Degree Type

Thesis

Date of Award

2017

Degree Name

Master of Science

Department

Biochemistry, Biophysics and Molecular Biology

Major

Biochemistry

First Advisor

Reuben J. Peters

Abstract

When confronted with the prospect of microbial infection, plants are armed with a mighty selection of antibiotic natural products. Rice’s selection of antibiotic natural products primarily consists in its production of diterpenoids. These diterpenoids have been identified from microbial infection, and have been characterized in vitro but remain to be extensively studied in planta. These diterpenoids are primarily synthesized into ent- or syn-copalyl diphosphate (CPP) by CPP synthases (CPS). OsCPS4, which makes syn-CPP, and OsCPS2, which makes ent-CPP, were both knocked out T-DNA insertions and infected with Magnaporthe oryzae and Xanthomonas oryzae. The Os-cps4 knockout is actually more resistant to X. oryzae, while the Os-cps2 knockout is more susceptible to pathogen attack. Further study indicated that Os-cps4 was susceptible to other non-host fungal strains. Though Os-cps2 was more susceptible to pathogen attack, the total amount of ent-derived diterpenoids was nearly equal to that of its wild type, leading to a hypothesis that there are spatial requirements for the production of phytocassanes and oryzalexins.

Using CRISPR/Cas9, knockouts of 8 other genes involved in phytoalexin biosynthesis were constructed and transformed into rice: Os-ksl10, Os-ksl7, Os-ksl4, Os-cyp76m7, Os-cyp76m7&m8, Os-cyp701a8, Os-cyp99a2, and Os-ms1. These lines will be used for future pathogenic experiments, and they provide new insight into the biosynthesis pathways even now.

Momilactone production was adversely affected in Os-cps4, Os-ksl4, Os-cyp76m7&m8, Os-cyp701a8, Os-cyp99a2, and Os-ms1. Oryzalexins were not reliably detectable in the wild typesamples analyzed for this thesis, though an alternative induction method has been developed for future experiments. An upregulation of the detectable oryzalexins (A, C, D, & E) Os-cyp99a2 gave way to hypothesize its activity in the production of oryzalexin F. Phytocassane production was adversely affected in Os-ksl7, Os-cyp76m7&m8, Os-cyp701a8, and Os-ms1. Thus, as exemplified here with rice diterpenoids, examination of metabolic allocation provides important clues into physiological function.

DOI

https://doi.org/10.31274/etd-180810-5952

Copyright Owner

Benjamin C Brown

Language

en

File Format

application/pdf

File Size

104 pages

Included in

Biochemistry Commons

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