Periodic Reporting for period 1 - MAVS in plants (Mitochondrial antiviral signaling in Plants)
Período documentado: 2023-05-01 hasta 2025-04-30
The systemic infections of agronomically important plants by viruses lead to significant economic losses in yield and quality of the crops. These losses are attributed to virus-induced developmental changes in the host plant, exhibited as disease symptoms. This argues for enhancing basic research to better understand and exploit the molecular mechanisms that underpin virus interference with plant development and disease resistance. Rhizomania is one of the most economically devastating disease of sugar beet and is caused by Beet necrotic yellow vein virus (BNYVV). BNYVV is a soil-borne virus transmitted by Polymyxa betae. Emerging evidence indicates that mitochondria plays essential role in plant immunity and thus represents a promising target for the development of sustainable broad- spectrum disease resistance in crops. Although, progress is being made in studies of the mitochondrial signaling in response to abiotic stress and bacterial/fungal pathogen, nothing is known about how plant viruses interfere with mitochondrial signaling. This project builds on the previous findings that BNYVV localize to mitochondria, and this occurrence is related to the assembly of the virus . Moreover, several nuclear genes encoding mitochondrial proteins appear to be repressed by a nuclear-localized BNYVV P25 virulence factor. In animals, mitochondrial signaling in response to virus infection has been well studied, but it has been overlooked in plants and still is an open field to find the potential targets for improved plant fitness against plant viruses. The study has been divided into following three objectives formulated as research questions (Objective1) how BNYVV is perceived by mitochondria, (Objective 2) how mitochondria transduce signal i.e. by production of ROS, which are produced as a result of broken electron transfer chain (ETC) and (Objective 3) does mitochondrial retrograde regulation imparts resistance to virus. The advanced knowledge on the mitochondrial antiviral signaling will have implications for the wider area of virus research and, in the long run, will provide new opportunities for improvement of plant fitness and immunity in agronomic applications.
The project comprehensively investigated the interaction between Beet necrotic yellow vein virus (BNYVV) and plant mitochondria, elucidating how viral infection perturbs mitochondrial homeostasis and triggers defense signaling pathways in Nicotiana benthamiana. The work was organized into several experimental packages addressing reactive oxygen species (ROS) dynamics, viral factor functions, mitochondrial stress responses, mitophagy regulation, and epigenetic modulation under mitochondrial retrograde signaling.
1. ROS Induction During BNYVV Infection
BNYVV infection was found to induce reactive oxygen species (ROS) production in plant cells, as visualized using DCHFA and mito-tracker staining. At 3 days post-infection (dpi), strong ROS signals originated from stomata and distinct dot-like structures at the cell periphery, which did not co-localize with mitochondria, suggesting alternative sources of ROS generation. Expression analysis further revealed that NbNPR1 was upregulated at 3 dpi, consistent with salicylic acid-mediated defense activation. These results establish a clear temporal correlation between viral infection and ROS-dependent immune signaling.
2. Viral P25-Mediated Repression of Host Promoters
The viral pathogenicity factor P25 was shown to repress host BvCyt c promoter activity directly, as demonstrated by dual luciferase and gel retardation assays. P25 bound specifically to the cyt c promoter fragment, confirming its role as a DNA-binding repressor. No direct interaction was detected with BvUGT promoter, suggesting indirect repression mechanisms. Yeast two-hybrid assays revealed no interaction between P25 and mitochondrial proteins (BvCyt c, BvTim13), and virus-induced gene silencing (VIGS) of these host factors did not affect viral accumulation. This constitutes the first evidence that BNYVV P25 functions as a DNA-binding protein capable of modulating host transcription.
3. Mitochondrial Stress and Electron Transport Chain Disruption
BNYVV infection induced mitochondrial stress, marked by the upregulation of NbAOX1a (3–8 fold higher than NbAOX1b), which remained elevated during late infection stages (17 dpi). Concomitantly, cytochrome c (cyt c) release into the cytosol was observed at 17 dpi, coinciding with necrosis onset, indicating mitochondrial dysfunction. GFP-tagged AtTOM7 effectively labeled mitochondria, enabling successful pull-down assays. This represents the first report of cyt c release in response to BNYVV infection, linking viral infection with mitochondrial stress and programmed cell death signaling.
4. Mitophagy and VDAC Induction
Western blot analysis revealed increased levels of the mitochondrial outer membrane protein VDAC at 17 dpi, while the mitochondrial-encoded protein CoxII remained unchanged. These findings suggest that BNYVV does not alter mitophagy but instead induces VDAC expression, likely as a host-driven signal promoting cell death to limit viral spread. The results highlight parallels with animal virus–induced apoptosis, reinforcing the role of mitochondria as central mediators of antiviral defense.
5. Viral Resistance Triggered by Disturbed Mitochondrial Homeostasis
Chemical disruption of the mitochondrial electron transport chain using Antimycin A activated mitochondrial retrograde regulation (MRR), leading to enhanced expression of the mitochondrial stress marker AOX1a and the defense-related protein PR10. Plants pretreated with Antimycin A exhibited significantly reduced BNYVV viral titers, demonstrating that mitochondrial dysfunction primes antiviral immunity. Furthermore, mitochondrial DNA (mtDNA) was detected in the cytosolic fraction at 17 dpi, suggesting its release acts as a damage-associated molecular pattern (DAMP) to trigger immune signaling.
6. Epigenetic Regulation Under Mitochondrial Stress
Chromatin immunoprecipitation (ChIP) analysis following Antimycin A treatment revealed a reduction in the repressive histone mark H3K9me2 across defense-related genes, indicating enhanced chromatin accessibility. The activating mark H3K4me3 decreased for most genes except NPR1, which maintained higher levels consistent with its pivotal role in salicylic acid–mediated immunity. These results demonstrate that MRR induces selective chromatin remodeling, fine-tuning defense gene transcription under mitochondrial stress.
Overall Achievements
• Discovered that viral P25 acts as a DNA-binding repressor of host promoters, a novel viral regulatory mechanism.
• Provided the first evidence of cytochrome c release during BNYVV infection, establishing a link between viral pathogenesis and mitochondrial apoptosis-like processes.
• Demonstrated that mitochondrial stress triggers antiviral defense through retrograde signaling, epigenetic modulation, and selective gene activation.
• Highlighted the non-involvement of mitophagy but a potential signaling role for VDAC induction during infection.
• Uncovered an epigenetic layer of defense regulation, connecting mitochondrial function with chromatin remodeling.
In conclusion, the project elucidates how BNYVV manipulates and is countered by mitochondrial signaling networks in plants, offering new insights into plant–virus interactions and mitochondrial involvement in antiviral defense.
1. ROS Induction During BNYVV Infection
BNYVV infection was found to induce reactive oxygen species (ROS) production in plant cells, as visualized using DCHFA and mito-tracker staining. At 3 days post-infection (dpi), strong ROS signals originated from stomata and distinct dot-like structures at the cell periphery, which did not co-localize with mitochondria, suggesting alternative sources of ROS generation. Expression analysis further revealed that NbNPR1 was upregulated at 3 dpi, consistent with salicylic acid-mediated defense activation. These results establish a clear temporal correlation between viral infection and ROS-dependent immune signaling.
2. Viral P25-Mediated Repression of Host Promoters
The viral pathogenicity factor P25 was shown to repress host BvCyt c promoter activity directly, as demonstrated by dual luciferase and gel retardation assays. P25 bound specifically to the cyt c promoter fragment, confirming its role as a DNA-binding repressor. No direct interaction was detected with BvUGT promoter, suggesting indirect repression mechanisms. Yeast two-hybrid assays revealed no interaction between P25 and mitochondrial proteins (BvCyt c, BvTim13), and virus-induced gene silencing (VIGS) of these host factors did not affect viral accumulation. This constitutes the first evidence that BNYVV P25 functions as a DNA-binding protein capable of modulating host transcription.
3. Mitochondrial Stress and Electron Transport Chain Disruption
BNYVV infection induced mitochondrial stress, marked by the upregulation of NbAOX1a (3–8 fold higher than NbAOX1b), which remained elevated during late infection stages (17 dpi). Concomitantly, cytochrome c (cyt c) release into the cytosol was observed at 17 dpi, coinciding with necrosis onset, indicating mitochondrial dysfunction. GFP-tagged AtTOM7 effectively labeled mitochondria, enabling successful pull-down assays. This represents the first report of cyt c release in response to BNYVV infection, linking viral infection with mitochondrial stress and programmed cell death signaling.
4. Mitophagy and VDAC Induction
Western blot analysis revealed increased levels of the mitochondrial outer membrane protein VDAC at 17 dpi, while the mitochondrial-encoded protein CoxII remained unchanged. These findings suggest that BNYVV does not alter mitophagy but instead induces VDAC expression, likely as a host-driven signal promoting cell death to limit viral spread. The results highlight parallels with animal virus–induced apoptosis, reinforcing the role of mitochondria as central mediators of antiviral defense.
5. Viral Resistance Triggered by Disturbed Mitochondrial Homeostasis
Chemical disruption of the mitochondrial electron transport chain using Antimycin A activated mitochondrial retrograde regulation (MRR), leading to enhanced expression of the mitochondrial stress marker AOX1a and the defense-related protein PR10. Plants pretreated with Antimycin A exhibited significantly reduced BNYVV viral titers, demonstrating that mitochondrial dysfunction primes antiviral immunity. Furthermore, mitochondrial DNA (mtDNA) was detected in the cytosolic fraction at 17 dpi, suggesting its release acts as a damage-associated molecular pattern (DAMP) to trigger immune signaling.
6. Epigenetic Regulation Under Mitochondrial Stress
Chromatin immunoprecipitation (ChIP) analysis following Antimycin A treatment revealed a reduction in the repressive histone mark H3K9me2 across defense-related genes, indicating enhanced chromatin accessibility. The activating mark H3K4me3 decreased for most genes except NPR1, which maintained higher levels consistent with its pivotal role in salicylic acid–mediated immunity. These results demonstrate that MRR induces selective chromatin remodeling, fine-tuning defense gene transcription under mitochondrial stress.
Overall Achievements
• Discovered that viral P25 acts as a DNA-binding repressor of host promoters, a novel viral regulatory mechanism.
• Provided the first evidence of cytochrome c release during BNYVV infection, establishing a link between viral pathogenesis and mitochondrial apoptosis-like processes.
• Demonstrated that mitochondrial stress triggers antiviral defense through retrograde signaling, epigenetic modulation, and selective gene activation.
• Highlighted the non-involvement of mitophagy but a potential signaling role for VDAC induction during infection.
• Uncovered an epigenetic layer of defense regulation, connecting mitochondrial function with chromatin remodeling.
In conclusion, the project elucidates how BNYVV manipulates and is countered by mitochondrial signaling networks in plants, offering new insights into plant–virus interactions and mitochondrial involvement in antiviral defense.
The project has contributed to the development and application of several cutting-edge molecular and cell biology techniques in plant systems. Notably, it established a rapid and efficient mitochondrial pull-down assay that enables the detection of cytochrome c release into the cytosol, a key marker of programmed cell death. This method provides a significant improvement in convenience, reproducibility, and time efficiency compared with conventional mitochondrial isolation procedures.
Furthermore, the study achieved several firsts in plant virology and cell biology. It enabled the detection of cytosolic mitochondrial DNA (mtDNA) and viral RNA localized on the mitochondrial surface, providing direct evidence for the involvement of mitochondria in antiviral signaling. In addition, proteomic analysis of the pulled-down mitochondria led to the identification of novel host and viral proteins associated with mitochondria during infection. This represents the first comprehensive study in plants to apply a streamlined mitochondria pull-down approach to elucidate organelle-associated immune processes.
The current work exemplifies how investment in fundamental plant pathology yields transformative outcomes across economic, societal, and industrial domains.
• Economic impact: Beet necrotic yellow vein virus (BNYVV) causes rhizomania, one of the most destructive viral diseases of sugar beet, leading to yield losses that can reach up to 100% under severe infection. By uncovering a previously unknown mitochondria-associated antiviral signaling pathway, this study opens new avenues for developing durable antiviral strategies and breeding programs aimed at resistance enhancement.
• Societal impact: Sugar beet is a major industrial crop, contributing approximately 20% of global sucrose production. Protecting this crop from BNYVV ensures a stable and sustainable sugar supply chain, thereby supporting food security, market stability, and rural livelihoods. Stabilizing sugar prices also mitigates food cost volatility, which disproportionately affects low-income populations.
Integrated Pest Management (IPM): Detailed knowledge of how BNYVV interacts with its host plants, policymakers can promote more effective IPM strategies that encourage the use of resistant crops to support the sustainable use of pesticides.
• European Green Deal- Sustainable agriculture: Understanding BNYVV infection mechanisms helps develop resistant sugar beet varieties, reducing reliance on pesticides and chemical soil treatments—key goals of the Green Deal’s Farm to Fork Strategy.
• Promoting the European way of life - Food security and safety: Protecting sugar beet—a major European crop—ensures a reliable food and bioenergy supply, reflecting the EU’s commitment to high standards of food quality and sustainability. Education and knowledge sharing: Research and innovation in plant virology strengthen European academic excellence and scientific literacy, integral to a knowledge-based society. Environmental stewardship: Sustainable control of BNYVV supports Europe’s values of responsible resource management and environmental care.
• A stronger Europe in the world - Global leadership in sustainable agriculture: Advancing molecular understanding of plant–virus interactions positions Europe as a global leader in crop protection science and biotechnology. Export competitiveness: Healthier beet crops enhance the competitiveness of European agricultural exports, supporting economic diplomacy and trade balance.
Furthermore, the study achieved several firsts in plant virology and cell biology. It enabled the detection of cytosolic mitochondrial DNA (mtDNA) and viral RNA localized on the mitochondrial surface, providing direct evidence for the involvement of mitochondria in antiviral signaling. In addition, proteomic analysis of the pulled-down mitochondria led to the identification of novel host and viral proteins associated with mitochondria during infection. This represents the first comprehensive study in plants to apply a streamlined mitochondria pull-down approach to elucidate organelle-associated immune processes.
The current work exemplifies how investment in fundamental plant pathology yields transformative outcomes across economic, societal, and industrial domains.
• Economic impact: Beet necrotic yellow vein virus (BNYVV) causes rhizomania, one of the most destructive viral diseases of sugar beet, leading to yield losses that can reach up to 100% under severe infection. By uncovering a previously unknown mitochondria-associated antiviral signaling pathway, this study opens new avenues for developing durable antiviral strategies and breeding programs aimed at resistance enhancement.
• Societal impact: Sugar beet is a major industrial crop, contributing approximately 20% of global sucrose production. Protecting this crop from BNYVV ensures a stable and sustainable sugar supply chain, thereby supporting food security, market stability, and rural livelihoods. Stabilizing sugar prices also mitigates food cost volatility, which disproportionately affects low-income populations.
Integrated Pest Management (IPM): Detailed knowledge of how BNYVV interacts with its host plants, policymakers can promote more effective IPM strategies that encourage the use of resistant crops to support the sustainable use of pesticides.
• European Green Deal- Sustainable agriculture: Understanding BNYVV infection mechanisms helps develop resistant sugar beet varieties, reducing reliance on pesticides and chemical soil treatments—key goals of the Green Deal’s Farm to Fork Strategy.
• Promoting the European way of life - Food security and safety: Protecting sugar beet—a major European crop—ensures a reliable food and bioenergy supply, reflecting the EU’s commitment to high standards of food quality and sustainability. Education and knowledge sharing: Research and innovation in plant virology strengthen European academic excellence and scientific literacy, integral to a knowledge-based society. Environmental stewardship: Sustainable control of BNYVV supports Europe’s values of responsible resource management and environmental care.
• A stronger Europe in the world - Global leadership in sustainable agriculture: Advancing molecular understanding of plant–virus interactions positions Europe as a global leader in crop protection science and biotechnology. Export competitiveness: Healthier beet crops enhance the competitiveness of European agricultural exports, supporting economic diplomacy and trade balance.