Plant Sciences Commons^™

Tal Effector-Nucleotide Targeter (Tale-Nt) 2.0: Tools For Tal Effector Design And Target Prediction, Erin L. Doyle, Nicholas J. Booher, Daniel S. Standage, Daniel F. Voytas, Volker P. Brendel, John K. Vandyk, Adam J. Bogdanove

Nicholas J. Booher

Transcription activator-like (TAL) effectors are repeat-containing proteins used by plant pathogenic bacteria to manipulate host gene expression. Repeats are polymorphic and individually specify single nucleotides in the DNA target, with some degeneracy. A TAL effector-nucleotide binding code that links repeat type to specified nucleotide enables prediction of genomic binding sites for TAL effectors and customization of TAL effectors for use in DNA targeting, in particular as custom transcription factors for engineered gene regulation and as site-specific nucleases for genome editing. We have developed a suite of web-based tools called TAL Effector-Nucleotide Targeter 2.0 (TALE-NT 2.0;https://boglab.plp.iastate.edu/) that enables design …

Determining The Functions Of Novel Genes Required For Photosynthesis, Gillian Gomer, Moshe Kafri, Martin Jonikas

Gillian Gomer

Determining The Functions Of Novel Genes Required For Photosynthesis, Gillian Gomer, Moshe Kafri, Martin Jonikas

Gillian Gomer

Gene Expression Programs During Shoot, Root, And Callus Development In Arabidopsis Tissue Culture, Ping Che, Sonia Lall, Dan Nettleton, Stephen H. Howell

Dan Nettleton

Shoots can be regenerated from Arabidopsis (Arabidopsis thaliana) root explants in tissue culture through a two-step process requiring preincubation on an auxin-rich callus induction medium. Regenerating tissues can be directed along different developmental pathways leading to the formation of shoots, new roots, or callus by transferring to the appropriate organ induction medium. Using gene-profiling methods, we identified groups of genes that serve as molecular signatures of the different developmental processes, i.e. genes that were specifically up- or down-regulated on one developmental pathway, but not on others. One transcription factor gene that was up-regulated during early shoot development was …

Hierarchical Modeling And Differential Expression Analysis For Rna-Seq Experiments With Inbred And Hybrid Genotypes, Andrew Lithio, Dan Nettleton

Dan Nettleton

The performance of inbred and hybrid genotypes is of interest in plant breeding and genetics. High-throughput sequencing of RNA (RNA-seq) has proven to be a useful tool in the study of the molecular genetic responses of inbreds and hybrids to environmental stresses. Commonly used experimental designs and sequencing methods lead to complex data structures that require careful attention in data analysis. We demonstrate an analysis of RNA-seq data from a split-plot design involving drought stress applied to two inbred genotypes and two hybrids formed by crosses between the inbreds. Our generalized linear modeling strategy incorporates random effects for whole-plot experimental …

Root Type-Specific Reprogramming Of Maize Pericycle Transcriptomes By Local High Nitrate Results In Disparate Lateral Root Branching Patterns, Peng Yu, Jutta A. Baldauf, Andrew Lithio, Caroline Marcon, Dan Nettleton, Chunjian Li, Frank Hochholdinger

Dan Nettleton

The adaptability of root system architecture to unevenly distributed mineral nutrients in soil is a key determinant of plant performance. The molecular mechanisms underlying nitrate dependent plasticity of lateral root branching across the different root types of maize are only poorly understood. In this study, detailed morphological and anatomical analyses together with cell type-specific transcriptome profiling experiments combining laser capture microdissection with RNA-seq were performed to unravel the molecular signatures of lateral root formation in primary, seminal, crown, and brace roots of maize (Zea mays) upon local high nitrate stimulation. The four maize root types displayed divergent branching …

Laser Microdissection Of Narrow Sheath Mutant Maize Uncovers Novel Gene Expression In The Shoot Apical Meristem, Xiaolan Zhang, Shahinez Madi, Lisa Borsuk, Dan Nettleton, Robert J. Elshire, Brent Buckner, Diane Janick-Buckner, Jon Beck, Marja Timmermans, Patrick S. Schnable, Michael J. Scanlon

Dan Nettleton

Microarrays enable comparative analyses of gene expression on a genomic scale, however these experiments frequently identify an abundance of differentially expressed genes such that it may be difficult to identify discrete functional networks that are hidden within large microarray datasets. Microarray analyses in which mutant organisms are compared to nonmutant siblings can be especially problematic when the gene of interest is expressed in relatively few cells. Here, we describe the use of laser microdissection microarray to perform transcriptional profiling of the maize shoot apical meristem (SAM), a ~100-μm pillar of organogenic cells that is required for leaf initiation. Microarray analyses …

Scanning Microarrays At Multiple Intensities Enhances Discovery Of Differentially Expressed Genes, David S. Skibbe, Xiujuan Wang, Xuefeng Zhao, Lisa A. Borsuk, Dan Nettleton, Patrick S. Schnable

Dan Nettleton

Motivation: Scanning parameters are often overlooked when optimizing microarray experiments. A scanning approach that extends the dynamic data range by acquiring multiple scans of different intensities has been developed.

Results: Data from each of three scan intensities (low, medium, high) were analyzed separately using multiple scan and linear regression approaches to identify and compare the sets of genes that exhibit statistically significant differential expression. In the multiple scan approach only one-third of the differentially expressed genes were shared among the three intensities, and each scan intensity identified unique sets of differentially expressed genes. The set of differentially expressed genes from …

The B73 Maize Genome: Complexity, Diversity, And Dynamics, Patrick S. Schnable, Doreen Ware, Kai Ying, Cheng-Ting Yeh, Scott J. Emrich, Yi Jia, Ananth Kalyanaraman, An-Ping Hsia, Yan Fu, Sanzhen Liu, Alan M. Myers, Dan Nettleton, Srinivas Aluru

Dan Nettleton

We report an improved draft nucleotide sequence of the 2.3-gigabase genome of maize, an important crop plant and model for biological research. Over 32,000 genes were predicted, of which 99.8% were placed on reference chromosomes. Nearly 85% of the genome is composed of hundreds of families of transposable elements, dispersed nonuniformly across the genome. These were responsible for the capture and amplification of numerous gene fragments and affect the composition, sizes, and positions of centromeres. We also report on the correlation of methylation-poor regions with Mu transposon insertions and recombination, and copy number variants with insertions and/or deletions, as well …

Loss Of Rna–Dependent Rna Polymerase 2 (Rdr2) Function Causes Widespread And Unexpected Changes In The Expression Of Transposons, Genes, And 24-Nt Small Rnas, Yi Jia, Damon R. Lisch, Kazuhiro Ohtsu, Michael J. Scanlon, Dan Nettleton, Patrick S. Schnable

Dan Nettleton

Transposable elements (TEs) comprise a substantial portion of many eukaryotic genomes and are typically transcriptionally silenced. RNA–dependent RNA polymerase 2 (RDR2) is a component of the RNA–directed DNA methylation (RdDM) silencing pathway. In maize, loss of mediator of paramutation1 (mop1) encoded RDR2 function results in reactivation of transcriptionally silenced Mu transposons and a substantial reduction in the accumulation of 24 nt short-interfering RNAs (siRNAs) that recruit RNA silencing components. An RNA–seq experiment conducted on shoot apical meristems (SAMs) revealed that, as expected based on a model in which RDR2 generates 24 nt siRNAs that suppress expression, most differentially …

Complementation Contributes To Transcriptome Complexity In Maize (Zea Mays L.) Hybrids Relative To Their Inbred Parents, Anja Paschold, Yi Jia, Caroline Marcon, Steve Lund, Nick B. Larson, Cheng-Ting Yeh, Stephan Ossowski, Christa Lanz, Dan Nettleton, Patrick S. Schnable, Frank Hochholdinger

Dan Nettleton

Typically, F₁-hybrids are more vigorous than their homozygous, genetically distinct parents, a phenomenon known as heterosis. In the present study, the transcriptomes of the reciprocal maize (Zea mays L.) hybrids B73×Mo17 and Mo17×B73 and their parental inbred lines B73 and Mo17 were surveyed in primary roots, early in the developmental manifestation of heterotic root traits. The application of statistical methods and a suitable experimental design established that 34,233 (i.e., 86%) of all high-confidence maize genes were expressed in at least one genotype. Nearly 70% of all expressed genes were differentially expressed between the two parents and 42%–55% …

The Maize Brown Midrib2 (Bm2) Gene Encodes A Methylenetetrahydrofolate Reductase That Contributes To Lignin Accumulation, Ho Man Tang, Sanzhen Liu, Sarah Hill-Skinner, Wei Wu, Danielle Reed, Cheng-Ting Yeh, Dan Nettleton, Patrick S. Schnable

Dan Nettleton

The midribs of maize brown midrib (bm) mutants exhibit a reddish‐brown color associated with reductions in lignin concentration and alterations in lignin composition. Here, we report the mapping, cloning, and functional and biochemical analyses of the bm2 gene. The bm2 gene was mapped to a small region of chromosome 1 that contains a putative methylenetetrahydrofolate reductase (MTHFR) gene, which is down‐regulated in bm2 mutant plants. Analyses of multiple Mu‐induced bm2‐Mu mutant alleles confirmed that this constitutively expressed gene is bm2. Yeast complementation experiments and a previously published biochemical characterization show that the bm2 gene encodes a …

Extreme‐Phenotype Genome‐Wide Association Study (Xp‐Gwas): A Method For Identifying Trait‐Associated Variants By Sequencing Pools Of Individuals Selected From A Diversity Panel, Jinliang Yang, Haiying Jiang, Cheng-Ting Yeh, Jianming Yu, Jeffrey A. Jeddeloh, Dan Nettleton, Patrick S. Schnable

Dan Nettleton

Although approaches for performing genome‐wide association studies (GWAS) are well developed, conventional GWAS requires high‐density genotyping of large numbers of individuals from a diversity panel. Here we report a method for performing GWAS that does not require genotyping of large numbers of individuals. Instead XP‐GWAS (extreme‐phenotype GWAS) relies on genotyping pools of individuals from a diversity panel that have extreme phenotypes. This analysis measures allele frequencies in the extreme pools, enabling discovery of associations between genetic variants and traits of interest. This method was evaluated in maize (Zea mays) using the well‐characterized kernel row number trait, which was …

Extensive Tissue-Specific Transcriptomic Plasticity In Maize Primary Roots Upon Water Deficit, Nina Opitz, Caroline Marcon, Anja Paschold, Waqas Ahmed Malik, Andrew Lithio, Ronny Brandt, Hans-Peter Piepho, Dan Nettleton, Frank Hochholdinger

Dan Nettleton

Water deficit is the most important environmental constraint severely limiting global crop growth and productivity. This study investigated early transcriptome changes in maize (Zea mays L.) primary root tissues in response to moderate water deficit conditions by RNA-Sequencing. Differential gene expression analyses revealed a high degree of plasticity of the water deficit response. The activity status of genes (active/inactive) was determined by a Bayesian hierarchical model. In total, 70% of expressed genes were constitutively active in all tissues. In contrast, <3% (50 genes) of water deficit-responsive genes (1915) were consistently regulated in all tissues, while >75% (1501 genes) were specifically regulated in a single root tissue. Water deficit-responsive genes were most numerous in the …

Transcriptomic And Anatomical Complexity Of Primary, Seminal, And Crown Roots Highlight Root Type-Specific Functional Diversity In Maize (Zea Mays L.), Huanhuan Tai, Xin Lu, Nin Opitz, Caroline Marcon, Anja Paschold, Andrew Lithio, Dan Nettleton, Frank Hochholdinger

Dan Nettleton

Maize develops a complex root system composed of embryonic and post-embryonic roots. Spatio-temporal differences in the formation of these root types imply specific functions during maize development. A comparative transcriptomic study of embryonic primary and seminal, and post-embryonic crown roots of the maize inbred line B73 by RNA sequencing along with anatomical studies were conducted early in development. Seminal roots displayed unique anatomical features, whereas the organization of primary and crown roots was similar. For instance, seminal roots displayed fewer cortical cell files and their stele contained more meta-xylem vessels. Global expression profiling revealed diverse patterns of gene activity across …

Stress Response To Co2 Deprivation By Arabidopsis Thaliana In Plant Cultures, Souvik Banerjee, Oskar Siemianowski, Meiling Liu, Kara R. Lind, Xinchun Tian, Dan Nettleton, Ludovico Cademartiri

Dan Nettleton

After being the standard plant propagation protocol for decades, cultures of Arabidopsis thaliana sealed with Parafilm remain common today out of practicality, habit, or necessity (as in co-cultures with microorganisms). Regardless of concerns over the aeration of these cultures, no investigation has explored the CO2 transport inside these cultures and its effect on the plants. Thereby, it was impossible to assess whether Parafilm-seals used today or in thousands of older papers in the literature constitute a treatment, and whether this treatment could potentially affect the study of other treatments.For the first time we report the CO2concentrations in Parafilm-sealed cultures of …

Non-Syntenic Genes Drive Rtcs-Dependent Regulation Of The Embryo Transcriptome During Formation Of Seminal Root Primordia In Maize (Zea Mays L.), Huanhuan Tai, Nina Opitz, Andrew Lithio, Xin Lu, Dan Nettleton, Frank Hochholdinger

Dan Nettleton

Seminal roots of maize are pivotal for early seedling establishment. The maize mutant rootless concerning crown and seminal roots (rtcs) is defective in seminal root initiation during embryogenesis. In this study, the transcriptomes of wild-type and rtcs embryos were analyzed by RNA-Seq based on histological results at three stages of seminal root primordia formation. Hierarchical clustering highlighted that samples of each genotype grouped together along development. Determination of their gene activity status revealed hundreds of genes specifically transcribed in wild-type or rtcs embryos, while K-mean clustering revealed changes in gene expression dynamics between wild-type and rtcs during embryo …

Interchromosomal Transfer Of Immune Regulation During Infection Of Barley With The Powdery Mildew Pathogen, Priyanka Surana, Ruo Xu, Gregory Fuerst, Antony V. E. Chapman, Dan Nettleton, Roger P. Wise

Dan Nettleton

Powdery mildew pathogens colonize over 9500 plant species, causing critical yield loss. The Ascomycete fungus, Blumeria graminis f. sp. hordei (Bgh), causes powdery mildew disease in barley (Hordeum vulgare L.). Successful infection begins with penetration of host epidermal cells, culminating in haustorial feeding structures, facilitating delivery of fungal effectors to the plant and exchange of nutrients from host to pathogen. We used expression Quantitative Trait Locus (eQTL) analysis to dissect the temporal control of immunity-associated gene expression in a doubled haploid barley population challenged with Bgh. Two highly significant regions possessing trans eQTL were identified near …

Substantial Contribution Of Genetic Variation In The Expression Of Transcription Factors To Phenotypic Variation Revealed By Erd-Gwas, Hung-Ying Lin, Qiang Liu, Xiao Li, Jinliang Yang, Sanzhen Liu, Yinlian Huang, Michael J. Scanlon, Dan Nettleton, Patrick S. Schnable

Dan Nettleton

Background: There are significant limitations in existing methods for the genome-wide identification of genes whose expression patterns affect traits.

Results: The transcriptomes of five tissues from 27 genetically diverse maize inbred lines were deeply sequenced to identify genes exhibiting high and low levels of expression variation across tissues or genotypes. Transcription factors are enriched among genes with the most variation in expression across tissues, as well as among genes with higher-than-median levels of variation in expression across genotypes. In contrast, transcription factors are depleted among genes whose expression is either highly stable or highly variable across genotypes. We developed a …

Spotted Cotton Oligonucleotide Microarrays For Gene Expression Analysis, Joshua A. Udall, Lex E. Flagel, Foo Cheung, Andrew W. Woodard, Ran Hovav, Ryan Adam Rapp, Jordan M. Swanson, Jinsuk J. Lee, Alan R. Gingle, Dan Nettleton, Christopher D. Town, Z. Jeffrey Chen, Jonathan F. Wendel

Dan Nettleton

Microarrays offer a powerful tool for diverse applications plant biology and crop improvement. Recently, two comprehensive assemblies of cotton ESTs were constructed based on three Gossypium species. Using these assemblies as templates, we describe the design and creation and of a publicly available oligonucleotide array for cotton, useful for all four of the cultivated species. Synthetic oligonucleotide probes were generated from exemplar sequences of a global assembly of 211,397 cotton ESTs derived from >50 different cDNA libraries representing many different tissue types and tissue treatments. A total of 22,787 oligonucleotide probes are included on the arrays, optimized to target the …

Parallel Genome-Wide Expression Profiling Of Host And Pathogen During Soybean Cyst Nematode Infection Of Soybean, Nagabhushana Ithal, Justin Recknor, Dan Nettleton, Leonard Hearne, Tom Maier, Thomas J. Baum, Melissa G. Mitchum

Dan Nettleton

Global analysis of gene expression changes in soybean (Glycine max) and Heterodera glycines (soybean cyst nematode [SCN]) during the course of infection in a compatible interaction was performed using the Affymetrix GeneChip soybean genome array. Among 35,611 soybean transcripts monitored, we identified 429 genes that showed statistically significant differential expression between uninfected and nematode-infected root tissues. These included genes encoding enzymes involved in primary metabolism; biosynthesis of phenolic compounds, lignin, and flavonoids; genes related to stress and defense responses; cell wall modification; cellular signaling; and transcriptional regulation. Among 7,431 SCN transcripts monitored, 1,850 genes showed statistically significant differential …

Sequence Mining And Transcript Profiling To Explore Cyst Nematode Parasitism, Axel A. Elling, Makedonka Mitreva, Xiaowu Gai, John Martin, Justin Recknor, Eric L. Davis, Richard S. Hussey, Dan Nettleton, James P. Mccarter, Thomas J. Baum

Dan Nettleton

Background: Cyst nematodes are devastating plant parasites that become sedentary within plant roots and induce the transformation of normal plant cells into elaborate feeding cells with the help of secreted effectors, the parasitism proteins. These proteins are the translation products of parasitism genes and are secreted molecular tools that allow cyst nematodes to infect plants.

Results: We present here the expression patterns of all previously described parasitism genes of the soybean cyst nematode, Heterodera glycines, in all major life stages except the adult male. These insights were gained by analyzing our gene expression dataset from experiments using the Affymetrix Soybean …

Mu Transposon Insertion Sites And Meiotic Recombination Events Co-Localize With Epigenetic Marks For Open Chromatin Across The Maize Genome, Sanzhen Liu, Cheng-Ting Yeh, Tieming Ji, Kai Ying, Haiyan Wu, Ho Man Tang, Yan Fu, Daniel S. Nettleton, Patrick S. Schnable

Dan Nettleton

The Mu transposon system of maize is highly active, with each of the ∼50–100 copies transposing on average once each generation. The approximately one dozen distinct Mutransposons contain highly similar ∼215 bp terminal inverted repeats (TIRs) and generate 9-bp target site duplications (TSDs) upon insertion. Using a novel genome walking strategy that uses these conserved TIRs as primer binding sites, Mu insertion sites were amplified from Mu stocks and sequenced via 454 technology. 94% of ∼965,000 reads carried Mu TIRs, demonstrating the specificity of this strategy. Among these TIRs, 21 novel Mu TIRs were discovered, revealing additional complexity of …

Flor Revisited (Again): Eqtl And Mutational Analysis Of Nb-Lrr Mediated Immunity To Powdery Mildew In Barley, Roger P. Wise, Priyanka Surana, Gregory S. Fuerst, Ruo Xu, Divya Mistry, Julie A. Dickerson, Dan Nettleton Dr.

Dan Nettleton

Genes encoding early signaling events in pathogen defense often are identified only by their phenotype. Such genes involved in barley-powdery mildew interactions include Mla, specifying race-specific resistance; Rar1 (Required for Mla12-specified resistance1), and Rom1 (Restoration of Mla-specified resistance1). The HSP90-SGT1-RAR1 complex appears to function as chaperone in MLA-specified resistance, however, much remains to be discovered regarding the precise signaling underlying plant immunity. Genetic analyses of fast-neutron mutants derived from CI 16151 (Mla6) uncovered a novel locus, designated Rar3 (Required for Mla6-specified resistance3). Rar3 segregates independent of Mla6 and Rar1, and rar3 mutants are susceptible to Blumeria graminis f. sp. hordei …

Duplicate Gene Expression In Allopolyploid Gossypium Reveals Two Temporally Distinct Phases Of Expression Evolution, Lex E. Flagel, Joshua A. Udall, Dan Nettleton, Jonathan F. Wendel

Dan Nettleton

Polyploidy has played a prominent role in shaping the genomic architecture of the angiosperms. Through allopolyploidization, several modern Gossypium (cotton) species contain two divergent, although largely redundant genomes. Owing to this redundancy, these genomes can play host to an array of evolutionary processes that act on duplicate genes. We compared homoeolog (genes duplicated by polyploidy) contributions to the transcriptome of a natural allopolyploid and a synthetic interspecific F1 hybrid, both derived from a merger between diploid species from the Gossypium A-genome and D-genome groups. Relative levels of A- and D-genome contributions to the petal transcriptome were determined for 1,383 gene …

Developmental Transcript Profiling Of Cyst Nematode Feeding Cells In Soybean Roots, Nagabhushana Ithal, Justin Recknor, Dan Nettleton, Tom Maier, Thomas J. Baum, Melissa G. Mitchum

Dan Nettleton

Cyst nematodes of the genus Heterodera are obligate, sedentary endoparasites that have developed highly evolved relationships with specific host plant species. Successful parasitism involves significant physiological and morphological changes to plant root cells for the formation of specialized feeding cells called syncytia. To better understand the molecular mechanisms that lead to the development of nematode feeding cells, transcript profiling was conducted on developing syncytia induced by the soybean cyst nematode Heterodera glycines in soybean roots by coupling laser capture microdissection with high-density oligonucleotide microarray analysis. This approach has identified pathways that may play intrinsic roles in syncytium induction, formation, and …

A Clade-Specific Arabidopsis Gene Connects Primary Metabolism And Senescence, Dallas C. Jones, Wenguang Zheng, Sheng Huang, Chuanlong Du, Xuefeng Zhao, Ragothaman M. Yennamalli, Taner Z. Sen, Dan Nettleton, Eve S. Wurtele, Ling Li

Dan Nettleton

Nearly immobile, plants have evolved new components to be able to respond to changing environments. One example is Qua Quine Starch (QQS, AT3G30720), an Arabidopsis thaliana-specific orphan gene that integrates primary metabolism with adaptation to environment changes. SAQR (Senescence-Associated and QQS-Related, AT1G64360), is unique to a clade within the family Brassicaceae; as such, the gene may have arisen about 20 million years ago. SAQR is up-regulated in QQS RNAi mutant and in the apx1 mutant under light-induced oxidative stress. SAQR plays a role in carbon allocation: overexpression lines of SAQR have significantly decreased starch content; …

The Subject Librarian Newsletter, Biology, Spring 2017, Sandy Avila

Sandy Avila

No abstract provided.

Tal Effector-Nucleotide Targeter (Tale-Nt) 2.0: Tools For Tal Effector Design And Target Prediction, Erin L. Doyle, Nicholas J. Booher, Daniel S. Standage, Daniel F. Voytas, Volker P. Brendel, John K. Vandyk, Adam J. Bogdanove

John K. VanDyk

Transcription activator-like (TAL) effectors are repeat-containing proteins used by plant pathogenic bacteria to manipulate host gene expression. Repeats are polymorphic and individually specify single nucleotides in the DNA target, with some degeneracy. A TAL effector-nucleotide binding code that links repeat type to specified nucleotide enables prediction of genomic binding sites for TAL effectors and customization of TAL effectors for use in DNA targeting, in particular as custom transcription factors for engineered gene regulation and as site-specific nucleases for genome editing. We have developed a suite of web-based tools called TAL Effector-Nucleotide Targeter 2.0 (TALE-NT 2.0;https://boglab.plp.iastate.edu/) that enables design of custom …

Phylogenetics Of Morus (Moraceae) Inferred From Its And Trnl-Trnf Sequence Data., Madhav Nepal

Madhav Nepal