Only minimal regions of tomato yellow leaf curl virus (TYLCV) are required for replication, expression and movement

Abstract

The IL-60 platform, consisting of a disarmed form of tomato yellow leaf curl virus (TYLCV) and auxiliary components, was previously developed as a nontransgenic universal vector system for gene expression and silencing that can express an entire operon in plants. IL-60 does not allow rolling-circle replication; hence, production of viral single-stranded (ss) DNA progeny is prevented. We used this double-stranded (ds) DNA-restricted platform (uncoupled from the dsDNA→ssDNA replication phase of progeny viral DNA) for functional genomics studies of TYLCV. We report that the noncoding 314-bp intergenic region (IR) is the only viral element required for viral dsDNA replication. None of the viral genes are required, suggesting recruitment of host factors that recognize the IR. We further show that IR-carrying reporter genes are also capable of replication but remain confined to the cells into which they were introduced. Only two sense-oriented viral genes (V1 and V2) need to be added to the IR-carrying construct for expression and movement. Hence, any IR-dsDNA construct supplemented with V1 and V2 becomes a replication-competent, mobile and expressing plant plasmid. All viral functions (replication, expression and movement) are determined by the IR and the sense-oriented genes. The complementary-oriented viral genes have auxiliary roles in the late phase of the virus "life cycle". The previously reported involvement of some viral genes in expression and movement is therefore revised.

Fig. 1

Illustrations (not to scale) of the various IR-carrying constructs. pT7 and SP6, sites of the T7 and SP6 promoters; *V2, the first 282 bp of ORF V2; C4*, the last 279 bp of the interrupted C1 ORF (the truncated N terminus of C4); C2, the entire ORF of TYLCV C2

Fig. 2

Stability of IR-carrying constructs in intact plants. All DNA samples were taken from the point of injection and were analyzed by PCR for the presence of GFP sequences. A. Stability of a GFP-harboring plasmid (pDRIVE–GFP) versus the same plasmid carrying the IR. Lanes 1 and 3, IR-carrying plasmids; lanes 2 and 4, IR-lacking plasmids; lane 6, negative control—DNA extract from untreated plants. B. Stability of 35S- vs. IR-carrying plasmids. PCR was performed at various times postinjection as specified

Fig. 3

Left frame: Replication of IR–GUS in protoplasts as indicated by real-time qPCR. The time after application of the construct is shown on the x-axis. Real-time qPCR (average of three repetitions) was performed on DNA extracted from 10⁵ protoplasts, and results were normalized to those for the 18S rRNA gene. DNA extracted from untransfected protoplasts served as a control for real-time PCR. Right frame: Slot-blot assay of IR–GFP. The slot-blot was carried out by applying 10⁵ lysed protoplasts to each slot and hybridizing to a GFP probe

Fig. 4

Evidence that the IR-GUS extracted from protoplasts is not methylated, indicating that it was replicated in the protoplasts and does not represent the input DNA. DNA extracted from protoplasts at various times following IR–GUS transfection was digested with DpnI, and the entire GUS sequence was PCR-amplified. Failure of DpnI digestion indicated that the DNA was not methylated. Lane 1, size markers; lane 2, control—DNA extracts from untransfected protoplasts; lane 3, digestion of bacterial-extracted IR–GUS; lanes 4–6, digestion of PCR-amplified DNA extracts from IR–GUS-transfected protoplasts at 16, 24 and 48 h posttransfection, respectively

Fig. 5

Western blot analysis for GUS. Lane 1, extracts of untreated plants; lane 2, extracts of plants treated with IL60+C2–IR–GUS–ter; lane 3: extracts of plants treated with IL60+35S:GUS–ter (no IR); lane 4, extract of plants treated with IL60+IR–GUS–ter; lane 5, extract of transgenic GUS-expressing plants

Fig. 6

GFP expression in protoplasts transfected with various IL-60 constructs. GFP (left-hand column) and chlorophyll (center column) fluorescence was detected using a confocal microscope supplemented with suitable filters. Superpositions of the left-hand column on the center column are presented in the right-hand column

Fig. 7

The effect of TYLCV infection on the expression and spread of IR-GFP. A. Detection of TYLCV in infected plant tissues by PCR, using TYLCV–CP primers. Lane 1, size markers; lane 2, DNA extract from untreated, uninoculated plants as template; lane 3, DNA extract from IR–GFP-treated, uninoculated plants as template; lanes 4, 6 and 7, DNA extracts from IR–GFP-treated, TYLCV-inoculated plants as template; lane 5, positive control—a plasmid carrying TYLCV CP served as the template. B. Expression and movement of IR–GFP and IR–GUS in TYLCV-infected plants. Confocal images were taken from leaves of IR–GFP-treated plants. I, TYLCV–inoculated, plasmid-devoid plant; II, plant treated with IR–GFP, 14 days after TYLCV inoculation; III and IV, plants treated with IR–GFP, 28 days after TYLCV inoculation. Bars represent 100 µm. V, image of a stained leaf section of an IR–GUS-treated plant, 14 days after TYLCV inoculation. VI, image of a stained leaf section of an untreated plant. C. Detection of GFP sequences (by PCR with GFP primers) in systemic leaves of IR–GFP-treated plants 28 days post-TYLCV inoculation. Lane 1, size markers; lane 2, DNA extract from a plasmid-devoid, TYLCV-infected plant as template; lane 3, positive control—a GFP-harboring plasmid served as a template; lane 4, DNA extract from IR–GFP-treated plant not inoculated with TYLCV as template; lanes 5 and 6, DNA extracts from IR–GFP-treated, TYLCV-inoculated plants as template; pD–IR–GFP, IR–GFP inserted in the plasmid pDRIVE

Fig. 8

Replication and movement of p1470–GFP in tomato plants. A. GFP detection in systemic leaves of a plant treated with p1470–GFP. B. GFP expression in parallel locations across the cell wall in two adjacent cells. C. PCR with primers for TYLCV CP confirms the spread of p1470. (+) and (−) indicate p1470–GFP-treated and untreated plants, respectively. D. RT-PCR with primers for TYLCV CP, confirming the expression of V1 (CP) from p1470. (+) and (−) indicate p1470–GFP-treated and untreated plants, respectively. Lane 8, negative control—PCR performed without template. E. PCR with GFP primers, corroborating the presence of p1470–GFP. (+) and (−) indicate p1470–GFP-treated and untreated plants, respectively. Lane 6, negative control—no template. F. PCR with primers for TYLCV C2. Two p1470–GFP-treated plants were analyzed for the presence of CP and C2. Lane 6, negative control—no template. The absence of C2 indicates a lack of TYLCV contamination. Lane 1 in frames C–F, size markers

Fig. 9

Immunocapture assays of plants treated with IR–PDS. Lane 1, PCR of the antibody-bound fraction from a TYLCV-infected, untreated plant; lane 2, PCR of the antibody-bound fraction from IR–PDS-treated plants with primers for TYLCV CP; lanes 3–6, same as in lane 2 but with primers for PDS amplification; lanes 7 and 8, negative controls—PCR with plant extracts in the absence of anti-TYLCV CP antibodies; lane 9, empty; lane 10, positive control—a PDS-carrying plasmid served as the template