Unravelling novel mechanisms of defense gene activation: a gateway to elevate disease resistance in plants

Project Information

R-ELEVATION

Grant agreement ID: 101039824

DOI

10.3030/101039824

EC signature date 29 April 2022

Start date 1 May 2022

End date 30 April 2027

Funded under

• European Research Council (ERC)

Total cost

€ 2 364 101,00

EU contribution

€ 2 364 101,00

Coordinated by

UNIVERSITEIT LEIDEN
Netherlands

Periodic Reporting for period 1 - R-ELEVATION (Unravelling novel mechanisms of defense gene activation: a gateway to elevate disease resistance in plants)

Reporting period: 2022-05-01 to 2024-10-31

Summary of the context and overall objectives of the project

Plant diseases significantly threaten global agriculture, impacting crop yields and food security. Understanding the molecular mechanisms underlying plant immunity is crucial for developing effective and sustainable strategies to combat these diseases. The R-ELEVATION project aims to advance our knowledge in this field by investigating the gene regulatory networks (GRNs) that govern plant immune responses. By leveraging cutting-edge technologies such as single-cell RNA sequencing, CRISPR/Cas9 gene editing, and comparative genomics, this project seeks to unravel the complex interactions between plants and pathogens.

The overarching objective of R-ELEVATION is to elucidate the regulatory mechanisms that enable plants to recognize and respond to pathogenic threats. Specifically, the project focuses on:

1. Developing and utilizing innovative inducible systems such as a copper-inducible system in tomatoes to profile ETI-specific GRNs, facilitating cross-species comparisons and enhancing our understanding of universal immune strategies in plants.

2. Employing a high-efficiency CRISPR/Cas9 system to create multi-gene knockouts, mainly targeting WRKY transcription factors involved in plant immunity.

3. Conducting single-cell RNA sequencing to address ambient RNA noise in single-nucleus RNA sequencing, ensuring accurate and reliable results.

4. Using deep learning and machine learning algorithms to decode intricate GRNs, incorporating additional methods for robust and reliable analyses.

The R-ELEVATION project is expected to significantly contribute to basic and applied plant sciences. By elucidating the molecular mechanisms of plant immunity, the project will provide valuable insights that can be translated into practical agricultural applications, including developing disease-resistant crops. This will develop sustainable strategies for enhancing crop resistance to diseases by reducing reliance on chemical pesticides, thereby contributing to global food security and agricultural sustainability. The project's innovative approaches and technological advancements will set new standards for research methodologies in plant biology. Establishing new RNA-seq library preparation methods and using inducible systems in non-model plants like tomatoes demonstrate the project's potential to drive research capabilities and applications forward. Moreover, the project's findings will be shared with the broader scientific community through publications, workshops, and collaborations, fostering knowledge exchange and capacity building. By addressing critical challenges in plant immunity, the R-ELEVATION project aims to contribute to sustainable agriculture, aligning with global efforts to ensure food security and environmental sustainability.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The R-ELEVATION project undertook a comprehensive approach to deepen our understanding of plant immunity, focusing on several key areas of research and development. Our efforts have resulted in significant scientific and technical achievements, outlined below:

1. We adapted a copper-inducible system in tomatoes, in collaboration with a research group in Taiwan, allowing precise control over ETI induction and facilitating cross-species comparisons with model plants like Arabidopsis. This was crucial for understanding ETI's regulatory mechanisms in different species.

2. We established a high-efficiency CRISPR/Cas9 system to generate multi-gene knockouts, mainly targeting WRKY transcription factors. This allowed us to create higher-order mutants and provided deeper insights into the specific roles and interactions of WRKY genes in plant immunity.

3. To mitigate ambient RNA contamination in single-nucleus RNA sequencing, we conducted single-cell RNA sequencing, ensuring accurate and reliable data. This enabled comprehensive single-cell transcriptomic analyses, uncovering spatially dynamic gene expression patterns in plant-pathogen interactions.

4. We used deep learning and machine learning algorithms to infer GRNs from high-throughput sequencing data. By incorporating additional methods, we enhanced the robustness and accuracy of our GRN inferences, providing detailed insights into the regulatory mechanisms underlying plant immune responses.

5. We developed a cost-effective RNA-seq library preparation method that improves gene coverage capacity and facilitates better validation of findings. This method has potential applications in broader genomic studies beyond the scope of this project.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The R-ELEVATION project has delivered several groundbreaking results that significantly advance the state of the art in plant immunity research, with profound potential impacts on both the scientific community and agricultural practices:

1. The copper-inducible system for ETI in tomatoes allows precise temporal control of gene expression, enabling detailed profiling of ETI-specific GRNs and cross-species comparisons. Further optimization and validation through demonstration projects and field trials are needed to ensure widespread adoption and commercialization.

2. The high-efficiency CRISPR/Cas9 multi-gene knockout system enables the dissection of complex gene networks involved in plant immunity. Adapting this technology to other crops and establishing regulatory guidelines will facilitate its commercialization and application in agriculture.

3. The single-cell RNA sequencing provides a comprehensive atlas of cell-type-specific responses to pathogen infection, guiding the breeding of crops with targeted immune traits. Expanding this approach to more plant species and developing cost-effective single-cell sequencing technologies will enhance its utility.

4. Our advanced computational methods integrated deep learning and machine learning algorithms for robust GRN inference providing reliable insights into plant immunity's regulatory mechanisms. Continuous improvement of computational algorithms and their validation are necessary for wider adoption.

5. Our cost-effective RNA-seq library preparation method reduces costs and improves gene coverage capacity, supporting large-scale transcriptomic studies. Scaling up and making it accessible to research institutions will maximize its impact.

The R-ELEVATION project has produced several high-impact results that extend beyond the current state of the art. Innovative systems and methodologies, such as the copper-inducible system for ETI and the high-efficiency CRISPR/Cas9 multi-gene knockout system, have provided powerful tools for plant immunity research. Single-cell RNA sequencing and advanced computational methods have yielded detailed insights into plant-pathogen interactions, while the new RNA-seq library preparation method has improved the efficiency and cost-effectiveness of transcriptomic studies.

To ensure the further uptake and success of these results, continued research, demonstration projects, and field trials are necessary. Access to markets, finance, robust intellectual property support, and a supportive regulatory framework will facilitate commercialization and widespread adoption. International collaborations and data-sharing platforms will promote knowledge exchange and accelerate the translation of research findings into practical agricultural applications. By addressing these key needs, the R-ELEVATION project’s outcomes can significantly contribute to sustainable agriculture and global food security.