Forward and reverse genetic approaches to investigate cellulose biosynthesis in Physcomitrium patens

Abstract

Cellulose biosynthesis is a common feature of land plants and involves multimeric complexes composed of cellulose synthase A (CESA) proteins and other structural proteins. The exact stoichiometry of CESA proteins and interactions among proteins within cellulose synthase complex (CSC) is not well understood. Therefore, cellulose biosynthesis inhibitors (CBIs) are useful tools in decoding fundamental aspects of cellulose biosynthesis. Here, I characterize the CBI indaziflam, with a unique mode of action for resistance management, which prevents plant growth by inhibiting cellulose biosynthesis. Arabidopsis thaliana indaziflam resistant mutants were identified through forward genetic screening. Since indaziflam is also active in moss Physcomitrium patens, a forward genetic approach to screen indaziflam resistance was applied on P. patens, and positional cloning combined with next-generation sequencing revealed two point mutations in CULLIN1 (CUL1) and AUXIN/INDOLE-3-ACETIC ACID INDUCED (Aux/IAA) which both are involved in auxin signaling pathways. The mutants were also cross-resistant to synthetic auxin 2,4-D. It is predicted that indaziflam affects plant growth and development and impacted the production and remodeling of plant cell wall directly or indirectly. Moreover, to gain insight into the nature of the protein composition of CSCs, I employed a strategy called Biotin identification (BioID), aimed at identifying proximate and vicinal proteins in vivo associated with CESAs in P. patens. I generated multiple BioID-CESA translational fusions by homologous recombination to identify biotinylated proximate proteins. Due to limitations, including but not limited to different behaviors of fused proteins tagged at the C- or N-terminus, decreased expression level, longer incubation time with biotin, higher incubation temperature, and large size BirA* tag, I was not able to identify any interacting proteins. Another finding of this thesis is that P. patens can be used to produce known natural products that are difficult to obtain by chemical synthesis. An in vivo combinatorial biosynthesis approach was pursued in P. patens to obtain rare cannabinoids with beneficial biological activity. The outcome was to produce rare cannabinoids and some pathway intermediates. This idea's other significant result is designing different drug-candidate-producing moss strains, especially within the chemical class of cannabinoids.