Ectopic expression of the p23 silencing suppressor of Citrus tristeza virus differentially modifies viral accumulation and tropism in two transgenic woody hosts

Abstract

Citrus tristeza virus (CTV), a phloem-restricted closterovirus infecting citrus, encodes three different silencing suppressors (p25, p20 and p23), one of which (p23) is a pathogenicity determinant that induces aberrations resembling CTV symptoms when expressed ectopically in transgenic citrus hosts. In this article, the effect of p23 ectopic expression on virus infection was examined in sweet orange (SwO), a highly susceptible host, and sour orange (SO), which severely restricts CTV cell-to-cell movement. Transgenic plants of both species ectopically expressing p23, or transformed with an empty vector, were graft inoculated with the mild CTV isolate T385 or with CTV-BC1/GFP, a clonal strain derived from the severe isolate T36 carrying the gene for the green fluorescent protein (GFP). CTV distribution in infected tissues was assessed by direct tissue blot immunoassay and fluorescence emission, and virus accumulation was estimated by quantitative real-time reverse transcriptase-polymerase chain reaction. CTV accumulation in p23-expressing and control SwO plants was similar, whereas the viral load in transgenic SO expressing p23 was 10-10(5) times higher than in the cognate control plants. Although few infection foci composed of a single cell were observed in the phloem of CTV-infected control SO, the number of foci in p23-expressing plants was higher and usually comprised two to six cells, indicating viral cell-to-cell movement. CTV was detected in mesophyll protoplasts and cells from infected SO and SwO expressing p23, but not in similar protoplasts and cells from infected control plants. Our results show that the ectopic expression of p23 enables CTV to escape from the phloem and, in addition, facilitates systemic infection of the resistant SO host. This is the first report of a viral-encoded protein that enhances virus accumulation and distribution in woody hosts.

Virus-Viroid Interactions: Citrus Tristeza Virus Enhances the Accumulation of Citrus Dwarfing Viroid in Mexican Lime via Virus-Encoded Silencing Suppressors

“…In this context, it is worth pointing out that the ectopic expression of RSSs enhances the accumulation of some viruses (28)(29)(30), most likely by suppressing the host antiviral RNA silencing. Consistent with this view, CTV also accumulates when p23 is ectopically expressed in sour orange (27). In contrast, expression of some RSSs has no effect on viroid accumulation in PSTVd-infected N. benthamiana (17).…”

“…Sequencing of RT-PCR amplicons from CDVd-infected plants indicated that the CDVd used as inoculum remained stable in all the treatments. The presence of p23, p20, and p25 was confirmed in equalized protein extracts of the transgenic lines, as well as in singly infected and coinfected nontransformed controls, by SDS-PAGE and Western blot analysis with p23 and p20 antisera (27) (Twentyfirst Century Biochemicals, Inc.) and with a mixture of 3DF1 and 3CA5 antibodies for detecting p25 (data not shown). Northern blot hybridization only detected the CDVd-sRNAs in transgenic lines expressing p23 (Fig.…”

“…However, our results show that the effect of p23 on viroid accumulation is host dependent, with the interaction between CTV and CDVd being perceptible in Mexican lime but not in etrog citron; this difference may result from the number of infected cells in each host or from distinct regulation of the replication/degradation pathways. Interestingly, the ectopic expression of p23 enhances CTV accumulation in sour orange (27) but not in Mexican lime (25), where the virus reaches low and high accumulation levels, respectively (31). Altogether, these data suggest that hosts in which the pathogen reaches high titers are not appropriate to study the effects of RSS.…”

“…Altogether, these data suggest that hosts in which the pathogen reaches high titers are not appropriate to study the effects of RSS. Finally, since p23 is an RNA-binding protein (32) with nucleolar (and plasmodesmatal) localization (33), which besides acting as an RSS also regulates viral plus-minus-strand accumulation (34) and movement (27), we cannot eliminate the possibility that some of these functions may also influence the observed effect on viroid accumulation. Intriguingly, Gambino et al found that Grapevine yellow speckle viroid 1, another viroid of the same genus, also accumulates in the nucleus and possibly in the nucleolus (35), although the plants in their study were not directly examined for CDVd.…”

Phloem restriction of viroids in three citrus hosts is overcome by grafting with Etrog citron: potential involvement of a translocatable factor

“…It should be noted that viroid titres in the leaf blades of C. karna, sour orange and trifoliate orange were rather low, suggesting that viroids were only present in the secondary veins -from where they were unable to exit and invade other cell types, including mesophyll cells -thus explaining why they could not be detected in protoplasts. Phloem-restricted infection is not exceptional in citrus and has been reported for viruses, like citrus tristeza virus (Fagoaga et al, 2011). The mature phloem contains the sieve tube, composed of a vertical column of elongated cells devoid of nuclei that are responsible for the longdistance movement of viruses and viroids, and companion cells with nuclei competent for virus and viroid replication.…”