Dissertation
Understanding the Molecular Basis of Fusarium solani Mediated Root Rot in Pisum sativum
Washington State University
Doctor of Philosophy (PhD), Washington State University
12/2020
DOI:
https://doi.org/10.7273/000005553
Abstract
Pea (Pisum sativum) is an important cool-season crop, which is gaining renewed prominence due to an increased interest in plant-derived proteins. Its high nutritional value, low production costs, and short life cycle make pea ideal for the plant-derived protein market. However, pea yields are jeopardized by the root rot fungus, Fusarium solani f. sp. pisi (Fsp). Green-seeded cultivars are susceptible to Fsp, while most of the wild, purple-seeded pea genotypes are highly resistant. A time course RNA-sequencing approach was undertaken to identify Fsp-responsive genes in four partially resistant and four susceptible green-seeded pea genotypes. Gene expression analysis resulted in the identification of 42,905 differentially expressed contigs (DECs). Fsp challenge produced a more intense and diverse overexpression of genes in the susceptible genotypes, while the partially resistant genotypes showed fewer changes in the expression of defense-related genes and a faster reset to a basal metabolic state. Genes involved in exocytosis, the anthocyanin synthesis, as well as the DRR230 pathogenesis-related gene were overexpressed in the partially resistant genotypes. Genes involved in endocytosis, sugar transport, salicylic acid synthesis, and cell death were overexpressed in the susceptible genotypes. Analysis of the 42,905 DECs resulted in the identification of 769 predicted Single Nucleotide Polymorphisms (SNPs), which were validated and used to screen two segregating populations and to perform quantitative trait loci (QTL) analyses. A new QTL WB.Fsp-Ps 5.1 explained 14.76% of the resistance to Fsp, while four other QTLs explained 5.26-8.05% of the variance. Lastly, the association between the anthocyanin biosynthesis pathway and Fsp resistance was studied via ectopic overexpression, antisense expression, and CRISPR/Cas9-mediated gene editing of the flavanone 3-hydroxylase gene in a highly resistant purple-seeded line. We report an efficient pea transformation protocol with a mean efficiency of 2.9%. Transgenic events exhibiting a range of variation in petal pigmentation were obtained representing CRISPR/Cas9, overexpression, and silencing constructs. To the best of our knowledge, this is the first demonstration of CRISPR/Cas9 mediated gene editing in pea. The transgenic lines will be used in subsequent pathogen challenge assays to determine if the pea anthocyanin biosynthesis pathway is critical for Fsp resistance.
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Details
- Title
- Understanding the Molecular Basis of Fusarium solani Mediated Root Rot in Pisum sativum
- Creators
- Bruce Andres Williamson Benavides
- Contributors
- Amit Dhingra (Chair)Kiwamu Tanaka (Committee Member) - Washington State University, Plant Pathology, Department ofLee A Hadwiger (Committee Member) - Washington State University, Plant Pathology, Department ofTheodore Kisha (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- Program in Molecular Plant Sciences
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Publisher
- Washington State University
- Number of pages
- 253
- Identifiers
- 99901054231501842
- Language
- English
- Resource Type
- Dissertation