The Conserved Proline18 in the Polerovirus P3a Is Important for Brassica Yellows Virus Systemic Infection

Abstract

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.

The Interaction Dynamics of Two Potato Leafroll Virus Movement Proteins Affects Their Localization to the Outer Membranes of Mitochondria and Plastids

“…This pattern was distinct from the one observed for the C-terminally-tagged homodimer. Consistent with the BrYV study [ 26 ], our results show that PLRV P3a also forms dimers in plant cells. However, our analysis also reveals that BiFC tag orientation influences the subcellular localization of the PLRV P3a homodimer.…”

“…To test this hypothesis, we used bimolecular fluorescence complementation (BiFC) to determine if PLRV P3a and P17 could self-interact and interact with each other in planta . Similar to what was reported for BrYV [ 26 ], when PLRV P3a was tagged at its C-terminus, the self-interaction was observed as three distinct phenotypes: as tiny, immobile YFP-fluorescent foci ( Movie S1 ), large aggregates within the cytosol, or as a faint “ER-like” network [ 44 ] that included the perinuclear space ( Figure 3 A). When tags were fused to the N-terminus of P3a, an orientation not tested in the BrYV publication [ 26 ], self-interaction was observed as highly mobile, dynamic, amorphous spots in the cytoplasm ( Figure 3 B and Movie S2 ).…”

“…This viral protein is small in size (4.8-5.3 kDa), highly conserved among divergent luteovirid species, and contains a putative transmembrane domain at its N-terminus. Null mutants of P3a [ 19 ] or a non-synonymous substitution of the conserved proline 18 residue [ 26 ] in Turnip yellows virus (TuYV, Polerovirus ) or Brassica yellows virus (BrYV, Polerovirus ), respectively, reduced or abolished the long-distance transport of virions. When fluorescent protein tags were fused to the C-termini of TuYV and BrYV P3a and transiently expressed in Nicotiana benthamiana epidermal cells, the fusion proteins localized to the ER, Golgi, and near PD [ 19 , 26 ].…”

“…For BrYV, these localization patterns were not affected by substitution of proline 18. In addition, homodimerization of BrYV P3a could also be observed in planta [ 26 ]. Ectopic expression of P3a proteins from different luteovirid species including the P3a protein from PLRV complimented the systemic movement of a BrYV P3a-null mutant indicating that the movement function of this protein is conserved within the family.…”

Development of polyclonal antisera against movement proteins of the three poleroviruses infecting cucurbits and their serological relationships

“…The intergenic-non-coding region (intergenic-NCR) approximately 80 nt in length locates between ORF2 and ORF3a. The P3a protein without AUG initiation encoded by ORF3a is indispensable for the long-distance movement of the virus [30][31].…”