Location: Sustainable Perennial Crops Laboratory2013 Annual Report
1a. Objectives (from AD-416):
There are three primary objectives of this research. The first objective is to sequence the genomes of Phytophthora palmivora and Phytophthora megakarya isolates pathogenic on cacao. The genomes of the two related species, both severe pathogens of Theobroma cacao, are to be compared once the sequencing is completed. The second objective is to identify cacao genes important in plant defense using microarray technologies. The third objective is to exploit genetic transformation technologies for the purpose of carrying out functional analysis of genes of potential importance in resistance of cacao to biotic and abiotic stresses, including plant disease. These studies seek to understand how cacao resists infection by plant pathogens and to exploit that understanding using traditional crop improvement techniques.
1b. Approach (from AD-416):
DNA sequencing technologies continue to become more cost effective. For example, pyrosequencing sequences small DNA fragments, but is capable of sequencing very large numbers of small sequences in a very short time. Bioinformatic techniques allow for the assembling of these short sequences into larger sequences and ultimately into linkage groups. The genome sequences of Phytophthora species will be determined using this and other available techniques as required. At the same time, ARS has access to the nearly complete sequence of the related genome of several Phytophthora species. Once the sequences of P. palmivora and P. megakarya have been collected, they will be overlaid onto each other and other available genome sequences to aid in assembly. Identification of genes involved in plant defense is a first critical step in understanding how plants resist infection from plant pathogens and respond to stress in general. ARS will use microarray technology to identify cacao transcripts responsive to pathogen infection in resistant and susceptible cacao clones. Total RNA will be isolated from cacao tissues. These samples will be hybridized to the full transcriptome microarray available at The Pennsylvania State University. Cacao genes showing differential expression patterns in association with the cacao resistance response will be identified and functionally analyzed. The functional analyses of specific genes involved in plant defense will be critical as the genomics capabilities available for cacao research expand. Identifying the exact function of genes considered important in plant defense will allow their exploitation in traditional and advanced breeding efforts to develop cacao materials resistant to the negative effects of stress. Cacao will be both constitutively and transiently transformed to overexpress genes of potential importance to plant defense. The transformed materials will be evaluated under conditions of stress, including disease to validate their importance in the defense process. The proposed research parallels objectives within the associated main project.
3. Progress Report:
Using Illumina sequencing, whole genome surveys and deep-transcriptome data were obtained for P. palmivora and P. megakarya. The pathogens were grown in purified liquid cultures to obtain enough biomass for extracting: 1) genomic DNA, 2) RNA from mycelium and 3) RNA from sporulating cultures. Preliminary data analysis has been conducted and confirms that a very deep and reasonably complete assembly of the genome has been produced. All assembly and quality statistics are good, although the number of contigs is still very large. Microarrays were used in a series of experiments to explore the gene expression during pathogen infection and in response to treatment with salicylic acid, a defense signaling molecule. A large set of genes that are co-regulated during these responses were identified. The promoters of three of these genes were isolated to study the mechanisms involved in the regulation of the defense response of cacao. Using a recently developed cacao transient gene-expression assay method, the functions of several candidate genes that function as regulators of the plant immune system (NPR1 and NPR3) were demonstrated. The efficacies of several anti-microbial proteins and peptides in enhancement of pathogen resistance were also demonstrated. A pipeline has been established allowing systematic testing of the function of additional candidate genes.