Date of Award

Spring 2017

Project Type

Dissertation

Program or Major

Genetics

Degree Name

Doctor of Philosophy

First Advisor

Louis S Tisa

Second Advisor

Feixia Chu

Third Advisor

Estelle Hrabak

Abstract

Globally, 20% of total cultivated and 33% of irrigated agricultural lands are affected by high salinity. By 2050, more than 50% of the arable land will be salinized. The hyper-ionic and hyper-osmotic stresses associated with salt-affected soils threaten the ability of cells to maintain optimal turgor pressure and intracellular ionic concentration for growth and functioning. The nitrogen-fixing soil actinobacterium Frankia shows marked variability in its tolerance to salinity. When in a symbiotic association with actinorhizal plants, Frankia enhances the tolerance of the plants to a range of abiotic stresses, including salinity. The Casuarina-Frankia association has been used to reclaim salt affected soils worldwide. Optimizing the use of the Casuarina-Frankia association for saline soil reclamation requires identifying salt-tolerant symbionts, unlocking the molecular mechanism behind the tolerance, and ultimately developing Frankia strains that combine the best symbiotic characteristics with high level of salt tolerance.

In this study, Frankia strains were screened for salt and osmotic stress tolerance under nitrogen-proficient and nitrogen-deficient conditions. Salt-tolerant and salt-sensitive strains were identified and the effect of salt and osmotic stress on the physiology of the strains and on their symbiotic performance was assessed. Tolerant strains were sequenced and comparative genomics, transcriptome profiling, proteomics, and physiological analysis were employed to identify potential mechanisms and candidate genes responsible for the contrasting phenotypes. An expression vector that stably replicates in Frankia was developed and used to constitutively express some of the candidate genes in the salt-sensitive strain.

Salt-tolerant Frankia strains (CcI6 and Allo2) that could withstand up to 1000 mM NaCl and a salt-sensitive Frankia strain (CcI3) which could withstand only up to 475 mM NaCl were identified. Comparative genomic analysis showed that all of the Casuarina isolates belonged to the same species (Frankia casuarinae at p=0.05 level). Pangenome analysis revealed a high abundance of singletons among all Casuarina isolates. The two salt-tolerant strains contained 153 shared unique genes (the majority of which code for hypothetical proteins) that were not found in the salt-sensitive strain. Transcriptome, proteome, and physiological analysis of the salt-tolerant and sensitive strains revealed vast differences in salt stress response with regards to cellular functions such as transcriptional regulation, cell envelope remodeling, osmolyte biosynthesis, and signal transduction. Among the 153 genes shared only between the salt-tolerant strains, seven, including a zinc peptidase, were responsive to salt stress. Constitutive expression of the zinc peptidase gene in the salt-sensitive strain (CcI3) led to increased salt-tolerance.

The comprehensive approach we took to analyze the complex trait of salt stress tolerance led to important findings that shape our understanding of the salt stress response. The tools and the data we generated in this study will serve as a springboard for future work in the area or in the broader field of Frankia genetics in general.

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