A genetic system for Citrus Tristeza Virus using the non-natural host Nicotiana benthamiana: an update

Abstract

In nature Citrus tristeza virus (CTV), genus Closterovirus, infects only the phloem cells of species of Citrus and related genera. Finding that the CTV T36 strain replicated in Nicotiana benthamiana (NB) protoplasts and produced normal virions allowed development of the first genetic system based on protoplast transfection with RNA transcribed from a full-genome cDNA clone, a laborious and uncertain system requiring several months for each experiment. We developed a more efficient system based on agroinfiltration of NB leaves with CTV-T36-based binary plasmids, which caused systemic infection in this non-natural host within a few weeks yielding in the upper leaves enough CTV virions to readily infect citrus by slash inoculation. Stem agroinoculation of citrus and NB plants with oncogenic strains of Agrobacterium tumefaciens carrying a CTV-T36 binary vector with a GUS marker, induced GUS positive galls in both species. However, while most NB tumors were CTV positive and many plants became systemically infected, no coat protein or viral RNA was detected in citrus tumors, even though CTV cDNA was readily detected by PCR in the same galls. This finding suggests (1) strong silencing or CTV RNA processing in transformed cells impairing infection progress, and (2) the need for using NB as an intermediate host in the genetic system. To maintain CTV-T36 in NB or assay other CTV genotypes in this host, we also tried to graft-transmit the virus from infected to healthy NB, or to mechanically inoculate NB leaves with virion extracts. While these trials were mostly unsuccessful on non-treated NB plants, agroinfiltration with silencing suppressors enabled for the first time infecting NB plants by side-grafting and by mechanical inoculation with virions, indicating that previous failure to infect NB was likely due to virus silencing in early infection steps. Using NB as a CTV host provides new possibilities to study virus-host interactions with a simple and reliable system.

Keywords: Agrobacterium tumefaciens; CTV; N. benthamiana protoplasts; RNA silencing suppressor; agroinoculation; graft transmission; infectious cDNA clones; oncogenic strains.

Figure 1

Outline of the binary plasmids used in this study. (A) Detail of the T-DNA region in the pCH20 binary vector with its size indicated at the right. RB and LB are the right and left borders, the NotI restriction sites for subcloning, the unique SwaI site of the T-DNA and the unique BamHI site present in the sacB gene are indicated. (B) Detail of the T-DNA region in the pCH20-GUSi binary vector. The cassette of the gus-intron selectable marker gene containing a catalase intron is shown. Shadowed boxes represent the 35S promoter of the Cauliflower mosaic virus (CaMV) and the nopaline synthase terminator (NOS-t) present in the cassette. Other details as in (A). (C) Schematic representation and relative orientation of the CTV-T36 expression cassette containing the full-genome cDNA clone CTV9R from the agroinfectious BAC CTV-vector (Ambrós et al., 2011). Shadowed boxes represent the double enhanced 35S promoter (35S × 2), the Hepatitis delta virus ribozyme (Rbz) and the NOS-t present in the cassette. The genome organization of the CTV gRNA is denoted by boxes indicating the open reading frames (ORFs) and their corresponding translation products. L1 and L2 mean the two leader papain-like proteases; MT, methyltransferase; HEL, helicase; RdRp, RNA-dependent RNA polymerase; CPm and CP, the minor and major coat proteins; intron, the ST-LS1 intron incorporated in some CTV clones (Ambrós et al., 2011). The NotI sites flanking the construct are indicated and the strategy for subcloning into the T-DNA of pCH20-GUSi is marked with dotted arrow lines.

Figure 2

Tumours incited by oncogenic A. tumefaciens strains in herbaceous and citrus hosts. (A) Left panel, tumours induced by A. tumefaciens C58 strain in Nicotiana species at 4 weeks post inoculation (wpi) (NB) or 5 wpi (N. occidentalis). Right panel, tumours induced by A. tumefaciens C58 and A281 strains in Mexican lime at 7 wpi. The rule on the left indicates the size of individual tumours. (B) GUS activity assay of stem tumours from individual lime plants agroinoculated with A. tumefaciens C58. Each dish contains slices from individual tumours from plants agroinoculated with C58 harboring the pCH20-GUSi (empty vector) or the pCH20-GUSi-CTV (CTV-T36 vector) plasmids and incubated with X-Gluc solution at 8 wpi. Controls consisted of lime bark tissue agroinoculated with the non-oncogenic A. tumefaciens Eha105 strain or with the C58 strain without plasmid. A detail of tumor slices showing many blue spots of high-GUS activity is presented on the right. (C) Confirmation of partial T-DNA integrations in the cell nucleus of agroinfected lime plants. PCR amplification products obtained from gDNA extracts of tumours incited by A. tumefaciens C58 harboring the pCH20-GUSi-CTV or the pCH20-GUSi vectors (line 10). Controls: PCR amplification products from pCH20-GUSi-CTV (containing an intron in the CTV ORF 1a) and pCH20-GUSi plasmids, or from distilled water. The T-DNA target regions amplified (from the right to the left border as indicated in Figure 1) and the size of the DNA fragments are indicated at the right. Each lane corresponds to an individual tumor sample from different plants in the same experiment and DNA bands under the same number in different gels correspond to the amplification products obtained using different primers and the same gDNA (obtained from two slices of the same tumor). M, 1 Kb Plus DNA marker (Invitrogen, Fisher), with relevant sizes of DNA fragments indicated at the left.

Figure 3

CTV accumulation in tumours of NB agroinoculated with A. tumefaciens C58 harboring the pCH20-GUSi-CTV vector. (A) Detail of tumor tissues induced by C58 in the stem of NB at 7 wpi showing three primary tumours (PT) and some tumorigenic leaves (ST, secondary tumours) that developed later beside the agroinoculation sites. (B) CTV accumulation in tumor tissues (or tumorigenic leaves) of NB plants agroinoculated with the C58 strain estimated by qRT-PCR (Ruiz-Ruiz et al., 2007) and expressed as number of CTV gRNA copies per ng of RNAt. Means and standard deviations were obtained from four technical replicates and two independent bioassays. Panels I and II show CTV accumulation in tumours of individual plants in bioassays 1 and 2, respectively. Asterisks above some bars indicate plants of these bioassays that became systemically infected by CTV. Right graphic in panel I shows the viral titer in tumorigenic leaves produced in three plants of bioassay 1.

Figure 4

Symptom expression and CTV distribution in NB plants systemically infected after agroinoculation with A. tumefaciens C58 harboring the pCH20-GUSi-CTV plasmid. (A) Detail of symptoms induced by CTV in agroinfected N. benthamiana. Left, stem tumours and vein clearing symptoms in a new young lateral shoot (right) at 7 wpi. Middle, epinasty in NB at 8 wpi. Right, new shoot in an old systemically infected NB (10 wpi) showing stunting, vein clearing and crinkly leaf. (B) Relative accumulation and distribution of CTV in shoots (first line), petioles (second line) and upper leaves of systemically infected NB. (C) Absolute accumulation of CTV in upper leaves from different NB plants estimated by qRT-PCR (Ruiz-Ruiz et al., 2007) as in Figure 3. (D) Northern blot analysis of RNAt (4 μg) extracted at 10 wpi from the upper leaves of a CTV-infected NB plant. Positions of the CTV genomic (gRNA) and subgenomic RNAs (sgRNAs) are indicated by arrows on the right. The blot was hybridized with a digoxigenin-labeled riboprobe specific for the 3′-terminal region of the CTV-T36 gRNA.

Figure 5

Northern blot analysis of RNAt from NB mesophyll protoplasts transfected with purified virions from citrus plants infected with the CTV isolate T318A at 1 through 5 days post-inoculation (dpi) (left panel), and the T36, T385, and T305 isolates at 5 dpi (right panel). Left lane, virion extract used to transfect protoplasts. Positions of the CTV gRNA and sgRNAs are indicated by arrows at both sides. A large dRNA present in T318A and its relative position is shown on the left. The blot was hybridized with a digoxigenin-labeled riboprobe specific for the 3′-terminal region of the CTV-T318A gRNA.

Figure 6

CTV systemic infection of NB plants after side-graft inoculation. Left, detail of side-graft inoculation of NB plants using CTV infected petioles (upper panel) or young shoots with vein clearing symptoms (lower panel). Right, severe stunting and crinkly leaf in a NB plant systemically infected (7 wpi); one surviving graft shows CTV symptoms.