Monica Britton

Transcriptome Profiling of Citrus Fruit Response to Huanglongbing Disease

Huanglongbing (HLB) or “citrus greening” is the most destructive citrus disease worldwide. In this work, we studied host responses of citrus to infection with Candidatus Liberibacter asiaticus (CaLas) using next-generation sequencing technologies. A deep mRNA profile was obtained from peel of healthy and HLB-affected fruit. It was followed by pathway and protein-protein network analysis and quantitative real time PCR analysis of highly regulated genes. We identified differentially regulated pathways and constructed networks that provide a deep insight into the metabolism of affected fruit. Data mining revealed that HLB enhanced transcription of genes involved in the light reactions of photosynthesis and in ATP synthesis. Activation of protein degradation and misfolding processes were observed at the transcriptomic level. Transcripts for heat shock proteins were down-regulated at all disease stages, resulting in further protein misfolding. HLB strongly affected pathways involved in source-sink communication, including sucrose and starch metabolism and hormone synthesis and signaling. Transcription of several genes involved in the synthesis and signal transduction of cytokinins and gibberellins was repressed while that of genes involved in ethylene pathways was induced. CaLas infection triggered a response via both the salicylic acid and jasmonic acid pathways and increased the transcript abundance of several members of the WRKY family of transcription factors. Findings focused on the fruit provide valuable insight to understanding the mechanisms of the HLB-induced fruit disorder and eventually developing methods based on small molecule applications to mitigate its devastating effects on fruit production.

Exploring the Transcriptome Landscape of Pomegranate Fruit Peel for Natural Product Biosynthetic Gene and SSR Marker Discovery

Pomegranate fruit peel is rich in bioactive plant natural products, such as hydrolyzable tannins and anthocyanins. Despite their documented roles in human nutrition and fruit quality, genes involved in natural product biosynthesis have not been cloned from pomegranate and very little sequence information is available on pomegranate in the public domain. Shotgun transcriptome sequencing of pomegranate fruit peel cDNA was performed using RNA-Seq on the Illumina Genome Analyzer platform. Over 100 million raw sequence reads were obtained and assembled into 9,839 transcriptome assemblies (TAs) (>200 bp). Candidate genes for hydrolyzable tannin, anthocyanin, flavonoid, terpenoid and fatty acid biosynthesis and/or regulation were identified. Three lipid transfer proteins were obtained that may contribute to the previously reported IgE reactivity of pomegranate fruit extracts. In addition, 115 SSR markers were identified from the pomegranate fruit peel transcriptome and primers were designed for 77 SSR markers. The pomegranate fruit peel transcriptome set provides a valuable platform for natural product biosynthetic gene and SSR marker discovery in pomegranate. This work also demonstrates that next-generation transcriptome sequencing is an economical and effective approach for investigating natural product biosynthesis, identifying genes controlling important agronomic traits, and discovering molecular markers in non-model specialty crop species.

The Oncogenes of Agrobacterium Tumefaciens and Agrobacterium Rhizogenes

The common soil bacteria Agrobacterium tumefaciens and Agrobacterium rhizogenes are unique genetic pathogens capable of fundamentally redirecting plant metabolism in order to generate macroscopic tissue masses (crown galls and hairy roots, respectively) which support the growth of large populations of Agrobacteria. Central to pathogenesis is the horizontal transfer of a suite of oncogenes from the tumor-inducing (Ti) plasmids of A. tumefaciens and the root-inducing (Ri) plasmids of A. rhizogenes into the plant cell genome. These oncogenes alter the synthesis, perception and/or transport of phytohormones in planta, leading to the development of the crown gall and hairy root structures from single genetically transformed plant cells. Crown galls and hairy roots become effective sinks that divert plant resources to produce opine compounds that can only be metabolized by the infecting strain of Agrobacterium. The basic genetic and biochemical mechanisms underlying A. tumefaciens tumorigenesis were initially described over 20 years ago, with the characterization of the ipt, iaaM and iaaH oncogenes. However, the simplistic San Marcos, CA 92096, USA Ave, Davis, CA 95616, USA; Department of Biological Sciences, 524 Monica T. Britton, Matthew A. Escobar and Abhaya M. Dandekar view of crown gall development as solely a function of ipt-driven cytokinin synthesis and iaaM/iaaH-driven auxin synthesis has recently given way to a more nuanced understanding of the roles of secondary oncogenes in modulating hormone perception and the complex hormone activation cascade in crown galls involving ethylene, abscisic acid and jasmonic acid. The biochemistry and functional significance of specific oncogenes in A. rhizogenesmediated hairy root development is less well understood, but recent work has substantially increased our understanding of the A. rhizogenes oncogenes, especially the rol genes. Expression of the rolA, B and C oncogenes in planta induces a subtle interaction with endogenous plant signal transduction pathways and transcription factors, affecting the local concentrations of several classes of plant hormones. These interactions lead to de novo meristem formation in transformed cells, with subsequent differentiation depending on the local hormone balance. This process most often results in the induction of highly branched non-geotropic adventitious roots, the “hairy root” phenotype. Further dissection of the molecular mechanisms underlying Agrobacterium pathogenesis should continue to yield broader insights into the understanding of endogenous hormone signaling pathways and tissue differentiation in plants.

Stacking resistance to crown gall and nematodes in walnut rootstocks

BackgroundCrown gall (CG) (Agrobacterium tumefaciens) and the root lesion nematodes (RLNs) (Pratylenchus vulnus) are major challenges faced by the California walnut industry, reducing productivity and increasing the cost of establishing and maintaining orchards. Current nematode control strategies include nematicides, crop rotation, and tolerant cultivars, but these methods have limits. Developing genetic resistance through novel approaches like RNA interference (RNAi) can address these problems. RNAi-mediated silencing of CG disease in walnut (Juglans regia L.) has been achieved previously. We sought to place both CG and nematode resistance into a single walnut rootstock genotype using co-transformation to stack the resistance genes. A. tumefaciens, carrying self-complimentary iaaM and ipt transgenes, and Agrobacterium rhizogenes, carrying a self-complimentary Pv010 gene from P. vulnus, were used as co-transformation vectors. RolABC genes were introduced by the resident T-DNA in the A. rhizogenes Ri-plasmid used as a vector for plant transformation. Pv010 and Pv194 (transgenic control) genes were also transferred separately using A. tumefaciens. To test for resistance, transformed walnut roots were challenged with P. vulnus and microshoots were challenged with a virulent strain of A. tumefaciens.ResultsCombining the two bacterial strains at a 1:1 rather than 1:3 ratio increased the co-transformation efficiency. Although complete immunity to nematode infection was not observed, transgenic lines yielded up to 79% fewer nematodes per root following in vitro co-culture than untransformed controls. Transgenic line 33-3-1 exhibited complete crown gall control and 32% fewer nematodes. The transgenic plants had thicker, longer roots than untransformed controls possibly due to insertion of rolABC genes. When the Pv010 gene was present in roots with or without rolABC genes there was partial or complete control of RLNs. Transformation using only one vector showed 100% control in some lines.ConclusionsCG and nematode resistance gene stacking controlled CG and RLNs simultaneously in walnuts. Silencing genes encoding iaaM, ipt, and Pv010 decrease CG formation and RLNs populations in walnut. Beneficial plant genotype and phenotype changes are caused by co-transformation using A. tumefaciens and A. rhizogenes strains. Viable resistance against root lesion nematodes in walnut plants may be accomplished in the future using this gene stacking technology.

Genome-wide SNP discovery in walnut with an AGSNP pipeline updated for SNP discovery in allogamous organisms

BackgroundA genome-wide set of single nucleotide polymorphisms (SNPs) is a valuable resource in genetic research and breeding and is usually developed by re-sequencing a genome. If a genome sequence is not available, an alternative strategy must be used. We previously reported the development of a pipeline (AGSNP) for genome-wide SNP discovery in coding sequences and other single-copy DNA without a complete genome sequence in self-pollinating (autogamous) plants. Here we updated this pipeline for SNP discovery in outcrossing (allogamous) species and demonstrated its efficacy in SNP discovery in walnut (Juglans regia L.).ResultsThe first step in the original implementation of the AGSNP pipeline was the construction of a reference sequence and the identification of single-copy sequences in it. To identify single-copy sequences, multiple genome equivalents of short SOLiD reads of another individual were mapped to shallow genome coverage of long Sanger or Roche 454 reads making up the reference sequence. The relative depth of SOLiD reads was used to filter out repeated sequences from single-copy sequences in the reference sequence. The second step was a search for SNPs between SOLiD reads and the reference sequence. Polymorphism within the mapped SOLiD reads would have precluded SNP discovery; hence both individuals had to be homozygous. The AGSNP pipeline was updated here for using SOLiD or other type of short reads of a heterozygous individual for these two principal steps. A total of 32.6X walnut genome equivalents of SOLiD reads of vegetatively propagated walnut scion cultivar ‘Chandler’ were mapped to 48,661 ‘Chandler’ bacterial artificial chromosome (BAC) end sequences (BESs) produced by Sanger sequencing during the construction of a walnut physical map. A total of 22,799 putative SNPs were initially identified. A total of 6,000 Infinium II type SNPs evenly distributed along the walnut physical map were selected for the construction of an Infinium BeadChip, which was used to genotype a walnut mapping population having ‘Chandler’ as one of the parents. Genotyping results were used to adjust the filtering parameters of the updated AGSNP pipeline. With the adjusted filtering criteria, 69.6% of SNPs discovered with the updated pipeline were real and could be mapped on the walnut genetic map. A total of 13,439 SNPs were discovered by BES re-sequencing. BESs harboring SNPs were in 677 FPC contigs covering 98% of the physical map of the walnut genome.ConclusionThe updated AGSNP pipeline is a versatile SNP discovery tool for a high-throughput, genome-wide SNP discovery in both autogamous and allogamous species. With this pipeline, a large set of SNPs were identified in a single walnut cultivar.

Effects of high temperatures on threatened estuarine fishes during periods of extreme drought.

ABSTRACT Climate change and associated increases in water temperatures may impact physiological performance in ectotherms and exacerbate endangered species declines. We used an integrative approach to assess the impact of elevated water temperature on two fishes of immediate conservation concern in a large estuary system, the threatened longfin smelt (Spirinchus thaleichthys) and endangered delta smelt (Hypomesus transpacificus). Abundances have reached record lows in California, USA, and these populations are at imminent risk of extirpation. California is currently impacted by a severe drought, resulting in high water temperatures, conditions that will become more common as a result of climate change. We exposed fish to environmentally relevant temperatures (14°C and 20°C) and used RNA sequencing to examine the transcriptome-wide responses to elevated water temperature in both species. Consistent with having a lower temperature tolerance, longfin smelt exhibited a pronounced cellular stress response, with an upregulation of heat shock proteins, after exposure to 20°C that was not observed in delta smelt. We detected an increase in metabolic rate in delta smelt at 20°C and increased expression of genes involved in metabolic processes and protein synthesis, patterns not observed in longfin smelt. Through examination of responses across multiple levels of biological organization, and by linking these responses to habitat distributions in the wild, we demonstrate that longfin smelt may be more susceptible than delta smelt to increases in temperatures, and they have little room to tolerate future warming in California. Understanding the species-specific physiological responses of sensitive species to environmental stressors is crucial for conservation efforts and managing aquatic systems globally. Highlighted Article: An integrative approach to assess the impact of elevated water temperature on fishes of immediate conservation concern highlights the vulnerability of longfin smelt relative to delta smelt.

Deep RNA-Seq profile reveals biodiversity, plant–microbe interactions and a large family of NBS-LRR resistance genes in walnut (Juglans regia) tissues

Deep RNA-Seq profiling, a revolutionary method used for quantifying transcriptional levels, often includes non-specific transcripts from other co-existing organisms in spite of stringent protocols. Using the recently published walnut genome sequence as a filter, we present a broad analysis of the RNA-Seq derived transcriptome profiles obtained from twenty different tissues to extract the biodiversity and possible plant–microbe interactions in the walnut ecosystem in California. Since the residual nature of the transcripts being analyzed does not provide sufficient information to identify the exact strain, inferences made are constrained to the genus level. The presence of the pathogenic oomycete Phytophthora was detected in the root through the presence of a glyceraldehyde-3-phosphate dehydrogenase. Cryptococcus, the causal agent of cryptococcosis, was found in the catkins and vegetative buds, corroborating previous work indicating that the plant surface supported the sexual cycle of this human pathogen. The RNA-Seq profile revealed several species of the endophytic nitrogen fixing Actinobacteria. Another bacterial species implicated in aerobic biodegradation of methyl tert-butyl ether (Methylibium petroleiphilum) is also found in the root. RNA encoding proteins from the pea aphid were found in the leaves and vegetative buds, while a serine protease from mosquito with significant homology to a female reproductive tract protease from Drosophila mojavensis in the vegetative bud suggests egg-laying activities. The comprehensive analysis of RNA-seq data present also unraveled detailed, tissue-specific information of ~400 transcripts encoded by the largest family of resistance (R) genes (NBS-LRR), which possibly rationalizes the resistance of the specific walnut plant to the pathogens detected. Thus, we elucidate the biodiversity and possible plant–microbe interactions in several walnut (Juglans regia) tissues in California using deep RNA-Seq profiling.

Probing functional polymorphisms in the dengue vector, Aedes aegypti

BackgroundDengue is the most prevalent arboviral disease world-wide and its primary vector is the mosquito Aedes aegypti. The current lack of commercially-available vaccines makes control of vector populations the only effective strategy to prevent dengue transmission. Aedes aegypti geographic populations exhibit great variability in insecticide resistance and susceptibility to dengue infection. The characterization of single nucleotide polymorphisms (SNPs) as molecular markers to study quantitatively this variation is needed greatly because this species has a low abundance of microsatellite markers and limited known restriction fragments length polymorphisms (RFLPs) and single-strand conformation polymorphism (SSCP) markers.ResultsWe used RNA-seq to characterize SNPs in three Ae. aegypti strains, including the Liverpool (LVP) strain, from which the current genome annotation is derived. We identified 131,764 unique genome locations with at least one alternative nucleotide to what is reported in the reference annotation. These comprised changes in both open-reading frames (ORFs) and untranslated regions (UTRs) of transcripts. An in depth-look at sequence variation in immunity genes revealed that those associated with autophagy, MD2-like receptors and Peptidoglycan Recognition Proteins had more sequence variation in their 3’UTRs than mutations associated with non-synonymous changes. This supports the conclusion that these genes had maintained their functional specificity while being adapted to different regulatory domains. In contrast, a number of peroxidases, serpins and Clip-domain serine proteases exhibited conservation of putative UTR regulatory sequences while displaying diversification of the ORFs. Transcriptome evidence also was found for ~2500 novel transcriptional units (NTUs) not annotated in the reference genome.ConclusionsThe transcriptome-wide assessment of within and inter-strain polymorphisms in Ae. aegypti adds considerably to the number of molecular markers available for genetic studies in this mosquito. Additionally, data supporting NTU discovery emphasizes the need for continuous amendments of the reference genome annotation.

RNA-seq analyses of changes in the Anopheles gambiae transcriptome associated with resistance to pyrethroids in Kenya: identification of candidate-resistance genes and candidate-resistance SNPs

BackgroundThe extensive use of pyrethroids for control of malaria vectors, driven by their cost, efficacy and safety, has led to widespread resistance. To favor their sustainable use, the World Health Organization (WHO) formulated an insecticide resistance management plan, which includes the identification of the mechanisms of resistance and resistance surveillance. Recognized physiological mechanisms of resistance include target site mutations in the para voltage-gated sodium channel, metabolic detoxification and penetration resistance. Such understanding of resistance mechanisms has allowed the development of resistance monitoring tools, including genotyping of the kdr mutation L1014F/S in the para gene.MethodsThe sequence-based technique RNA-seq was applied to study changes in the transcriptome of deltamethrin-resistant and -susceptible Anopheles gambiae mosquitoes from the Western Province of Kenya. The resulting gene expression profiles were compared to data in the most recent literature to derive a list of candidate resistance genes. RNA-seq data were analyzed also to identify sequence polymorphisms linked to resistance.ResultsA total of five candidate-resistance genes (AGAP04177, AGAP004572, AGAP008840, AGAP007530 and AGAP013036) were identified with altered expression between resistant and susceptible mosquitoes from West and East Africa. A change from G to C at position 36043997 of chromosome 3R resulting in A101G of the sulfotransferase gene AGAP009551 was significantly associated with the resistance phenotype (odds ratio: 5.10). The kdr L1014S mutation was detected at similar frequencies in both phenotypically resistant and susceptible mosquitoes, suggesting it is no longer fully predictive of the resistant phenotype.ConclusionsOverall, these results support the conclusion that resistance to pyrethroids is a complex and evolving phenotype, dependent on multiple gene functions including, but not limited to, metabolic detoxification. Functional convergence among metabolic detoxification genes may exist, with the role of each gene being modulated by the life history and selection pressure on mosquito populations. As a consequence, biochemical assays that quantify overall enzyme activity may be a more suitable method for predicting metabolic resistance than gene-based assays.

YeATS - a tool suite for analyzing RNA-seq derived transcriptome identifies a highly transcribed putative extensin in heartwood/sapwood transition zone in black walnut.

The transcriptome provides a functional footprint of the genome by enumerating the molecular components of cells and tissues. The field of transcript discovery has been revolutionized through high-throughput mRNA sequencing (RNA-seq). Here, we present a methodology that replicates and improves existing methodologies, and implements a workflow for error estimation and correction followed by genome annotation and transcript abundance estimation for RNA-seq derived transcriptome sequences (YeATS - Yet Another Tool Suite for analyzing RNA-seq derived transcriptome). A unique feature of YeATS is the upfront determination of the errors in the sequencing or transcript assembly process by analyzing open reading frames of transcripts. YeATS identifies transcripts that have not been merged, result in broken open reading frames or contain long repeats as erroneous transcripts. We present the YeATS workflow using a representative sample of the transcriptome from the tissue at the heartwood/sapwood transition zone in black walnut. A novel feature of the transcriptome that emerged from our analysis was the identification of a highly abundant transcript that had no known homologous genes (GenBank accession: KT023102). The amino acid composition of the longest open reading frame of this gene classifies this as a putative extensin. Also, we corroborated the transcriptional abundance of proline-rich proteins, dehydrins, senescence-associated proteins, and the DNAJ family of chaperone proteins. Thus, YeATS presents a workflow for analyzing RNA-seq data with several innovative features that differentiate it from existing software.