ARGONAUTE4 is required for resistance to Pseudomonas syringae in Arabidopsis

Abstract

Here, we report the characterization of the Arabidopsis thaliana ocp11 (for overexpressor of cationic peroxidase11) mutant, in which a beta-glucuronidase reporter gene under the control of the H(2)O(2)-responsive Ep5C promoter is constitutively expressed. ocp11 plants show enhanced disease susceptibility to the virulent bacterium Pseudomonas syringae pv tomato DC3000 (P.s.t. DC3000) and also to the avirulent P.s.t. DC3000 carrying the effector avrRpm1 gene. In addition, ocp11 plants are also compromised in resistance to the nonhost pathogen P. syringae pv tabaci. Genetic and molecular analyses reveal that ocp11 plants are not affected in salicylic acid perception. We cloned OCP11 and show that it encodes ARGONAUTE4 (AGO4), a component of the pathway that mediates the transcriptional gene silencing associated with small interfering RNAs that direct DNA methylation at specific loci, a phenomenon known as RNA-directed DNA methylation (RdDM). Thus, we renamed our ocp11 mutant ago4-2, as it represents a different allele to the previously characterized recessive ago4-1. Both mutants decrease the extent of DNA cytosine methylation at CpNpG and CpHpH (asymmetric) positions present at different DNA loci and show commonalities in all of the molecular and phenotypic aspects that we have considered. Interestingly, we show that AGO4 works independently of other components of the RdDM pathway in mediating resistance to P.s.t. DC3000, and loss of function in other components of the pathway operating upstream of AGO4, such as RDR2 and DCL3, or operating downstream, such as DRD1, CMT3, DRM1, and DRM2, does not compromise resistance to this pathogen.

Figure 1.

Characterization of ocp11 Plants. (A) Comparison of the macroscopic appearance of a 5-week-old wild-type plant (left) and an ocp11 plant (right). (B) Comparative histochemical analysis of GUS activity in fully expanded rosette leaves from a parental wild-type plant carrying the PEp5C:GUS transgene (left), an ocp11 mutant plant (center), and an F1 ocp11/OCP11 plant (right). (C) Histograms of P.s.t. DC3000 growth rate in wild-type (Col-0), ocp11, npr1, and dth9 plants. The growth of the bacteria was determined at 0 (white bars), 3 (black bars), and 5 (gray bars) DPI. Analysis of variance indicates differences with a significance level of 0.05. FW, fresh weight.

Figure 2.

Effect of SA on P.s.t. DC3000 Growth Rate in ocp11 Plants. (A) Effect of SA on the growth rate of P.s.t. DC3000 in wild-type (Col-0) and ocp11 plants. Two days prior to the inoculation with P.s.t. DC3000, plants were sprayed once with either water or a 200 μM solution of SA until runoff. (B) Induced expression of GST-6 and PR-1 following inoculation of wild-type and ocp11 plants with P.s.t. DC3000 (avrRpm1). h.p.i., hours after inoculation. (C) Histograms of P.s.t. DC3000 growth rate in wild-type (Col-0), ocp11, sid2-1, eds5-3, ocp11 sid2-1, and ocp11 eds5-3 plants. (D) Histograms of P.s. tabaci growth rate in wild-type (Col-0), ocp11, and nho1 plants. (E) Histograms of P.s.t. DC3000 (avrRpm1) growth rate in wild-type (Col-0), ocp11, and rpm1-1 plants. Bacterial growth was determined at 0 (white bars), 3 (black bars), and 5 (gray bars) DPI. Analysis of variance indicates differences with a significance level of 0.05.

Figure 3.

Positional Cloning of OCP11. (A) Location of OCP11 on the BAC clone T20P8. OCP11 was positioned in a 137-kb region between BACs F12C20 and T20P8 and corresponds to the gene At2g27040. Lowercase letters mark nucleotide sequences at the beginning of the corresponding PIWI domain of the protein encoded by the At2g27040 gene. The mutant allele is indicated below the wild-type sequence. The G-to-A transition is indicated in boldface uppercase letters. The deduced amino acid sequences are indicated in uppercase single-letter code below each nucleotide triplet, and the boldface letters mark the amino acid changes (E to K) in the protein sequences. (B) Histochemical staining for GUS activity in fully expanded rosette leaves obtained from wild-type transgenic plants carrying PEp5C:GUS upon transformation with P35S empty vector (left), construct P35S:ocp11 (center), or construct P35S:OCP11 (right).

Figure 4.

Comparison of ago4-1 and ago4-2 Phenotypes. (A) Quantification of 5mC at positions CpG (left), CpHpH (center), and CpNpG (right) present at the At SN1 locus, as detected by bisulfite sequencing of DNA samples from ago4-2, ago4-1, and the corresponding wild-type controls, Col-0 and Ler, respectively. (B) Histochemical staining of GUS activity in fully expanded rosette leaves obtained from wild-type transgenic PEp5C:GUS plants (left), homozygous ago4-1 PEp5C:GUS plants (center), and heterozygous F1 ago4-1/AGO4-1 plants (right). (C) Histograms of P.s.t. DC3000 growth rate in ago4-2, ago4-1, and the corresponding wild-type plants, Col-0 and Ler, respectively. (D) Histograms of P.s.t. DC3000 (avrRpm1) growth rate in wild-type (Ler), ago4-1, and nho1 plants. (E) Histograms of P.s. tabaci growth rate in wild-type (Ler), ago4-1, and nho1 plants. Bacterial growth was determined at 0 (white bars), 3 (black bars), and 5 (gray bars) DPI. Analysis of variance indicates differences with a significance level of 0.05.

Figure 5.

P.s.t. DC3000 Growth Rate in Different Mutants of the RdDM Pathway. (A) Histograms of P.s.t. DC3000 growth rate in wild-type (Col-0), dcl3-1, rdr2-1, and ago4-2 plants. (B) Histograms of P.s.t. DC3000 growth rate in wild-type (Ler), ago4-1, and cmt3-7 plants. (C) Histograms of P.s.t. DC3000 growth rate in wild-type (Col-0), ago4-2, dmr1-2 dmr2-2, and drd1-6 plants. Growth of the bacteria was determined at 0 (white bars), 3 (black bars), and 5 (gray bars) DPI. Analysis of variance indicates differences with a significance level of 0.05.

Figure 6.

Methylation Status of the Ep5C 5′ Promoter Region in Arabidopsis and Tomato Plants. (A) Quantification of 5mC at positions CpG (left), CpHpH (center), and CpNpG (right) in a 188-nucleotide 5′ promoter region of PEp5C:GUS in Arabidopsis transgenic plants, as detected by bisulfite sequencing of DNA samples from ago4-1 PEp5C:GUS and the corresponding wild-type control P_Ep5C:GUS. (B) Quantification of 5mC at positions CpG (left), CpHpH (center), and CpNpG (right) in a 188-nucleotide 5′ promoter region of the Ep5C locus in tomato. DNA samples were obtained from leaf tissue at 4 h after inoculation with P.s.t. DC3000. Control plants were inoculated with 10 mM MgSO₄ (mock).

Figure 7.

Schematic Representation of the Arabidopsis RdDM Pathway and Proposed Mode of Action of AGO4 in Plant Defense. RdDM (solid lines) requires RNA polymerase IV (Pol IVa) and RNA-dependant RNA polymerase 2 (RDR2) to form double-stranded RNA (dsRNA) and Dicer-Like3 (DCL3) to form 24-nucleotide siRNAs. The 24-nucleotide siRNAs guide AGO4 to its DNA target, and Chromomethylase3 (CMT3) and Domains Rearranged Methyltransferase2 (DRM2), dependent on Defective in RNA-directed DNA methylation (DRD1), mediate DNA methylation. For the AGO4 mode of action in plant defense, it is proposed (dashed lines) that a source of 24-nucleotide siRNAs different from that generated by DCL3 or a source of miRNAs would be required. This would require a DCL (DCL?) protein different from DCL3. It is also possible that other methyltransferases (¿?), different from DRM2 and CMT3, would be necessary for AGO4 action in plant defense. nt, nucleotide.