Selective Bacterial Community Enrichment between the Pitcher Plants Sarracenia minor and Sarracenia flava.

The interconnected and overlapping habitats present in natural ecosystems remain a challenge in determining the forces driving microbial community composition. The cuplike leaf structures of some carnivorous plants, including those of the family Sarraceniaceae, are self-contained ecological habitats that represent systems for exploring such microbial ecology questions. We investigated whether Sarracenia minor and Sarracenia flava cultivate distinct bacterial communities when sampled at the same geographic location and time. This sampling strategy eliminates many abiotic environmental variables present in other studies that compare samples harvested over time, and it could reveal biotic factors driving the selection of microbes. DNA extracted from the decomposing detritus trapped in each Sarracenia leaf pitcher was profiled using 16S rRNA amplicon sequencing. We identified a surprising amount of bacterial diversity within each pitcher, but we also discovered bacteria whose abundance was specifically enriched in one of the two Sarracenia species. These differences in bacterial community representation suggest some biotic influence of the Sarracenia plant on the bacterial composition of their pitchers. Overall, our results suggest that bacterial selection due to factors other than geographic location, weather, or prey availability is occurring within the pitchers of these two closely related plant species. This indicates that specific characteristics of S. minor and S. flava may play a role in fostering distinct bacterial communities. These confined, naturally occurring microbial ecosystems within Sarracenia pitchers may provide model systems to answer important questions about the drivers of microbial community composition, succession, and response to environmental perturbations. IMPORTANCE This study uses amplicon sequencing to compare the bacterial communities of environmental samples from the detritus of the leaf cavities of Sarracenia minor and Sarracenia flava pitcher plants. We sampled the detritus at the same time and in the same geographic location, eliminating many environmental variables present in other comparative studies. This study revealed that different species of Sarracenia contain distinct bacterial members within their pitchers, suggesting that these communities are not randomly established based on environmental factors and the prey pool but are potentially enriched for by the plants' chemical or physical environment. This study of these naturally occurring, confined microbial ecosystems will help further establish carnivorous pitcher plants as a model system for answering important questions about the development and succession of microbial communities.

Investigational Assay for Haplotype Phasing of the Huntingtin Gene.

Novel treatments for Huntington's disease (HD), a progressive neurodegenerative disorder, include selective targeting of the mutant allele of the huntingtin gene (mHTT) carrying the abnormally expanded disease-causing cytosine-adenine-guanine (CAG) repeat. WVE-120101 and WVE-120102 are investigational stereopure antisense oligonucleotides that enable selective suppression of mHTT by targeting single-nucleotide polymorphisms (SNPs) that are in haplotype phase with the CAG repeat expansion. Recently developed long-read sequencing technologies can capture CAG expansions and distant SNPs of interest and potentially facilitate haplotype-based identification of patients for clinical trials of oligonucleotide therapies. However, improved methods are needed to phase SNPs with CAG repeat expansions directly and reliably without need for familial genotype/haplotype data. Our haplotype phasing method uses single-molecule real-time sequencing and a custom algorithm to determine with confidence bases at SNPs on mutant alleles, even without familial data. Herein, we summarize this methodology and validate the approach using patient-derived samples with known phasing results. Comparison of experimentally measured CAG repeat lengths, heterozygosity, and phasing with previously determined results showed improved performance. Our methodology enables the haplotype phasing of SNPs of interest and the disease-causing, expanded CAG repeat of the huntingtin gene, enabling accurate identification of patients with HD eligible for allele-selective clinical studies.

Genes Encoding Recognition of the Cladosporium fulvum Effector Protein Ecp5 Are Encoded at Several Loci in the Tomato Genome.

The molecular interactions between tomato and Cladosporium fulvum have been an important model for molecular plant pathology. Complex genetic loci on tomato chromosomes 1 and 6 harbor genes for resistance to Cladosporium fulvum, encoding receptor like-proteins that perceive distinct Cladosporium fulvum effectors and trigger plant defenses. Here, we report classical mapping strategies for loci in tomato accessions that respond to Cladosporium fulvum effector Ecp5, which is very sequence-monomorphic. We screened 139 wild tomato accessions for an Ecp5-induced hypersensitive response, and in five accessions, the Ecp5-induced hypersensitive response segregated as a monogenic trait, mapping to distinct loci in the tomato genome. We identified at least three loci on chromosomes 1, 7 and 12 that harbor distinct Cf-Ecp5 genes in four different accessions. Our mapping showed that the Cf-Ecp5 in Solanum pimpinellifolium G1.1161 is located at the Milky Way locus. The Cf-Ecp5 in Solanum pimpinellifolium LA0722 was mapped to the bottom arm of chromosome 7, while the Cf-Ecp5 genes in Solanum lycopersicum Ontario 7522 and Solanum pimpinellifolium LA2852 were mapped to the same locus on the top arm of chromosome 12. Bi-parental crosses between accessions carrying distinct Cf-Ecp5 genes revealed putative genetically unlinked suppressors of the Ecp5-induced hypersensitive response. Our mapping also showed that Cf-11 is located on chromosome 11, close to the Cf-3 locus. The Ecp5-induced hypersensitive response is widely distributed within tomato species and is variable in strength. This novel example of convergent evolution could be used for choosing different functional Cf-Ecp5 genes according to individual plant breeding needs.

Alterations in airway microbiota in patients with PaO2/FiO2 ratio ≤ 300 after burn and inhalation injury.

BACKGROUND: Injury to the airways after smoke inhalation is a major mortality risk factor in victims of burn injuries, resulting in a 15-45% increase in patient deaths. Damage to the airways by smoke may induce acute respiratory distress syndrome (ARDS), which is partly characterized by hypoxemia in the airways. While ARDS has been associated with bacterial infection, the impact of hypoxemia on airway microbiota is unknown. Our objective was to identify differences in microbiota within the airways of burn patients who develop hypoxemia early after inhalation injury and those that do not using next-generation sequencing of bacterial 16S rRNA genes. RESULTS: DNA was extracted from therapeutic bronchial washings of 48 patients performed within 72 hours of hospitalization for burn and inhalation injury at the North Carolina Jaycee Burn Center. DNA was prepared for sequencing using a novel molecule tagging method and sequenced on the Illumina MiSeq platform. Bacterial species were identified using the MTToolbox pipeline. Patients with hypoxemia, as indicated by a PaO2/FiO2 ratio ≤ 300, had a 30% increase in abundance of Streptococcaceae and Enterobacteriaceae and 84% increase in Staphylococcaceae as compared to patients with a PaO2/FiO2 ratio > 300. Wilcoxon rank-sum test identified significant enrichment in abundance of OTUs identified as Prevotella melaninogenica (p = 0.042), Corynebacterium (p = 0.037) and Mogibacterium (p = 0.048). Linear discriminant effect size analysis (LefSe) confirmed significant enrichment of Prevotella melaninognica among patients with a PaO2/FiO2 ratio ≤ 300 (p<0.05). These results could not be explained by differences in antibiotic treatment. CONCLUSIONS: The airway microbiota following burn and inhalation injury is altered in patients with a PaO2/FiO2 ratio ≤ 300 early after injury. Enrichment of specific taxa in patients with a PaO2/FiO2 ratio ≤ 300 may indicate airway environment and patient changes that favor these microbes. Longitudinal studies are necessary to identify stably colonizing taxa that play roles in hypoxemia and ARDS pathogenesis.

Development of a 63K SNP Array for Cotton and High-Density Mapping of Intraspecific and Interspecific Populations of Gossypium spp.

High-throughput genotyping arrays provide a standardized resource for plant breeding communities that are useful for a breadth of applications including high-density genetic mapping, genome-wide association studies (GWAS), genomic selection (GS), complex trait dissection, and studying patterns of genomic diversity among cultivars and wild accessions. We have developed the CottonSNP63K, an Illumina Infinium array containing assays for 45,104 putative intraspecific single nucleotide polymorphism (SNP) markers for use within the cultivated cotton species Gossypium hirsutum L. and 17,954 putative interspecific SNP markers for use with crosses of other cotton species with G. hirsutum. The SNPs on the array were developed from 13 different discovery sets that represent a diverse range of G. hirsutum germplasm and five other species: G. barbadense L., G. tomentosum Nuttal × Seemann, G. mustelinum Miers × Watt, G. armourianum Kearny, and G. longicalyx J.B. Hutchinson and Lee. The array was validated with 1,156 samples to generate cluster positions to facilitate automated analysis of 38,822 polymorphic markers. Two high-density genetic maps containing a total of 22,829 SNPs were generated for two F2 mapping populations, one intraspecific and one interspecific, and 3,533 SNP markers were co-occurring in both maps. The produced intraspecific genetic map is the first saturated map that associates into 26 linkage groups corresponding to the number of cotton chromosomes for a cross between two G. hirsutum lines. The linkage maps were shown to have high levels of collinearity to the JGI G. raimondii Ulbrich reference genome sequence. The CottonSNP63K array, cluster file and associated marker sequences constitute a major new resource for the global cotton research community.

The antipsychotic olanzapine interacts with the gut microbiome to cause weight gain in mouse.

The second-generation antipsychotic olanzapine is effective in reducing psychotic symptoms but can cause extreme weight gain in human patients. We investigated the role of the gut microbiota in this adverse drug effect using a mouse model. First, we used germ-free C57BL/6J mice to demonstrate that gut bacteria are necessary and sufficient for weight gain caused by oral delivery of olanzapine. Second, we surveyed fecal microbiota before, during, and after treatment and found that olanzapine potentiated a shift towards an "obesogenic" bacterial profile. Finally, we demonstrated that olanzapine has antimicrobial activity in vitro against resident enteric bacterial strains. These results collectively provide strong evidence for a mechanism underlying olanzapine-induced weight gain in mouse and a hypothesis for clinical translation in human patients.

MT-Toolbox: improved amplicon sequencing using molecule tags.

BACKGROUND: Short oligonucleotides can be used as markers to tag and track DNA sequences. For example, barcoding techniques (i.e. Multiplex Identifiers or Indexing) use short oligonucleotides to distinguish between reads from different DNA samples pooled for high-throughput sequencing. A similar technique called molecule tagging uses the same principles but is applied to individual DNA template molecules. Each template molecule is tagged with a unique oligonucleotide prior to polymerase chain reaction. The resulting amplicon sequences can be traced back to their original templates by their oligonucleotide tag. Consensus building from sequences sharing the same tag enables inference of original template molecules thereby reducing effects of sequencing error and polymerase chain reaction bias. Several independent groups have developed similar protocols for molecule tagging; however, user-friendly software for build consensus sequences from molecule tagged reads is not readily available or is highly specific for a particular protocol. RESULTS: MT-Toolbox recognizes oligonucleotide tags in amplicons and infers the correct template sequence. On a set of molecule tagged test reads, MT-Toolbox generates sequences having on average 0.00047 errors per base. MT-Toolbox includes a graphical user interface, command line interface, and options for speed and accuracy maximization. It can be run in serial on a standard personal computer or in parallel on a Load Sharing Facility based cluster system. An optional plugin provides features for common 16S metagenome profiling analysis such as chimera filtering, building operational taxonomic units, contaminant removal, and taxonomy assignments. CONCLUSIONS: MT-Toolbox provides an accessible, user-friendly environment for analysis of molecule tagged reads thereby reducing technical errors and polymerase chain reaction bias. These improvements reduce noise and allow for greater precision in single amplicon sequencing experiments.

Draft Genome Sequences of a Phylogenetically Diverse Suite of Pseudomonas syringae Strains from Multiple Source Populations.

Here, we report the draft genome sequences for 7 phylogenetically diverse isolates of Pseudomonas syringae, obtained from numerous environmental sources and geographically proximate crop species. Overall, these sequences provide a wealth of information about the differences (or lack thereof) between isolates from disease outbreaks and those from other sources.

Variable suites of non-effector genes are co-regulated in the type III secretion virulence regulon across the Pseudomonas syringae phylogeny.

Pseudomonas syringae is a phylogenetically diverse species of Gram-negative bacterial plant pathogens responsible for crop diseases around the world. The HrpL sigma factor drives expression of the major P. syringae virulence regulon. HrpL controls expression of the genes encoding the structural and functional components of the type III secretion system (T3SS) and the type three secreted effector proteins (T3E) that are collectively essential for virulence. HrpL also regulates expression of an under-explored suite of non-type III effector genes (non-T3E), including toxin production systems and operons not previously associated with virulence. We implemented and refined genome-wide transcriptional analysis methods using cDNA-derived high-throughput sequencing (RNA-seq) data to characterize the HrpL regulon from six isolates of P. syringae spanning the diversity of the species. Our transcriptomes, mapped onto both complete and draft genomes, significantly extend earlier studies. We confirmed HrpL-regulation for a majority of previously defined T3E genes in these six strains. We identified two new T3E families from P. syringae pv. oryzae 1_6, a strain within the relatively underexplored phylogenetic Multi-Locus Sequence Typing (MLST) group IV. The HrpL regulons varied among strains in gene number and content across both their T3E and non-T3E gene suites. Strains within MLST group II consistently express the lowest number of HrpL-regulated genes. We identified events leading to recruitment into, and loss from, the HrpL regulon. These included gene gain and loss, and loss of HrpL regulation caused by group-specific cis element mutations in otherwise conserved genes. Novel non-T3E HrpL-regulated genes include an operon that we show is required for full virulence of P. syringae pv. phaseolicola 1448A on French bean. We highlight the power of integrating genomic, transcriptomic, and phylogenetic information to drive concise functional experimentation and to derive better insight into the evolution of virulence across an evolutionarily diverse pathogen species.

Practical innovations for high-throughput amplicon sequencing.

We describe improvements for sequencing 16S ribosomal RNA (rRNA) amplicons, a cornerstone technique in metagenomics. Through unique tagging of template molecules before PCR, amplicon sequences can be mapped to their original templates to correct amplification bias and sequencing error with software we provide. PCR clamps block amplification of contaminating sequences from a eukaryotic host, thereby substantially enriching microbial sequences without introducing bias.

Defining the core Arabidopsis thaliana root microbiome.

Land plants associate with a root microbiota distinct from the complex microbial community present in surrounding soil. The microbiota colonizing the rhizosphere (immediately surrounding the root) and the endophytic compartment (within the root) contribute to plant growth, productivity, carbon sequestration and phytoremediation. Colonization of the root occurs despite a sophisticated plant immune system, suggesting finely tuned discrimination of mutualists and commensals from pathogens. Genetic principles governing the derivation of host-specific endophyte communities from soil communities are poorly understood. Here we report the pyrosequencing of the bacterial 16S ribosomal RNA gene of more than 600 Arabidopsis thaliana plants to test the hypotheses that the root rhizosphere and endophytic compartment microbiota of plants grown under controlled conditions in natural soils are sufficiently dependent on the host to remain consistent across different soil types and developmental stages, and sufficiently dependent on host genotype to vary between inbred Arabidopsis accessions. We describe different bacterial communities in two geochemically distinct bulk soils and in rhizosphere and endophytic compartments prepared from roots grown in these soils. The communities in each compartment are strongly influenced by soil type. Endophytic compartments from both soils feature overlapping, low-complexity communities that are markedly enriched in Actinobacteria and specific families from other phyla, notably Proteobacteria. Some bacteria vary quantitatively between plants of different developmental stage and genotype. Our rigorous definition of an endophytic compartment microbiome should facilitate controlled dissection of plant-microbe interactions derived from complex soil communities.

Development and mapping of SNP assays in allotetraploid cotton.

A narrow germplasm base and a complex allotetraploid genome have made the discovery of single nucleotide polymorphism (SNP) markers difficult in cotton (Gossypium hirsutum). To generate sequence for SNP discovery, we conducted a genome reduction experiment (EcoRI, BafI double digest, followed by adapter ligation, biotin-streptavidin purification, and agarose gel separation) on two accessions of G. hirsutum and two accessions of G. barbadense. From the genome reduction experiment, a total of 2.04 million genomic sequence reads were assembled into contigs with an N(50) of 508 bp and analyzed for SNPs. A previously generated assembly of expressed sequence tags (ESTs) provided an additional source for SNP discovery. Using highly conservative parameters (minimum coverage of 8× at each SNP and 20% minor allele frequency), a total of 11,834 and 1,679 non-genic SNPs were identified between accessions of G. hirsutum and G. barbadense in genome reduction assemblies, respectively. An additional 4,327 genic SNPs were also identified between accessions of G. hirsutum in the EST assembly. KBioscience KASPar assays were designed for a portion of the intra-specific G. hirsutum SNPs. From 704 non-genic and 348 genic markers developed, a total of 367 (267 non-genic, 100 genic) mapped in a segregating F(2) population (Acala Maxxa × TX2094) using the Fluidigm EP1 system. A G. hirsutum genetic linkage map of 1,688 cM was constructed based entirely on these new SNP markers. Of the genic-based SNPs, we were able to identify within which genome ('A' or 'D') each SNP resided using diploid species sequence data. Genetic maps generated by these newly identified markers are being used to locate quantitative, economically important regions within the cotton genome.