Date of Graduation

8-2023

Document Type

Thesis

Degree Name

Master of Science in Crop, Soil & Environmental Sciences (MS)

Degree Level

Graduate

Department

Crop, Soil & Environmental Sciences

Advisor/Mentor

Srivastava, Vibha

Committee Member

Rojas, Clemencia M.

Second Committee Member

Shakiba, Ehsan

Third Committee Member

Sakon, Joshua

Keywords

SnRK1; Rice; Knockout mutants; Phenotypic characterization; Transcriptomic analysis; Defense response; Metabolic processes; Rice blast; Sheath blight; Bacterial panicle blight

Abstract

SnRK1 is a heterotrimeric protein kinase that is composed of a catalytic subunit (α) and two regulatory subunits (β and βγ), and it has a main role in regulating energy homeostasis in the plant by modulating anabolic and catabolic process. SnRK1 phosphorylates and alters the activities of enzymes involved in metabolism and regulates gene expression by altering the activity of chromatin-remodeling enzymes or the transcription factors. Rice contains three functional paralogs of SnRK1α: SnRK1αa (LOC_Os03g17980), SnRK1αb (LOC_Os08g37800), and SnRK1αc (LOC_Os05g45420). This study focused on the function of these SnRK1 paralogs by evaluating the phenotypic and transcriptomic characteristics and the disease response of the knockout mutants developed by CRISPR/Cas9 mediated targeted mutagenesis. These knockout lines consisted of the double-mutant snrk1αa+b lines and the single mutant snrk1αc lines. The phenotypic characterization of early-stage seedlings in ½ MS media showed that snrka+b mutants had lower seedling length compared with the WT but snrk1αc mutants were similar to the WT. The snrk1αc mutants showed phenotypic differences in the late developmental stages in the greenhouse, mainly in the yield parameters such as number of seeds per panicle and total weight of seeds per plant. This is in agreement with previous studies that showed that SnRK1αa and SnRK1αb are primarily expressed in the early seedling stages and SnRK1αc is significantly expressed in the later vegetative and reproductive phases. Transcriptomic analysis on 7 days old seedlings showed that the defense response and the secondary metabolic process were upregulated in WT Kitaake seedlings exposed to extended darkness mimicking starvation. In contrast, the dark-exposed snrk1αa+b mutant showed downregulation of these biological processes and upregulation of light-induced processes such as ribosome biogenesis, translation, and DNA replication. However, not many biological processes were found to be significantly up or down regulated in the snrk1αc mutant. Therefore, stress response during early stages of seedling growth is controlled by SnRK1αa and/or SnRK1αb, and SnRK1αc does not play a major role in the seedling growth or stress response during early phases of the development. Next, the study examined the response of snrk1 mutants against three different diseases: rice blast caused by Magnaporthe oryzae, sheath blight caused by Rhizoctonia solani, and bacterial panicle blight caused by Burkholderia glumae. Previous studies have shown that mutation in the SnRK1 gene increases susceptibility to pathogens. However, in our experiments we did not observe an increase of susceptibility in the snrk1αa+b or snrk1αc mutants to bacterial panicle blight and sheath blight diseases. However, regarding blast fungus caused by M. oryzae, we noted that snrk1 mutants were more susceptible, which is in accordance with the literature and our transcriptomic results that shows downregulation of defense response in snrk1 mutants.

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