Fig 1.
Organization of geminivirus DNA-A.
The circular diagram depicts the dsDNA RF of a typical bipartite begomovirus (e.g., TGMV, CaLCuV), with expanded views of the intergenic region (IR). Viral genes, indicated by solid arrows, encode replication initiator protein (Rep/AL1/AC1); transcriptional activator protein (TrAP/AL2/AC2); replication enhancer; (REn/AL3/AC3); and coat protein (CP/AR1/AV1). AL4/AC4 is a pathogenicity factor encoded within the Rep ORF in a different reading frame. Within the IR, the common region (CR) present in both DNA-A and DNA-B is represented by a light blue ellipse. The replication origin core includes the conserved hairpin and Rep binding sites. The Rep and CP transcription start sites (TSS) are indicated by right angle arrows. The conserved late element (CLE), CAAT, and TATAA box sequences are indicated in light blue boxes. The region used in the yeast one-hybrid screen is also shown. Diagrams are not to scale.
Fig 2.
A class II TCP binding site (CLE) is necessary for efficient TCP24 binding to CP promoter sequences.
GST-tagged or 6xHis-tagged TCP24 protein was isolated from E. coli Rosetta cells and purified as described in Methods. (A) Increasing volumes (6, 12, 18 μl) of purified GST-TCP24 protein were incubated with a TGMV CP promoter fragment containing a wild type (Probe 1) or mutated (Probe 2) CLE sequence. Protein-DNA complexes were separated on 2% agarose gel in 0.5X TB buffer, stained with ethidium bromide, and the positions of unbound probe (P) and TCP24 protein-probe DNA complexes (**) detected by UV illuminator. (B) GST-TCP24 was incubated with a CaLCuV CP promoter fragment containing a wild type (Probe 3) or mutated (Probe 4) CLE sequence. Protein-DNA complexes were separated and visualized as in (A). (C) Purified 6xHis-TCP24 protein (0.5 μg) was incubated with an FITC-labeled, wild type TGMV CP promoter fragment (Probe 1) in the presence (+) or absence (-) of a 50-fold molar excess of unlabeled competitor DNAs (Probes 1–4, as indicated). Protein-DNA complexes were separated on 6% polyacrylamide TBE gels and the positions of unbound probe and TCP24 protein-probe DNA complexes (**) detected with the iBright 1500 Imaging System. (D) Purified 6xHis-TCP24 protein (0.5 μg) was incubated with an FITC-labeled, wild type CaLCuV CP promoter fragment (Probe 3) in the presence (+) or absence (-) of a 50-fold molar excess of unlabeled competitor DNAs (Probes 1–4, as indicated). Protein-DNA complexes were separated and visualized as in (C). GST protein (A and B) and pENTR DNA (C and D) were negative controls. The images in (A) and (B) are representative of two independent experiments, and images in (C) and (D) of three independent experiments.
Fig 3.
TCP24 interacts with the TGMV CP promoter in vivo.
(A) The linear map represents the previously characterized TGMV A55M transgene [8]. Right angle arrows above the diagram indicate the positions of transcription start sites and arrows below the diagram indicate the positions of primers used for qPCR after ChIP. The CP coding region is replaced by GUS (CP/GUS) and AL2 is inactivated by mutation (AL2x). (B) Transgenic A55M N. benthamiana plants were infiltrated with Agrobacterium containing a TMV-based vector capable of expressing either GST-tagged TCP24 (GST-TCP24) or AtERF13 (GST-ERF13, negative control). Protein-DNA complexes were obtained by ChIP, and qPCR was performed on DNA in immune complexes using a primer set specific for the TGMV CP promoter. Fold change represents the difference in the amount of product detectable in qPCR reactions from samples immunoprecipitated using anti-GST antibody (GST Ab) relative to anti-RFP antibody (RFP Ab, negative control). (C) As in (B), except protein-DNA complexes from A55M plants expressing GST-TCP24 were captured by ChIP using anti-GST or anti-GUS (negative control).
Fig 4.
TCP24 interacts with TGMV AL2 protein.
Protein-protein (Far Western) gel blots are shown. Samples of 6xHis-TCP24, 6xHis-AL2, and 6xHis-GFP were resolved on 4–20% protein gels and gel blots were overlayed with a soluble fraction from E. coli expressing either (A) GST-TCP24, (B) GST, or (C) buffer only (Mock). Membranes overlayed with GST-TCP24 or GST were incubated with an anti-GST monoclonal antibody. The mock was incubated with an anti-6xHis monoclonal antibody. The position of protein standards is shown on the left and asterisks indicate the AL2 protein dimer. The images are representative results from 9 independent experiments.
Fig 5.
Bimolecular Fluorescence Complementation (BiFC) analysis of TCP24-AL2 complexes in N. benthamiana epidermal cells.
Constructs expressing full length AL2 protein or TCP24 fused to the N- or C-terminal portion of YFP were delivered to N. benthamiana leaves by agroinfiltration. Images were examined for fluorescence indicative of interaction with a 40x objective using FITC (eGFP signal) and Rhodamine (RFP-Histone H4 signal) filter sets. Photographs represent merged images from the two filter sets. H4-RFP localizes to the nucleus and is identified by red fluorescence, while reconstituted YFP is identified by green fluorescence. Protein combinations are indicated on each image, with the protein listed first fused to the N-terminal portion of YFP, and the second fused to the C-terminal portion. Controls expressed either nYFP or cYFP. Panels including AL2-AL2 and TCP24-TCP24 are tests of self-interactions. Scale bars indicate 75 μm. The images are representative of results from three independent experiments.
Fig 6.
TCP24 co-immunoprecipitates with TGMV AL2 protein.
Constructs to express FLAG-AL2, GST-AL2, GST-TCP24, GST (control), or TRBO (empty vector) were agroinfiltrated to N. benthamiana leaves. Cultures were mixed prior to infiltration so that all GST-tagged proteins were separately co-expressed with FLAG-AL2. Tissues were obtained from infiltration zones and extracts used for Co-IP reactions. Protein (Western) gel blots are shown. Rightmost lanes in each blot contained protein from non-infiltrated tissue. (A) Protein blot probed with GST antibody. (B) IP with FLAG antibody followed by protein blot probed anti-FLAG. (C) IP with FLAG antibody followed by protein blot probed with anti-GST. Asterisks indicate dimeric AL2*. Triangles indicate cross-reacting (background) proteins.
Table 1.
Infectivity of CaLCuV and TGMV wild type and cle- mutant viruses in N. benthamiana.
Fig 7.
Viral DNA loads in N. benthamiana plants infected with wild type or cle- mutant TGMV or CaLCuV.
Total DNA was isolated from systemic tissue infected with wild type or cle- mutant viruses and qPCR performed using primers specific for the (A) TGMV or (B) CaLCuV CP ORF. The bars illustrate the average amount of viral DNA (log10 scale) present in systemic leaf tissue away from the site of inoculation in individual plants that were symptomatic (S) or asymptomatic (AS) following inoculation with wild type (WT) or cle- mutant viruses. Viral DNA copy numbers were calculated by comparison to a standard curve. Statistical differences between pairs were determined by ANOVA followed by a Tukey-Kramer post-hoc comparison. Individual values for viral DNA amounts (ng) and number of copies of the viral genome are shown in S1 and S2 Tables.
Fig 8.
Quantitation of symptom severity, viral DNA, and CP mRNA in systemically infected Arabidopsis.
(A) Representative photograph illustrating symptom severity range for wild type (WT) CaLCuV in Arabidopsis (ecotype Col-0). Photos were taken at 22 dpi. Red boxes highlight deformed bolts and numbers indicate severity scores: Asymptomatic = 0; mild deformation of floral heads and siliques on a single bolt = 1; mild deformation of floral heads and siliques on multiple bolts = 2; moderate deformation of floral heads and siliques on multiple bolts coupled with mild to moderate stunting = 3; severe deformation of floral heads and siliques coupled with severe stunting = 4. (B) Representative photograph of CaLCuV cle- symptom severity range. (C) Quantitation of symptom severity scores for both CaLCuV WT (n = 44 plants) and CaLCuV cle- (n = 45 plants). Percentage of plants with each score is illustrated. (D) Viral DNA levels. Total viral DNA was quantified by qPCR using a primer set specific for the CP coding region and normalized to 18S ribosomal DNA. Wild type CaLCuV was set to a value of 1.0. Significance was calculated using an unpaired Student’s t-test. Bars indicate standard error of the mean of three biological replicates. S, symptomatic tissue, AS, asymptomatic tissue. (E) CP mRNA levels. Total RNA was extracted from the same samples as (D) and CP mRNA measured by RT-qPCR using primers specific for the CP coding region. Viral RNA was normalized to total viral DNA as measured by qPCR, then normalized to PP2A.
Table 2.
Infectivity of CaLCuV wild type and cle- mutant viruses in Arabidopsis.
Fig 9.
Multiple class II TCP transcript levels are altered by CaLCuV infection.
(A) Total RNA was obtained from uninfected Arabidopsis plants, and TCP2, 3, 4, 10, and 24 transcript levels were determined by RT-qPCR. Transcript levels were normalized to PP2A. (B) Total RNA was obtained from Arabidopsis plants 22 days after agroinoculation with WT CaLCuV DNA-A and DNA-B. Transcript levels in infected plants (I) were normalized to PP2A and compared to levels in uninfected plants (U). (C) Arabidopsis seedlings were vacuum infiltrated with Agrobacterium cultures to deliver CaLCuV DNA-A or empty Ti plasmid vector (pMON521). Total RNA was isolated one- and two-days post-infiltration and TCP24 transcript levels determined by RT-qPCR, and normalized to PP2A. Experiments included at least three replicates, and bars indicate standard error. Asterisks indicate significant differences (p < 0.05) as determined by Students t-test.
Fig 10.
Time course of viral DNA and CP mRNA accumulation in N. benthamiana plants infiltrated with wild type or cle- mutant TGMV or CaLCuV.
Leaves from N. benthamiana plants were infiltrated with Agrobacterium cultures containing wild type (WT) or cle- TGMV or CaLCuV DNA-A and total DNA isolated 24- to 72 hours post-inoculation (hpi). Images shown are scans of Southern blots hybridized to probes specific for TGMV (A) or CaLCuV (B) DNA-A as detected by chemiluminescence (Lanes 1–6). Lanes 1a and 2a are images of longer exposures to detect viral DNA at 24 hpi. Covalently closed circular (ccc), open circular (oc) and linear (lin) double-stranded DNA forms, as well as single-stranded (ss) viral DNA forms, are indicated. Residual inoculum DNA (Inoc) is also indicated. Relative levels and absolute chemiluminescence units for each viral DNA form are given in S3 Table. (C) Samples were isolated from the same tissue as the DNA shown above (A and B) and CP mRNA copy number was determined by RT-qPCR using a standard curve. The graph illustrates the number of copies of TGMV CP mRNA detected in total RNA isolated 48 and 72-hpi from leaves of N. benthamiana plants infused with Agrobacterium cultures containing wild type (WT) or cle- mutant TGMV DNA A. Levels of CP mRNA were adjusted for dsDNA levels and data are shown in S4 Table.
Fig 11.
CLE mutation decreases H3K27me3 levels on the viral IR.
ChIP-qPCR experiments were performed with H3K27me3 antibody using nuclear extracts from N. benthamiana or Arabidopsis plants systemically infected with wild type (WT) or cle- TGMV or CaLCuV. Tissue from symptomatic plants was pooled for analysis. Data were normalized to input DNA, with signal from the negative control IgG immunoprecipitate subtracted. Values were also normalized to ChIP-qPCRs performed with histone H3 antibody using the same extracts. Amplicon regions (~100 bp) are depicted on diagrams that show the positions of the intergenic region (IR), conserved hairpin, the common region, and the Rep and CP ORFs with transcription start sites (right angle arrows). (A) ChIP with extracts from N. benthamiana (Nb) plants infected with wild type TGMV or TGMV cle-. Three replicates are shown, with standard error of the mean. (B) ChIP with extracts from Arabidopsis (At) infected with wild type CaLCuV or CaLCuV cle-. Two replicates are shown, with standard error of the mean.