FUNCTIONAL ANALYSIS OF NON EXPRESSOR OF PR1 (NPR1) AND ITS PARALOG NPR3 IN THEOBROMA CACAO AND ARABIDOPSIS THALIANA

Open Access
- Author:
- Shi, Zi
- Graduate Program:
- Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 01, 2010
- Committee Members:
- Mark Guiltinan, Dissertation Advisor/Co-Advisor
Mark Guiltinan, Committee Chair/Co-Chair
Siela Maximova, Committee Member
Sarah Mary Assmann, Committee Member
Hong Ma, Committee Member
Yinong Yang, Committee Member - Keywords:
- Cacao
Arabidopsis
plant defense
NPR3 - Abstract:
- Arabidopsis NON EXPRESSOR OF PR1 (NPR1) is a key transcription regulator of the salicylic acid (SA) mediated defense signaling pathway. The NPR gene family consists of NPR1 and five other NPR1-like genes in Arabidopsis. This research focuses on the functional analysis of an NPR1 ortholog from Theobroma cacao L. and characterization of one of the NPR1 paralogs, NPR3, in both Arabidopsis and cacao. To identify the function of NPR3 in Arabidopsis, I first examined the gene expression pattern of NPR3 and found it to be strongly expressed in developing flower tissues. Interestingly, an npr3 knockout mutant displayed enhanced resistance to Pseudomonas syringae tomato pv. DC3000 (P.s.t.) infection of immature flowers. Gene expression analysis also revealed increased basal and induced levels of PR1 transcripts in npr3 developing flowers. To investigate the possible mechanism of NPR3-dependent negative regulation of defense response, I tested the physical interactions of NPR3 with both TGA2 and NPR1 in vivo, which suggests that NPR3 represses NPR1-dependent transcription by inhibiting the nuclear localization of NPR1 through direct binding to TGA2 and NPR1. To characterize the NPR1 ortholog from cacao, I isolated TcNPR1 gene from genotype of Scavina6, and demonstrated that it expresses constitutively in all the tested tissues. To functionally analyze this gene, a bacterial growth assay was carried out with npr1-2 transgenic lines overexpressing TcNPR1, and a reduced level of bacterial growth demonstrated that TcNPR1 can partially complement Arabidopsis the npr1-2 mutation. In addition, TcNPR1 was shown to translocate into nuclei upon SA treatment in a manner identical to Arabidopsis native NPR1. To further explore the NPR gene family in cacao, I identified a total of four NPR-like genes from the cacao genome, and phylogenetic analysis indicated that the duplications of three clades in this gene family occurred before the divergence of Arabidopsis and cacao. To identify the functional ortholog of Arabidopsis NPR3, I isolated a putative TcNPR3 gene and demonstrated that its expression level was higher in un-open flowers and older leaves, a pattern similar to Arabidopsis NPR3. A complementation test of TcNPR3 expressed in the Arabidopsis npr3-3 null mutant showed that TcNPR3 can functionally substitute for the Arabidopsis NPR3 gene, demonstrating that TcNPR3 is the functional ortholog of AtNPR3. To obtain the genome-wide transcriptional responses of SA treatment in cacao, I used microarray analysis to measure gene expression in two cacao genotypes (ICS1 and Scavina6), three leaf developmental stages (A, C and E) and two treatments (water and SA). After validating the microarray results with RT-PCR, I identified differentially expressed genes from all twenty-four pair-wise comparisons. Interestingly, chloroplast and mitochondrial genes are enriched in SA-induced Scavina6 but those genes are underrepresented in ICS1, suggesting that the oxidative burst and hypersensitive response during defense response may vary between the two genotypes. In all, this research will not only offer us the knowledge of defense response mechanism and signal transduction regulation in Arabidopsis and cacao, but also provide molecular tools for selecting cultivars with enhanced disease resistance for cacao breeders and farmers.