Volker Brendel

TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction

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 TAL effector repeat arrays for desired targets and prediction of TAL effector binding sites, ranked by likelihood, in a genome, promoterome or other sequence of interest. Search parameters can be set by the user to work with any TAL effector or TAL effector nuclease architecture. Applications range from designing highly specific DNA targeting tools and identifying potential off-target sites to predicting effector targets important in plant disease.

PlantGDB: a resource for comparative plant genomics

PlantGDB (http://www.plantgdb.org/) is a genomics database encompassing sequence data for green plants (Viridiplantae). PlantGDB provides annotated transcript assemblies for >100 plant species, with transcripts mapped to their cognate genomic context where available, integrated with a variety of sequence analysis tools and web services. For 14 plant species with emerging or complete genome sequence, PlantGDBs genome browsers (xGDB) serve as a graphical interface for viewing, evaluating and annotating transcript and protein alignments to chromosome or bacterial artificial chromosome (BAC)-based genome assemblies. Annotation is facilitated by the integrated yrGATE module for community curation of gene models. Novel web services at PlantGDB include Tracembler, an iterative alignment tool that generates contigs from GenBank trace file data and BioExtract Server, a web-based server for executing custom sequence analysis workflows. PlantGDB also hosts a plant genomics research outreach portal (PGROP) that facilitates access to a large number of resources for research and training.

PlantGDB, plant genome database and analysis tools

PlantGDB (http://www.plantgdb.org/) is a database of molecular sequence data for all plant species with significant sequencing efforts. The database organizes EST sequences into contigs that represent tentative unique genes. Contigs are annotated and, whenever possible, linked to their respective genomic DNA. Genome sequence fragments are assembled similarly. The goal of the PlantGDB web site is to establish the basis for identifying sets of genes common to all plants or specific to particular species by integrating a number of bioinformatics tools that facilitate gene prediction and cross- species comparisons. For species with large-scale genome sequencing efforts, PlantGDB provides genome browsing capabilities that integrate all available EST and cDNA evidence for current gene models (for Arabidopsis thaliana, see the AtGDB site at http://www.plantgdb.org/AtGDB/).

MaizeGDB, the community database for maize genetics and genomics

The Maize Genetics and Genomics Database (MaizeGDB) is a central repository for maize sequence, stock, phenotype, genotypic and karyotypic variation, and chromosomal mapping data. In addition, MaizeGDB provides contact information for over 2400 maize cooperative researchers, facilitating interactions between members of the rapidly expanding maize community. MaizeGDB represents the synthesis of all data available previously from ZmDB and from MaizeDB-databases that have been superseded by MaizeGDB. MaizeGDB provides web-based tools for ordering maize stocks from several organizations including the Maize Genetics Cooperation Stock Center and the North Central Regional Plant Introduction Station (NCRPIS). Sequence searches yield records displayed with embedded links to facilitate ordering cloned sequences from various groups including the Maize Gene Discovery Project and the Clemson University Genomics Institute. An intuitive web interface is implemented to facilitate navigation between related data, and analytical tools are embedded within data displays. Web-based curation tools for both designated experts and general researchers are currently under development. MaizeGDB can be accessed at http://www.maizegdb.org/.

Optimal spliced alignment of homologous cDNA to a genomic DNA template

MOTIVATION Supplementary cDNA or EST evidence is often decisive for discriminating between alternative gene predictions derived from computational sequence inspection by any of a number of requisite programs. Without additional experimental effort, this approach must rely on the occurrence of cognate ESTs for the gene under consideration in available, generally incomplete, EST collections for the given species. In some cases, particular exon assignments can be supported by sequence matching even if the cDNA or EST is produced from non-cognate genomic DNA, including different loci of a gene family or homologous loci from different species. However, marginally significant sequence matching alone can also be misleading. We sought to develop an algorithm that would simultaneously score for predicted intrinsic splice site strength and sequence matching between the genomic DNA template and a related cDNA or EST. In this case, weakly predicted splice sites may be chosen for the optimal scoring spliced alignment on the basis of surrounding sequence matching. Strongly predicted splice sites will enter the optimal spliced alignment even without strong sequence matching. RESULTS We designed a novel algorithm that produces the optimal spliced alignment of a genomic DNA with a cDNA or EST based on scoring for both sequence matching and intrinsic splice site strength. By example, we demonstrate that this combined approach appears to improve gene prediction accuracy compared with current methods that rely only on either search by content and signal or on sequence similarity. AVAILABILITY The algorithm is available as a C subroutine and is implemented in the SplicePredictor and GeneSeqer programs. The source code is available via anonymous ftp from ftp. zmdb.iastate.edu. Both programs are also implemented as a Web service at http://gremlin1.zool.iastate.edu/cgi-bin/s p.cgiand http://gremlin1.zool.iastate.edu/cgi-bin/g s.cgi, respectively. CONTACT vbrendel@iastate.edu

Evolutionary Comparisons of RecA-Like Proteins Across All Major Kingdoms of Living Organisms

Abstract. Protein sequences with similarities to Escherichia coli RecA were compared across the major kingdoms of eubacteria, archaebacteria, and eukaryotes. The archaeal sequences branch monophyletically and are most closely related to the eukaryotic paralogous Rad51 and Dmc1 groups. A multiple alignment of the sequences suggests a modular structure of RecA-like proteins consisting of distinct segments, some of which are conserved only within subgroups of sequences. The eukaryotic and archaeal sequences share an N-terminal domain which may play a role in interactions with other factors and nucleic acids. Several positions in the alignment blocks are highly conserved within the eubacteria as one group and within the eukaryotes and archaebacteria as a second group, but compared between the groups these positions display nonconservative amino acid substitutions. Conservation within the RecA-like core domain identifies possible key residues involved in ATP-induced conformational changes. We propose that RecA-like proteins derive evolutionarily from an assortment of independent domains and that the functional homologs of RecA in noneubacteria comprise an array of RecA-like proteins acting in series or cooperatively.

Two New Complete Genome Sequences Offer Insight into Host and Tissue Specificity of Plant Pathogenic Xanthomonas spp.

Xanthomonas is a large genus of bacteria that collectively cause disease on more than 300 plant species. The broad host range of the genus contrasts with stringent host and tissue specificity for individual species and pathovars. Whole-genome sequences of Xanthomonas campestris pv. raphani strain 756C and X. oryzae pv. oryzicola strain BLS256, pathogens that infect the mesophyll tissue of the leading models for plant biology, Arabidopsis thaliana and rice, respectively, were determined and provided insight into the genetic determinants of host and tissue specificity. Comparisons were made with genomes of closely related strains that infect the vascular tissue of the same hosts and across a larger collection of complete Xanthomonas genomes. The results suggest a model in which complex sets of adaptations at the level of gene content account for host specificity and subtler adaptations at the level of amino acid or noncoding regulatory nucleotide sequence determine tissue specificity.

The ASRG database: identification and survey of Arabidopsis thaliana genes involved in pre-mRNA splicing

A total of 74 small nuclear RNA (snRNA) genes and 395 genes encoding splicing-related proteins were identified in the Arabidopsis genome by sequence comparison and motif searches, including the previously elusive U4atac snRNA gene. Most of the genes have not been studied experimentally. Classification of these genes and detailed information on gene structure, alternative splicing, gene duplications and phylogenetic relationships are made accessible as a comprehensive database of Arabidopsis Splicing Related Genes (ASRG) on our website.

The Maize Genome Contains a Helitron Insertion

The maize mutation sh2-7527 was isolated in a conventional maize breeding program in the 1970s. Although the mutant contains foreign sequences within the gene, the mutation is not attributable to an interchromosomal exchange or to a chromosomal inversion. Hence, the mutation was caused by an insertion. Sequences at the two Sh2 borders have not been scrambled or mutated, suggesting that the insertion is not caused by a catastrophic reshuffling of the maize genome. The insertion is large, at least 12 kb, and is highly repetitive in maize. As judged by hybridization, sorghum contains only one or a few copies of the element, whereas no hybridization was seen to the Arabidopsis genome. The insertion acts from a distance to alter the splicing of the sh2 pre-mRNA. Three distinct intron-bearing maize genes were found in the insertion. Of most significance, the insertion bears striking similarity to the recently described DNA helicase–bearing transposable elements termed Helitrons. Like Helitrons, the inserted sequence of sh2-7527 is large, lacks terminal repeats, does not duplicate host sequences, and was inserted between a host dinucleotide AT. Like Helitrons, the maize element contains 5′ TC and 3′ CTRR termini as well as two short palindromic sequences near the 3′ terminus that potentially can form a 20-bp hairpin. Although the maize element lacks sequence information for a DNA helicase, it does contain four exons with similarity to a plant DEAD box RNA helicase. A second Helitron insertion was found in the maize genomic database. These data strongly suggest an active Helitron in the present-day maize genome.

Comparison of RNA expression profiles based on maize expressed sequence tag frequency analysis and micro-array hybridization

Assembly of 73,000 expressed sequence tags (ESTs) representing multiple organs and developmental stages of maize (Zea mays) identified approximately 22,000 tentative unique genes (TUGs) at the criterion of 95% identity. Based on sequence similarity, overlap between any two of nine libraries with more than 3,000 ESTs ranged from 4% to 20% of the constituent TUGs. The most abundant ESTs were recovered from only one or a minority of the libraries, and only 26 EST contigs had members from all nine EST sets (presumably representing ubiquitously expressed genes). For several examples, ESTs for different members of gene families were detected in distinct organs. To study this further, two types of micro-array slides were fabricated, one containing 5,534 ESTs from 10- to 14-d-old endosperm, and the other 4,844 ESTs from immature ear, estimated to represent about 2,800 and 2,500 unique genes, respectively. Each array type was hybridized with fluorescent cDNA targets prepared from endosperm and immature ear poly(A+) RNA. Although the 10- to 14-d-old postpollination endosperm TUGs showed only 12% overlap with immature ear TUGs, endosperm target hybridized with 94% of the ear TUGs, and ear target hybridized with 57% of the endosperm TUGs. Incomplete EST sampling of low-abundance transcripts contributes to an underestimate of shared gene expression profiles. Reassembly of ESTs at the criterion of 90% identity suggests how cross hybridization among gene family members can overestimate the overlap in genes expressed in micro-array hybridization experiments.