CRISPR cacao: 42% smaller Phytophthora lesions in lab tests

Researchers at Penn State, led by Mark Guiltinan and Siela Maximova, used CRISPR-Cas9 to edit the TcNPR3 gene in cacao (Theobroma cacao L.) and reduce its susceptibility to Phytophthora, the pathogens behind black pod disease. The results were published in Plant Biotechnology Journal doi.org. Genome-edited plants showed a 42% reduction in lesion size after Phytophthora challenge and were phenotypically comparable to wild-type controls doi.org. Field trials are underway doi.org.

Editing TcNPR3 and removing the transgene

TcNPR3 is a known negative regulator of plant defence. The team produced transgenic T0 lines with mutations predicted to disrupt its function. In in vitro foliar assays, these lines were less susceptible to Phytophthora infection doi.org. The T0 plants were grown to maturity and outcrossed with non-transgenic cacao to eliminate the T-DNA sequences associated with the CRISPR-Cas9 transgene doi.org.

Whole-genome sequencing of the T0 parents and 22 progeny revealed a single T-DNA insertion site in each T0 line. Seven progeny retained the edited npr3 alleles but had no T-DNA insertions doi.org. According to Guiltinan, in one case only two bases changed out of roughly 450 million in the plant's genome. The researchers state that, under U.S. and EU regulatory frameworks, the resulting cacao is no longer considered a GMO.

Gene expression and field testing

Transcriptome analysis of the mutant lines showed upregulation of genes associated with reactive oxygen species generation, defence-related transcription factors and pathogenesis-related proteins doi.org. Several genes were downregulated. This suggests that TcNPR3 may act as both a repressor and an activator in regulating basal transcriptional states doi.org.

Black pod disease is estimated to reduce cacao yields by roughly 30% worldwide. The laboratory results do not yet show whether the protection will hold under commercial growing conditions. Field evaluations are being carried out with the U.S. Department of Agriculture in Hawaii and Puerto Rico, and are intended to assess long-term agronomic performance under natural conditions doi.org.

Penn State has licensed the commercial rights to plants carrying the mutation to AgroNetica, a cacao biotechnology company. The company plans to continue testing and expand production.

Penn State edits TcNPR3 in cacao: 42% smaller Phytophthora lesions in lab tests

Editing TcNPR3 and removing the transgene

Gene expression and field testing

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