Arabidopsis double-stranded RNA binding protein DRB3 participates in methylation-mediated defense against geminiviruses

Abstract

Arabidopsis encodes five double-stranded RNA binding (DRB) proteins. DRB1 and DRB2 are involved in microRNA (miRNA) biogenesis, while DRB4 functions in cytoplasmic posttranscriptional small interfering RNA (siRNA) pathways. DRB3 and DRB5 are not involved in double-stranded RNA (dsRNA) processing but assist in silencing transcripts targeted by DRB2-associated miRNAs. The goal of this study was to determine which, if any, of the DRB proteins might also participate in a nuclear siRNA pathway that leads to geminivirus genome methylation. Here, we demonstrate that DRB3 functions with Dicer-like 3 (DCL3) and Argonaute 4 (AGO4) in methylation-mediated antiviral defense. Plants employ repressive viral genome methylation as an epigenetic defense against geminiviruses, using an RNA-directed DNA methylation (RdDM) pathway similar to that used to suppress endogenous invasive DNAs such as transposons. Chromatin methylation inhibits virus replication and transcription, and methylation-deficient host plants are hypersusceptible to geminivirus infection. Using a panel of drb mutants, we found that drb3 plants uniquely exhibit a similar hypersensitivity and that viral genome methylation is substantially reduced in drb3 compared to wild-type plants. In addition, like dcl3 and ago4 mutants, drb3 plants fail to recover from infection and cannot accomplish the viral genome hypermethylation that is invariably observed in asymptomatic, recovered tissues. Small RNA analysis, bimolecular fluorescence complementation, and coimmunoprecipitation experiments show that DRB3 acts downstream of siRNA biogenesis and suggest that it associates with DCL3 and AGO4 in distinct subnuclear compartments. These studies reveal that in addition to its previously established role in the miRNA pathway, DRB3 also functions in antiviral RdDM.

Importance: Plants use RNA-directed DNA methylation (RdDM) as an epigenetic defense against geminiviruses. RNA silencing pathways in Arabidopsis include five double-stranded RNA binding proteins (DRBs) related to Drosophila R2D2 and mammalian TRBP and PACT. While DRB proteins have defined roles in miRNA and cytoplasmic siRNA pathways, a role in nuclear RdDM was elusive. Here, we used the geminivirus system to show that DRB3 is involved in methylation-mediated antiviral defense. Beginning with a panel of Arabidopsis drb mutants, we demonstrated that drb3 plants uniquely show enhanced susceptibility to geminiviruses. Further, like dcl3 and ago4 mutants, drb3 plants fail to hypermethylate the viral genome, a requirement for host recovery. We also show that DRB3 physically interacts with the RdDM pathway components DCL3 and AGO4 in the nucleus. This work highlights the utility of geminiviruses as models for de novo RdDM and places DRB3 protein in this fundamental epigenetic pathway.

FIG 1

Arabidopsis drb3 mutants show enhanced susceptibility to geminivirus infection. (A) Photographs illustrate CaLCuV disease symptoms in wild-type (Col-0 ecotype) and drb3 mutant plants at 14 days postinoculation. (B) BCTV symptoms at 21 days postinoculation. BCTV has an inherently longer latent period than CaLCuV. (C) Histograms show the percentages of cytosines methylated in CaLCuV IR DNA isolated from wild-type and drb3 plants, as determined by bisulfite sequencing.

FIG 2

Arabidopsis drb3 and dcl3 mutants do not recover from infection with BCTV L2⁻ mutant virus. (A and B) The photographs show secondary tissue of wild-type (Col-0), drb2, drb3, drb4, and drb5 plants, as well as dcl2, dcl3, and dcl4 plants, infected with BCTV L2⁻ virus. Note severe disease symptoms in the drb3 and dcl3 mutants and recovery (near absence of symptoms) in the wild type and all other mutants. Although deformation of floral tissue was not observed in secondary tissue of infected drb4 and drb5 mutants, plants were occasionally stunted. Examples of stunted drb4 and drb5 plants are shown in this figure. (C) Close-up views of infected shoots from drb3, drb4, dcl3, and dcl4 plants.

FIG 3

Arabidopsis drb3 and dcl3 mutants fail to hypermethylate the viral genome. Methylation of BCTV L2⁻ IR DNA was assessed by bisulfite sequencing. (A) Histograms indicate the percentages of cytosine residues methylated in different sequence contexts in viral DNA obtained from secondary tissues of wild-type (Col-0 or Ler-0) or mutant plants. The drb and dcl mutants are in Col-0, whereas ago4 is in the Ler-0 ecotype background. Bars indicate the mean ± standard error (SE). Also shown are cytosine methylation profiles of BCTV L2⁻ DNA obtained from recovered wild-type Col-0 (B), nonrecovered drb3 (C), recovered drb4 (D), nonrecovered dcl3 (E), recovered dcl4 (F), recovered wild-type Ler-0 (G), and nonrecovered ago4 (H) plants. A second, independent experiment included recovered wild-type Col-0 (I), nonrecovered drb3 (J), and nonrecovered dcl3 (K) plants. The dots represent all cytosines in the IR and are color coded according to sequence context (red, CG; blue, CNG; green, CHH). Filled circles indicate methylation, and each line represents the sequence of an individual clone, arranged from most to least methylated. Significant reductions in methylation were noted for drb3 (P = 0.013) compared to wild-type controls, as determined by the Mann-Whitney rank sum test.

FIG 4

Hypermethylated viral genomes are associated with H3K9me2. ChIP-BS was performed with extracts from BCTV L2⁻-infected, nonrecovered primary tissue using antibodies to H3Ac or H3K9me2. Input (extract without ChIP) and ChIP DNAs were bisulfite treated, and PCR primers spanning the viral IR were used to amplify associated DNA. Products were cloned and sequenced. (A) Histograms indicate the percentages of cytosine residues methylated in different sequence contexts. (B) Cytosine methylation profiles. (C and D) In a second, independent experiment, ChIP-BS was performed with extracts from BCTV L2⁻ infected, recovered secondary tissue, where the input DNA was already hypermethylated. The dots represent all cytosines in the IR and are color coded according to sequence context (red, CG; blue, CNG; green, CHH). Filled circles indicate methylation, and each line represents the sequence of an individual clone, arranged from most to least methylated. The difference between the nonrecovered input and H3K9me2 fractionated samples was significant (P = 0.007), as determined by the Mann-Whitney rank sum test.

FIG 5

DRB3 interacts with DCL3 and AGO4 in distinct subnuclear bodies. BiFC analysis of the indicated DCL, DRB, and AGO proteins in N. benthamiana epidermal cells was performed. Constructs expressing proteins fused to the N- or C-terminal portion of YFP were delivered by agroinfiltration to N. benthamiana leaves. Cells were photographed at 36 h postinfiltration using a confocal laser scanning microscope. RFP-histone 2B (RFP-H2B) and RFP-fibrillarin were used as markers for the nucleus and nucleolus, respectively. Protein combinations are indicated above each photograph. The photographs in panel A show lower-magnification views (×20), while panel B shows high-magnification views of nuclei (×100).

FIG 6

DRB3 coimmunoprecipitates with DCL3 and AGO4. Immunoprecipitation (IP) was performed with FLAG antibody (α-FLAG) or ADK antibody (α-ADK, negative control), and proteins in immune complexes were detected by Western blotting using HA antibody (α-HA), α-FLAG, or α-ADK. (A) Extracts from plants expressing DCL3-FLAG or HA₂His₆-DRB3 were mixed at ∼10:1 to compensate for extremely low levels of DCL3-FLAG expression (below the level of detection in input samples). (B) Extracts were obtained from plants coexpressing FLAG-AGO4 and HA₂His₆-DRB3.

FIG 7

DRB3 is not required for biogenesis of geminivirus-derived siRNAs. RNA blot hybridization shows the accumulation of virus-specific siRNAs in BCTV-infected wild-type (Col-0 and Ler-0) and mutant (drb3, drb4, dcl3, dcl4, and ago4) plants. Ethidium bromide-stained rRNA served as a loading control. The positions of oligonucleotide size markers are indicated at the left.