Transcriptome-guided combinatorial gene editing to unlock the homeostasis of plants and microbiota

• Applicants: Professor Dr. Paul Schulze-Lefert; Professorin Dr. Alga Zuccaro
• Subject Area: Organismic Interactions, Chemical Ecology and Microbiomes of Plant Systems
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 565892813

Plants in nature are associated with microbial communities collectively known as the plant microbiota. Although mounting evidence has demonstrated the importance of the plant microbiota for plant growth and health, it has recently been shown that a few microbiota members are opportunistic pathogens that can cause infections under favorable environmental conditions. It is therefore not only of interest to understand principles underlying microbiota assembly, but also how the plant host and beneficial microbiota members keep opportunistic pathogens in check. However, most of the plant genetic determinants underlying homoeostatic interactions in healthy plants with the microbiota remain undefined. We have identified more than 400 genes in Arabidopsis thaliana (hereafter Arabidopsis) roots that respond differentially to colonization by synthetic bacterial communities with contrasting immunomodulatory traits under immune-stimulating conditions. Mutational studies of few tested host genes from these expression clusters confirm that these genes are not only markers, but are needed for proper microbiota assembly. We hypothesize that microbiota-mediated host transcriptomic changes are the result of reciprocal host-microbiota interactions. Here we develop a method to construct combinatorial CRISPR libraries for Arabidopsis gene editing to facilitate functional studies of gene expression clusters. Specifically, we have shown by proof-of-principle experiments that it is possible to generate a combinatorial CRISPR library in two restriction-ligation reactions. The CRISPR library can simultaneously target up to eight plant genes randomly selected from a pool of approximately one hundred synthesized guide RNA (gRNA) sequences. The combinatorial Arabidopsis mutant libraries will be inoculated with defined bacterial communities alone or with additional opportunistic or host-adapted bacterial or fungal pathogens to identify single or higher-order mutant(s) that have an altered microbiota profile. These data will be complemented with the microbial profiles of Arabidopsis gene edited mutants grown in natural soil. We will develop additional tools, including i) engineered bacterial microbiota members that express a quantifiable fluorescence or bioluminescence marker, and ii) transgenic reporter plants with proxies for inducible plant resistance responses to various pathogens in the presence of defined microbial communities. This project will inform how genetic perturbations of the host impact microbiota composition and plant health and uncover host co-expression modules needed for plant microbiota homeostasis that keeps pathogens in check.

• DFG Programme: Research Grants
• International Connection: Taiwan
• Partner Organisation: National Science and Technology Council (NSTC)
• Cooperation Partner: Ka-Wai Ma, Ph.D.