Abstract
Theobroma cacao, the source of cocoa, suffers significant losses to a variety of pathogens resulting in reduced incomes for millions of farmers in developing countries. Development of disease resistant cacao varieties is an essential strategy to combat this threat, but is limited by sources of genetic resistance and the slow generation time of this tropical tree crop. In this study, we present the first application of genome editing technology in cacao, using Agrobacterium-mediated transient transformation to introduce CRISPR/Cas9 components into cacao leaves and cotyledon cells. As a first proof of concept, we targeted the cacao Non-Expressor of Pathogenesis-Related 3 (TcNPR3) gene, a suppressor of the defense response. After demonstrating activity of designed single-guide RNAs (sgRNA) in vitro, we used Agrobacterium to introduce a CRISPR/Cas9 system into leaf tissue, and identified the presence of deletions in 27% of TcNPR3 copies in the treated tissues. The edited tissue exhibited an increased resistance to infection with the cacao pathogen Phytophthora tropicalis and elevated expression of downstream defense genes. Analysis of off-target mutagenesis in sequences similar to sgRNA target sites using high-throughput sequencing did not reveal mutations above background sequencing error rates. These results confirm the function of NPR3 as a repressor of the cacao immune system and demonstrate the application of CRISPR/Cas9 as a powerful functional genomics tool for cacao. Several stably transformed and genome edited somatic embryos were obtained via Agrobacterium-mediated transformation, and ongoing work will test the effectiveness of this approach at a whole plant level.
Highlights
Theobroma cacao, the tropical tree which produces cocoa beans, is the centerpiece of the multi-billion dollar chocolate industry and is a vital export for many developing countries
The cacao Non-expressor of Pathogenesis-Related 1 (TcNPR1) gene, known as the master regulator of the immune system (Cao et al, 1997; Fu and Dong, 2013), was characterized by Shi et al (2010) and its overexpression resulted in reduction of Phythophthora spp. infection in cacao leaf tissue (Fister et al, 2015), Functional genomics analysis of several other cacao candidate defense related genes was conducted
Using the CRISPR site finder toolset in Geneious v9 (Kearse et al, 2012), we identified potential single-guide RNAs (sgRNA) target sites within the the cacao Non-Expressor of Pathogenesis-Related 3 (TcNPR3) gene (Criollo gene ID Tc06_g011480)
Summary
Theobroma cacao, the tropical tree which produces cocoa beans, is the centerpiece of the multi-billion dollar chocolate industry and is a vital export for many developing countries. Infection of cacao by a variety of pathogens severely impacts global production with 20–30% of pods destroyed pre-harvest (Ploetz, 2016). The major classes of cacao pathogen receptor genes and other components of the downstream pathways were first globally described through whole genome sequencing and phylogenetic analysis with model plant systems (Argout et al, 2011). The cacao Non-expressor of Pathogenesis-Related 1 (TcNPR1) gene, known as the master regulator of the immune system (Cao et al, 1997; Fu and Dong, 2013), was characterized by Shi et al (2010) and its overexpression resulted in reduction of Phythophthora spp. infection in cacao leaf tissue (Fister et al, 2015), Functional genomics analysis of several other cacao candidate defense related genes was conducted. An artificial miRNAmediated knockdown of TcNPR3 resulted in increased resistance to infection (Shi et al, 2013b)
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