Transient Expression of CRISPR/Cas9 Machinery Targeting TcNPR3 Enhances Defense Response in Theobroma cacao

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.

Chromosome Engineering in Tropical Cash Crops

“…Cacao tree plantations require a large area of land and labor force [16]. Because diseases are a persistent problem for cacao, improved disease resistance via breeding is imperative [17]. Each year, infection with a variety of pathogens severely cripples global cacao production, with about 30% of all pods destroyed before harvesting [17].…”

“…In West Africa, severe outbreaks of Phytophthora spp. can destroy all cacao pods on a farm [17]. The fungal pathogen Moniliophthora perniciosa causes Witches' broom disease, which seriously cripples cacao yields in Ecuador and Brazil [16].…”

“…The genome is believed to contain 21,784 protein-coding genes [23]. Agrobacterium-mediated transient transformation with CRISPR/Cas9 of cacao leaves and cotyledon cells [17].…”

Development of Improved Fruit, Vegetable, and Ornamental Crops Using the CRISPR/Cas9 Genome Editing Technique

“…The authors hypothesized that cells in which NPR3 was mutated activated their defense responses. Subsequently, they generated stably transformed and genome-edited somatic embryos for future testing the effectiveness of CRISPR/Cas9 at a whole plant level in cacao [80]. Banana streak virus (BSV) is one of the most important limitations for banana production.…”

Clinical applications of CRISPR‐based genome editing and diagnostics

“…[6][7][8][9][10][11][12][13] Since then, CRISPR technology has opened the door to brand new avenues of biologic research in diverse organisms like ferrets, coral, and cacao. [14][15][16] Scientists can manipulate the genomes of cultured cells to knock out genes, fluorescently tag endogenous proteins, make specific sets of mutations to model disease, and more. 17,18 CRISPR has revolutionized biological research and the next frontier is medicine.…”