Winters, Noah P. Evolutionary and functional genetics of disease resistance in theobroma cacao and its wild relatives. A Dissertation in Ecology Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2022
Abstract: Plants have complex and dynamic immune systems that have evolved over millennia to help them resist pathogen invasion. Humans have worked to incorporate these evolved defenses into crops through breeding. However, many crop cultivars only harness a fraction of the overall genetic diversity available to a given species, or have such a long history of domestication that most diversity has been lost. Evaluating previously neglected germplasm for desirable traits, such as disease resistance, is therefore an essential step towards breeding crops that are adapted to both current and emerging threats. In this dissertation, we examine the evolution of defense response across populations of Theobroma cacao L. and wild species of Theobroma, with the goal of identifying genetic elements essential for protection against the cacao pathogen Phytophthora palmivora. Download PDF.
Knollenberg, Benjamin. Characterization of Specialized Metabolites Involved in Black Pod Rot Resistance in Theobroma Cacao. A Dissertation in Plant Biology Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy June 2020. Download PDF.
Abstract: Cacao (Theobroma cacao, chocolate tree) is an important tropical crop that provides the raw ingredients for chocolate production and provides an important source of income to over five million farmers worldwide. Production of cacao is continually limited by diseases, with global annual crop losses estimated between thirty and forty percent. Oomycete (water mold) pathogens in the genus Phytophthora are the most widespread and economically destructive of the cacao pathogens, causing a disease known as black pod rot (BPR). Increasing cacao resistance to BPR is the most promising strategy for reducing yield losses due to the disease but requires an understanding of cacao defense mechanisms of cacao against BPR. Plant specialized metabolites are well known to contribute to plant defense and have been underexploited as breeding targets in cacao. In this work, the metabolite composition of a BPR tolerant and a BPR susceptible genotype were compared by untargeted metabolomics using liquid chromatography – tandem mass spectrometry (LC-MS/MS). This analysis revealed that the tolerant genotype ‘Scavina 6’ (‘Sca6’) accumulates clovamide, a hydroxycinnamic acid amide (HCAA), and several related compounds in leaf tissue at much higher concentrations that the susceptible genotype ‘Imperial College Selection 1’ (‘ICS1’). Both genotypes tested produced equivalent amounts of clovamide in fruit peel, but sulfated HCAAs accumulated to high levels only in ‘Sca6’ in this tissue. These results implicated clovamide and HCAAs as important contributors to the difference in resistance between the two genotypes, so clovamide was synthesized and characterized for its role in defense. Clovamide was found to be a substrate for cacao polyphenol oxidase (PPO), which is involved in reactive quinone formation, protein cross linking, and oxidative browning. Furthermore, clovamide inhibited protease and pectinase in vitro, activities associated with pathogenicity of Phytophthora spp. Clovamide also inhibited growth of three Phytophthora species in vitro. Based on these activities of clovamide and the high relative accumulation of clovamide in ‘Sca6’ I determined that clovamide is an important resistance factor in cacao and a promising breeding target for enhancing resistance. Since clovamide appears important for BPR resistance in cacao, I sought to elucidate cacao genes involved in its biosynthesis by combining metabolomics (LC-MS/MS) and transcriptomics (RNA-Seq) analysis of ‘Sca6’ and ‘ICS1’ leaves of three developmental stages and functional characterization of candidate clovamide biosynthetic genes. By correlating clovamide abundance with that of over 1,000 metabolites identified in extracts by LC-MS/MS, I found a strong negative correlation between clovamide and several flavonoid glucosides. Based on this observation I hypothesized that clovamide biosynthesis directs flux of UDP-glucose away from flavonoid glucoside production and therefore proceeds through a pathway requiring UDP-glucose dependent glycosyltransferase (UGT) and serine carboxypeptidase-like acyltransferase (SCPL-AT) activities. Following this hypothesis, I identified three cacao UGT and three SCPL-AT candidate genes based on expression patterns consistent with HCAA accumulation. All possible UGT/SCPL-AT candidate combinations were co-expressed in N. benthamiana to identify only one combination that resulted in clovamide production: cacao gene IDs (Criollo v2 reference genome) Tc04v2_g000570 (UGT) and Tc02v2_g013480 (SCPL-AT). The UGT candidate, Tc04v2_g000570 was more highly expressed in ‘Sca6’ than ‘ICS1’ (~5.6-fold, p < 0.00001) and was also found to have a premature stop codon in the coding sequence of the ‘ICS1’ allele, resulting in a truncated predicted protein sequence lacking a conserved UGT domain identified by InterProScan. Taken together, these results demonstrate that differential expression and allelic variation in the UGT Tc04v2_g000570 are the likely cause of the difference in leaf clovamide content between ‘Sca6’ and ‘ICS1’. The proposed UGT/SCPLAT pathway for clovamide biosynthesis in cacao is in contrast to the 4-CL/BAHD-AT pathway reported in red clover. BAHD- and SCPL-acyltransferases are evolutionarily distinct protein families, meaning that the two species evolved clovamide biosynthesis completely independently. In order to identify other genes controlling leaf clovamide content that were not detected using the transcriptomics/metabolomics approach, a quantitative trait loci (QTL) analysis for clovamide content was performed in an F2 population derived from ‘Sca6’ and ‘ICS1’. Leaf clovamide content was quantified in 116 F2 trees by HPLC-DAD and clovamide was found to be segregating. Another hydroxycinnamic acid derivative, chlorogenic acid (CGA), was also segregating. Clovamide and CGA content had a slight but significant negative correlation in the F2 population, so CGA was included as a covariate in QTL analyses. Two QTL were identified for clovamide, including one on chromosome 1 (“QTL1”) and one on chromosome 4 (“QTL4”). A three-factor model including QTL1, QTL4, and CGA explained 20.91% of variation in leaf clovamide content (p = 8.34E-05), suggesting that environmental factors or other undetected loci are also contributing to the variation. RNA-Seq data from ‘Sca6’ and ‘ICS1’ leaves was analyzed to identify differentially expressed genes (DEGs) in the two QTL. QTL4 contained several DEGs including Tc04v2_g000570, the UDP-glucose dependent glycosyltransferase (UGT) previously identified as a clovamide biosynthetic gene using the metabolomics/transcriptomics approach described above. QTL4 also contained an uncharacterized chloroplastic ABC transporter with a suspected role in clovamide precursor transport that was more highly expressed in ‘Sca6’ than ‘ICS1’ (~4-fold higher, p < 0.05). QTL1 also contained several DEGs including one uncharacterized serine carboxypeptidase-like acyltransferase (SCPL-AT) that had slightly higher expression in ‘Sca6’ than ‘ICS1’ (~0.9-fold, p < 0.05). A different cacao SCPL-AT was previously identified as a clovamide biosynthetic gene, described above. Surprisingly, the two clovamide QTL overlapped perfectly with two previously identified self-incompatibility loci in cacao, suggesting a potential but currently tenuous connection between clovamide biosynthesis and self-incompatibility in cacao. Overall, this work identifies a new resistance factor in cacao, clovamide, that should be explored as a breeding target and metabolic selectable marker in cacao breeding programs. Furthermore, it contributes to our broader understanding of plant specialized metabolite biosynthesis by identifying an example of convergent evolution in clovamide biosynthesis, which proceeds by an SCPL-AT in cacao and a BAHD-AT in red clover. This work also generates new questions about the potential role of clovamide in self-incompatibility in cacao that warrant further exploration. The integration of metabolomics, transcriptomics, and QTL analysis presented in this work may provide a road map for future attempts to identify important defense metabolites and elucidate their biosynthetic pathways in other systems.
Fister, A. S. (2016). Genomics of the theobroma cacao L. defense response (Order No. 10300628). Available From ProQuest Dissertations & Theses A&I. (1848683680). Retrieved from http://ezaccess.libraries.psu.edu/login?url=https://search-proquest-com.ezaccess.libraries.psu.edu/docview/1848683680?accountid=13158
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 qRTPCR 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.
Zhang, Y. (2014). Functional genomics of theobroma cacao fatty acid biosynthesis: Convergence of fatty acid desaturation, embryo development, and defense signaling responses (Order No. 3690183). Available From ProQuest Dissertations & Theses A&I. (1658228179). Retrieved from http://ezaccess.libraries.psu.edu/login?url=https://search-proquest-com.ezaccess.libraries.psu.edu/docview/1658228179?accountid=13158
Abstract: Theobroma cacao L. (chocolate tree) is an important cash crop for 40-50 million farmers and their families in its tropical growing regions worldwide. Cocoa butter and cocoa powder extracted from cacao seeds provide the main raw ingredients for chocolate manufacturing, supporting a $80 billion global business. A unique fatty acid composition of cocoa butter makes its melting temperature close to the human body temperature, which is not only of particular importance for industrial uses, but also a valuable quality trait targeted by breeding programs. My Ph.D. dissertation focused mainly on the fatty acid biosynthesis pathway in cacao seeds. I identified a key desaturase gene TcSAD1 from a large stearoyl-acyl carrier protein-desaturase gene family in cacao that plays a crucial role in converting stearic acid (18:0, saturated fatty acid) into oleic acid (18:1, unsaturated fatty acid). The expression of TcSAD1 was highly correlated with the change of fatty acid composition during cacao seed development. The activity of TcSAD1 rescued all the Arabidopsis ssi2 (a fatty acid desaturase) related mutant phenotypes, further supporting its in vivo functions. The discovery of the critical function of TcSAD1 offers a new strategy for screening for novel genotypes with desirable fatty acid compositions, and for use in breeding programs, to help pyramid genes for quality traits such as cocoa butter content. Moreover, because of the significance of fatty acid biosynthesis and lipid accumulation during cacao seed development, to further explore the regulatory mechanism, I functionally characterized of a master regulator, TcLEC2 gene, which controls both zygotic and somatic embryo development of cacao. Transient overexpression of TcLEC2 induced the expression of a variety of seed specific genes in cacao leaves. Furthermore, functions of TcLEC2 were explored during somatic embryogenesis, which is an in vitro propagation system for cacao. My results suggested that the activity of TcLEC2 determines the embryogenic capacity of the cacao tissue explants and correlated with embryogenic capacity of cultured cells. Transgenic embryos overexpressing TcLEC2 produced a significantly higher number of embryos compared to non-transgenic embryos; however, most of these transgenic somatic embryos exhibited abnormal phenotypes, and the development normally ceased at globular stage. This discovery may have future applications in increasing the efficiency of cacao mass propagation programs. Notably, in addition to major storage compounds in cacao seeds, fatty acids also function as signals involved in defense responses. I found that the endogenous level of 18:1 was modulated by exogenous glycerol application. Glycerol application on cacao leaves increased the level of glycerol-3-phosphate and lowered the level of 18:1 through an acylation reaction, which further triggered the defense responses. 100mM glycerol was sufficient to induce the accumulation of ROS, activate the expression of a variety of pathogen-related genes, and confer enhanced resistance against fungal pathogen Phytophthora capsici. My results demonstrated the potential of foliar glycerol application to become an environmentally safe means to induce the plant defense responses and fight important plant diseases in the field. Together, my Ph.D. dissertation makes major contributions to three important research areas in cacao: (1) identification of the key gene regulating fatty acid composition in cocoa butter, (2) improvement of large-scale propagation system (somatic embryogenesis) of cacao, (3) enhancement of cacao foliar disease resistance. This thesis not only provides useful knowledge of the regulatory mechanisms of important quality traits at the molecular and genetic levels, but also demonstrates the potential of taking advantage of cacao genomic resources to accelerate cacao basic research and breeding programs. <pdf>
Shi, Z. (2010). Functional analysis of non expressor of PR1 (NPR1) and its paralog NPR3 in theobroma cacao and arabidopsis thaliana (Order No. 3442953). Available From ProQuest Dissertations & Theses A&I. (853752677). Retrieved from http://ezaccess.libraries.psu.edu/login?url=https://search-proquest-com.ezaccess.libraries.psu.edu/docview/853752677?accountid=13158
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 PRI 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. <pdf>
Liu, Y. (2010). Molecular analysis of genes involved in the synthesis of proanthocyanidins in theobroma cacao (Order No. 3420238). Available From ProQuest Dissertations & Theses A&I. (750368282). Retrieved from http://ezaccess.libraries.psu.edu/login?url=https://search-proquest-com.ezaccess.libraries.psu.edu/docview/750368282?accountid=13158
The flavonoids catechin and epicatechin, and their polymerized oligomers, the proanthocyanidins (PAs, also called condensed tannins), accumulate to levels of up to 15% of the total weight of dry seeds of Theobroma cacao L. These compounds have been associated with several health benefits in humans including antioxidant activity, improvement of cardiovascular health and reduction of cholesterol levels. They also play important roles in pest and disease defense throughout the plant. This research focuses on molecularly dissecting the proanthocyanidin biosynthetic pathway of Theobroma cacao. To this end, I first isolated candidate genes from T.cacao (Tc) encoding key structural enzymes of this pathway which include, anthocyanidin reductase (ANR), leucoanthocyanidin dioxygenase (LDOX, also called anthocyanidin synthase, ANS) and leucoanthocyanidin reductase (LAR). I performed gene expression profiling of candidate TcANR, TcANS and TcLAR in various tissues through different developmental stages and also evaluated PA accumulation levels in those tissues. My results suggested that all PA candidate genes are co-regulated and positively correlated with PA synthesis. To functionally analyze the candidate genes, I used the model plants Arabidopsis and tobacco as expression platforms. Results from Arabidopsis mutant complementation tests and transgenic tobacco plants constitutively overexpressing cacao genes demonstrate that the candidate structural genes isolated from cacao are true ANS, ANR and LAR genes and all actively involved in PA synthesis in cacao. To further explore the transcriptional regulation of the PA synthesis pathway, I then isolated and characterized an R2R3 type MYB transcription factor TcMYBPA from cacao. I examined the spatial and temporal gene expression patterns of TcMYBPA in cacao and found it to be developmentally expressed in a manner consistent with its involvement in PAs as well as anthocyanin synthesis. Complementation test of TcMYBPA in Arabidopsis tt2 mutant suggested that TcMYBPA could functionally substitute Arabidopsis TT2 gene. Interestingly, except PA accumulation in seeds, I also observed an obvious increase of anthocyanidin accumulation in hypocotyls of transgenic Arabidopsis plants. This is consistent with gene expression analysis which showed that the entire PA pathway could be induced by overexpression of TcMYBPA gene, including DFR, LDOX (ANS) and BAN (ANR). Therefore I concluded that the isolated TcMYBPA gene encodes an R2R3 type MYB transcription factor and is involved in the regulation of both anthocyanin and PA synthesis in cacao. This research will not only offer us the knowledge of secondary metabolites production in cacao, but also provides molecular tools for breeding of cacao varieties with improved disease resistance and enhanced flavonoid profiles for nutritional and pharmaceutical applications. <pdf>
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