Summary P0 protein of some polerovirus members can target ARGONAUTE 1 ( AGO 1) to suppress RNA silencing. Although P0 harbors an F‐box‐like motif reported to be essential for interaction with S phase kinase‐associated protein 1 ( SKP 1) and RNA silencing suppression, it is the autophagy pathway that was shown to contribute to AGO 1 degradation. Therefore, the role of P0– SKP 1 interaction in silencing suppression remains unclear. We conducted global mutagenesis and comparative functional analysis of P0 encoded by Brassica yellows virus (BrYV) (P0 Br ). We found that several residues within P0 Br are required for local and systemic silencing suppression activities. Remarkably, the F‐box‐like motif mutant of P0 Br , which failed to interact with SKP 1, is destabilized in vivo . Both the 26S proteasome system and autophagy pathway play a role in destabilization of the mutant protein. Furthermore, silencing of a Nicotiana benthamiana SKP 1 ortholog leads to the destabilization of P0 Br . Genetic analyses indicated that the P0 Br – SKP 1 interaction is not directly required for silencing suppression activity of P0 Br , but it facilitates stability of P0 Br to ensure efficient RNA silencing suppression. Consistent with these findings, efficient systemic infection of Br YV requires P0 Br . Our results reveal a novel strategy used by BrYV for facilitating viral suppressors of RNA silencing stability against degradation by plant cells.
The Brassica yellows virus genotype A P0 protein interacts with Rubisco assembly factor 2 in tobacco, affecting its nuclear accumulation and promoting viral infection.
As a core subunit of the SCF complex that promotes protein degradation through the 26S proteasome, S-phase kinase-associated protein 1 (SKP1) plays important roles in multiple cellular processes in eukaryotes, including gibberellin (GA), jasmonate, ethylene, auxin and light responses. P7-2 encoded by Rice black streaked dwarf virus (RBSDV), a devastating viral pathogen that causes severe symptoms in infected plants, interacts with SKP1 from different plants. However, whether RBSDV P7-2 forms a SCF complex and targets host proteins is poorly understood. In this study, we conducted yeast two-hybrid assays to further explore the interactions between P7-2 and 25 type I Oryza sativa SKP1-like (OSK) proteins, and found that P7-2 interacted with eight OSK members with different binding affinity. Co-immunoprecipitation assay further confirmed the interaction of P7-2 with OSK1, OSK5 and OSK20. It was also shown that P7-2, together with OSK1 and O. sativa Cullin-1, was able to form the SCF complex. Moreover, yeast two-hybrid assays revealed that P7-2 interacted with gibberellin insensitive dwarf2 (GID2) from rice and maize plants, which is essential for regulating the GA signaling pathway. It was further demonstrated that the N-terminal region of P7-2 was necessary for the interaction with GID2. Overall, these results indicated that P7-2 functioned as a component of the SCF complex in rice, and interaction of P7-2 with GID2 implied possible roles of the GA signaling pathway during RBSDV infection.
ORF3a, a newly identified non-AUG-initiated ORF encoded by members of genera Polerovirus and Luteovirus, is required for long-distance movement in plants. However, the mechanism of action of P3a in viral systemic movement is still not clear. In this study, sequencing of a brassica yellows virus (BrYV) mutant defective in systemic infection revealed two-nucleotide variation at positions 3406 and 3467 in the genome. Subsequent nucleotide substitution analysis proved that only the non-synonymous substitution (C→U) at position 3406, resulting in P3aP18L, abolished the systemic infection of BrYV. Preliminary investigation showed that wild type BrYV was able to load into the petiole of the agroinfiltrated Nicotiana benthamiana leaves, whereas the mutant displayed very low efficiency. Further experiments revealed that the P3a and its mutant P3aP18L localized to the Golgi apparatus and near plasmodesmata, as well as the endoplasmic reticulum. Both P3a and P3aP18L were able to self-interact in vivo, however, the mutant P3aP18L seemed to form more stable dimer than wild type. More interestingly, we confirmed firstly that the ectopic expression of P3a of other poleroviruses and luteoviruses, as well as co-infection with Pea enation mosaic virus 2 (PEMV 2), restored the ability of systemic movement of BrYV P3a defective mutant, indicating that the P3a is functionally conserved in poleroviruses and luteoviruses and is redundant when BrYV co-infects with PEMV 2. These observations provide a novel insight into the conserved function of P3a and its underlying mechanism in the systemic infection.
To counteract host antiviral RNA silencing, plant viruses encode numerous viral suppressors of RNA silencing (VSRs). P0 proteins have been identified as VSRs in many poleroviruses. However, their suppressor function has not been fully characterized. Here, we investigated the function of P0 from pea mild chlorosis virus (PMCV) in the suppression of local and systemic RNA silencing via green fluorescent protein (GFP) co-infiltration assays in wild-type and GFP-transgenic Nicotiana benthamiana (line 16c). Amino acid deletion analysis showed that N-terminal residues Asn 2 and Val 3, but not the C-terminus residues from 230–270 aa, were necessary for PMCV P0 (P0PM) VSR activity. P0PM acted as an F-box protein, and triple LPP mutation (62LPxx79P) at the F-box-like motif abolished its VSR activity. In addition, P0PM failed to interact with S-phase kinase-associated protein 1 (SKP1), which was consistent with previous findings of P0 from potato leafroll virus. These data further support the notion that VSR activity of P0 is independent of P0–SKP1 interaction. Furthermore, we examined the effect of P0PM on ARGONAUTE1 (AGO1) protein stability, and co-expression analysis showed that P0PM triggered AGO1 degradation. Taken together, our findings suggest that P0PM promotes degradation of AGO1 to suppress RNA silencing independent of SKP1 interaction.
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