Doctoral Dissertations (FSCI)

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Browsing Doctoral Dissertations (FSCI) by Author "Bonetta, Dario"

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    Forward and reverse genetic approaches to investigate cellulose biosynthesis in Physcomitrium patens
    (2021-04-01) Behnami, Sara; Bonetta, Dario
    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.
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    Forward screening for herbicide resistance in Arabidopsis thaliana and the monocot species Triticum aestivum
    (2019-06-01) Reavell-Roy, Elysabeth; Bonetta, Dario
    Cellulose is the primary structural component of plant cell walls and is composed of β-1,4-glucan chains. The cellulose synthesis complex is composed of multiple cellulose synthases (CESAs), which work together to extend the cellulose molecule. Although extensively studied, cellulose biosynthesis is not yet fully understood. One way to elucidate the role of CESAs is through their interactions with cellulose biosynthesis inhibitors (CBIs). CBI resistant Arabidopsis thaliana mutants were identified from previous forward genetic screens. Some identified CBI resistant mutants demonstrated altered cellulose crystallinity, and cell wall saccharification. In a larger crop species, these characteristics would allow for plant cell wall material to be efficiently converted into cellulosic ethanol. Therefore, a forward genetic screen has been carried out in Triticum aestivum, bread wheat. Three CBI resistant mutants have been identified in wheat, and were found to contain point mutations in the wheat ortholog for AtCESA3, TaCESA1. These CBI resistant mutants demonstrated altered cell wall saccharification, and cellulose crystallinity. The identification of CBI resistant mutants in wheat demonstrates that traits identified in A. thaliana can be reproduced in a monocot crop species. The second focus of this thesis was to develop new herbicide resistant lines in A. thaliana. Indaziflam is a potent herbicide used to control annual grasses and broadleaf weeds. To better understand the plant pathways affected by indaziflam, a forward genetic screen for indaziflam resistance was completed in A. thaliana. Approximately 750, 000 A. thaliana seeds were screened for indaziflam resistance, and only one allele, indaziflam resistant 1 (izr1), was identified to cause weak indaziflam resistance. Positional cloning coupled with next-generation sequencing revealed that a point mutation in the CULLIN1 (CUL1) encoding gene caused the decrease in indaziflam sensitivity. CUL1 is an E3 ligase involved in a variety of plant signalling pathways, including auxin response. Along with decreased sensitivity to indaziflam, izr1 seedlings demonstrated reduced sensitivity to auxin mimicking herbicides. Comparing izr1 to a known auxin resistant CUL1 mutant, cul1-6, and the known CBI resistant CESA mutant, ixr1-1, revealed that alterations to the CUL1 protein or CESA3 can affect indaziflam sensitivity.