Search

The digital imaging and vision applications in science (DIVAS) program was built to improve the computational self-efficacy and skill of first- and second-year college students majoring in biological and chemical sciences. Our three-year pilot study showed that the program could be successful in both fronts. The scholars, faculty, and staff who participated formed a community of practice that became the heart of the DIVAS program. Through this community, we expanded access to the image processing workshop in collaboration with The Carpentries, supported faculty and secondary educators in developing computing modules for their classrooms, and created and staffed a “writing center for computing” on the host campus. Overall, the DIVAS program has sparked a local computing culture. DIVAS interventions and resources are freely available for adoption by other institutions. We hope to grow the community in a way that builds student access and opportunities and supports educators in the process.

Two Gordonia bacteriophages, GrootJr and NovumRegina, were discovered, sequenced, and annotated. These phages were isolated from distinct soil and water samples, respectively, on Gordonia terrae 3612. These phages belong to the CR2 subcluster and are similar in genome size and gene content.

Two mycobacteriophage genomes were newly sequenced and annotated. Duggie and Hocus were discovered, enriched, and isolated from soil using Mycobacterium smegmatis mc2155. The bacteriophages are lytic Siphoviridae and belong to the B1 subcluster. The Hocus and Duggie genomes are highly similar to one another in both nucleotide sequence and gene content.

Three mycobacteriophages, Bipolarisk, Bread, and FudgeTart, were isolated from enriched soil samples found in Crete, NE. All three phages are lytic, belong to subcluster C1, and infect Mycobacterium smegmatis mc2155. The structures of the three genomes are similar, with slight variations in gene number and content.

TAL effectors delivered by phytopathogenic Xanthomonas species are DNA-sequence-specific transcriptional activators of host susceptibility genes and sometimes resistance genes. The modularity of DNA recognition by TAL effectors makes them important also as tools for gene targeting and genome editing. Effector binding elements (EBEs) recognized by native TAL effectors in plants have been identified only on the forward strand of target promoters. Here, we demonstrate that TAL effectors can drive plant transcription from EBEs on either strand and in both directions. Furthermore, we show that a native TAL effector from Xanthomonas oryzae pv. oryzicola drives expression of a target with an EBE on each strand of its promoter. By inserting that promoter and derivatives between two reporter genes oriented head to head, we show that the TAL effector drives expression from either EBE in the respective orientations, and that activity at the reverse-strand EBE also potentiates forward transcription driven by activity at the forward-strand EBE. Our results reveal new modes of action for TAL effectors, suggesting the possibility of yet unrecognized targets important in plant disease, expanding the search space for off-targets of custom TAL effectors, and highlighting the potential of TAL effectors for probing fundamental aspects of plant transcription.

Four bacteriophages infecting Mycobacterium smegmatis mc 2 155 (three belonging to subcluster P1 and one belonging to subcluster P2) were isolated from soil and sequenced. All four phages are similar in the left arm of their genomes, but the P2 phage differs in the right arm. All four genomes contain features of temperate phages.

Transcription activator-like effectors (TALEs) are proteins found in the genus Xanthomonas of phytopathogenic bacteria. These proteins enter the nucleus of cells in the host plant and can induce the expression of susceptibility genes (S genes), triggering disease. TALEs bind the promoter region of S genes following a specific code, which allows the prediction of binding sites based on TALEs amino acid sequences. New candidate S genes can then be discovered by finding the intersection between genes induced in the presence of TALEs and genes containing predicted effector binding elements. By contrasting differential expression data and binding site predictions across different datasets, patterns of TALE diversification or convergence may be unveiled, but this requires the seamless integration of different genomic and transcriptomic data. With this in mind, we present daTALbase, a curated relational database that integrates TALE-related data including bacterial TALE sequences, plant promoter sequences, predicted TALE binding sites, transcriptomic data of host plants in response to TALE-harboring bacteria, and other associated data. The database can be explored to uncover new candidate S genes as well as to study variation in TALE repertories and their corresponding targets. The first version of the database here presented includes data for Oryza sp.–Xanthomonas pv. oryzae interactions. Future versions of the database will incorporate information for other pathosystems involving TALEs.

Plant root exudates are important mediators in the interactions that occur between plants and microorganisms in the soil, yet much remains to be learned about spatial and temporal variation in their production. This work outlines a method utilizing a novel colorimetric paper to detect spatial and temporal changes in the production of nitrogen-containing compounds on the root surface. While existing methods have made it possible to conduct detailed analysis of root exudate composition, relatively less is known about where in the root system exudates are produced and how this localization changes as the root grows. Furthermore, there is much to learn about how exudate localization and composition varies in response to stress. Root exudates are chemically diverse secretions composed of organic acids, amino acids, proteins, sugars, and other metabolites. The sensor utilized for the method, ninhydrin, is a colorless substance in solution that reacts with free amino groups to form a purple dye. A detection paper was developed by formulating ninhydrin into a print solution that was uniformly deposited onto paper with a commercial ink jet printer. This “ninhydrin paper” was used to analyze the chemical makeup of root surfaces from maize seedlings grown vertically on germination paper. Through contact between the ninhydrin paper and seedling root surfaces, combined with images of both the seedlings and dried ninhydrin papers captured using a standard flatbed scanner, nitrogen-containing substances on the root surface can be localized and concentration of signal estimated for over two weeks of development. The method was found to be non-inhibiting to plant growth over the analysis period although damage to root hairs was observed. The method is sensitive in the detection of free amines at concentrations as little as 140 µM. Furthermore, ninhydrin paper is stable, showing consistent color changes up to two weeks after printing. The method is expandable to the detection of other relevant compounds produced during root development. This relatively simple, low-cost method could contribute to a better understanding of root exudates and mechanisms used by plants to interact with the complex soil environment during growth and development.

Engineered nucleases that cleave specific DNA sequences in vivo are valuable reagents for targeted mutagenesis. Here we report a new class of sequence-specific nucleases created by fusing transcription activator-like effectors (TALEs) to the catalytic domain of the FokI endonuclease. Both native and custom TALE-nuclease fusions direct DNA double-strand breaks to specific, targeted sites.

Transcription activator-like (TAL) effectors are transcription factors injected into plant cells by pathogenic bacteria of the genus Xanthomonas. They function as virulence factors by activating host genes important for disease, or as avirulence factors by turning on genes that provide resistance. DNA-binding specificity is encoded by polymorphic repeats in each protein that correspond one-to-one with different nucleotides. This code has facilitated target identification and opened new avenues for engineering disease resistance. It has also enabled TAL effector customization for targeted gene control, genome editing, and other applications. This article reviews the structural basis for TAL effector-DNA specificity, the impact of the TAL effector-DNA code on plant pathology and engineered resistance, and recent accomplishments and future challenges in TAL effector-based DNA targeting.

Xanthomonas transcription activator-like (TAL) effectors promote disease in plants by binding to and activating host susceptibility genes. Plants counter with TAL effector-activated executor resistance genes, which cause host cell death and block disease progression. We asked whether the functional specificity of an executor gene could be broadened by adding different TAL effector binding elements (EBEs) to it. We added six EBEs to the rice Xa27 gene, which confers resistance to strains of the bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) that deliver the TAL effector AvrXa27. The EBEs correspond to three other effectors from Xoo strain PXO99(A) and three from strain BLS256 of the bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc). Stable integration into rice produced healthy lines exhibiting gene activation by each TAL effector, and resistance to PXO99(A) , a PXO99(A) derivative lacking AvrXa27, and BLS256, as well as two other Xoo and 10 Xoc strains virulent toward wildtype Xa27 plants. Transcripts initiated primarily at a common site. Sequences in the EBEs were found to occur nonrandomly in rice promoters, suggesting an overlap with endogenous regulatory sequences. Thus, executor gene specificity can be broadened by adding EBEs, but caution is warranted because of the possible coincident introduction of endogenous regulatory elements.

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.

Most of the scientific journals require published microarray experiments to meet Minimum Information About a Microarray Experiment (MIAME) standards. This ensures that other researchers have the necessary information to interpret the results or reproduce them. Required MIAME information includes raw experimental data, processed data, and data processing procedures. However, the normalization method is often reported inaccurately or not at all. It may be that the scaling factor is not even known except to experienced users of the normalization software. We propose that using a seeded clustering algorithm, researchers can identify or verify previously unknown or doubtful normalization information. For that, we generate descriptive statistics (mean, variance, quantiles, and moments) for normalized expression data from gene chip experiments available in the ArrayExpress database and cluster chips based on these statistics. To verify that clustering grouped chips by normalization method, we normalize raw data for chips chosen from experiments in ArrayExpress using multiple methods. We then generate the same descriptive statistics for the normalized data and cluster the chips using these statistics. We use this dataset of known pedigree as seeding data to identify normalization methods used in unknown or doubtful situations.

TAL effectors are re-targetable transcription factors used for tailored gene regulation and, as TAL effector-nuclease fusions (TALENs), for genome engineering. Their hallmark feature is a customizable central string of polymorphic amino acid repeats that interact one-to-one with individual DNA bases to specify the target. Sequences targeted by TAL effector repeats in nature are nearly all directly preceded by a thymine (T) that is required for maximal activity, and target sites for custom TAL effector constructs have typically been selected with this constraint. Multiple crystal structures suggest that this requirement for T at base 0 is encoded by a tryptophan residue (W232) in a cryptic repeat N-terminal to the central repeats that exhibits energetically favorable van der Waals contacts with the T. We generated variants based on TAL effector PthXo1 with all single amino acid substitutions for W232. In a transcriptional activation assay, many substitutions altered or relaxed the specificity for T and a few were as active as wild type. Some showed higher activity. However, when replicated in a different TAL effector, the effects of the substitutions differed. Further, the effects differed when tested in the context of a TALEN in a DNA cleavage assay, and in a TAL effector-DNA binding assay. Substitution of the N-terminal region of the PthXo1 construct with that of one of the TAL effector-like proteins of Ralstonia solanacearum, which have arginine in place of the tryptophan, resulted in specificity for guanine as the 5' base but low activity, and several substitutions for the arginine, including tryptophan, destroyed activity altogether. Thus, the effects on specificity and activity generated by substitutions at the W232 (or equivalent) position are complex and context dependent. Generating TAL effector scaffolds with high activity that robustly accommodate sites without a T at position 0 may require larger scale re-engineering.