Date on Master's Thesis/Doctoral Dissertation

8-2022

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Biology

Degree Program

Biology, PhD

Committee Chair

Running, Mark P.

Committee Co-Chair (if applicable)

Perlin, Michael H.

Committee Member

Perlin, Michael H.

Committee Member

Schultz, David J.

Committee Member

Palmer, Kenneth E.

Committee Member

Hwangbo, Dae-Sung

Author's Keywords

Physcomitrium patens; Arabidopsis; prenylation; gene knockdown; sterilization; SARS-CoV-2

Abstract

Protein prenylation, a common lipid post-translational modification, is required for growth and development in eukaryotes. Rab geranylgeranylation involves the addition of one or two 20-carbon geranylgeranyl moieties to Rab-GTPase target proteins, which regulate intracellular vesicle trafficking. The reaction is carried out by heterodimeric Rab geranylgeranyltransferase (Rab-GGT), which is composed of two associated α- and β-subunits, with the assistance of an additional protein called Rab escort protein (REP). Loss of function of the Rab-GGT α subunit RGTA1 has not been reported in any plant. While knockout of either of the two β subunits RGTB1 or RGTB2 results in no phenotype in the moss Physcomitrium patens (P. patens, formerly Physcomitrella patens), in the flowering plant Arabidopsis thaliana, knockout of RGTB1 results in loss of apical dominance and photomorphogenic and gravitropic defects, and knockout of RGTB2 results in a subtle growth defect in certain cells. These results showed that both Rab-GGT β subunits in P. patens are redundant, but RGTB2 in Arabidopsis could not fully compensate for the loss of function of RGTB1 and vice versa. Previous studies in our lab showed that complete knockout of any P. patens Rab-GGT components (RGTA1, RGTB1 & RGTB2, REP) appears to be lethal, since no viable single mutant plants of RGTA1 or REP and double mutant plants of RGTB1 and RGTB2 were recovered. Therefore, the biological function of Rab-GGT remains largely unknown. Here we have generated P. patens transgenic plants containing artificial miRNA constructs targeting each Rab-GGT component by an inducible knockdown system, and systematically analyzed the phenotypes upon induction. The results showed that knockdown of either RGTA1 or REP, or knockdown of RGTB1 in a rgtb2 knockout background (or vice versa) resulted in defects in tip growth (polar cell elongation), reduced or incomplete caulonema differentiation, and an altered response towards exogenous phytohormones. This dissertation may help to elucidate the functional role of Rab-GGT in P. patens, as well as provide fundamental insights into key P. patens developmental processes and environmental responses.

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