Date

2018

Department or Program

Biological Sciences

Primary Wellesley Thesis Advisor

Kaye Peterman

Additional Advisor(s)

Andrea Sequeira

Additional Advisor

Andrew Webb

Additional Advisor

Patricia Berman

Abstract

Polar growth, or asymmetrical elongation of the cell, is a highly conserved developmental process that occurs in the moss, Physcomitrella patens, during protonemal and rhizoid growth. While several studies have examined the molecular underpinnings of polar growth in vascular plants, those of polar growth in the moss have yet to be fully elucidated. Current research suggests that phosphatidylinositol-4-phosphate (PI4P), a regulatory phospholipid that controls a variety of cellular processes including membrane trafficking, cytoskeleton organization, and intracellular signaling, plays a role in polar growth of plant cells. Knockout of genes that encode phosphatidylinositol-4-kinases (PI4Ks), the enzymes that synthesize PI4P, results in defects in polar growth of root hairs in Arabidopsis thaliana. Studies in nonvascular plants coincide with this finding. In P. patens, overexpression of a GFP-tagged PI4P-binding domain resulted in wavy protonemal cells, the result of an inability to maintain a polar growth axis. Additionally, pi4kα1 knockout resulted in stunted rhizoid growth in the bryophyte, Marchantia. These lines of evidence strongly support the role of the PI4P in plant cell polar growth. Our project sought to further elucidate the role of PI4P in protonemal growth by using the Clustered Regularly Interspaced Palindromic Repeats (CRISPR) method to knock out PI4Kα1 and PI4Kα2. Using the webtool CRISPOR V1, a complementary target sequence for each of the PI4K variants was acquired to target each of the genes of interest. Under control of the U6 promoter, these sgRNA sequences were cloned into a plasmid and used in combination with a positive selection marker and the Cas9 plasmid to transform P. patens protoplasts. Sequencing analysis of plants that regenerated revealed that CRISPR-mediated genomic editing was successful. Of the plants sequenced, 48% were single mutants, while fewer plants were found to be double mutants (20%). Furthermore, frameshift-causing mutations that resulted in truncated PI4K proteins were identified among mutant sequences. pi4kα1 and pi4kα2 putative null mutant lines exhibited different phenotypes. pi4kα2 null mutants closely resembled wild-type moss lines in measures of polar growth and overall development. pi4kα1 null mutant lines too lacked a polar growth defect; however, a defect in gametophore development was observed. The latter finding has not been reported in previous literature and offers an opportunity for further study. Ultimately, these findings will provide valuable insight into how the roles of PI4P and PI4Ks have evolved in different plant lineages.

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