Periodic Reporting for period 1 - ViROSCa (Deciphering the roles of reactive oxygen species and calcium during viral infection in Arabidopsis)
Período documentado: 2023-10-01 hasta 2025-09-30
Resumen del contexto y de los objetivos generales del proyecto
Viral diseases are a major and growing threat to global food security, responsible for severe crop losses and rising pesticide use. One such emerging pathogen is Plantago asiatica mosaic virus (PlAMV), a potexvirus capable of infecting a wide range of hosts and spreading rapidly between plant cells. Once a virus enters a leaf, it moves from cell to cell through tiny channels called plasmodesmata, eventually reaching the whole plant. How plants detect viral invasion at this early stage — and how viruses overcome these defenses — remains poorly understood.
The ViROSCa project addresses this knowledge gap by investigating how PlAMV infection affects two critical host defense signals: calcium (Ca²⁺) and reactive oxygen species (ROS). These signals act as early messengers in plant immunity, triggering cellular responses that can contain or slow down pathogen spread. The project’s main objective is to identify how these signals are activated and coordinated at the cell membrane during infection, which genes are involved, and how this knowledge can be used to support the development of more virus-resistant and resilient crops.
This research contributes directly to European Union priorities, including the Green Deal and Farm to Fork strategies, by providing scientific knowledge that can help reduce dependence on chemical pesticides and enable more sustainable agricultural practices.
Work performed and main achievements
The project combined transcriptomic analysis, live-cell imaging, and genetic approaches to study early host responses to PlAMV infection in Arabidopsis thaliana.
• Gene expression profiling: Infection by PlAMV triggered strong activation of membrane-associated genes involved in calcium influx and ROS metabolism.
• Calcium imaging: Using a fluorescent calcium biosensor (R-GECO1), the team visualized a rapid wave of calcium release starting at the infection site and spreading to neighboring cells. This calcium signal occurred before the virus accumulated, indicating it is an early step in infection.
• Functional genetics: Mutants lacking GLR3, CNGC, or CPK3 allowed the virus to spread faster between cells, confirming that these genes are key components of the antiviral response.
• ROS imaging: A second biosensor (HyPer7-kRas) revealed a striking spatial pattern in ROS accumulation: ROS were suppressed in infected cells but increased in surrounding cells, creating a defensive perimeter. This defense pattern required the genes RBOHD, CPK3, and MOCA1.
• Signaling hierarchy: RBOHD-derived ROS production acted upstream of MOCA1-mediated lipid remodeling, and MOCA1 function was dominant over CPK3. This revealed a hierarchical membrane signaling cascade coordinating early antiviral defense.
• Systemic control: RBOHD was essential not only for local defense but also for preventing the virus from spreading through the whole plant, while MOCA1 played a more local role at the membrane.
• PTI independence: Core pattern-triggered immunity (PTI) components such as SERK co-receptors and HIR proteins were not required for PlAMV restriction, revealing a distinct, PTI-independent signaling pathway.
This integrated approach uncovered how viruses interact with host membrane signaling and how plants activate targeted defenses at infection sites.
Progress beyond the state of the art and expected results
Before this project, the molecular events linking calcium influx, ROS production, and viral movement were not clearly defined. ViROSCa provides the first detailed picture of this interaction:
• It identified key host genes (GLR3, CNGC, CPK3, RBOHD/F, MOCA1) that control early defense signaling and viral spread.
• It demonstrated that Ca²⁺ elevation is an early infection signal, preceding virus movement.
• It revealed a spatial ROS defense gradient around infected cells and its genetic dependencies.
• It mapped the signaling hierarchy linking Ca²⁺ channels, ROS-producing enzymes, and membrane lipid remodeling.
• It showed that this defense mechanism functions independently of classical PTI pathways.
These discoveries set the stage for new strategies to enhance natural antiviral defenses in crops through breeding or biotechnological approaches. They also provide new biosensor tools and imaging methods that can be adopted by other research groups and industries.
Societal and economic impact
Plant viruses are a major driver of crop loss and pesticide use worldwide. By uncovering how early membrane signaling restricts viral spread, ViROSCa provides a biological alternative to chemical control.
This work directly supports:
• EU Green Deal and Farm to Fork Strategy, by reducing reliance on pesticides and promoting resilient agriculture.
• EU Plant Health Regulations, through identifying new genetic markers for resistance.
• Horizon Europe goals (Cluster 6), by linking fundamental research with applied agricultural innovation.
The project also trained a highly skilled researcher in advanced imaging and molecular techniques, strengthened international collaborations (France–UK), and generated tools and knowledge that will remain available to the scientific community. In the longer term, these results may contribute to virus-resistant crops, improved food security, and more sustainable farming systems in Europe and globally.
The ViROSCa project addresses this knowledge gap by investigating how PlAMV infection affects two critical host defense signals: calcium (Ca²⁺) and reactive oxygen species (ROS). These signals act as early messengers in plant immunity, triggering cellular responses that can contain or slow down pathogen spread. The project’s main objective is to identify how these signals are activated and coordinated at the cell membrane during infection, which genes are involved, and how this knowledge can be used to support the development of more virus-resistant and resilient crops.
This research contributes directly to European Union priorities, including the Green Deal and Farm to Fork strategies, by providing scientific knowledge that can help reduce dependence on chemical pesticides and enable more sustainable agricultural practices.
Work performed and main achievements
The project combined transcriptomic analysis, live-cell imaging, and genetic approaches to study early host responses to PlAMV infection in Arabidopsis thaliana.
• Gene expression profiling: Infection by PlAMV triggered strong activation of membrane-associated genes involved in calcium influx and ROS metabolism.
• Calcium imaging: Using a fluorescent calcium biosensor (R-GECO1), the team visualized a rapid wave of calcium release starting at the infection site and spreading to neighboring cells. This calcium signal occurred before the virus accumulated, indicating it is an early step in infection.
• Functional genetics: Mutants lacking GLR3, CNGC, or CPK3 allowed the virus to spread faster between cells, confirming that these genes are key components of the antiviral response.
• ROS imaging: A second biosensor (HyPer7-kRas) revealed a striking spatial pattern in ROS accumulation: ROS were suppressed in infected cells but increased in surrounding cells, creating a defensive perimeter. This defense pattern required the genes RBOHD, CPK3, and MOCA1.
• Signaling hierarchy: RBOHD-derived ROS production acted upstream of MOCA1-mediated lipid remodeling, and MOCA1 function was dominant over CPK3. This revealed a hierarchical membrane signaling cascade coordinating early antiviral defense.
• Systemic control: RBOHD was essential not only for local defense but also for preventing the virus from spreading through the whole plant, while MOCA1 played a more local role at the membrane.
• PTI independence: Core pattern-triggered immunity (PTI) components such as SERK co-receptors and HIR proteins were not required for PlAMV restriction, revealing a distinct, PTI-independent signaling pathway.
This integrated approach uncovered how viruses interact with host membrane signaling and how plants activate targeted defenses at infection sites.
Progress beyond the state of the art and expected results
Before this project, the molecular events linking calcium influx, ROS production, and viral movement were not clearly defined. ViROSCa provides the first detailed picture of this interaction:
• It identified key host genes (GLR3, CNGC, CPK3, RBOHD/F, MOCA1) that control early defense signaling and viral spread.
• It demonstrated that Ca²⁺ elevation is an early infection signal, preceding virus movement.
• It revealed a spatial ROS defense gradient around infected cells and its genetic dependencies.
• It mapped the signaling hierarchy linking Ca²⁺ channels, ROS-producing enzymes, and membrane lipid remodeling.
• It showed that this defense mechanism functions independently of classical PTI pathways.
These discoveries set the stage for new strategies to enhance natural antiviral defenses in crops through breeding or biotechnological approaches. They also provide new biosensor tools and imaging methods that can be adopted by other research groups and industries.
Societal and economic impact
Plant viruses are a major driver of crop loss and pesticide use worldwide. By uncovering how early membrane signaling restricts viral spread, ViROSCa provides a biological alternative to chemical control.
This work directly supports:
• EU Green Deal and Farm to Fork Strategy, by reducing reliance on pesticides and promoting resilient agriculture.
• EU Plant Health Regulations, through identifying new genetic markers for resistance.
• Horizon Europe goals (Cluster 6), by linking fundamental research with applied agricultural innovation.
The project also trained a highly skilled researcher in advanced imaging and molecular techniques, strengthened international collaborations (France–UK), and generated tools and knowledge that will remain available to the scientific community. In the longer term, these results may contribute to virus-resistant crops, improved food security, and more sustainable farming systems in Europe and globally.
Trabajo realizado desde el comienzo del proyecto hasta el final del período abarcado por el informe y los principales resultados hasta la fecha
The ViROSCa project aimed to elucidate how Plantago asiatica mosaic virus (PlAMV) interacts with host calcium (Ca²⁺) and reactive oxygen species (ROS) signaling pathways at the plasma membrane to facilitate or restrict viral movement. To address this, we combined gene expression analysis, live-cell biosensor imaging, and targeted genetic approaches in Arabidopsis thaliana.
1. Transcriptional reprogramming of Ca²⁺ and ROS pathways during viral infection
Our findings demonstrate that PlAMV infection elicits extensive reprogramming of host calcium (Ca²⁺) and reactive oxygen species (ROS) signaling pathways, suggesting a tight coordination between these two central signaling networks during compatible virus–host interactions. The upregulation of CIPK and OSCA genes, together with GLR-type channels, indicates activation of Ca²⁺ influx and sensing components that likely modulate early stress signaling at the plasma membrane (Yuan et al., 2014; Toyota, 2018). Conversely, the suppression of CPK, CML, and CNGC family members implies viral interference with Ca²⁺-dependent defense activation, possibly to dampen downstream immune responses such as hypersensitive cell death or defense gene expression (Ranty et al., 2016; Bredow & Monaghan, 2019).
Consistent with this, GO enrichment analysis revealed strong overrepresentation of processes linked to ROS biosynthesis and hydrogen peroxide response, aligning with the observed spatial accumulation of ROS in tissues distal to infection foci. This pattern supports a model where PlAMV suppresses local ROS generation at infection sites—potentially to prevent antiviral cell death—while promoting ROS signaling in neighboring cells to facilitate intercellular communication and systemic signaling (Love et al., 2012; Fichman & Mittler, 2020). The elevated expression of ROS-related genes, together with altered Ca²⁺ signaling components, highlights the reciprocal regulation between Ca²⁺ and ROS during viral infection, as Ca²⁺ fluxes are known to activate NADPH oxidases (RBOHs), the main enzymatic source of apoplastic ROS (Kadota et al., 2015; Gilroy et al., 2016).
2. Early Ca²⁺ signaling wave precedes viral accumulation
Using transgenic Arabidopsis lines expressing the cytosolic Ca²⁺ biosensor R-GECO1, we visualized real-time Ca²⁺ dynamics upon PlAMV inoculation. A rapid and transient increase in Ca²⁺ signal was detected at the initial infection site and propagated to neighboring cells before viral GFP fluorescence became visible. This demonstrates that Ca²⁺ elevation is an early event in the infection process, potentially priming cells for virus movement.
3. Identification of Ca²⁺ signaling components restricting viral spread
We performed PlAMV-GFP cell-to-cell movement assays in glr, cpk3, cngc, and hpca1 mutant lines. Mutants lacking GLR, CNGC, or CPK3 exhibited significantly larger infection foci than wild-type controls, indicating enhanced virus movement. No effect was observed in hpca1 mutants, demonstrating pathway specificity. These results identify a subset of Ca²⁺ signaling genes that act as negative regulators of viral spread.
4. Spatial organization of ROS during infection
To map ROS responses, we developed transgenic plants expressing the plasma membrane-targeted HyPer7-kRas biosensor. PlAMV infection induced a striking spatial pattern: ROS levels were suppressed in infected cells (local area, LA) but elevated in neighboring distal cells (DA). This reveals a defense perimeter around the infection site, likely aimed at limiting viral propagation.
5. Genetic dissection of ROS signaling pathways
ROS accumulation at the PM was lost in moca1, rbohD, and cpk3 mutants, demonstrating that MOCA1 (a lipid remodeling factor), RBOHD (an NADPH oxidase), and CPK3 (a calcium-dependent protein kinase) are required for infection-induced ROS signaling. Movement assays further showed that rbohD and rbohF mutants support larger infection foci, confirming their role in restricting viral spread. In contrast, aquaporin triple mutants (pip) displayed no effect, indicating specificity in ROS regulation.
6. Signaling hierarchy between Ca²⁺, ROS, and lipid remodeling
Epistasis analyses revealed that RBOHD acts upstream of MOCA1, as the rbohD × moca1 double mutant phenocopied the rbohD single mutant. Additionally, MOCA1 was genetically dominant over CPK3, indicating that these pathways act in distinct but interconnected layers. This establishes a hierarchical model in which Ca²⁺-dependent RBOH activation leads to ROS production, which in turn modulates PM lipid organization via MOCA1.
7. Differential roles in local vs systemic defense
Whole-plant imaging demonstrated that rbohD mutants displayed extensive systemic spread of PlAMV, highlighting the importance of RBOHD for both local and long-distance antiviral defense. In contrast, moca1 mutants showed enhanced local spread but reduced systemic movement, indicating a spatially distinct role in viral regulation.
8. PTI pathway components are dispensable for PlAMV restriction
Mutants in canonical PTI regulators (e.g. SERK1, BAK1/BKK1, HIR) showed no significant change in viral propagation, demonstrating that PlAMV restriction operates independently of classical pattern-triggered immunity. This identifies a novel defense mechanism distinct from PTI.
Main scientific achievements:
• Generation of stable Arabidopsis biosensor lines for Ca²⁺ and ROS imaging during viral infection.
• Identification of Ca²⁺ signaling components (GLR3, CNGC, CPK3) and ROS regulators (RBOHD/F, MOCA1) that restrict viral movement.
• Discovery of a spatial ROS defense gradient at the plasma membrane surrounding infection foci.
• Definition of a signaling hierarchy linking Ca²⁺ influx and RBOH activation in antiviral defense.
• Demonstration that this mechanism functions independently of canonical PTI.
• Establishment of a robust imaging and genetic platform for future antiviral studies in plants.
1. Transcriptional reprogramming of Ca²⁺ and ROS pathways during viral infection
Our findings demonstrate that PlAMV infection elicits extensive reprogramming of host calcium (Ca²⁺) and reactive oxygen species (ROS) signaling pathways, suggesting a tight coordination between these two central signaling networks during compatible virus–host interactions. The upregulation of CIPK and OSCA genes, together with GLR-type channels, indicates activation of Ca²⁺ influx and sensing components that likely modulate early stress signaling at the plasma membrane (Yuan et al., 2014; Toyota, 2018). Conversely, the suppression of CPK, CML, and CNGC family members implies viral interference with Ca²⁺-dependent defense activation, possibly to dampen downstream immune responses such as hypersensitive cell death or defense gene expression (Ranty et al., 2016; Bredow & Monaghan, 2019).
Consistent with this, GO enrichment analysis revealed strong overrepresentation of processes linked to ROS biosynthesis and hydrogen peroxide response, aligning with the observed spatial accumulation of ROS in tissues distal to infection foci. This pattern supports a model where PlAMV suppresses local ROS generation at infection sites—potentially to prevent antiviral cell death—while promoting ROS signaling in neighboring cells to facilitate intercellular communication and systemic signaling (Love et al., 2012; Fichman & Mittler, 2020). The elevated expression of ROS-related genes, together with altered Ca²⁺ signaling components, highlights the reciprocal regulation between Ca²⁺ and ROS during viral infection, as Ca²⁺ fluxes are known to activate NADPH oxidases (RBOHs), the main enzymatic source of apoplastic ROS (Kadota et al., 2015; Gilroy et al., 2016).
2. Early Ca²⁺ signaling wave precedes viral accumulation
Using transgenic Arabidopsis lines expressing the cytosolic Ca²⁺ biosensor R-GECO1, we visualized real-time Ca²⁺ dynamics upon PlAMV inoculation. A rapid and transient increase in Ca²⁺ signal was detected at the initial infection site and propagated to neighboring cells before viral GFP fluorescence became visible. This demonstrates that Ca²⁺ elevation is an early event in the infection process, potentially priming cells for virus movement.
3. Identification of Ca²⁺ signaling components restricting viral spread
We performed PlAMV-GFP cell-to-cell movement assays in glr, cpk3, cngc, and hpca1 mutant lines. Mutants lacking GLR, CNGC, or CPK3 exhibited significantly larger infection foci than wild-type controls, indicating enhanced virus movement. No effect was observed in hpca1 mutants, demonstrating pathway specificity. These results identify a subset of Ca²⁺ signaling genes that act as negative regulators of viral spread.
4. Spatial organization of ROS during infection
To map ROS responses, we developed transgenic plants expressing the plasma membrane-targeted HyPer7-kRas biosensor. PlAMV infection induced a striking spatial pattern: ROS levels were suppressed in infected cells (local area, LA) but elevated in neighboring distal cells (DA). This reveals a defense perimeter around the infection site, likely aimed at limiting viral propagation.
5. Genetic dissection of ROS signaling pathways
ROS accumulation at the PM was lost in moca1, rbohD, and cpk3 mutants, demonstrating that MOCA1 (a lipid remodeling factor), RBOHD (an NADPH oxidase), and CPK3 (a calcium-dependent protein kinase) are required for infection-induced ROS signaling. Movement assays further showed that rbohD and rbohF mutants support larger infection foci, confirming their role in restricting viral spread. In contrast, aquaporin triple mutants (pip) displayed no effect, indicating specificity in ROS regulation.
6. Signaling hierarchy between Ca²⁺, ROS, and lipid remodeling
Epistasis analyses revealed that RBOHD acts upstream of MOCA1, as the rbohD × moca1 double mutant phenocopied the rbohD single mutant. Additionally, MOCA1 was genetically dominant over CPK3, indicating that these pathways act in distinct but interconnected layers. This establishes a hierarchical model in which Ca²⁺-dependent RBOH activation leads to ROS production, which in turn modulates PM lipid organization via MOCA1.
7. Differential roles in local vs systemic defense
Whole-plant imaging demonstrated that rbohD mutants displayed extensive systemic spread of PlAMV, highlighting the importance of RBOHD for both local and long-distance antiviral defense. In contrast, moca1 mutants showed enhanced local spread but reduced systemic movement, indicating a spatially distinct role in viral regulation.
8. PTI pathway components are dispensable for PlAMV restriction
Mutants in canonical PTI regulators (e.g. SERK1, BAK1/BKK1, HIR) showed no significant change in viral propagation, demonstrating that PlAMV restriction operates independently of classical pattern-triggered immunity. This identifies a novel defense mechanism distinct from PTI.
Main scientific achievements:
• Generation of stable Arabidopsis biosensor lines for Ca²⁺ and ROS imaging during viral infection.
• Identification of Ca²⁺ signaling components (GLR3, CNGC, CPK3) and ROS regulators (RBOHD/F, MOCA1) that restrict viral movement.
• Discovery of a spatial ROS defense gradient at the plasma membrane surrounding infection foci.
• Definition of a signaling hierarchy linking Ca²⁺ influx and RBOH activation in antiviral defense.
• Demonstration that this mechanism functions independently of canonical PTI.
• Establishment of a robust imaging and genetic platform for future antiviral studies in plants.
Avances que van más allá del estado de la técnica e impacto potencial esperado (incluida la repercusión socioeconómica y las implicaciones sociales más amplias del proyecto hasta la fecha)
Before the start of the ViROSCa project, the molecular mechanisms linking calcium (Ca²⁺) signaling, reactive oxygen species (ROS) production, and viral movement in plants were largely undefined. Previous studies had suggested that both signals are involved in plant defense, but their temporal sequence, spatial organization, and genetic control during viral infection remained unknown.
ViROSCa has made several key scientific advances that significantly push the field beyond the current state of the art:
1. Early signaling events during virus infection
The project provides the first evidence that cytosolic calcium signaling is triggered rapidly and locally after PlAMV entry, preceding viral accumulation. This was demonstrated through live-cell imaging with the R-GECO1 biosensor, revealing calcium waves initiating at infection sites and propagating to neighboring cells. This early signaling defines a new temporal framework for antiviral defense.
2. Identification of key signaling genes restricting virus movement
Functional genetics showed that the GLR3, CNGC, and CPK3 genes are essential for restricting PlAMV spread between cells. This directly implicates specific Ca²⁺ influx channels and kinases in antiviral signaling, identifying new molecular targets for future breeding and biotechnology efforts.
3. Discovery of a spatial ROS defense pattern
Using a plasma membrane–targeted ROS biosensor (HyPer7-kRas), we discovered a distinct ROS defense gradient: infected cells suppress ROS, while surrounding cells exhibit elevated ROS accumulation, creating a protective perimeter. This spatial organization of ROS during viral infection had not been previously described in plants.
4. Signaling hierarchy linking calcium, ROS, and lipid remodeling
Epistasis analysis revealed that RBOHD-derived ROS production acts upstream of MOCA1-dependent lipid remodeling, and MOCA1 is genetically dominant over CPK3. This established a clear hierarchical cascade:
Ca²⁺ → CPK3 → RBOHD (ROS) → MOCA1 (Sphingolipid regulator).
This is the first detailed molecular model connecting these pathways in antiviral defense.
5. Local vs systemic defense mechanisms
The project demonstrated that RBOHD is required for both local containment and systemic restriction of PlAMV, whereas MOCA1 is mainly involved in local signaling. This functional differentiation provides a mechanistic basis for designing resistance strategies that combine early local containment and systemic immunity.
6. PTI-independent antiviral pathway
Unlike many other pathogens, PlAMV does not depend on classical pattern-triggered immunity (PTI) signaling. Mutants in SERK co-receptors and HIR proteins showed no significant phenotype. This reveals an alternative immune route that could be exploited to enhance resistance in crops where PTI is less effective.
Potential impacts and future uptake
The results open up several avenues for application and innovation:
• Crop improvement: The identified signaling components (GLR3, CNGC, CPK3, RBOHD, MOCA1) represent potential targets for molecular breeding or gene editing to generate virus-resistant crop varieties.
• Non-chemical protection strategies: By enhancing natural antiviral signaling, it may be possible to reduce pesticide use, supporting EU sustainability goals.
• Technological platform: The Ca²⁺ and ROS biosensor imaging pipelines developed in this project offer transferable research tools for studying early immune signaling in other crop-pathogen systems.
• Mechanistic foundation for innovation: The defined signaling hierarchy provides a biological blueprint for designing immune modulators, biostimulants, or novel resistance markers.
Key needs for further uptake
To maximize the impact of these findings, the following next steps are recommended:
1. Further research:
o Translation of these findings into major crop species (e.g. tomato, potato, rice).
o Exploration of how these pathways interact with other plant defense mechanisms and environmental factors.
2. Demonstration and validation:
o Testing gene-edited or marker-assisted breeding lines under greenhouse and field conditions.
o Assessing performance against multiple virus species to confirm broad-spectrum resistance.
3. Innovation and IP strategy:
o Protection of key molecular targets and biosensor technologies through intellectual property frameworks where appropriate.
o Partnerships with breeding companies and biostimulant developers to transfer knowledge to practice.
4. Policy and standardization support:
o Integration of biological resistance strategies into EU plant health and integrated pest management (IPM) policies.
o Supporting regulatory frameworks that facilitate the adoption of resistant varieties and new sustainable plant protection tools.
ViROSCa delivers a fundamental shift in how we understand early plant antiviral defense, establishing a new signaling framework that links calcium influx, ROS production, and lipid remodeling. These discoveries have high translational potential for agriculture, support EU Green Deal and Farm to Fork goals, and provide a strong foundation for future research, innovation, and sustainable crop protection.
ViROSCa has made several key scientific advances that significantly push the field beyond the current state of the art:
1. Early signaling events during virus infection
The project provides the first evidence that cytosolic calcium signaling is triggered rapidly and locally after PlAMV entry, preceding viral accumulation. This was demonstrated through live-cell imaging with the R-GECO1 biosensor, revealing calcium waves initiating at infection sites and propagating to neighboring cells. This early signaling defines a new temporal framework for antiviral defense.
2. Identification of key signaling genes restricting virus movement
Functional genetics showed that the GLR3, CNGC, and CPK3 genes are essential for restricting PlAMV spread between cells. This directly implicates specific Ca²⁺ influx channels and kinases in antiviral signaling, identifying new molecular targets for future breeding and biotechnology efforts.
3. Discovery of a spatial ROS defense pattern
Using a plasma membrane–targeted ROS biosensor (HyPer7-kRas), we discovered a distinct ROS defense gradient: infected cells suppress ROS, while surrounding cells exhibit elevated ROS accumulation, creating a protective perimeter. This spatial organization of ROS during viral infection had not been previously described in plants.
4. Signaling hierarchy linking calcium, ROS, and lipid remodeling
Epistasis analysis revealed that RBOHD-derived ROS production acts upstream of MOCA1-dependent lipid remodeling, and MOCA1 is genetically dominant over CPK3. This established a clear hierarchical cascade:
Ca²⁺ → CPK3 → RBOHD (ROS) → MOCA1 (Sphingolipid regulator).
This is the first detailed molecular model connecting these pathways in antiviral defense.
5. Local vs systemic defense mechanisms
The project demonstrated that RBOHD is required for both local containment and systemic restriction of PlAMV, whereas MOCA1 is mainly involved in local signaling. This functional differentiation provides a mechanistic basis for designing resistance strategies that combine early local containment and systemic immunity.
6. PTI-independent antiviral pathway
Unlike many other pathogens, PlAMV does not depend on classical pattern-triggered immunity (PTI) signaling. Mutants in SERK co-receptors and HIR proteins showed no significant phenotype. This reveals an alternative immune route that could be exploited to enhance resistance in crops where PTI is less effective.
Potential impacts and future uptake
The results open up several avenues for application and innovation:
• Crop improvement: The identified signaling components (GLR3, CNGC, CPK3, RBOHD, MOCA1) represent potential targets for molecular breeding or gene editing to generate virus-resistant crop varieties.
• Non-chemical protection strategies: By enhancing natural antiviral signaling, it may be possible to reduce pesticide use, supporting EU sustainability goals.
• Technological platform: The Ca²⁺ and ROS biosensor imaging pipelines developed in this project offer transferable research tools for studying early immune signaling in other crop-pathogen systems.
• Mechanistic foundation for innovation: The defined signaling hierarchy provides a biological blueprint for designing immune modulators, biostimulants, or novel resistance markers.
Key needs for further uptake
To maximize the impact of these findings, the following next steps are recommended:
1. Further research:
o Translation of these findings into major crop species (e.g. tomato, potato, rice).
o Exploration of how these pathways interact with other plant defense mechanisms and environmental factors.
2. Demonstration and validation:
o Testing gene-edited or marker-assisted breeding lines under greenhouse and field conditions.
o Assessing performance against multiple virus species to confirm broad-spectrum resistance.
3. Innovation and IP strategy:
o Protection of key molecular targets and biosensor technologies through intellectual property frameworks where appropriate.
o Partnerships with breeding companies and biostimulant developers to transfer knowledge to practice.
4. Policy and standardization support:
o Integration of biological resistance strategies into EU plant health and integrated pest management (IPM) policies.
o Supporting regulatory frameworks that facilitate the adoption of resistant varieties and new sustainable plant protection tools.
ViROSCa delivers a fundamental shift in how we understand early plant antiviral defense, establishing a new signaling framework that links calcium influx, ROS production, and lipid remodeling. These discoveries have high translational potential for agriculture, support EU Green Deal and Farm to Fork goals, and provide a strong foundation for future research, innovation, and sustainable crop protection.