Decoding calcium pathway activated by plant intracellular immune receptors

Project Information

DeCaETI

Grant agreement ID: 101063732

DOI

10.3030/101063732

Project closed

EC signature date 19 August 2022

Start date 3 July 2023

End date 2 July 2025

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 203 464,32

Coordinated by

UNIVERSITEIT LEIDEN
Netherlands

Periodic Reporting for period 1 - DeCaETI (Decoding calcium pathway activated by plant intracellular immune receptors)

Reporting period: 2023-07-03 to 2025-07-02

Summary of the context and overall objectives of the project

Plant diseases are a major challenge to global agriculture, food security, and economic stability, causing significant crop losses worldwide. With increasing demands for sustainable agricultural practices, understanding the molecular mechanisms that drive plant immunity is crucial for developing long-lasting disease resistance strategies. This project aims to address a fundamental question in plant immunity: how do plants decode and respond to pathogen attacks through calcium signaling?

Calcium signalling is a well-conserved second messenger system in all living organisms, playing a critical role in plant defence responses. However, despite its well-established role in immunity, little is known about how intracellular plant immune receptors, known as nucleotide-binding leucine-rich repeat receptors (NLRs), activate calcium signalling upon pathogen recognition. Furhow calcium signals regulate large-scale transcriptional reprogramming to trigger effective defence responses remains unclear responses.

To bridge these knowledge gaps, this project investigates the intricate relationship between NLR immune receptors and calcium signalling. The research is structured around three key objectives:

Decoding compartmentalized calcium dynamics triggered by intracellular immune receptors (NLRs) upon pathogen detection.
Characterizing the role of helper NLRs in generating calcium signatures and orchestrating immune-related gene expression.
Identifying calmodulins (calcium-binding proteins) that interact with key transcription factors to interpret calcium signals and activate defence responses.
By integrating advanced molecular biology, live-cell imaging, and genetic approaches, this project aims to unravel how calcium signals are precisely regulated and decoded during plant immune responses. The findings will provide valuable insights into plant immunity, paving the way for innovative breeding strategies that enhance crop resilience to disease.

This project has the potential to drive significant advancements in plant breeding, particularly in developing crops with durable resistance to pathogens. Understanding how calcium signaling contributes to plant immunity will enable researchers and breeders to identify genetic targets for improving disease resistance in various crops. This knowledge can be applied to develop climate-resilient and sustainable agricultural practices, reducing reliance on chemical pesticides and improving global food security.

The results of this project will be disseminated through scientific publications, conferences, and outreach activities to maximize impact. Additionally, collaboration with plant breeding programs and biotechnology industries will facilitate the translation of research findings into practical applications. By uncovering new molecular pathways in plant immunity, this project will contribute to the long-term goal of ensuring global food production stability in the face of increasing agricultural challenges.

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

1) Screening and Analysis of Inducible Helper NLRs Transgenic Lines
Two groups of helper NLRs, ADRs and NRGs, play critical roles in plant immunity. To investigate their specific functions in effector-triggered immunity (ETI), we developed and screened estradiol-inducible transgenic lines expressing active forms of ADR1L2 and NRG1.1. These lines were validated through molecular analysis for further functional characterization.

We assessed the expression dynamics of a key ETI marker gene to compare the immune responses triggered by ADR1 and NRG1 pathways. Initial observations suggested distinct temporal expression patterns, potentially linked to differences in protein activation timing or pathway-specific regulatory mechanisms. To further investigate these dynamics, we conducted time-series transcriptome profiling under mock and estradiol-induced conditions. This analysis identified several genes associated with cellular stress responses, immune signalling, and calcium-binding proteins. A complementary transcriptome study of the ADR1L2 inducible line is currently underway to provide comparative insights.

In addition, we initiated studies to determine whether helper NLR oligomerization influences subcellular localization and protein interactions. For this purpose, inducible fluorescently tagged NRG1.1 and ADR1L2 constructs were developed, and stable transgenic lines have been generated for future imaging and interaction studies.

2) Development of a Nuclear/Cytoplasmic Dual Calcium Indicator System
To visualize calcium dynamics in vivo at a subcellular resolution, we designed a dual calcium indicator system incorporating nucleus-localized and cytoplasm-localized fluorescent calcium sensors. These sensors were codon-optimized for efficient expression in Arabidopsis thaliana. The final construct has been successfully validated and introduced into wild-type and mutant backgrounds for functional studies. Screening of transgenic lines is in progress to identify optimal fluorescence expression for live imaging of calcium signaling dynamics during immune responses.

These efforts aim to provide novel insights into the regulation of calcium signalling during plant immune activation, contributing to a deeper understanding of how intracellular immune receptors orchestrate defense responses at the molecular and cellular levels.

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)

Our research advances the understanding of plant immune mechanisms by integrating molecular genetics, time-series transcriptomics, and real-time calcium signaling analysis. These findings provide novel insights into plant defence responses and offer practical applications for improving crop resilience.

Key Scientific Advances
1. Unraveling Helper NLR Functions: We identified distinct immune signalling pathways regulated by different helper NLRs, highlighting their specific roles in plant defence.
2. Real-Time Calcium Imaging in Immunity: The development of a nuclear/cytoplasmic dual calcium indicator system enables high-resolution tracking of calcium flux during immune activation, providing new insights into intracellular signaling dynamics.
3. Multi-Omics Integration: By combining transcriptomic, proteomic, and calcium-imaging data, we refined our understanding of immune responses, enhancing the potential for engineering disease-resistant crops.

Potential Applications and Impact
1. Breeding Disease-Resistant Crops: Identifying key immune regulators and calcium signaling components offers new targets for developing more resilient crop varieties.
2. Synthetic Biology for Immune Modulation: Insights from immune signalling pathways can inform the design of synthetic immune circuits, enabling precise control over plant defences.Agricultural and 3. Industrial Use: The transgenic lines and molecular tools developed in this project provide valuable resources for both academic research and commercial crop protection strategies.
4. Regulatory and Policy Implications: Our findings contribute to discussions on the regulation of genome-edited crops, supporting their role in sustainable agriculture.

Future Directions
Future work will focus on validating these findings in diverse crop species, integrating them into breeding programs, and collaborating with industry partners for commercialization. Intellectual property protection and regulatory alignment will be key to ensuring successful implementation. This research bridges fundamental discoveries with applied solutions, supporting the development of more resilient and sustainable agricultural systems.