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Proc Natl Acad Sci U S A. Nov 21, 2006; 103(47): 18002–18007.
Published online Oct 27, 2006. doi:  10.1073/pnas.0608258103
PMCID: PMC1693862
Plant Biology

A pathogen-inducible endogenous siRNA in plant immunity

Abstract

RNA interference, mediated by small interfering RNAs (siRNAs), is a conserved regulatory process that has evolved as an antiviral defense mechanism in plants and animals. It is not known whether host cells also use siRNAs as an antibacterial defense mechanism in eukaryotes. Here, we report the discovery of an endogenous siRNA, nat-siRNAATGB2, that is specifically induced by the bacterial pathogen Pseudomonas syringae carrying effector avrRpt2. We demonstrate that the biogenesis of this siRNA requires DCL1, HYL1, HEN1, RDR6, NRPD1A, and SGS3. Its induction also depends on the cognate host disease resistance gene RPS2 and the NDR1 gene that is required for RPS2-specified resistance. This siRNA contributes to RPS2-mediated race-specific disease resistance by repressing PPRL, a putative negative regulator of the RPS2 resistance pathway.

Keywords: antibacterial defense, DCL1, RDR6, RPS2-specific

Endogenous small interfering RNAs (siRNAs) and microRNAs (miRNAs) have emerged as important regulators of eukaryotic gene expression by guiding mRNA cleavage, translational inhibition, or chromatin modification (1, 2). In Arabidopsis, >100 miRNAs have been reported and shown to be important for plant development (3, 4) and abiotic stress tolerance (57). One miRNA was recently shown to contribute to basal defense against bacteria by regulating auxin signaling (8). In contrast to the relatively limited number of miRNAs, thousands of endogenous siRNAs have been sequenced (6, 911). However, their biological roles are largely unknown except for the functions of transacting siRNAs (ta-siRNA) in plant development and hormone signaling (4) and the roles of some chromatin-associated siRNAs in DNA methylation and transcriptional gene silencing (4). Borsani et al. (12) recently discovered a new class of endogenous siRNAs derived from the overlapping region of a pair of natural antisense transcripts (NATs) (12). These so-called nat-siRNAs regulate salt stress response in Arabidopsis (12). Despite large intergenic spaces, a significant proportion of eukaryotic genomes are arranged as NATs (13). More than 1,000 pairs of NATs exist in Arabidopsis (14, 15). Our analysis of transcript profiles from an Arabidopsis microarray database (16) has revealed that, in many cases, one transcript of a NAT pair is specifically induced under certain abiotic or biotic conditions. The induced transcript may pair with the existing antisense transcript and trigger the nat-siRNA formation, resulting in the silencing of the antisense transcript in cis or other homologous loci in trans. NATs may serve as one of the major sources of endogenous siRNAs for gene regulation in response to different environmental conditions. This hypothesis is well supported by the presence of >100 nat-siRNAs in the Massively Parallel Signature Sequencing (MPSS) and Arabidopsis Small RNA Project (ASRP) databases (9, 17). In this study, we identified a nat-siRNA that is specifically induced by the bacterial pathogen Pseudomonas syringae (Ps) carrying effector avrRpt2 (18). We demonstrate that its induction depends on a novel biogenesis pathway that requires the cognate host disease resistance (R) gene RPS2 (19) and the NDR1 (20) gene that is also required for RPS2-specified resistance. This siRNA represses a negative regulator of the RPS2 resistance pathway.

Results

Induction of a nat-siRNA by Bacterial Pathogen Ps Carrying avrRpt2.

Pathogen effectors can be recognized by R proteins and can trigger a series of disease resistance responses, including activating and repressing a large array of genes (21). To address whether endogenous siRNAs play a role in gene expression reprogramming in R gene-mediated disease resistance, we searched the small RNA databases (9, 17) and examined nat-siRNAs generated from NAT pairs that are potentially regulated by bacterial pathogenesis. Excitingly, we discovered that a 22-nt nat-siRNA (ASRP1957), derived from the overlapping region of a Rab2-like small GTP-binding protein gene (ATGB2, At4g35860) and a PPR (pentatricopeptide repeats) protein-like gene (PPRL, At4g35850), is strongly induced by Ps pathovar tomato (Pst) carrying avirulence (avr) gene avrRpt2 but not avrRpm1, avrRps4, or avrPphB (Fig. 1A). We named it nat-siRNAATGB2. We used Pst strain DC3000 for all of the experiments in this study.

Fig. 1.
A nat-siRNA is induced by Pst (avrRpt2). (A) Detection of the nat-siRNA by Northern blot analysis. The nat-siRNA sequence is shown under the panel. Small RNA was extracted from the leaves harvested at 15 hpi of Pst (2 × 107 cfu/ml) carrying various ...

The nat-siRNAATGB2 sequence is complementary to the 3′ UTR region of the antisense gene PPRL and thus could potentially induce silencing of PPRL. We examined the expression of PPRL as well as the sense gene ATGB2 upon Pst challenge. The ATGB2 transcript was strongly induced by both Pst (avrRpt2) and Pst (avrRpm1) (Fig. 1B), whereas the PPRL mRNA was substantially down regulated only by Pst (avrRpt2) infection where the nat-siRNAATGB2 was strongly induced (Fig. 1 A and C). The result suggests that down-regulation of PPRL depends on the induction of the nat-siRNAATGB2 and the induction of ATGB2 alone is not sufficient for inducing nat-siRNAATGB2. To determine whether the induction of ATGB2 is necessary for inducing nat-siRNAATGB2, we obtained a T-DNA insertion line (Salk_083103) of ATGB2 from the Salk collection (22). The homozygous line is a partial knock-down mutant with T-DNA inserted in the 3rd intron (Fig. 1D). We detected less induction of nat-siRNAATGB2 and less repression of PPRL mRNA expression in this knock-down line than in the WT plants after Pst (avrRpt2) challenge (Fig. 1 D and E). These results suggest that the induction of sense transcript ATGB2 is necessary but not sufficient for nat-siRNAATGB2 accumulation (Fig. 1 A, B, and D), implying that the induction of this siRNA is under multiple layers of control and requires other factors associated with Pst (avrRpt2) infection.

Biogenesis of nat-siRNAATGB2.

To define the components required for its biogenesis, we examined nat-siRNAATGB2 in Pst (avrRpt2)-challenged small RNA biogenesis mutants and the corresponding WT ecotypes. The Arabidopsis genome has four DICER-like (DCL) proteins (23). Interestingly, the induction of nat-siRNAATGB2 could be detected in dcl2-1, dcl3-1, and dcl4-2 mutants but not in the dcl1-9 mutant (Fig. 2A). The result indicates that the miRNA biogenesis component DCL1 is required for the formation of nat-siRNAATGB2. This observation differs from the biogenesis of the 24-nt nat-siRNASRO5 that requires DCL2, 21-nt nat-siRNAs that require both DCL1 and DCL2 (12), and ta-siRNAs that require DCL1 and DCL4 (24). We did not detect any other siRNAs generated from the overlapping region of PPRL and ATGB2 or siRNA that is complementary to nat-siRNAATGB2 (data not shown). Thus, this nat-siRNA is generated from a specific site of the overlapping region of PPRL and ATGB2 transcripts and is strand-specific.

Fig. 2.
Accumulation of nat-siRNAATGB2 depends on DCL1, HYL1, and RDR6, and also requires HEN1, NRPD1a, and SGS3. (A) Northern blot analysis of nat-siRNAATGB2 in various Pst (avrRpt2)-treated small RNA biogenesis mutants and their corresponding WT controls. MiR171 ...

Mutations in the dsRNA-binding protein HYL1 and RNA-dependent RNA polymerase (RDR) 6 also totally blocked the accumulation of nat-siRNAATGB2, whereas a mutation in RDR2 had no effect (Fig. 2A). RDR6 is required for virus-induced gene silencing, transgene silencing, and ta-siRNA production (25). HYL1 has been indicated to interact with DCL1 (26) and affects the accumulation of several miRNAs (27) and ta-siRNAs (24). The level of nat-siRNAATGB2 was reduced in sgs3, the RNA methyltransferase mutant hen1, and the RNA polymerase IVa mutant nrpd1a (Fig. 2A), which is similar to that of salt-induced nat-siRNAs (12). These results suggest a biogenesis pathway for nat-siRNAATGB2 in which nat-siRNAATGB2 is processed by the DCL1-HYL1 complex, stabilized by HEN1-mediated methylation, and amplified by RDR6-, SGS3-, and RNA polymerase IVa-mediated reactions.

To confirm that the down-regulation of PPRL depends on the induction of nat-siRNAATGB2, we examined the PPRL mRNA level in the mutants that failed to generate nat-siRNAATGB2 upon Pst (avrRpt2) infection. The down-regulation of PPRL was abolished in dcl1-9, hyl1, and rdr6 compared with WT Landsberg erecta (Ler), Nossen-0 (No) and C24, respectively (Fig. 2B). The sgs3 and nrpd1a mutants, where the accumulation of nat-siRNAATGB2 is significantly reduced, also accumulate 2- to 3-fold more PPRL mRNA than that in their corresponding controls (Fig. 2B). Thus, the down-regulation of PPRL is mediated by nat-siRNAATGB2.

To test whether the overlapping region is sufficient for generating nat-siRNAATGB2, the full-length or overlapping region of ATGB2 was coexpressed with PPRL transiently in Nicotiana benthamiana leaves. Flag-tagged PPRL cDNA with its 3′ UTR was cloned into a binary vector driven by the CaMV 35S promoter. Full-length (F) or only the overlapping region (O) of ATGB2 cDNA was cloned into the inducible expression vector pTA7002 (28). Substantial down-regulation of PPRL was observed at both the RNA and protein levels after induction of either full length (F) or only the overlapping region of ATGB2 (O) by dexamethasone (Dex) (Fig. 2C), as was the induction of nat-siRNAATGB2. The results suggest that the overlapping region alone is sufficient to give rise to the nat-siRNA and to induce antisense gene silencing.

Induction of nat-siRNAATGB2 Depends on RPS2 and NDR1.

Pathogen-derived effectors are recognized directly or indirectly by specific plant R proteins and trigger rapid race-specific resistance responses. The effector avrRpt2 of Ps is specifically recognized by the coiled-coil NBS-LRR type R protein RPS2 (19) and triggers a series of resistance responses, including the generation of reactive oxygen species, reprogramming of gene expression, and induction of hypersensitive responses (HR), which limit bacterial growth. The specific induction of nat-siRNAATGB2 by avrRpt2 implies a functional involvement of the siRNA in RPS2-mediated race-specific disease resistance. To further understand the regulation of nat-siRNAATGB2 by pathogen infection, we examined its accumulation in various mutants of resistance signaling components. Although the induction level of ATGB2 by avrRpt2 was not substantially different in these mutants, the accumulation of nat-siRNAATGB2 differed considerably (Fig. 3 A and B). nat-siRNAATGB2 was not detected in rps2 (101C) and ndr1 mutants, which indicates that nat-siRNAATGB2 induction requires the functional resistance protein RPS2 and NDR1, both of which are required for avrRpt2-induced resistance (20). Mutations in other resistance signaling components, SCF ubiquitin ligase complex component SGT1b, systemic acquired resistance signaling component NPR1, and ethylene signaling component EIN2 also reduced the level of nat-siRNAATGB2. Consistent with the silencing of PPRL by nat-siRNAATGB2, the mutants with no or reduced levels of nat-siRNAATGB2 showed no or less suppression of PPRL expression (Fig. 3C). These mutations had no effect on the accumulation of miR173, which demonstrates a specific biogenesis regulation of nat-siRNAATGB2 by the disease resistance signaling pathways. The jasmonic acid (JA) signaling mutant jar1, salicylic acid (SA) signaling mutant pad4, SA biosynthesis mutant eds16, and SGT1b homologue SGT1a had no effect on nat-siRNAATGB2 accumulation (Fig. 3A). These results suggest that some components in basal defense and ethylene signaling may interfere with nat-siRNAATGB2 regulation.

Fig. 3.
Accumulation of nat-siRNAATGB2 is controlled by RPS2 and some components of the disease resistance signaling pathway. Northern blot analysis of nat-siRNAATGB2 (A) and ATGB2 (B) was performed on Pst (avrRpt2)-treated defense-signaling mutants and WT Col-0 ...

PPRL Acts as a Negative Regulator of RPS2 Resistance Pathway.

Based on the down-regulation of PPRL by nat-siRNAATGB2 in response to Pst (avrRpt2) challenge, we hypothesized that PPRL may negatively regulate RPS2-mediated resistance. PPRL is an atypical PPR protein with an unknown function and is localized in mitochondria (29). To assess its function in disease resistance, we isolated T-DNA insertion lines of PPRL (Salk_013843 and Salk_071137) from the Salk collection (22) and also generated PPRL cDNA-Flag (without UTR) overexpression lines (Fig. 4A) for loss- and gain-of-function studies. Complete knockout of PPRL expression may lead to enhanced disease resistance to avrRpt2 because the PPRL gene is silenced after pathogen infection in the WT resistance plants. No difference was observed in the growth of both virulent Pst (EV) and avirulent Pst (avrRpt2) between PPRL knockout lines and the WT control (data not shown). It is likely that the possible enhanced resistance was masked by the existing strong resistance to avrRpt2 and, therefore, was difficult to score. However, when PPRL-Flag overexpression plants were inoculated with a high concentration (1 × 107 cfu/ml) of Pst (avrRpt2), delayed HR was observed (Fig. 4B) and the transgenic plants displayed considerably less electrolyte leakage at 24 h postinoculation (hpi) (Fig. 4C), which indicates a reduced level of cell death in the overexpression plants. Bacterial growth was measured on the plants infected with a low concentration (2 × 105 cfu/ml) of Pst carrying EV, avrRpm1, or avrRpt2. The Pst (avrRpt2) titer of the overexpression line 32 containing a high level of PPRL was ≈6- to 8-fold higher than that of the WT at 4 days postinoculation (dpi). Line 33, with a low level of PPRL overexpression, had about a 4- to 5-fold increase in Pst (avrRpt2) bacterial growth than that in the WT (Fig. 4D). No difference was observed in the growth of Pst (EV) or Pst (avrRpm1) between PPRL overexpression line and WT control. These results show that overexpression of PPRL attenuates RPS2-mediated disease resistance and suggest that PPRL may function as a negative regulator of the RPS2 pathway.

Fig. 4.
Overexpression of PPRL attenuates RPS2-mediated resistance in Arabidopsis plants. (A) Western blot analysis of transgenic Arabidopsis plants overexpressing PPRL (Sigma anti-FLAG, 1:2,000 dilution). Shown is the Rubisco large subunit from a gel that was ...

The induction of nat-siRNAATGB2 is blocked in dcl1-9, hyl1, and rdr6. Because the dcl1-9 mutation has strong pleiotropic phenotypes, we chose to examine bacterial growth in rdr6 and hyl1 mutants. An 8-fold increase of Pst (avrRpt2) bacterial growth was observed in rdr6 at 4 dpi compared with that in the WT C24 plants, whereas no significant difference in Pst (avrRpm1) growth was detected (Fig. 4E). Most strikingly, we observed a complete loss of RPS2-mediated resistance in hyl1, whereas RPM1-mediated disease resistance was not affected (Fig. 4E). hyl1 may affect the biogenesis of an array of small RNAs induced by Pst (avrRpt2), and the elimination of nat-siRNAATGB2 in hyl1 may contribute a portion of the observed pathogen susceptibility phenotype. As expected, we did not detect any obvious difference in pathogen growth between Col-0 WT control and dcl3-1 mutant (Fig. 4E), which affects the accumulation only of siRNAs associated with chromatin modification, but not nat-siRNAATGB2 (Fig. 2A). No difference in pathogen growth was observed between the rdr6, hyl1, or dcl3 mutants and their corresponding WT plants after Pst (avrRpm1) inoculation (Fig. 4E). Thus, RDR6 and HYL1 play critical roles in RPS2-mediated resistance pathway by controlling the biogenesis of nat-siRNAATGB2 and possibly of other endogenous siRNAs.

Discussion

Here we identified an endogenous siRNA, nat-siRNAATGB2, which is specifically induced by Pst (avrRpt2). This nat-siRNA is produced by a unique biogenesis pathway that requires DCL1, HYL1, HEN1, RDR6, SGS3, and RNA polymerase IVa (Fig. 5). Its formation not only requires the induction of the sense transcript ATGB2, but also depends on the host resistance gene RPS2 and its resistance signaling components, including NDR1 (Fig. 5). The biogenesis pathway of this 22-nt nat-siRNA suggests an intricate regulation of endogenous siRNA formation. The specific induction of nat-siRNAATGB2 leads to the silencing of the antisense gene PPRL. Our results suggest that PPRL is a negative regulator of RPS2 signaling pathway and silencing of PPRL by nat-siRNAATGB2 plays a positive role in disease resistance. More than 450 PPR proteins, characterized by the presence of tandem pentatricopeptide repeats, exist in Arabidopsis and the majority of them have unknown functions (30). A few studies point to an involvement of PPR proteins in posttranscriptional processes mainly in organelles, including RNA editing (31), mRNA silencing by cleavage (32) and translational regulation (33), etc. The PPRL protein contains five atypical PPR motifs and is mitochondrial localized (29, 30). Mitochondrion is the major organelle involved in oxidative burst and hypersensitive responses in plant disease resistance and leads to local cell death. How these events are regulated and how the signal is transduced are still largely unknown. A recent study shows that a mitochondrial-localized PPR-containing protein interacts with inhibitor of apoptosis proteins and regulates caspase activity and programmed cell death in mammalian cells (34). We speculate that PPRL may regulate avrRpt2-triggered oxidative burst, hypersensitive responses, or programmed cell death, possibly through specific protein–RNA or protein–protein interactions. Future biochemical analysis on PPRL and identification of its interaction proteins and RNAs will elucidate the mechanism of its function in RPS2-mediated bacteria resistance.

Fig. 5.
Model for nat-siRNAATGB2 biogenesis and function. Components in red are required for nat-siRNAATGB2 formation. RISC, RNA-induced silencing complex.

siRNA-mediated gene silencing plays an essential role in antiviral defense in both plant and animal systems (35, 36). However, these siRNAs generated from viral RNAs are extragenomic in origin. Defense regulation mediated by endogenous small RNAs has been reported in only a few cases thus far, all of which involve only miRNAs. In mammals, miRNA-mediated antiviral defense has been reported (37), but the biological roles of endogenous siRNAs have not been explored. In plants, miRNA miR393 regulates plant basal defense by targeting auxin signaling components (8). A direct connection between endogenous siRNAs and defense responses has not been reported previously in any organism. Our study here provides the first example of endogenous siRNAs that play a role in bacterial disease resistance in Arabidopsis. Gene expression profiling studies indicate that the defense responses are mediated by activation and repression of a large array of genes, but how the regulation of gene expression is achieved is largely unknown. Our data suggest that endogenous siRNA-mediated gene silencing may serve as one important mechanism for gene expression reprogramming in plant defense responses. Our finding of induction of a nat-siRNA in responses to bacterial infection opens up many new questions and provides new opportunities to elucidate the molecular events controlling plant disease resistance.

Materials and Methods

Plant Material and Growth Conditions.

Arabidopsis thaliana mutants rdr2-1, dcl2-1, dcl3-1, and dcl4-2, were provided by Jim Carrington (Center for Genome Research and Biocomputing, Oregon State University, Corvallis). dcl1-9 and hen1-1 were a gift from Xuemei Chen. sde1 (rdr6 in this study) and sde4/nrpd1a were provided by David Baulcombe (Sainsbury Laboratory, Norwich, U.K.). sgs3 was a gift from Herve Vaucheret (Institut National de la Recherche Agronomique, Versailles, France). hyl1 was a gift from Nina Federoff (The Huck Institutes of Life Science, Pennsylvania State University, University Park). npr1 and pad4 were gifts from Xinnian Dong (Duke University, Durham, NC). sgt1a and sgt1b were provided by Jane Parker (Max-Planck-Institut fur Zuchtungsforschung, Cologne, Germany). ein2 was a gift from Athanasios Theologis (Plant Gene Expression Center, Albany, CA). eds16 was provided by Mary Wildermuth (University of California, Berkeley). jar1 was provided by Linda Walling. These mutants were in the Columbia (Col-0), Landsberg erecta (Ler), Nossen-0 (No), or C24 genetic backgrounds as indicated in the text and figures. The mutant rps2 (101C) has a stop codon at amino acid 235 of RPS2. Arabidopsis plants were grown at 23°C ± 1°C at 12-h light/12-h dark photoperiod. N. benthamiana plants were grown at 23°C ± 1°C at 16-h light/8-h dark photoperiod.

Plasmid DNA Constructs.

For generating PPRL overexpression lines, full-length PPRL cDNA without 3′ UTR was amplified with primers 5′-CAC CAT GAA GTT CCT CAT GCA ATC CAT T-3′ and 5′-ACG CCT ATT AGG TAA TGT CCC T-3′ and cloned into the plant expression GATEWAY destination vector p35SGATFH with C-terminal Flag tag to avoid disruption of the signal peptide at the N terminus of the protein.

Isolation and Northern Blot Analysis of Small RNAs.

Leaves harvested at 15 hpi of Pst (2 × 107 cfu/ml) were used for RNA extraction and Northern blot analysis of both high and low molecular weight RNAs. For enrichment of small RNAs, the total RNA was dissolved in 4 M lithium chloride and precipitated. About 75–120 μg of low-molecular-weight RNA was used and separated by 17% denaturing polyacrylamide gel. The blots were probed and washed as described (12).

Real-time RT-PCR was performed as in ref. 38. PPRL1 was amplified with primers that locate outside of the overlapping region: 5′-GCT TCA TCG CCG GAG GAA ATC-3′ and 5′-TTA ACC GAG CAC CCT TCA TCG T-3′. Transcript levels were normalized to that of ubiquitin (5′-CGG AAA GAC CAT TAC TCT GGA-3′ and 5′-CAA GTG TGC GAC CAT CCT CAA-3′). Each experiment was repeated three times. The comparative Ct method was applied (ABI User Bulletin No. 2, Applied Biosystems, West Chester, PA).

Transient Expression Studies in N. benthamiana.

A 564-bp overlapping region was amplified with the primers 5′-ACG CGT CGA CAT GTG GAG CCA CCC GCA GTT CGA AAA ACG TAC TCA AGG TGC AGC TGG AGG A-3′ and 5′-GAA GGG GAA CTA GTG TTA GTG ACG CGA ACA TAC AAT AAC TTG CG-3′. Full-length ATGB2 was amplified by using primers 5′-ACG CGT CGA CAT GTG GAG CCA CCC GCA GTT CGA AAA ATC TTA CGA TTA TCT CTT CAA G-3′ and 5′-GAA GGG GAA CTA GTG TTA GTG A C GCG AAC ATA CAA TAA CTT GCG-3′. A strep tag sequence was included in the forward primers. The amplified products were cloned in XhoI and SpeI sites of the pTA7002 (28). Agrobacterium tumefaciens strain GV3101 cells harboring PPRL or ATGB2 constructs (OD600 = 1.0) were mixed at 1:1 ratio and coinfiltrated into 3-week-old N. benthamiana leaves. The expression of full-length or overlapping region of ATGB2 was induced by infiltration of 30 μM Dex at 48 hpi, and leaf tissue was collected at 24 h after Dex induction.

Bacterial Growth Assays.

Pst carrying EV (pVSP61) or avrRpt2, avrRpm1, avrRps4 and avrPphB were used to infect 4-week-old Arabidopsis leaves by infiltration at a concentration of ≈2 × 105 cfu/ml. The bacterial titer was measured at 0 and 4 dpi as in ref. 38.

HR Assay and Electrolyte Leakage Measurements.

Leaves of 4-week-old Arabidopsis plants were infiltrated with 2 × 107 cfu/ml Pst (avrRpt2) for HR assay. Leaves were infiltrated with either 10 mM MgCl2 (mock) or 1 × 107 cfu/ml Pst (avrRpt2) for electrolyte leakage assay. The leaf disks were washed in water for 50 min and then transferred into 15 ml of water incubating for 16 h. The tubes containing leaf disks and water were then autoclaved. Conductivity was measured before and after autoclave by an EC meter (VWR Scientific, West Chester, PA). The percentage of ion leakage before and after autoclave was calculated and plotted. Four replicates were conducted in each treatment.

Acknowledgments

We thank Shou-Wei Ding and Xuemei Chen (University of California, Riverside, CA) and Jim Carrington for stimulating discussion on the manuscript and for providing seeds of various mutants; David Baulcombe, Nina Federoff, Herve Vaucheret, Xinnian Dong, Jane Parker, Athanasios Theologis, Mary Wildermuth, and Linda Walling (University of California, Riverside) for providing seeds of various genotypes; Thomas Girke for bioinformatics assistance; Julia Bailey-Serres (University of California, Riverside) for binary plasmids; and James Borneman for access to a real-time Icycler in his laboratory. This work was supported by U.S. Department of Agriculture, State Agricultural Experiment Station Research Allocation Award PPA-7517H from the University of California, Riverside (to H.J.), Department of Energy Grant DE-FG02-88ER13917, and National Institutes of Health Grants R01-FM069680-01 (to B.J.S.) and R01GM59138 and R01GM070795 (to J.-K.Z.).

Abbreviations

miRNA
microRNA
ta-siRNA
transacting siRNA
NAT
natural antisense transcript
Ps
Pseudomonas syringae
Pst
Ps pathovar tomato
avr
avirulence
DCL
DICER-like
RDR
RNA-dependent RNA polymerase
Dex
dexamethasone
hpi
h postinoculation
dpi
days postinoculation
PPR
pentatricopeptide repeat
PPRL
PPR protein-like
HR
hypersensitive responses.

Footnotes

The authors declare no conflict of interest.

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