial sequence identity with the cultivated strawberry (Fragaria × ananassa) and other economically important rosaceous plants. Here we report the draft F. vesca genome, which was sequenced to ×39 coverage using second-generation technology, assembled de novo and then anchored to the genetic linkage map into seven pseudochromosomes. This diploid strawberry sequence lacks the large genome duplications seen in other rosids. Gene prediction modeling identified 34,809 genes, with most being supported by transcriptome mapping. Genes critical to valuable horticultural traits including flavor, nutritional value and flowering time were identified. Macrosyntenic relationships between Fragaria and Prunus predict a hypothetical ancestral Rosaceae genome that had nine chromosomes. New phylogenetic analysis of 154 protein-coding genes suggests that assignment of Populus to Malvidae, rather than Fabidae, is warranted.

Multi-platform next-generation sequencing of the domestic turkey (Meleagris gallopavo): genome assembly and analysis.

A synergistic combination of two next-generation sequencing platforms with a detailed comparative BAC physical contig map provided a cost-effective assembly of the genome sequence of the domestic turkey (Meleagris gallopavo). Heterozygosity of the sequenced source genome allowed discovery of more than 600,000 high quality single nucleotide variants. Despite this heterozygosity, the current genome assembly (∼1.1 Gb) includes 917 Mb of sequence assigned to specific turkey chromosomes. Annotation identified nearly 16,000 genes, with 15,093 recognized as protein coding and 611 as non-coding RNA genes. Comparative analysis of the turkey, chicken, and zebra finch genomes, and comparing avian to mammalian species, supports the characteristic stability of avian genomes and identifies genes unique to the avian lineage. Clear differences are seen in number and variety of genes of the avian immune system where expansions and novel genes are less frequent than examples of gene loss. The turkey genome sequence provides resources to further understand the evolution of vertebrate genomes and genetic variation underlying economically important quantitative traits in poultry. This integrated approach may be a model for providing both gene and chromosome level assemblies of other species with agricultural, ecological, and evolutionary interest.

Transcriptome sequencing of the Microarray Quality Control (MAQC) RNA reference samples using next generation sequencing.

BACKGROUND: Transcriptome sequencing using next-generation sequencing platforms will soon be competing with DNA microarray technologies for global gene expression analysis. As a preliminary evaluation of these promising technologies, we performed deep sequencing of cDNA synthesized from the Microarray Quality Control (MAQC) reference RNA samples using Roche's 454 Genome Sequencer FLX. RESULTS: We generated more that 3.6 million sequence reads of average length 250 bp for the MAQC A and B samples and introduced a data analysis pipeline for translating cDNA read counts into gene expression levels. Using BLAST, 90% of the reads mapped to the human genome and 64% of the reads mapped to the RefSeq database of well annotated genes with e-values

Targeted development of registries of biological parts.

BACKGROUND: The design and construction of novel biological systems by combining basic building blocks represents a dominant paradigm in synthetic biology. Creating and maintaining a database of these building blocks is a way to streamline the fabrication of complex constructs. The Registry of Standard Biological Parts (Registry) is the most advanced implementation of this idea. METHODS/PRINCIPAL FINDINGS: By analyzing inclusion relationships between the sequences of the Registry entries, we build a network that can be related to the Registry abstraction hierarchy. The distribution of entry reuse and complexity was extracted from this network. The collection of clones associated with the database entries was also analyzed. The plasmid inserts were amplified and sequenced. The sequences of 162 inserts could be confirmed experimentally but unexpected discrepancies have also been identified. CONCLUSIONS/SIGNIFICANCE: Organizational guidelines are proposed to help design and manage this new type of scientific resources. In particular, it appears necessary to compare the cost of ensuring the integrity of database entries and associated biological samples with their value to the users. The initial strategy that permits including any combination of parts irrespective of its potential value leads to an exponential and economically unsustainable growth that may be detrimental to the quality and long-term value of the resource to its users.

Genome sequence of Brucella abortus vaccine strain S19 compared to virulent strains yields candidate virulence genes.

The Brucella abortus strain S19, a spontaneously attenuated strain, has been used as a vaccine strain in vaccination of cattle against brucellosis for six decades. Despite many studies, the physiological and molecular mechanisms causing the attenuation are not known. We have applied pyrosequencing technology together with conventional sequencing to rapidly and comprehensively determine the complete genome sequence of the attenuated Brucella abortus vaccine strain S19. The main goal of this study is to identify candidate virulence genes by systematic comparative analysis of the attenuated strain with the published genome sequences of two virulent and closely related strains of B. abortus, 9-941 and 2308. The two S19 chromosomes are 2,122,487 and 1,161,449 bp in length. A total of 3062 genes were identified and annotated. Pairwise and reciprocal genome comparisons resulted in a total of 263 genes that were non-identical between the S19 genome and any of the two virulent strains. Amongst these, 45 genes were consistently different between the attenuated strain and the two virulent strains but were identical amongst the virulent strains, which included only two of the 236 genes that have been implicated as virulence factors in literature. The functional analyses of the differences have revealed a total of 24 genes that may be associated with the loss of virulence in S19. Of particular relevance are four genes with more than 60 bp consistent difference in S19 compared to both the virulent strains, which, in the virulent strains, encode an outer membrane protein and three proteins involved in erythritol uptake or metabolism.

Early blood profiles of virus infection in a monkey model for Lassa fever.

Acute arenavirus disease in primates, like Lassa hemorrhagic fever in humans, begins with flu-like symptoms and leads to death approximately 2 weeks after infection. Our goal was to identify molecular changes in blood that are related to disease progression. Rhesus macaques (Macaca mulatta) infected intravenously with a lethal dose of lymphocytic choriomeningitis virus (LCMV) provide a model for Lassa virus infection of humans. Blood samples taken before and during the course of infection were used to monitor gene expression changes that paralleled disease onset. Changes in blood showed major disruptions in eicosanoid, immune response, and hormone response pathways. Approximately 12% of host genes alter their expression after LCMV infection, and a subset of these genes can discriminate between virulent and non-virulent LCMV infection. Major transcription changes have been given preliminary confirmation by quantitative PCR and protein studies and will be valuable candidates for future validation as biomarkers for arenavirus disease.

Open architecture expression profiling of plant transcriptomes and gene discovery using GeneCalling technology.

The recent rapid developments in genomics tools, technologies, and bioinformatics have revolutionized gene expression analysis. It is now routine to measure gene expression modulation at the genomic level. GeneCalling technology is an open architecture system capable of assaying more than 95% of genes expressed in a tissue. Unlike the closed systems, GeneCalling is not dependent upon an existing sequence or clone database. GeneCalling uses as low as 50 pg of the cDNA from samples and identifies cDNA fragments that are differentially modulated within a set of samples. With the use of 96 pairs of restriction enzymes, more than 30,000 cDNA fragments are routinely assayed to identify those that are differentially modulated. Specific processes, such as SeqCalling, Trace Poisoning, and GeneCall Poisoning, are set up to not only confirm the known genes, but also to clone and analyze unknown and novel genes that have an interesting expression profile. GeneCalling has been successfully applied to expression profiling of several plant and fungal species, and resulted in identification and characterization of genes that are useful in commercial applications towards improving agriculturally important traits in plants.

Overexpression of a gene encoding hydrogen peroxide-generating oxalate oxidase evokes defense responses in sunflower.

Oxalate oxidase (OXO) converts oxalic acid (OA) and O(2) to CO(2) and hydrogen peroxide (H(2)O(2)), and acts as a source of H(2)O(2) in certain plant-pathogen interactions. To determine if the H(2)O(2) produced by OXO can function as a messenger for activation of defense genes and if OXO can confer resistance against an OA-producing pathogen, we analyzed transgenic sunflower (Helianthus annuus cv SMF3) plants constitutively expressing a wheat (Triticum aestivum) OXO gene. The transgenic leaf tissues could degrade exogenous OA and generate H(2)O(2). Hypersensitive response-like lesion mimicry was observed in the transgenic leaves expressing a high level of OXO, and lesion development was closely associated with elevated levels of H(2)O(2), salicylic acid, and defense gene expression. Activation of defense genes was also observed in the transgenic leaves that had a low level of OXO expression and had no visible lesions, indicating that defense gene activation may not be dependent on hypersensitive response-like cell death. To further understand the pathways that were associated with defense activation, we used GeneCalling, an RNA-profiling technology, to analyze the alteration of gene expression in the transgenic plants. Among the differentially expressed genes, full-length cDNAs encoding homologs of a PR5, a sunflower carbohydrate oxidase, and a defensin were isolated. RNA-blot analysis confirmed that expression of these three genes was significantly induced in the OXO transgenic sunflower leaves. Furthermore, treatment of untransformed sunflower leaves with jasmonic acid, salicylic acid, or H(2)O(2) increased the steady-state levels of these mRNAs. Notably, the transgenic sunflower plants exhibited enhanced resistance against the OA-generating fungus Sclerotinia sclerotiorum.

Comprehensive transcript profiling of Pto- and Prf-mediated host defense responses to infection by Pseudomonas syringae pv. tomato.

The disease resistance gene Pto encodes a serine/threonine protein kinase that confers resistance in tomato to Pseudomonas syringae pv. tomato strains that express the effector protein AvrPto. Pto-mediated resistance to bacterial speck disease also requires Prf, a protein with leucine-rich repeats and a putative nucleotide-binding site, although the role of Prf in the defense pathway is not known. We used GeneCalling, an open-architecture, mRNA-profiling technology, to identify genes that are either induced or suppressed in leaves 4 h after bacterial infection in the Pto- and Prf-mediated tomato-Pseudomonas(avrPto) interaction. Over 135 000 individual cDNA fragments representing an estimated 90% of the transcripts expressed in tomato leaves were examined and 432 differentially expressed genes were identified. The genes encode over 25 classes of proteins including 11 types of transcription factors and many signal transduction components. Differential expression of 91% of the genes required both Pto and Prf. Interestingly, differential expression of 32 genes did not require Pto but was dependent on Prf. Thus, our data support a role for Prf early in the Pto pathway and indicate that Prf can also function as an independent host recognition determinant of bacterial infection. Comprehensive expression profiling of the Pto-mediated defense response allows the development of many new hypotheses about the molecular basis of resistance to bacterial speck disease.