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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Science. Author manuscript; available in PMC Feb 15, 2009.
Published in final edited form as:
PMCID: PMC2570098
NIHMSID: NIHMS69388

Suppression of the microRNA pathway by bacterial effector proteins

Abstract

Plants and animals sense pathogen-associated molecular patterns (PAMPs) and in turn differentially regulate a subset of microRNAs (miRNAs). However, the extent to which the miRNA pathway contributes to innate immunity remains unknown. Here, we show that miRNA-deficient mutants of Arabidopsis partly restore growth of a type-three secretion-defective mutant of Pseudomonas syringae. These mutants also sustained growth of non-pathogenic Pseudomonas fluorescens and Escherichia coli strains, implicating miRNAs as key components of plant basal defense. Accordingly, we have identified P. syringae effectors that suppress transcriptional activation of some PAMP-responsive miRNAs, miRNA biogenesis, stability or activity. These results provide compelling evidence that, like viruses, bacteria have evolved to suppress RNA silencing to cause disease.

In RNA silencing, double-stranded (ds)RNA is processed into small RNAs through the action of RNase-III-like Dicer enzymes. The small RNAs guide Argonaute (AGO)-containing RNA-induced silencing complexes (RISCs) to inhibit gene expression at the transcriptional or post-transcriptional levels (1). In the Arabidopsis thaliana miRNA pathway, miRNA precursors (pre-miRNAs) are excised from non-coding primary transcripts (pri-miRNAs) and processed into mature miRNA duplexes by Dicer-like 1 (DCL1). Upon HEN1-catalyzed 2′-O-methylation (2), one miRNA strand incorporates an AGO1-containing RISC to direct endonucleolytic cleavage or translational repression of target transcripts (1). DCL4 and DCL2 have major defensive functions by processing viral-derived dsRNA into small interfering (si)RNAs, which, like miRNAs, are loaded into AGO1-RISC. As a counter-defensive strategy, viruses deploy viral suppressors of RNA-silencing, or VSRs (3). RNA-silencing also contributes to resistance against bacterial pathogens (4-7), which elicit an innate immune response upon perception of PAMPs by host-encoded pattern recognition receptors (PRRs). For example, the Arabidopsis miR393 is PAMP-responsive (4, 8) and contributes to resistance against virulent P. syringae pv. tomato strain DC3000 (Pto DC3000) (4). Nonetheless, the full extent to which cellular small RNAs, including miRNAs, participate to PAMP-triggered immunity (PTI) in plants remains unknown.

To address this issue, Arabidopsis mutants defective for siRNA or miRNA accumulation were challenged with Pto DC3000 hrcC-, a mutant that lacks a functional type-III secretion system required for effector protein delivery into host cells (9). This bacterium elicits, but cannot suppress PTI, and consequently multiplies poorly on wild-type Col-0- and La-er-inoculated leaves (Fig. 1A, S1). However, Pto DC3000 hrcC-growth was specifically enhanced in the miRNA-deficient dcl1-9 and hen1-1 mutants (Fig. 1A), in which induction of the basal defense marker gene WRKY30 was also compromised ((10), Fig. S2A).

Fig. 1
The Arabidopsis miRNA pathway promotes basal and non-host resistances to bacteria. (A) Six-week-old plants were inoculated by syringe-infiltration using a Pto DC3000 hrcC- concentration of 106 colony-forming units (cfu) per ml. Error bars: standard error ...

Because PTI is also a major component of non-host resistance (10, 11), we challenged dcl1-9 and hen1-1 mutants with P. syringae pv. phaseolicola (Psp) strain NPS3121, which infects bean but not Arabidopsis. Both dcl1-9 and hen1-1 mutants sustained Psp NPS3121 growth (Fig. 1B) and displayed compromised WRKY30 induction (Fig. S2B). Enhanced bacterial growth was also observed with the non-pathogenic P. fluorescens Pf-5 and E. coli W3110 strains (Fig. 1C/D). Furthermore, the above non-virulent bacteria all induced chlorosis and necrosis on miRNA-deficient mutants resembling bacterial disease symptoms triggered by virulent Pto DC3000 (Fig. 1E, S3A-D). However, we cannot exclude the participation of other endogenous small RNAs in this process (6, 7) because the hen1-1 mutant, which is additionally impaired in the accumulation of many types of small RNAs (12), consistently displayed more disease symptoms than the dcl1-9 mutant did (Fig. S3). Despite this possibility, our results indicate that the miRNA pathway is likely to be an essential component of plant basal defense. As a corollary, some bacterial effectors must have evolved to suppress host miRNA functions to enable disease.

In principle, suppression of the miRNA pathway could affect miRNA transcription, biogenesis or activity. To test the first possibility, we challenged wild-type plants with Pto DC3000 or Pto DC3000 hrcC-, and analyzed the levels of several pri-miRNAs. In virulent Pto DC3000-treated plants, induction of the PAMP-responsive pri-miR393a/b and pri-miR396b (4, 8) was significantly suppressed, as was induction of WRKY30 and Flagellin Receptor Kinase 1 (FRK1) (13) used as internal controls (Fig. 2A). By contrast, the PAMP-insensitive pri-miR166a and pri-miR173 remained unaffected. We then used the previously described miR393a-p::eGFP and miR393b-p::eGFP transgenic lines, which report miR393a and miR393b transcriptional activity (4). Pto DC3000 hrcC- caused an increase in eGFP mRNA levels in both transgenic lines, indicating the presence of PAMP-responsive elements upstream of miR393a and miR393b (Fig. 2B). However, this induction was compromised by Pto DC3000, as was induction of the FRK1 control (Fig. 2B). These results suggest that some Pto DC3000 effectors suppress PAMP-triggered transcriptional activation of pri-miR393a/b.

Fig. 2
Transcriptional repression of PAMP-responsive miRNAs by Pto DC3000 and AvrPtoB. (A) Five-week-old plants were syringe-infiltrated with a concentration of 2 × 107 cfu/ml of either Pto DC3000 or Pto DC3000 hrcC- and pri-miRNA expression monitored ...

To test this hypothesis, we engineered T-DNA constructs to deliver distinct Pto DC3000 effectors into leaves of the Arabidopsis efr mutant, which sustains efficient Agrobacterium-mediated transient transformation (14) without affecting pri-miRNA, mature miRNA or miRNA target levels (Fig. S4). Bacterial effector expression was confirmed (Fig. S5A), and pri-miRNA levels subsequently monitored. AvrPtoB, an effector with E3-ubiquitin ligase activity (15), down-regulated pri-miR393a and pri-miR393b accumulation without affecting the PAMP-insensitive pri-miR166a (Fig. 2C). This effect occurs, at least in part, at the transcriptional level because AvrPtoB delivery into miR393a-p::eGFP/efr and miR393b-p::eGFP/efr leaves inhibited both basal expression and PAMP-triggered induction of eGFP (Fig. 2E/F, S5B). Similar effects were obtained with AvrPtoBF525A, a stable mutant in which the E3-ligase activity is abolished (Fig. 2D-F, S5B). Therefore, AvrPtoB suppresses miR393a and miR393b transcription independently of its E3-ligase activity, as previously shown for AvrPtoB-mediated suppression of PAMP-responsive FRK1 (16).

To assay for interference with miRNA biogenesis or stability, levels of several PAMP-insensitive and -sensitive miRNAs were monitored upon transient effector delivery into efr leaves. Three bacterial effectors significantly reduced accumulation of unrelated miRNAs (Fig. 3A, S5A-C, S6), among which AvrPto is a well-characterized Pto DC3000 effector with demonstrated virulence function ((17), Fig. S9). Further molecular analysis revealed that AvrPto-mediated reduction in miRNA accumulation may occur, at least in part, at the post-transcriptional level because AvrPto did not alter pri-miRNA transcript levels (Fig. S7A). Accordingly, conditional AvrPto expression in stable transgenic lines stabilizes miR393 precursors and concomitantly decreases accumulation of mature miR393, with no or little effects on pri-miR393 transcript levels (Fig. 3B/C, S7B). Therefore, AvrPto possibly interferes with processing of some miRNA precursors, a phenomenon also observed during Pto DC3000 infection (Fig. 3C, S8).

Fig. 3
Suppression of miRNA accumulation or activity by bacterial effectors. (A). Four-week-old efr plants were syringe-infiltrated with A. tumefaciens carrying AvrPto, AvrPtoY89D, AvrPtoG2A or GUS-intron (GUS) constructs. Five days post-infiltration, accumulation ...

AvrPto interacts with, and inhibits the kinase activity of, multiple transmembrane PRRs (18). Moreover, AvrPto strongly interacts with BAK1 (BRI1-Associated receptor-kinase 1), a shared signaling partner of the brassinosteroid receptor BRI1 (Brassinosteroid-Insenstive 1) and the flagellin receptor FLS2 (Flagellin Sensing 2) (19-21). The AvrPto-BAK1 interaction compromises the ligand-dependent FLS2-BAK1 association resulting in suppression of PTI (19). Accordingly, AvrPtoY89D, which is unable to interact with PRRs and BAK1 (18, 19), displays compromised virulence function (18). Transient delivery of AvrPtoY89D did not alter miRNA accumulation, nor did delivery of AvrPtoG2A, carrying a mutated myristoylation site that disrupts AvrPto host plasma membrane localization ((16, 22), Fig. 3A, S5C). Conditional expression of an N-terminal histidine-tagged version of AvrPto (6xHis-AvrPto) displaying similarly compromised subcellular localization and virulence function gave comparable results (Fig. 3B/C, S9). We conclude that AvrPto interferes with miRNA accumulation and this interference is linked with its virulence function (SOM text).

Finally, we tested whether Pto DC3000 effectors could also suppress miRNA activity. We used the SUC-SUL (SS) reporter line in which phloem-specific expression of an inverted-repeat transgene triggers non-cell-autonomous RNAi of the endogenous SULPHUR (SUL) transcript, causing vein-centered chlorosis (23). Of the 21nt (DCL4-dependent) and 24nt (DCL3-dependent) SUL siRNA species, only the former is required for RNAi in an AGO1-dependent manner (23). Stable transgenic lines overexpressing HopT1-1 (Fig. S10) exhibited attenuated chlorosis (Fig. 3E) and accumulated higher SUL mRNA levels (Fig. 3G). However, the SUL siRNA levels remained unchanged (Fig. 3D), mimicking the reported effects of the ago1-12 mutation in SS (23). Also as in ago1-12, canonical miRNAs accumulated normally in HopT1-1-overexpressing lines, despite higher levels of miRNA target transcripts (Fig. 3F/G, S11) suggesting that HopT1-1 likely suppresses slicing mediated by AGO1. Interestingly, further transient overexpression of HopT1-1 in efr plants showed a dramatic increase in the protein, but not mRNA, levels of the miR834 target COP1-interacting protein 4 (CIP4) (Fig. 3H, S12A/B). Thus, HopT1-1 additionally, and perhaps predominantly, suppresses miRNA-directed translational inhibition, which is consistent with the involvement of AGO1 in this second process (24). Similarly, higher protein levels of CIP4 and of the copper/zinc superoxide dismutase 1 (CSD1-miR398 target) were detected in plants infected with virulent Pto DC3000 (Fig. 3H, S12C), with no effect on CSD1, CIP4 and some other miRNA target transcript levels (Fig. S13).

We show here that the miRNA pathway plays a major role in antibacterial basal defense and, accordingly, we have identified bacterial suppressors of RNA silencing or ‘BSRs’. This finding provides a plausible explanation for the synergistic interactions observed in the field between some viral and bacterial phytopathogens. Consistent with this idea, we found that infection by Turnip Mosaic Virus (TuMV), which produces the P1-HcPro suppressor of siRNA and miRNA functions (25, 26) reduces basal and non-host resistances to promote growth and disease-like symptoms from non-virulent Pto DC3000 hrcC- and Psp NPS3121 bacteria (Fig. 4). It will now be important to elucidate how silencing factors are modified by VSRs and BSRs, and whether such modifications are sensed by specific Resistance (R) proteins, as postulated by the ‘guard hypothesis’ (27).

Fig. 4
TuMV infection enhances growth of and rescues symptoms induced by Pto DC3000 hrcC- and Psp NPS3121. (A) Five-week-old Col-0 plants were infected for 7 days with TuMV and further challenged with Pto DC3000 hrcC- at a concentration of 106 cfu/ml. The picture ...

The implication of the miRNA pathway in innate immunity is not specific to plants. For example, human MiR146 is induced by several microbial components (28). Because type III secretion systems are widespread across Gram-negative bacteria (29), the intriguing possibility emerges that human pathogenic bacteria also have evolved to suppress RNA silencing to cause disease.

Supplementary Material

Supp

Acknowledgments

We thank Y. Yamamoto, A. Collmer, J. Alfano, G. Martin, C. Zipfel, E. Cascales, G. Preston, P. Hauck and Y. B. Kwack for materials. Supported by a Fellowship from the Federation of European Biochemical Societies (L.N); an Action Thématique Incitative sur Programme grant from the CNRS and a grant from the trilateral génoplante–German Plant Genome Research Program–Spanish ministry of Research (F.J and O.V); and US NIH grant #5R01AI060761 (K.N and SY.H).

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