Genomics of the Theobroma cacao L. Defense Response
Open Access
- Author:
- Fister, Andrew Sean
- Graduate Program:
- Genetics
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 07, 2016
- Committee Members:
- Mark Guiltinan, Dissertation Advisor/Co-Advisor
Majid R Foolad, Committee Chair/Co-Chair
Siela Maximova, Committee Member
Charles T Anderson, Committee Member
James Harold Marden, Outside Member - Keywords:
- Cacao
Plant
Genetics
Disease
Defense
Transformation
Chocolate
Tree
Pathogenesis-Related - Abstract:
- Theobroma cacao, the source of cocoa and a cash crop of global economic importance, suffers significant annual losses due to several pathogens. While study of the molecular mechanisms of defense in cacao has been limited, the recent sequencing of two cacao genomes has greatly expedited the ability to study genes and gene families with roles in defense. Here, the pathogenesis-related (PR) gene families were bioinformatically identified, and family size and gene organization were compared to other plant species, revealing significant conservation throughout higher monocots and dicots. Expression of the PR families was also analyzed using a whole genome microarray to measure transcriptomic regulation in leaves after treatment of cacao seedlings with two pathogens, identifying the induced PR genes within each family. We found significant overlap between the PR genes induced by the pathogens, and subsequent qRT-PCR revealed up to 5000-fold induction of specific PR family members. Next, the regulation of the defense response in cacao by salicylic acid, a major defense hormone, was analyzed. The study focused on two genotypes, the broadly resistant Scavina 6 and the widely susceptible ICS1. First, treatment of leaves of two cacao genotypes with salicylic acid was shown to enhance resistance of both. Moreover, overexpression of TcNPR1, a master regulator of systemic acquired resistance, is also shown to enhance the defense response, supporting the importance of salicylic acid and its downstream targets in cacao immunity. Microarray analysis of the transcriptomic response to salicylic acid revealed genotype-specific responses to hormone treatment. ICS1 appeared to show a more canonical response to salicylic acid, with more PR genes induced, while Scavina 6 exhibited increased expression of chloroplastic and mitochondrial genes. It was hypothesized that this induction was linked to increased ROS production, and subsequent ROS staining experiments confirmed higher concentration of superoxide in salicylic acid-treated Scavina 6 leaf tissue. Third, a pilot study was performed to quantify genetic variability within defense genes. Using DNA samples representing three populations of cacao – Peruvian, Ecuadorian, and French Guianan – we amplified three genes involved in defense, two predicted to be more variable (cysteine-rich repeat secretory peptide 38 and a polygalacturonase inhibitor) and one predicted to harbor less polymorphism (pathogenesis-related 1). Population genetic analysis of variability suggested that the gene predicted to be more variable may be under diversifying selection, suggesting that they may directly interact with rapidly evolving pathogen proteins. The experiment validated previously described observations about the populations, in particular that the French Guianan population was less variable than the others. The study also supported the predictions regarding gene variability, indicating that our strategy for identifying genes with more variation appears to be applicable but will require further validation. The Guiltinan-Maximova lab developed a protocol for transient transformation of cacao leaf tissue, which has been applied to characterizing gene function in several published analyses. Here the highly efficient protocol is presented in full, along with data collected in a series of optimization experiments. We also use the protocol to demonstrate the effect of overexpression of a cacao chitinase after subsequent infection with Phytophthora mycelia. A preliminary study describing a strategy for selection of high-priority candidate genes for functional characterization is described. Six genes were cloned and overexpressed using the transient transformation protocol; and while the study showed the ability of our protocol to significantly increase transcript abundance of the gene of interest, it did not validate the role of any of the genes in defense by showing decreased susceptibility. This dissertation contributes to the study of genomics and molecular mechanisms of defense in four key ways: 1) 15 classes of defense genes are identified and their expression dynamics are characterized, 2) genotype-specific differences in defense response are identified, providing insight into different strategies for survival, 3) variability within defense genes is discovered, differentiating populations of cacao and providing evidence for diversifying selection, and 4) a rapid and efficient strategy for gene functional analysis, which will enhance future genetic analyses in cacao, is presented.