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.
Highlights
Plant viruses are obligate intracellular parasites living exclusively in the symplast of their plant host
In order to establish effectively systemic infection, the viruses that transport in the phloem should fulfill four processes: (1) amplification in the preliminary infection site; (2) short-distance movement between mesophyll cells through the plasmodesmata (PD) (Schoelz et al, 2011); (3) loading into sieve elements after passing through several vasculature-associated cell types and longdistance transport along the vascular bundle system (Carrington et al, 1996; Hipper et al, 2013); Brassica yellows virus (BrYV) Systemic Infection Requires P3a Proline18 (4) virus unloading from sieve elements into distal sink tissues and reestablishment new infection site
No RNA accumulation of AGC or BrYVP18L were detected in upper leaves of inoculated plants when they were co-infiltrated with P3aP18L (Figure 3B). These results showed that transient expression of P3a can efficiently rescue systemic movement of BrYVP18L or AGC mutants, indicating that the Pro18 residue in P3a is crucial for the long distance movement of BrYV in N. benthamiana
Summary
Plant viruses are obligate intracellular parasites living exclusively in the symplast of their plant host. In order to establish effectively systemic infection, the viruses that transport in the phloem should fulfill four processes: (1) amplification in the preliminary infection site; (2) short-distance movement between mesophyll cells through the plasmodesmata (PD) (Schoelz et al, 2011); (3) loading into sieve elements after passing through several vasculature-associated cell types and longdistance transport along the vascular bundle system (Carrington et al, 1996; Hipper et al, 2013); BrYV Systemic Infection Requires P3a Proline. Brassica yellows virus (BrYV) is a tentative species in the genus Polerovirus Viruses in this genus and Luteovirus are limited to the phloem and transmitted by aphid (King et al, 2012; Zhou et al, 2017). As the BrYV is a phloem-restricted virus, an umbravirus Pea enation mosaic virus 2 (PEMV 2) can help BrYV to be mechanically transmitted and invade non-vascular tissue in Nicotiana benthamiana (Zhou et al, 2017)
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