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 <i>Polerovirus</i> and <i>Luteovirus</i>, 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 P3a^P18L, abolished the systemic infection of BrYV. Preliminary investigation showed that wild type BrYV was able to load into the petiole of the agroinfiltrated <i>Nicotiana benthamiana</i> leaves, whereas the mutant displayed very low efficiency. Further experiments revealed that the P3a and its mutant P3a^P18L localized to the Golgi apparatus and near plasmodesmata, as well as the endoplasmic reticulum. Both P3a and P3a^P18L were able to self-interact <i>in vivo</i>, however, the mutant P3a^P18L 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 <i>Pea enation mosaic virus 2</i> (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.