Geminivirus-encoded TrAP suppressor inhibits the histone methyltransferase SUVH4/KYP to counter host defense
- Texas A&M University, United States
- Chinese Academy of Sciences, China
- Zhejiang University, China
- Yunnan Academy of Tobacco Agricultural Sciences, China
- Heilongjiang Bayi Agricultural University, China
- Jiangsu Academy of Agricultural Sciences, China
Figures
Figure 1 with 1 supplement
TrAP caused developmental abnormalities in Arabidopsis but not through miRNA pathway.
(A) Western blot analysis of 35S-TrAP-3HA and 35S-TrAP-CFP transgenic lines. Arrows indicate the locations of the tagged TrAP proteins; * Cross-reaction band serves a loading control. (B) …
Figure 1—figure supplement 1
sRNA blot analysis of additional miRNAs and siRNA in the TrAP overexpression transgenic plants.
Total RNA was prepared from a pool of T2 transgenic plants (n > 50 for each line). sRNA blots were probed using 5′ end 32P-labeled oligonucleotide probes complementary to the indicated miRNAs or …
Figure 2
TrAP is genetically involved in the TGS pathway.
(A) 35S-TrAP transgenic plants phenocopied lhp1 mutants. Photographs were taken of 15-day seedlings. (B, C) Microarray results were validated by qRT-PCR analysis. Only 12 randomly selected loci were …
Figure 3 with 1 supplement
TrAP interacts with KYP in vivo.
(A) Schematic representation of the luciferase complementation imaging assay shows the different combinations of infiltrated constructs that were fused either to N-terminal (NLuc) and C-terminal …
Figure 3—figure supplement 1
Mass spectrometry analyses confirmed endogenous KYP as a bona fide TrAP-interacting partner.
(A) β-estradiol Inducible expression of Flag-4Myc-TrAP protein in Arabidopsis transgenic lines (B) 31 peptides (green) uniquely match to the KYP sequence. (C) Description of recovered peptides …
Figure 4 with 2 supplements
TrAP interacts directly with KYP through the SET domain.
(A, B) In vitro pull-down assays showed that KYP specifically interacted with TGMV TrAP (A) and CaLCuV TrAP (B). Left panel, Coomassie brilliant blue R250 staining of the proteins shows their …
Figure 4—figure supplement 1
TrAP does not interact with LHP1 in vitro.
In vitro pull down assays of GST-TrAP by MBP-LHP1 is shown. Left panel, Coomassie brilliant blue R250 staining of the proteins shows their mobility. Right panel, output of in vitro pull-down assays. …
Figure 4—figure supplement 2
TrAP directly interacts with KYP paralogs SUVH2, 5, and 6.
In vitro pull down assays. Left panel, coomassie brilliant blue R250 staining of the proteins shows their mobility. Right panel, output of in vitro pull-down assays. The recovered MBP-tagged bait …
Figure 5
TrAP inhibited HMTase activity of KYP in vitro.
(A) Coomassie staining of purified proteins uses for the assays. (B) In vitro HMTase reconstitution assays with different molar ratio of MBP and MBP-TrAP proteins (0–10×) relative to GST-KYP. The …
Figure 6 with 2 supplements
ChIP-qPCR analyses of H3K4me3, H3K9me2, and H3K27me3 in TrAP-regulated loci in vivo.
(A) TrAP-activated transposons in heterochromatic regions contained reduced H3K9me2 and H3K27me3 but did not show consistent variation in H3K4me3 marks. (B) TrAP-upregulated flowering genes showed …
Figure 6—figure supplement 1
Western blot analysis to show specificity of antibodies used for ChIP assays in the study.
Crude extract (A) and isolated nuclei (B) were probed with antibodies against histone 3, H3K9me2 (Abcam Cat# ab1220), H3K27me3 (Millipore Cat# 07-449) and H3K4me3 (Millipore Cat# 04-745), …
Figure 6—figure supplement 2
ChIP-PCR assays for selected flowering genes and heterochromatic loci confirm ChIP-qPCR.
(A) ChIP-PCR analysis of various histone 3 modifications in flowering genes in different genetic backgrounds. (B) ChIP-PCR analysis of various histone 3 modifications in TEs in different genetic …
Figure 7 with 1 supplement
TrAP reduces CHH DNA methylation in vivo.
(A) Genome-wide heatmap of DNA methylation levels in Col-0, kyp mutant, and TrAP transgenic plants (Left). Sequence context of all cytosine, CG, CHG, and CHH methylation was depicted as black, …
Figure 7—figure supplement 1
Gene ontology of CHH hypomethylated genes in TrAP transgenic plants and kyp mutant.
The genes associated to the CHH hypomethylated DMRs in both TrAP transgenic and kyp mutant plants underwent Gene Ontology analysis using AgriGo tool with TAIR10 as reference.
Figure 8 with 1 supplement
KYP methylates Geminivirus chromatin as a host defense.
(A) Western blot analysis of kyp complementation lines expressing PKYP- or 35S-Flag-4Myc-KYP using anti-myc antibody. *, a cross-reaction band serves as a loading control. (B) Representative CaLCuV …
Figure 8—figure supplement 1
Virus chromatin contains H3K9me2 marks.
ChIP-PCR assays of H3K9me2 marks on the CaLCuV DNA A. ChIP assays were conducted on 9-day-old seedlings using antibodies specific for H3K9me2 (Abcam Cat# ab1220), H3K27me3 (Millipore Cat# 07-449), …
Figure 9 with 1 supplement
Infectivity of CaLCuV lacking functional TrAP protein.
(A) Sequence alignment of CaLCuV Δtrap and CaLCuV sequences. The translated amino acids are shown for each sequence, and the XbaI restriction site resulting from the T to A point mutation is …
Figure 9—figure supplement 1
CaLCuV lacking functional TrAP protein cannot cause systemic infection in wild-type plants.
The region corresponding to TrAP gene was amplified by PCR using the primers AL3_cterm_F and AL1_cterm_R (Supplementary file 8) from total DNA extracted from plants infected with CaLCuV or CaLCuV Δtr…
Author response image 1
Additional files
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Supplementary file 1
Microarray analysis of 35S-TrAP transgenic plants.
- https://doi.org/10.7554/eLife.06671.021
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Supplementary file 2
Transcriptome comparison of the genes deregulated by TrAP overexpression and lhp1 loss-of-function mutant.
- https://doi.org/10.7554/eLife.06671.022
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Supplementary file 3
Expression levels of genes encoding for TGS components in TrAP transgenic plants.
- https://doi.org/10.7554/eLife.06671.023
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Supplementary file 4
List of proteins tested for interaction with TrAP.
- https://doi.org/10.7554/eLife.06671.024
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Supplementary file 5
CG methylation analysis of Col-0 wild type, kyp mutant, and TrAP transgenic plants.
- https://doi.org/10.7554/eLife.06671.025
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Supplementary file 6
CHG methylation analysis of Col-0 wild type, kyp mutant, and TrAP transgenic plants.
- https://doi.org/10.7554/eLife.06671.026
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Supplementary file 7
CHH methylation analysis of Col-0 wild type, kyp mutant, and TrAP transgenic plants.
- https://doi.org/10.7554/eLife.06671.027
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Supplementary file 8
List of primers used in this article.
- https://doi.org/10.7554/eLife.06671.028
