Dataset Information

Protocol: transient expression system for functional genomics in the tropical tree Theobroma cacao L.

ABSTRACT: BACKGROUND:Theobroma cacao L., the source of cocoa, is a crop of significant economic value around the world. To facilitate the study of gene function in cacao we have developed a rapid Agrobacterium-mediated transient genetic transformation protocol. Here we present a detailed methodology for our transformation assay, as well as an assay for inoculation of cacao leaves with pathogens. RESULTS:Agrobacterium tumefaciens cultures are induced then vacuum-infiltrated into cacao leaves. Transformation success can be gauged 48 h after infiltration by observation of green fluorescent protein and by qRT-PCR. We clarify the characteristics of cacao leaf stages and demonstrate that our strategy efficiently transforms leaves of developmental stage C. The transformation protocol has high efficacy in stage C leaves of four of eight tested genotypes. We also present the functional analysis of cacao chitinase overexpression using the transient transformation system, which resulted in decreased pathogen biomass and lesion size after infection with Phytophthora tropicalis. CONCLUSIONS:Leaves expressing transgenes of interest can be used in subsequent functional genetic assays such as pathogen bioassay, metabolic analysis, gene expression analysis etc. This transformation protocol can be carried out in 1 day, and the transgenes expressing leaf tissue can be maintained in petri dishes for 5-7 days, allowing sufficient time for performance of additional downstream gene functional analysis. Application of these methods greatly increases the rapidity with which candidate genes with roles in defense can be tested.

SUBMITTER: Fister AS

PROVIDER: S-EPMC4788949 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Protocol: transient expression system for functional genomics in the tropical tree Theobroma cacao L.

Fister Andrew S AS Shi Zi Z Zhang Yufan Y Helliwell Emily E EE Maximova Siela N SN Guiltinan Mark J MJ

Plant methods 20160311

<h4>Background</h4>Theobroma cacao L., the source of cocoa, is a crop of significant economic value around the world. To facilitate the study of gene function in cacao we have developed a rapid Agrobacterium-mediated transient genetic transformation protocol. Here we present a detailed methodology for our transformation assay, as well as an assay for inoculation of cacao leaves with pathogens.<h4>Results</h4>Agrobacterium tumefaciens cultures are induced then vacuum-infiltrated into cacao leaves ...[more]

PMID: 26973706

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Project description:BACKGROUND: The Arabidopsis thaliana NPR1 gene encodes a transcription coactivator (NPR1) that plays a major role in the mechanisms regulating plant defense response. After pathogen infection and in response to salicylic acid (SA) accumulation, NPR1 translocates from the cytoplasm into the nucleus where it interacts with other transcription factors resulting in increased expression of over 2000 plant defense genes contributing to a pathogen resistance response. RESULTS: A putative Theobroma cacao NPR1 cDNA was isolated by RT-PCR using degenerate primers based on homologous sequences from Brassica, Arabidopsis and Carica papaya. The cDNA was used to isolate a genomic clone from Theobroma cacao containing a putative TcNPR1 gene. DNA sequencing revealed the presence of a 4.5 kb coding region containing three introns and encoding a polypeptide of 591 amino acids. The predicted TcNPR1 protein shares 55% identity and 78% similarity to Arabidopsis NPR1, and contains each of the highly conserved functional domains indicative of this class of transcription factors (BTB/POZ and ankyrin repeat protein-protein interaction domains and a nuclear localization sequence (NLS)). To functionally define the TcNPR1 gene, we transferred TcNPR1 into an Arabidopsis npr1 mutant that is highly susceptible to infection by the plant pathogen Pseudomonas syringae pv. tomato DC3000. Driven by the constitutive CaMV35S promoter, the cacao TcNPR1 gene partially complemented the npr1 mutation in transgenic Arabidopsis plants, resulting in 100 fold less bacterial growth in a leaf infection assay. Upon induction with SA, TcNPR1 was shown to translocate into the nucleus of leaf and root cells in a manner identical to Arabidopsis NPR1. Cacao NPR1 was also capable of participating in SA-JA signaling crosstalk, as evidenced by the suppression of JA responsive gene expression in TcNPR1 overexpressing transgenic plants. CONCLUSION: Our data indicate that the TcNPR1 is a functional ortholog of Arabidopsis NPR1, and is likely to play a major role in defense response in cacao. This fundamental knowledge can contribute to breeding of disease resistant cacao varieties through the application of molecular markers or the use of transgenic strategies.

Project description:We present a typology of cacao agroforest systems in Colombian Amazonia. These systems had yet to be described in the literature, especially their potential in terms of biodiversity conservation. The systems studied are located in a post-conflict area, and a deforestation front in Colombian Amazonia. Cacao cropping systems are of key importance in Colombia: cacao plays a prime role in post conflict resolution, as cacao is a legal crop to replace illegal crops; cacao agroforests are expected to be a sustainable practice, promoting forest-friendly land use.We worked in 50 x 2000 m2 agroforest plots, in Colombian Amazonia. A cluster analysis was used to build a typology based on 28 variables characterised in each plot, and related to diversity, composition, spatial structure and light availability for the cacao trees. We included variables related to light availability to evaluate the amount of transmitted radiation to the cacao trees in each type, and its suitability for cacao ecophysiological development.We identified 4 types of cacao agroforests based on differences concerning tree species diversity and the impact of canopy spatial structure on light availability for the cacao trees in the understorey. We found 127 tree species in the dataset, with some exclusive species in each type. We also found that 3 out of the 4 types identified displayed an erosion of tree species diversity. This reduction in shade tree species may have been linked to the desire to reduce shade, but we also found that all the types described were compatible with good ecophysiological development of the cacao trees.Cacao agroforest systems may actually be achieving biodiversity conservation goals in Colombian Amazonia. One challenging prospect will be to monitor and encourage the conservation of tree species diversity in cacao agroforest systems during the development of these cropping systems, as a form of forest-friendly management enhancing sustainable peace building in Colombia.

Dataset Information

Protocol: transient expression system for functional genomics in the tropical tree Theobroma cacao L.

Publications

Protocol: transient expression system for functional genomics in the tropical tree Theobroma cacao L.

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