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Restricted Access Thesis

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First Advisor

Bruce D. Kohorn


Wall associated kinases are receptor proteins found in the plasma membrane of angiosperms that bind to both native cell wall pectin and pectin fragments (oligogalacturonides) to control cell growth and stress response pathways. WAKs have five protein isoforms that are encoded on a 30kb locus on chromosome 1 of Arabidopsis thaliana, with WAK1 and WAK2 being the most ubiquitously expressed isoforms. Mutations in individual WAKs have only weak phenotypes but the generation of WAK double or multiple mutations has been hampered by the tight linkage of the WAK genes. Using CRISPR-Cas9, a null point mutation in the kinase domain coding region of WAK1 was created in an Arabidopsis line containing a WAK2 null mutation, thereby generating a WAK1/WAK2 double mutant line. Cas9 was expressed using either the 35S or pYAO promoters. When the 35S promoter was used to drive Cas9 activity only somatic WAK1 mutations in the wak2(-/-) line were produced, and these had no phenotypic impact upon the growth and development of this transgenic plant line. WAK2 null plants transformed with the pYAO promoted Cas9 showed an increased level of somatic mutant activity in the first generation of transformed plants. The progeny of plants transformed with the pYAO promoted Cas9 had high levels of mutation in WAK1 and displayed high rates of seedling lethality and retarded development, suggesting that WAK1 and WAK2 are essential for plant development and that plants lacking WAK1 and WAK2 are unable to survive.


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