Arabidopsis RNA-dependent RNA polymerases and dicer-like proteins in antiviral defense and small interfering RNA biogenesis during Turnip Mosaic Virus infection

Abstract

Plants respond to virus infections by activation of RNA-based silencing, which limits infection at both the single-cell and system levels. Viruses encode RNA silencing suppressor proteins that interfere with this response. Wild-type Arabidopsis thaliana is immune to silencing suppressor (HC-Pro)-deficient Turnip mosaic virus, but immunity was lost in the absence of DICER-LIKE proteins DCL4 and DCL2. Systematic analysis of susceptibility and small RNA formation in Arabidopsis mutants lacking combinations of RNA-dependent RNA polymerase (RDR) and DCL proteins revealed that the vast majority of virus-derived small interfering RNAs (siRNAs) were dependent on DCL4 and RDR1, although full antiviral defense also required DCL2 and RDR6. Among the DCLs, DCL4 was sufficient for antiviral silencing in inoculated leaves, but DCL2 and DCL4 were both involved in silencing in systemic tissues (inflorescences). Basal levels of antiviral RNA silencing and siRNA biogenesis were detected in mutants lacking RDR1, RDR2, and RDR6, indicating an alternate route to form double-stranded RNA that does not depend on the three previously characterized RDR proteins.

Figure 1.

TuMV HC-Pro Containing the AS9 Substitution Lacks RNA Silencing Suppressor Activity in a miR171-Guided Transient Assay in N. benthamiana Leaves. Parental and mutant forms of HC-Pro (HC), or two other suppressors (p19 and p21), were coinfiltrated with 35S:miR171-precursor and 35S:SCL6-IV. Leaves were collected 48 h after infiltration. SCL6-IV mRNA (a) and its miR171-guided 3′ cleavage product (b) were analyzed in three independent samples by RNA gel blot assays using randomly primed ³²P-radiolabeled probes. For each treatment, the average (a)/(b) ratio for three replicates is indicated. Note that the parental, but not mutant, forms of the suppressors inhibited target cleavage.

Figure 2.

Propagation of TuMV-AS9-GFP in a Transient Infection System. (A) Infection of N. benthamiana leaves by TuMV-AS9-GFP after launching by Agrobacterium infiltration in the presence of p19-HA at the indicated cell densities. Infection was measured as the number of multicellular infection foci at 5 DAI under UV light in a 4-cm² area in the middle of the leaf. (B) Effect of p19-HA on infection efficiency of N. benthamiana leaves by TuMV-AS9-GFP and TuMV-GFP. Cultures expressing p19-HA were provided at constant cell density (OD₆₀₀ = 5 × 10⁻¹). Cultures transformed with pCB-TuMV-AS9-GFP or pCB-TuMV-GFP were provided at 10-fold dilutions of OD₆₀₀ = 5 × 10⁻¹. Empty vector was used to normalize the cell density to OD₆₀₀ = 1.0. Images were taken at 5 DAI under UV light. Infection foci were plotted for samples receiving cultures at OD = 5 × 10⁻⁴. The histogram shows the average and se for 16 leaves per treatment. (C) Time course of accumulation of TuMV-AS9-GFP CP in N. benthamiana leaves infiltrated with Agrobacterium cultures containing pP19-HA and pCB-TuMV-AS9-GFP (OD₆₀₀ = 5 × 10⁻¹). Relative accumulation was plotted using 6 DAI measurements equal to 1.0. The histogram shows the average and se for three replicates (individual leaves) per treatment.

Figure 3.

Local and Systemic Infection of Arabidopsis by TuMV-GFP and TuMV-AS9-GFP. (A) Infection efficiency and CP accumulation in inoculated leaves at 7 DAI. Images were taken at 7 DAI under UV light. The number of infection foci for TuMV-GFP and TuMV-AS9-GFP was expressed relative to those in Col-0 (2.6 ± 1 foci per leaf) or dcl2-1 dcl3-2 dcl4-2 (4.6 ± 1.5), respectively. The histogram shows the average and se for 52 leaves and 13 plants per treatment. For each virus, bars with the same letter are not statistically different (Tukey's test with α = 0.01). CP accumulation values (average ± se) were normalized to the average number of infection foci. M, mock inoculated. (B) CP accumulation in inflorescence clusters at 7 and 15 DAI, relative to TuMV-AS9-GFP in dcl2-1 dcl3-2 dcl4-2 plants. Average and se for each virus-Arabidopsis genotype combination are indicated at the bottom. (C) Col-0 and dcl2-1 dcl3-2 dcl4-2 triple mutant plants inoculated with TuMV-AS9-GFP or TuMV-GFP (10 DAI) or mock-inoculated. (D) Bioassay of TuMV-AS9-GFP from systemically infected dcl2-1 dcl3-2 dcl4-2 mutant source plants (15 DAI). Average number of infection foci (+ se) at 7 DAI were plotted. Bars with the same letter are not statistically different (Tukey's test with α = 0.01).

Figure 4.

Profile of TuMV-Derived siRNAs in Whole Arabidopsis Plants at 10 DAI. Values are averages and se from three replicate libraries, normalized to reads/million. Sense and antisense polarity reads were plotted on the y axis in the positive and negative directions, respectively. (A) Abundance, by size class and polarity, of TuMV-derived small RNAs at 10 DAI in three plant genotypes. (B) Genome-wide distribution of 21-nucleotide TuMV-derived siRNAs at 10 DAI. The scale was capped at 150 reads. (C) Distribution of 21-nucleotide siRNAs mapping to the 5′ UTR region of TuMV RNA at 10 DAI. Numbers in parenthesis indicate the percentage of antisense TuMV-derived reads that mapped to the 5′ UTR. Based on length, a random distribution of siRNA along the TuMV genome would yield 1.3% mapping to the 5′ UTR.

Figure 5.

Local and Systemic Infection of Arabidopsis dcl Mutants by TuMV-GFP and TuMV-AS9-GFP. (A) Local infection foci in inoculated rosette leaves at 6 DAI. The histogram shows average (+ se) number of foci from 14 plants, each with four inoculated leaves. For each virus, bars with the same letter are not statistically different (Tukey's test with α = 0.01). TuMV-GFP and TuMV-AS9-GFP infection efficiency is expressed relative to Col-0 and to dcl2-1 dcl3-2 dcl4-2, respectively. (B) GFP fluorescence in noninoculated rosette and cauline leaves from plants inoculated with TuMV-AS9-GFP at 15 DAI. TuMV-GFP infection of Col-0 is shown for comparison. (C) TuMV-GFP accumulation (CP, average + se) in leaves (7 and 15 DAI) and inflorescence (15 DAI), expressed relative to accumulation of TuMV-AS9-GFP CP in dcl2-1 dcl3-2 dcl4-2. (D) TuMV-AS9-GFP accumulation (CP, average + se) in leaves (7 and 15 DAI) and inflorescence (15 DAI), relative to dcl2-1 dcl3-2 dcl4-2. Within each tissue, bars with the same letter are not statistically different (Tukey's test with α = 0.01).

Figure 6.

Accumulation of TuMV-GFP– and TuMV-AS9-GFP–Derived siRNAs in Arabidopsis dcl Mutants. Virus-derived siRNA were detected using ³²P-radiolabeled probes made by random priming of cDNA corresponding to the CI protein coding region. Virus-derived siRNA signals were normalized to U6 RNA signals from the same blots. In dcl4-2 single and dcl3-1 dcl4-2 double mutants, siRNAs were 22 nucleotides long. In dcl2-1 dcl4-2 double mutants, siRNAs were 24 nucleotides long. (A) Inoculated rosette leaves at 7 DAI. (B) Noninoculated cauline leaves at 15 DAI. (C) Inflorescence clusters at 15 DAI. (D) Average (+ se) TuMV-GFP–derived siRNA signal intensity in four independent replicates in each genotype. Within each tissue, bars with the same letter are not statistically different (Tukey's test with α = 0.01). (E) Accumulation of TuMV-AS9-GFP–derived siRNAs.

Figure 7.

Local and Systemic Infection Arabidopsis rdr Mutants by TuMV-GFP and TuMV-AS9-GFP. (A) Local infection foci in inoculated rosette leaves at 6 DAI. The histogram shows average (+ se) number of foci from 14 plants, each with four inoculated leaves. For each virus, bars with the same letter are not statistically different (Tukey's test with α = 0.01). TuMV-GFP and TuMV-AS9-GFP infection efficiency is expressed relative to Col-0 and to dcl2-1 dcl3-2 dcl4-2, respectively. (B) GFP fluorescence in noninoculated rosette and cauline leaves from plants inoculated with TuMV-AS9-GFP, at 15 DAI. TuMV-GFP infection of Col-0 is shown for comparison. (C) rdr1-1 rdr2-1 rdr6-15 triple mutant plants (10 DAI) inoculated with TuMV-AS9-GFP, TuMV-GFP, or mock inoculated. (D) TuMV-GFP accumulation (CP, average + se) in leaves (7 and 15 DAI) and inflorescence (15 DAI), relative to CP levels in Col-0. Within each tissue type, no statistically significant differences (Tukey's test with α = 0.01) were detected between genotypes. (E) TuMV-AS9-GFP CP accumulation in leaves (7 and 15 DAI) and inflorescence (15 DAI), relative to dcl2-1 dcl3-2 dcl4-2. Within each tissue, bars with the same letter are not statistically different (Tukey's test with α = 0.01).

Figure 8.

Accumulation of TuMV-GFP– and TuMV-AS9-GFP–Derived siRNAs in Arabidopsis rdr Mutants. Blot assays were done as described in Figure 6. (A) Inoculated leaves at 7 DAI. (B) Noninoculated cauline leaves at 15 DAI. (C) Inflorescence clusters at 15 DAI. Both TuMV-GFP and TuMV-AS9-GFP-inoculated samples were tested. (D) Average (+ se) TuMV-GFP–derived siRNA signal intensity in four independent replicates in each genotype. Within each tissue, bars with the same letter are not statistically different (Tukey's test with α = 0.01). (−) and (+) indicate Col-0 mock-inoculated or TuMV-GFP infected, respectively.