Plant viruses, obligate parasites, severely threaten crop production. Many rely on hemipteran insect vectors (whiteflies, aphids, leafhoppers) for inter-plant transmission. The vector ingests the virus, then transmits it during feeding. This intricate interaction between insect, virus, and plant determines transmission efficiency. Plant responses to hemipteran infestation include activation of the SA signaling pathway, crucial for antiviral defenses. However, how plant viruses overcome these defenses remains largely unknown. Viral proteins often manipulate plant processes, including immunity, by targeting hormonal pathways. For example, begomovirus betasatellites' βC1 protein downregulates JA signaling to enhance vector efficiency. This study aimed to determine whether and how viruses adapt to hemipteran-induced SA-mediated defenses, focusing on the interaction between begomoviruses, their associated betasatellites, and their whitefly vector.

Previous research has established the importance of arthropod vectors in plant virus transmission and the role of plant hormones, particularly SA and JA, in plant defense responses to both insects and viruses. Studies have shown that some viral proteins can suppress plant immunity by targeting hormonal pathways. For instance, the betasatellite-encoded βC1 protein has been shown to suppress JA signaling, promoting vector efficiency. However, the mechanisms by which plant viruses counteract the SA-mediated antiviral defenses induced by hemipteran vectors remained largely unexplored prior to this study.

The researchers investigated the interaction between the whitefly vector *Bemisia tabaci*, tobacco (*Nicotiana tabacum*) or *N. benthamiana* plants, tobacco curly shoot virus (TbCSV), and its associated betasatellite (TbCSB). They used various techniques, including: 1. **Whitefly infestation experiments:** Plants were infested with whiteflies for 48 hours to induce SA accumulation. Subsequently, plants were inoculated with TbCSV, TbCSV+TbCSB, or TbCSV+mutant TbCSB (lacking βC1). 2. **SA treatment:** Plants were sprayed with various concentrations of SA to mimic whitefly-induced SA accumulation and study the effects on virus infection. 3. **Transgenic plants:** *N. benthamiana* plants expressing the TbCSB βC1 protein or the *NahG* gene (which degrades SA) were used to investigate the role of βC1 and SA in antiviral resistance. 4. **Transcriptomic analysis:** Gene expression levels of SA-responsive genes (PR1a, PR2) were analyzed to assess SA signaling activation. 5. **Biochemical analyses:** Phytohormone levels (SA, JA, JA-Ile) were quantified using HPLC-MS. 6. **Virological analyses:** Virus titers were measured using qPCR. 7. **Protein-protein interaction studies:** GST pull-down assays, BiFC assays, and yeast two-hybrid assays were employed to identify proteins interacting with βC1 and examine the interaction between βC1, NbHSP90s, and NbNPR3. 8. **Semi-in vivo and in vitro protein degradation assays:** To examine the effect of βC1 on NbNPR3 degradation, researchers used cycloheximide (CHX) to inhibit protein synthesis and analyzed NbNPR3 levels in the presence or absence of SA and βC1. They also used proteasome inhibitors to assess the role of the proteasome in NbNPR3 degradation. 9. **Virus-induced gene silencing (VIGS):** To explore the roles of NbHSP90s and NbNPR3 in βC1-mediated suppression of SA signaling, VIGS was used to silence their expression in *N. benthamiana* plants. 10. **Similar experiments were conducted with aphid-borne viruses (CMV and TuMV), focusing on the interaction of their viral proteins (2b and HC-Pro, respectively) with NbHSP90s and NbNPR3.**

The study revealed the following key findings: 1. **Whitefly infestation significantly increased SA accumulation in both tobacco and *N. benthamiana* plants.** This increased SA enhanced plant resistance to TbCSV. 2. **Co-infection with TbCSV and TbCSB significantly reduced the whitefly-induced increase in plant resistance to TbCSV.** The reduction in resistance was associated with lower levels of SA accumulation. 3. **Exogenous SA application enhanced plant resistance to TbCSV but less so when TbCSB was present.** This confirmed the role of SA in antiviral defense and the ability of TbCSB to suppress this defense. 4. **The βC1 protein encoded by TbCSB was crucial for mitigating the negative effects of SA on begomovirus infection.** Plants expressing βC1 showed reduced resistance to TbCSV even under SA treatment. 5. **βC1 interacted with NbHSP90s, molecular chaperones involved in protein folding and stability.** BiFC, yeast two-hybrid, and co-immunoprecipitation (Co-IP) assays confirmed these interactions. 6. **NbHSP90s interacted with NbNPR3, a negative regulator of SA signaling.** This interaction was also demonstrated through BiFC and Co-IP assays. 7. **βC1 suppressed SA signaling by inhibiting SA-induced degradation of NbNPR3 in an NbHSP90-dependent manner.** The absence of NbHSP90s reversed the suppressive effect of βC1 on SA signaling. 8. **SA-induced NbNPR3 degradation was proteasome-dependent, and βC1 inhibited this degradation.** 9. **Viral proteins from aphid-borne viruses (CMV 2b and TuMV HC-Pro) also interacted with NbHSP90s and inhibited SA-induced NbNPR3 degradation**, suggesting a conserved mechanism for suppressing SA signaling across different virus families. 10. **A proposed model for the interaction shows that whitefly infestation induces SA accumulation, leading to NPR3 degradation and activation of antiviral defenses. However, viral proteins (βC1, 2b, HC-Pro) interact with HSP90s, preventing NbNPR3 degradation and suppressing SA signaling, promoting viral replication.**

This study uncovered a novel mechanism employed by begomoviruses and other plant viruses to evade the plant's SA-mediated antiviral defense triggered by hemipteran vector infestation. The viral proteins (βC1, CMV 2b, TuMV HC-Pro) target the HSP90-NPR3 interaction, a critical node in SA signaling, to maintain NbNPR3 stability and suppress SA-mediated immunity. This is a significant advancement in understanding plant-virus-vector interactions, as it identifies a conserved strategy across different virus families for manipulating host defenses. The findings highlight the intricate interplay between plant immunity, viral counter-defense strategies, and vector-mediated virus transmission. The evolutionary advantage of this strategy is likely driven by the selective pressure to overcome the host's SA-mediated antiviral response, facilitating efficient virus transmission by hemipteran vectors.

This research demonstrates that betasatellite-encoded βC1 proteins and other viral proteins from different virus families employ a conserved mechanism to suppress SA-mediated antiviral defenses in plants. By interacting with NbHSP90s and modulating the stability of the SA signaling repressor NbNPR3, these viral proteins effectively counter the plant's defense response triggered by hemipteran vector infestation. Future research could explore the molecular details of the interactions between viral proteins, HSP90s, and NbNPR3, investigating potential modifications or conformational changes induced by the viral proteins. Expanding the study to other hemipteran-vectored viruses and plant species could further elucidate the generality and significance of this mechanism.

While this study provides compelling evidence for the role of βC1 and other viral proteins in suppressing SA signaling, some limitations should be considered. The study primarily focused on *N. benthamiana* and tobacco plants, and the generalizability of these findings to other plant species requires further investigation. Additionally, the precise molecular mechanism by which βC1 interacts with NbHSP90s to prevent NbNPR3 degradation remains to be fully elucidated. The focus on specific viral proteins and host factors limits the full understanding of the complex interactions within the virus-vector-plant system. Further investigation is needed to thoroughly characterize the interplay of other factors and pathways that might be involved in this intricate system.