Computational and experimental analysis of TAL effector-DNA binding

dc.contributor.advisor Adam J. Bogdanove
dc.contributor.advisor Daniel Nettleton
dc.contributor.author Doyle, Erin
dc.contributor.department Plant Pathology and Microbiology
dc.date 2018-08-11T16:25:32.000
dc.date.accessioned 2020-06-30T02:47:34Z
dc.date.available 2020-06-30T02:47:34Z
dc.date.copyright Tue Jan 01 00:00:00 UTC 2013
dc.date.embargo 2013-01-01
dc.date.issued 2013-01-01
dc.description.abstract <p>TAL effectors, from the plant-pathogenic bacterial genus Xanthomonas, are DNA binding proteins that can be engineered to bind to almost any sequence of interest. The DNA target of the TAL effector is encoded by a modular central repeat region, with each repeat specifying a single binding site nucleotide. TAL effectors can be targeted to novel DNA sequences by assembling the corresponding repeat sequence. Therefore, custom TAL effectors have become important tools for manipulating gene expression and creating site-specific DNA modifications. This dissertation explores TAL effector-DNA binding through computational and experimental analyses.</p> <p>I identified positional and composition biases in known TAL effector-target pairs and proposed guidelines for designing custom TAL effectors and TAL effector nucleases (TALENs). Using these guidelines, I created a software tool for TAL effector design. We expanded this tool into a suite of tools for TAL effector/TALEN design and target site prediction. Target site predictions can be used to estimate potential off-target binding of custom TAL effector constructs or to identify unknown targets of natural TAL effectors.</p> <p>Next, I present a case study in engineering disease resist rice plants. Inserting multiple TAL effector binding elements (EBEs) into the promoter of a rice resistance gene conferred resistance to diverse strains of Xanthomonas oryzae. Analysis of the EBE sequences revealed that TAL effectors have evolved to target specific host regulatory sequences, and caution is warranted when introducing such sequences into the promoter of an executor resistance gene.</p> <p>Finally, I examine the role of the TAL effector N terminus in DNA binding. Most natural TAL effector binding sites are preceded by a T at the 5' end (T0). Structural data suggests T0 is encoded by tryptophan 232 (W232) in the cryptic -1st repeat. We show that substitutions for W232 alter TAL effector activity and specificity for T0. However, we find that the TAL effector-T0 interaction is complex and may depend on other residues in the -1st repeat, the 0th cryptic repeat, or repeat sequence context. Better understanding of TAL effector-DNA binding will improve TAL effector design and target prediction and enhance understanding of the role of TAL effectors in plant disease.</p>
dc.format.mimetype application/pdf
dc.identifier archive/lib.dr.iastate.edu/etd/13132/
dc.identifier.articleid 4139
dc.identifier.contextkey 4250779
dc.identifier.doi https://doi.org/10.31274/etd-180810-2733
dc.identifier.s3bucket isulib-bepress-aws-west
dc.identifier.submissionpath etd/13132
dc.identifier.uri https://dr.lib.iastate.edu/handle/20.500.12876/27321
dc.language.iso en
dc.source.bitstream archive/lib.dr.iastate.edu/etd/13132/Doyle_iastate_0097E_13457.pdf|||Fri Jan 14 19:45:21 UTC 2022
dc.subject.disciplines Agricultural Science
dc.subject.disciplines Agriculture
dc.subject.disciplines Bioinformatics
dc.subject.disciplines Biology
dc.subject.disciplines Plant Pathology
dc.subject.keywords TAL effectors
dc.subject.keywords TALENs
dc.subject.keywords Xanthomonas
dc.subject.keywords Xanthomonas oryzae
dc.title Computational and experimental analysis of TAL effector-DNA binding
dc.type article
dc.type.genre dissertation
dspace.entity.type Publication
relation.isOrgUnitOfPublication a26b5928-54bb-4a0b-a973-95d649d1ad83
thesis.degree.discipline Bioinformatics and Computational Biology
thesis.degree.level dissertation
thesis.degree.name Doctor of Philosophy
File
Original bundle
Now showing 1 - 1 of 1
Name:
Doyle_iastate_0097E_13457.pdf
Size:
4.9 MB
Format:
Adobe Portable Document Format
Description: