The Taxonomic and Functional Nature of Plant-Associated Microbiomes

Microbes live in close association with eukaryotes and have substantial impacts on fitness and well-being of their hosts. In plants, microbes can colonize soil adjacent to plant roots and can even survive inside of plant tissues. They can have either positive or negative effects on plant fitness and therefore show potential for use as an agricultural tool. However, our current understanding of how these microbial communities are formed and how they function is limited. The work described in this dissertation reports novel insights regarding plant-associated microbiomes along with new and improved methods for observing their associations with plants. Chapter 1 gives a brief introduction to plant associated microbiomes and the common methods used to study them. Chapter 2 outlines colonization patterns of Arabidopsis thaliana associated microbiomes. The taxa that colonize Arabidopsis roots endophytically are distinct from those colonizing the soil surrounding the roots (rhizosphere) and unplanted bulk soil. This suggests that plants modulate these communities and possibly select for microbes that provide specific fitness advantages. Endophytic communities of plants grown in different soils exemplify how soil type is the primary factor in determining the taxa found in these communities. However, there are core taxa that are consistently found in the endophyte compartment regardless of soil type and other factors. These core microbes are potential candidates that are actively selected by plants. Communities associated with different Arabidopsis ecotypes and ages have only minor differences. Chapter 3 presents improved methods for profiling taxa in plant-associated microbiomes by utilizing two techniques. First, PCR amplification clamps designed from peptide nucleic acids are used to reduce plant chloroplast contamination. Removing unwanted chloroplast contamination reduces the cost of sequencing by increasing the yield of usable, bacterial 16S reads. Second, 16S amplicons are tagged with a unique DNA oligo (i.e. molecule tag) prior to PCR amplification. After PCR amplification and sequencing, reads having the same molecule tag likely originated from the same DNA template. Therefore, discrepancies between these reads are presumably sequencing errors and can be corrected bioinformatically. Identifying and correcting these sequencing errors can be performed using the MTToolbox software described in Chapter 4. Correcting sequencing errors using molecule tagged reads and MTToolbox substantially reduces the number of spurious singleton OTUs. Chapter 5 compares the functional profiles of Arabidopsis rhizospheres against those in the bulk soil and also describes novel methods for comparing across metagenomes. These methods can report functional differences between microbiomes at a global level or for specific taxa. For example, transcription-related functions were identified as frequently enriched in the rhizosphere across a broad diversity of taxa. Alternatively, synthesis of cyclic beta-1,2-glucans were identified as rhizosphere enriched in multiple taxa among a small group of betaproteobacteria despite other betaproteobacteria having different enrichment patterns. Additionally, rare functions in these metagenomes were more likely to be classified as rhizosphere enriched suggesting that microbes are constantly evolving new mechanisms for rhizosphere colonization. Therefore, identifying taxa specific enrichments patterns is important for understanding mechanisms associated with rhizosphere colonization. Collectively, the methods and insights described in this document expand our understanding of plant microbiomes and generate important hypotheses for future examination.Doctor of Philosoph

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 (Gossypiumhirsutum). 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 N50 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 F2 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

MT-Toolbox: improved amplicon sequencing using molecule tags

Abstract 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

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

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

<div><p>Background</p><p>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.</p><p>Results</p><p>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 <i>Streptococcaceae</i> and <i>Enterobacteriaceae</i> and 84% increase in <i>Staphylococcaceae</i> as compared to patients with a PaO₂/FiO₂ ratio > 300. Wilcoxon rank-sum test identified significant enrichment in abundance of OTUs identified as <i>Prevotella melaninogenica (p</i> = 0.042), <i>Corynebacterium</i> (<i>p</i> = 0.037) and <i>Mogibacterium</i> (<i>p</i> = 0.048). Linear discriminant effect size analysis (LefSe) confirmed significant enrichment of <i>Prevotella melaninognica</i> among patients with a PaO₂/FiO₂ ratio ≤ 300 (<i>p</i><0.05). These results could not be explained by differences in antibiotic treatment.</p><p>Conclusions</p><p>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.</p></div

CumbieJasonBotanyPlantPathologyVariableSuitesNon-Effector_SupportingInformation.zip

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