Author:
Wei Wei,Kim Doogie,Koehler Naio,Bendl Ashley,Cho Myeong-Je,Krasileva Ksenia
Abstract
AbstractPlant diseases pose a significant threat to global crop production. Most disease resistance genes used in crop breeding programs encode nucleotide-binding leucine-rich repeat receptors (NLRs) that are limited in pathogen specificity and durability. In this study, we leveraged synthetic biology to develop an inducible broad-spectrum resistance in tomatoes. Constitutive expression of autoactive NLRs in plants leads to robust resistance against multiple pathogens but significantly stunts growth. We expressed autoactive NLRs under the control of pathogen-inducible (PI) promoters to mitigate the fitness costs. Taking advantage of extensive, new genomic and transcriptomic resources, we identified PI promoters that responded to multiple pathogens but not abiotic stress. We further validated functionality of predicted elements through a promoter luciferase assay. We generated significant resistance in transgenic tomatoes but we also encountered unwanted expression induction of the native promoter regions in flowers which led to lethal fruit development. Thus, we pursued promoter engineering for fine-tuning the induction. We identified cis-regulatory regions responsible for pathogen-inducibility through promoter bashing experiments and recombined the native promoter with the inducible part and the core promoter. Furthermore, we rationally created synthetic promoters showing a gradient of expression levels, which will allow for selection for transgenic tomatoes with the best performance. We found that the spacing between functional sequences, repeat number of inducible sequences, and core promoters all influence the outcome of engineering. Our study outlines a framework for developing broad-spectrum synthetic immune constructs with reduced fitness cost and provides examples of pathogen-inducible promoter engineering.
Publisher
Cold Spring Harbor Laboratory
Reference63 articles.
1. Magical Mystery Tour: MLO Proteins in Plant Immunity and beyond;The New Phytologist,2014
2. Pathogenesis Related Protein-1 (PR-1) Genes in Tomato (Solanum Lycopersicum L.): Bioinformatics Analyses and Expression Profiles in Response to Drought Stress;Genomics,2020
3. A Fruitful Decade Using Synthetic Promoters in the Improvement of Transgenic Plants;Frontiers in Plant Science,2019
4. Resistance to ‘Candidatus Liberibacter Asiaticus,’ the Huanglongbing Associated Bacterium, in Sexually And/or Graft-Compatible Citrus Relatives;Frontiers in Plant Science,2020
5. Comprehensive Construction Strategy of Bidirectional Green Tissue-Specific Synthetic Promoters;Plant Biotechnology Journal,2020