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.
