1 Pathogen resistance in Theobroma cacao Dan Lordan College of Agricultural Sciences Undergraduate Research Proposal I.

Introduction and Background Information Theobroma cacao, or the chocolate tree, is a vital crop of incredible economic value to many countries around the globe !he "vory Coast and #hana are t$o of the largest cacao producing countries in the $orld and most global cacao is gro$n in Africa and South America "ts use as a stable source of income, ho$ever, is complicated by its susceptibility to some destructive pathogens %aving se&uenced the Theobroma cacao genome in previous years, the focus of this part of the lab no$ is to investigate the resistance of the cocoa plant and attempt to identify the genes that control the response there$ith Cocoa plants $ith naturally high levels of pathogen resistance are being studied to determine $hich genes contribute to their above average ability to maintain gro$th !here are several documented $ays that plants have developed to fight pathogens !he main path that is being studied here, a reactionary one, involves signal transduction cascades follo$ing pathogen recognition that set off hormonal cascades to begin the elimination of the disease causing agent !he identification of these resistance genes could lead to the breeding of Theobroma cacao to better sustain a population !he loci responsible for these traits are vital for understanding their mechanisms and for the propagation of pathogen resistant plants II. Project Objectives !he ob'ective of this pro'ect is to identify all relevant genes of interest regarding pathological resistance in the Theobroma cacao genome and understand their function Disease resistance specific to the area in $hich the plants are farmed can then be selected for through breeding III. Significance of the Project !he outcomes of this pro'ect $ill be beneficial to both science as a $hole and the cocoa industry (rom a purely scientific standpoint, the identification of pathogen resistant genes and the study of evolved genetic resistance is pertinent to medical research, molecular biology, and many other fields "n relation to Theobroma cacao itself, the breeding of resistant strains could revolutioni)e the cocoa industry and reduce ris* involved in cultivation +any of the countries gro$ing cocoa are underdeveloped and this could help stabili)e the economy and provide a long term established crop for the people there IV. Describe Your Role !hroughout the (all ,-1. semester, my role in this process $ill be functional testing of genes of interest " $ill be learning techni&ues including transient leaf transformation, pathogen application assays, and R/A e0tractions After mastering the techni&ues, " $ill $or* $ith graduate students to perform Agrobacterium transformation

Pathogen resistance in Theobroma cacao , Dan Lordan of leaf tissue " $ill then perform R/A e0tractions and use &R!1PCR to measure the collected R/A to understand the effectiveness of the gene over e0pression "n this $ay " can help identify the loci that $e are searching for and their ability to fight particular pathogens V. aterials and ethods Several cacao defense genes have already been cloned and are ready to be further studied !o do so, leaf tissue $ill then be treated $ith agrobacterium containing a vector $hich $ill allo$ overe0pression of candidate defense genes After transforming leaves, pathogens of cacao $ill also be applied to the leaf tissue and infection $ill be monitored for ,1. days !he lesion areas $ill be collected for D/A and R/A e0traction !he D/A $ill be used in &PCR to measure pathogen gro$th $hile the R/A $ill be used in &R!1 PCR to ensure the resistance genes of interest $ere overe0pressed, and to test if upregulating those genes affected other genes in important defense path$ays VI. !o" Data #ill Be $nal%&ed and 'sed !he measurements of effects of gene function $ill sho$ the loci responsible for pathogenic response +easurement of lesion si)e and pathogen D/A accumulation in defense gene1overe0pressing tissue $ill be compared to controls to sho$ that the genes2 activity decreases pathogen gro$th Using &R!1PCR, $e $ill identify other genes that are activated by the increased activity of the genes of interest in order to develop genetic mar*ers to improve breeding !his $or* is part of a larger pro'ect that tests effectiveness of many plant defense genes All $or* leads to$ards the goal of breeding healthier crops and decreasing hunger and poverty VII. (iterature )ited Roberts, A , % Pimentel, C !rapnell, and L Pachter, Identification of novel transcripts in annotated genomes using RNA-Seq. 3ioinformatics, ,-11 *+41567 p ,8,91 ,8,: Risterucci, A + , D Paulin, + Ducamp, ; A < /=#oran, and C Lanaud, Identification of QTLs related to cocoa resistance to three species of h!tophthora. !heoretical and Applied #enetics, ,--8 ,-.4167 p 1>?115. Lapin, D and # @an den Ac*erve*en, Susceptibilit! to plant disease" more than a failure of host immunit!. !rends in plant science, ,-18 ,.41-67 p 9.>199.