Date

2018

Department or Program

Biological Sciences

Primary Wellesley Thesis Advisor

Kaye Peterman

Abstract

Polarized cell expansion has been observed in pollen tubes, root hairs, neurons, yeast, moss rhizoids and protonemata. Polar growth is highly regulated by complex signaling systems, where the key molecular players are conserved from system to system, but have yet to be fully elucidated. Phosphoinositides have been shown to play a key role in polar growth regulation involved in membrane trafficking and the regulation of the actin cytoskeleton among other things. Current research suggests that the regulatory phospholipid, phosphatidylinositol-4-phosphate (PtdIns4P), is involved in a number of these cellular processes. PtdIns4P is synthesized by Phosphatidylinositol-4-Kinase, which may provide a point of genetic regulation for the purpose of identifying the role of PtdIns4P in polar growth. The Clustered Regularly Interspaced Palindromic Repeats (CRISPR) system was used to generate loss of function PI4KIIIβ1 and β2 mutants for testing our hypothesis that PI4Kb1 and b2 function in the production of PtdIns4P, which plays a critical role in polarized cell expansion in Physcomitrella. Vectors for expression of sgRNAs that target the PI4KIIIβ1 and β2 genes were constructed using the Gateway® cloning system. P. patens protoplasts were transformed with the PI4KIII b1 and b2 sgRNA expression vectors, along with pCas9 and pBPNRF. CRISPR target sites of the resulting transformed plants were sequenced to identify pi4kIIIb1 and b2 null mutants. Site-specific PCR and sequencing validated that no off-target sites were mutated. P. patens pi4kIIIb1b2 mutants colonies, which developed initially by tip growth, were grown and characterized for a reduction in colony size. Gametophytes were isolated from the colonies and the rhizoids, which also grow by polarized cell expansion, and were observed for changes in rhizoid length. There was no significant difference in colony size of the pi4kIIIb1, pi4kIIIb2 or pi4kIIIb1 pi4kIIIb2 mutants when compared to wild-type. In addition there were no observable differences in rhizoid length for either of the mutants. These results suggest that PI4KIIIb1 and b2 do not function in polar growth in Physcomitrella patens.

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