Most plant viruses counter the RNA silencing-based antiviral defense by expressing viral suppressors of RNA silencing (VSRs). In this sense, VSRs may be regarded as virulence effectors that can be recognized by the host as avirulence (avr) factors to induce R-mediated resistance. We made use of Agrobacterium-mediated transient coexpression of VSRs in combination with Potato virus X (PVX) to recapitulate in local tissues the systemic necrosis (SN) caused by PVX-potyvirus synergistic infections in Nicotiana benthamiana. The hypersensitive response (HR)-like response was associated with an enhanced accumulation of PVX subgenomic RNAs. We further show that expression of P25, the VSR of PVX, in the presence of VSR from different viruses elicited an HR-like response in Nicotiana spp. Furthermore, the expression of P25 by a Plum pox virus (PPV) vector was sufficient to induce an increase of PPV pathogenicity that led to necrotic mottling. A frameshift mutation in the P25 open reading frame (ORF) of PVX did not lead to necrosis when coexpressed with VSRs. These findings indicate that P25 is the main PVX determinant involved in eliciting a systemic HR-like response in PVX-associated synergisms. Moreover, we show that silencing of SGT1 and RAR1 attenuated cell death in both PVX-potyvirus synergistic infection and the HR-like response elicited by P25. Our study underscores that P25 variants that have impaired ability to suppress RNA silencing cannot act as elicitors when synergized by the presence of other VSRs. These findings highlight the importance of RNA silencing suppression activity in the HR-like response elicited by VSRs in certain hosts. IMPORTANCEThe work presented here describes how the activity of the PVX suppressor P25 elicits an HR-like response in Nicotiana spp. when overexpressed with other VSR proteins. This finding suggests that the SN response caused by PVX-associated synergisms is a delayed immune response triggered by P25, once it reaches a threshold level by the action of other VSRs. Moreover, this work supports the contention that the silencing suppressor activity of PVX P25 protein is a prerequisite for HR elicitation. We propose that unidentified avr determinants could be involved in other cases of viral synergisms in which heterologous "helper" viruses encoding strong VSRs exacerbate the accumulation of the avr-encoding virus. I n host-virus interactions, RNA silencing triggered by viral double-stranded RNA (dsRNA) is a general mechanism involved in immunity against viruses (1). By analogy with the zigzag model that describes plant-microbe interactions, dsRNA and RNA silencing could be regarded as a viral pathogen-associated molecular pattern (PAMP) and PAMP-triggered immunity, respectively (2). Most viruses counter the RNA silencing-based antiviral defense by expressing viral suppressors of RNA silencing (VSRs). Therefore, VSRs may be regarded as virulence effectors that facilitate viral infection in plants. As suggested by the zigzag model, plants may have developed a countermeasure agai...

Infections of plants by multiple viruses are common in nature and may result in synergisms in pathologies. Several environmental factors influence plant-virus interactions and act on virulence and host defense responses. Mixed viral infections may be more frequent under environmental conditions associated with global warming. Here, we address how changes in the two main parameters behind global warming, carbon dioxide concentrations ([CO₂]) and temperature, may affect virulence of Potato virus X (PVX)/potyvirus-associated synergism compared with single infections in Nicotiana benthamiana. Elevated [CO₂] resulted in attenuated virulence of single infection by PVX, which correlated with a lower accumulation of virus. In contrast, virulence of PVX/potyvirus-associated synergism was maintained at elevated [CO₂]. On the other hand, elevated temperature decreased markedly both virulence and virus titers in the synergistic infection. We also show that the HR-like response elicited by transient coexpression of PVX P25 together with the potyviral helper component-proteinase protein was significantly enhanced by elevated temperature, whereas it was reduced by elevated [CO₂]. Both proteins are main pathogenicity determinants in PVX-associated synergisms. These findings indicate that, under environmental conditions associated with global warming, virulence of PVX/potyvirus-associated synergisms is expected to vary relative to single infections and, thus, may have pathological consequences in the future.

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