Innovations in cloning genes identified in genetic screens and their application to cytokinin signaling in Arabidopsis thaliana

The plant hormone cytokinin is responsible for regulation of a diverse set of biological and developmental phenomena in plants such as leaf senescence, organ formation, nutrient distribution, abiotic stress response, and others. The core components of the cytokinin signaling pathway have been identified and characterized in Arabidopsis thaliana and other plant species, but questions remain about whether biological factors exist that may participate in or regulate the pathway. In this work, I present the results of a sensitized genetic screen for enhancers of ahp (eah) mutants which are hyposensitive to exogenously supplied cytokinin signals. The eah screen identified several new alleles of ahk4, one of the cytokinin receptors in Arabidopsis, as well as implicating the light-signaling transcription factor HY5. As part of the screen, the “mutagenomics” strategy for analyzing uncharacterized mutants in a screen was developed. Mutagenomics involves high-throughput resequencing of a small number of mutant plants with the goal of detecting protein-altering single nucleotide polymorphisms (SNPs). The SNPs detected across the experiment are then assessed to determine if any genes were mutated more frequently than would be expected by chance alone. A second aspect of mutagenomics involves resequencing plant lines of common descent, referred to as siblings, to determine which mutations are homozygous in all related plants. The causative mutation should be contained in the list of shared homozygous mutations. The mutagenomics strategies have the potential to accelerate the pace of genetic screens. I also present a primer design tool, indCAPS, developed to aid the search for novel CRISPR/Cas9-derived mutant alleles. The indCAPS platform is intended to be used for Cleaved Amplified Polymorphic Sequence (CAPS) or derived CAPS (dCAPS) restriction-digest genotyping assays. Tools exist for designing dCAPS primers but they assume the researcher is genotyping a SNP allele. CRISPR/Cas9-derived alleles are typically insertion/deletion (indel) events that result in a frameshift of a protein, and the previously developed primer design tools often fail when given sequences with indels. The indCAPS platform was developed with the ability to distinguish indel alleles as well as SNP alleles.Doctor of Philosoph

indCAPS: A tool for designing screening primers for CRISPR/Cas9 mutagenesis events

Genetic manipulation of organisms using CRISPR/Cas9 technology generally produces small insertions/deletions (indels) that can be difficult to detect. Here, we describe a technique to easily and rapidly identify such indels. Sequence-identified mutations that alter a restriction enzyme recognition site can be readily distinguished from wild-type alleles using a cleaved amplified polymorphic sequence (CAPS) technique. If a restriction site is created or altered by the mutation such that only one allele contains the restriction site, a polymerase chain reaction (PCR) followed by a restriction digest can be used to distinguish the two alleles. However, in the case of most CRISPR-induced alleles, no such restriction sites are present in the target sequences. In this case, a derived CAPS (dCAPS) approach can be used in which mismatches are purposefully introduced in the oligonucleotide primers to create a restriction site in one, but not both, of the amplified templates. Web-based tools exist to aid dCAPS primer design, but when supplied sequences that include indels, the current tools often fail to suggest appropriate primers. Here, we report the development of a Python-based, species-agnostic web tool, called indCAPS, suitable for the design of PCR primers used in dCAPS assays that is compatible with indels. This tool should have wide utility for screening editing events following CRISPR/Cas9 mutagenesis as well as for identifying specific editing events in a pool of CRISPR-mediated mutagenesis events. This tool was field-tested in a CRISPR mutagenesis experiment targeting a cytokinin receptor (AHK3) in Arabidopsis thaliana. The tool suggested primers that successfully distinguished between wild-type and edited alleles of a target locus and facilitated the isolation of two novel ahk3 null alleles. Users can access indCAPS and design PCR primers to employ dCAPS to identify CRISPR/Cas9 alleles at http://indcaps.kieber.cloudapps.unc.edu/

A Regulatory Network for Coordinated Flower Maturation

For self-pollinating plants to reproduce, male and female organ development must be coordinated as flowers mature. The Arabidopsis transcription factors AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8 regulate this complex process by promoting petal expansion, stamen filament elongation, anther dehiscence, and gynoecium maturation, thereby ensuring that pollen released from the anthers is deposited on the stigma of a receptive gynoecium. ARF6 and ARF8 induce jasmonate production, which in turn triggers expression of MYB21 and MYB24, encoding R2R3 MYB transcription factors that promote petal and stamen growth. To understand the dynamics of this flower maturation regulatory network, we have characterized morphological, chemical, and global gene expression phenotypes of arf, myb, and jasmonate pathway mutant flowers. We found that MYB21 and MYB24 promoted not only petal and stamen development but also gynoecium growth. As well as regulating reproductive competence, both the ARF and MYB factors promoted nectary development or function and volatile sesquiterpene production, which may attract insect pollinators and/or repel pathogens. Mutants lacking jasmonate synthesis or response had decreased MYB21 expression and stamen and petal growth at the stage when flowers normally open, but had increased MYB21 expression in petals of older flowers, resulting in renewed and persistent petal expansion at later stages. Both auxin response and jasmonate synthesis promoted positive feedbacks that may ensure rapid petal and stamen growth as flowers open. MYB21 also fed back negatively on expression of jasmonate biosynthesis pathway genes to decrease flower jasmonate level, which correlated with termination of growth after flowers have opened. These dynamic feedbacks may promote timely, coordinated, and transient growth of flower organs

A Regulatory Network for Coordinated Flower Maturation

For self-pollinating plants to reproduce, male and female organ development must be coordinated as flowers mature. The Arabidopsis transcription factors AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8 regulate this complex process by promoting petal expansion, stamen filament elongation, anther dehiscence, and gynoecium maturation, thereby ensuring that pollen released from the anthers is deposited on the stigma of a receptive gynoecium. ARF6 and ARF8 induce jasmonate production, which in turn triggers expression of MYB21 and MYB24, encoding R2R3 MYB transcription factors that promote petal and stamen growth. To understand the dynamics of this flower maturation regulatory network, we have characterized morphological, chemical, and global gene expression phenotypes of arf, myb, and jasmonate pathway mutant flowers. We found that MYB21 and MYB24 promoted not only petal and stamen development but also gynoecium growth. As well as regulating reproductive competence, both the ARF and MYB factors promoted nectary development or function and volatile sesquiterpene production, which may attract insect pollinators and/or repel pathogens. Mutants lacking jasmonate synthesis or response had decreased MYB21 expression and stamen and petal growth at the stage when flowers normally open, but had increased MYB21 expression in petals of older flowers, resulting in renewed and persistent petal expansion at later stages. Both auxin response and jasmonate synthesis promoted positive feedbacks that may ensure rapid petal and stamen growth as flowers open. MYB21 also fed back negatively on expression of jasmonate biosynthesis pathway genes to decrease flower jasmonate level, which correlated with termination of growth after flowers have opened. These dynamic feedbacks may promote timely, coordinated, and transient growth of flower organs

Evaluation of a Sample Concentration Procedure in the Analysis of Dermal Exposure to 1,6-Hexamethylene Diisocyanate and its Oligomers

Dermal exposure to 1,6-hexamethylene diisocyanate (HDI) and its oligomers HDI biuret and HDI isocyanurate has been implicated in occupation asthma in automotive spray-painters. Dermal exposure assessment for these compounds has been hampered due to low sample concentrations for these compounds. A sample concentration process, where samples are dried under a stream of nitrogen gas and resuspended in a smaller volume of liquid, was developed and tested in order to improve sensitivity and specificity for sample quantification and exposure assessment. The method was evaluated by comparing tape-strips samples spiked with derivatized standard or clearcoat solutions before and after concentration. In addition, the method was evaluated using 412 dermal tape-strip samples collected from automotive refinishing workers occupationally exposed to diisocyanates. Concentrations of the urea derivatives of HDI, biuret, and isocyanurate were determined using LC-MS analysis via selective ion monitoring. The results showed that both the dry-down process and skin exposure produced significant variation in the spiked sample concentrations. In the worker samples, the dry-down process increased the number of samples with detectable levels of isocyanurate from 125 to 302 while the number of samples for biuret increased from 8 to 70. Interference from unknown compounds impaired analysis of HDI after drying. In summary, the dry-down procedure has a potential to increase the sensitivity and specificity of isocyanurate in the tape-strip samples collected from the exposed workers. However, the potential biases introduced in the sample processing and the increased analytical cost indicate that this method is not suitable in its current state for adaptation to the processing of samples collected from exposed workers.Bachelor of Science in Public Healt

indCAPS: A tool for designing screening primers for CRISPR/Cas9 mutagenesis events.

Genetic manipulation of organisms using CRISPR/Cas9 technology generally produces small insertions/deletions (indels) that can be difficult to detect. Here, we describe a technique to easily and rapidly identify such indels. Sequence-identified mutations that alter a restriction enzyme recognition site can be readily distinguished from wild-type alleles using a cleaved amplified polymorphic sequence (CAPS) technique. If a restriction site is created or altered by the mutation such that only one allele contains the restriction site, a polymerase chain reaction (PCR) followed by a restriction digest can be used to distinguish the two alleles. However, in the case of most CRISPR-induced alleles, no such restriction sites are present in the target sequences. In this case, a derived CAPS (dCAPS) approach can be used in which mismatches are purposefully introduced in the oligonucleotide primers to create a restriction site in one, but not both, of the amplified templates. Web-based tools exist to aid dCAPS primer design, but when supplied sequences that include indels, the current tools often fail to suggest appropriate primers. Here, we report the development of a Python-based, species-agnostic web tool, called indCAPS, suitable for the design of PCR primers used in dCAPS assays that is compatible with indels. This tool should have wide utility for screening editing events following CRISPR/Cas9 mutagenesis as well as for identifying specific editing events in a pool of CRISPR-mediated mutagenesis events. This tool was field-tested in a CRISPR mutagenesis experiment targeting a cytokinin receptor (AHK3) in Arabidopsis thaliana. The tool suggested primers that successfully distinguished between wild-type and edited alleles of a target locus and facilitated the isolation of two novel ahk3 null alleles. Users can access indCAPS and design PCR primers to employ dCAPS to identify CRISPR/Cas9 alleles at http://indcaps.kieber.cloudapps.unc.edu/

Number of productive primers generated for tested loci.

<p>Simulated amplicons were made using generated primers. Non-productive primers did not amplify sequences capable of being distinguished with a restriction digest. Problematic primers amplify sequences capable of being distinguished, but the reaction would likely not amplify DNA due to primer defects such as 3’ mismatches or gaps in alignment to provided sequence. Productive primers are expected to successfully amplify DNA capable of being distinguished by a restriction digestion.</p

CAPS/dCAPS markers can distinguish alleles, but output of dCAPS Finder 2.0 can be flawed.

<p>(A) Diagram of CAPS technique. An amplicon centered on a restriction site (blue bar) disrupted by a SNP or indel (red bar) is differentially cleaved by a restriction enzyme (RE) in the wild-type vs mutant. (B) Diagram of the dCAPS technique. A restriction site can be introduced into either the wild-type or mutant target sequences using mismatched oligonucleotide primers to discriminate two sequences. The mutation (green bar) disrupts the introduced restriction site such that it is not cleaved by the restriction enzyme (RE). Gel electrophoresis can be used to identify the size difference between the wild-type and mutant fragments in both the CAPS and dCAPS methods. (C-F) A sequence with a two base pair deletion at a CRISPR cut site, chosen using CRISPR-Plant [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188406#pone.0188406.ref012" target="_blank">12</a>], was supplied to dCAPS Finder 2.0 with a mismatch allowance of 1 base pair. A minority of proposed assays are viable (C), but others possess too many mismatches for successful amplification by PCR or do not introduce diagnostic restriction sites (D-F).</p

Homozygous editing events in <i>AHK3</i> were identified.

<p>(A) The indCAPS package was used to generate a primer recognizing a <i>Bsa</i> BI site spanning the CRISPR cut site (between the green bases). A single mismatch was required in the primer (indicated in red). The genomic locus for <i>AHK3</i> is shown. Boxes indicate exons, red bars—transmembrane domains, black region—CHASE domain, grey region—histidine kinase domain, yellow region—receiver domain, blue region– 3’ UTR. Locations of T-DNA insertion sites (<i>ahk3-1</i>, <i>ahk3-3</i>, and <i>ahk3-7</i>) and targeted editing site are indicated. (B,C) The assay was used to screen <i>A</i>. <i>thaliana</i> plants stably transformed with a pCUT binary vector system [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188406#pone.0188406.ref008" target="_blank">8</a>] and sgRNA constructs targeting <i>AHK3</i>. (B) Wild-type controls at edit location. (C) T2 plants from two representative independent transformation events are shown. The uncut amplicon is 90 bp and the wild-type allele is cleaved to produce 36 bp and 54 bp fragments. (D) Progeny from two T2 plants heterozygous for editing events were selected and analyzed for editing. (E) <i>A</i>. <i>thaliana</i> seedlings imaged at 2.5 weeks of growth. Shown are Col-0; <i>ahk3-3</i>; <i>ahk2</i>,<i>4</i>; <i>ahk2-5</i> <i>ahk3-7</i> <i>cre1-12</i>; <i>ahk2-1/+</i> <i>ahk3-1</i> <i>ahk4-1</i>; <i>ahk2-7</i> <i>ahk3-3/+</i> <i>cre1-12</i>; <i>ahk2-7</i> <i>ahk3-9</i> <i>cre1-12</i>; <i>ahk2-7</i> <i>ahk3-10</i> <i>cre1-12</i>. All plants at same scale.</p

Three Auxin Response Factors Promote Hypocotyl Elongation

The hormone auxin regulates growth largely by affecting gene expression. By studying Arabidopsis (Arabidopsis thaliana) mutants deficient in AUXIN RESPONSE FACTORS (ARFs), we have identified three ARF proteins that are required for auxin-responsive hypocotyl elongation. Plants deficient in these factors have reduced responses to environmental conditions that increase auxin levels, including far-red-enriched light and high temperature. Despite having decreased auxin responses, the ARF-deficient plants responded to brassinosteroid and gibberellin, indicating that different hormones can act partially independently. Aux/IAA proteins, encoded by IAA genes, interact with ARF proteins to repress auxin response. Silencing expression of multiple IAA genes increased hypocotyl elongation, suggesting that Aux/IAA proteins modulate ARF activity in hypocotyls in a potential negative feedback loop