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Endogenous small RNAs and antibacterial immunity in plants
FEBS Letters 582 (2008) 2679–2684
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Endogenous small RNAs and antibacterial immunity in plants Hailing Jin Department of Plant Pathology and Microbiology, Center for Plant Cell Biology and Institute for Integrative Genome Biology, University of California, Riverside, CA 92521, USA Received 10 June 2008; revised 26 June 2008; accepted 30 June 2008 Available online 11 July 2008 Edited by Shou-Wei Ding
Abstract Small RNAs are non-coding regulatory RNA molecules that control gene expression by mediating mRNA degradation, translational inhibition, or chromatin modification. Virusderived small RNAs induce silencing of viral RNAs and are essential for antiviral defense in both animal and plant systems. The role of host endogenous small RNAs on antibacterial immunity has only recently been recognized. Host disease resistance and defense responses are achieved by activation and repression of a large array of genes. Certain endogenous small RNAs in plants, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are induced or repressed in response to pathogen attack and subsequently regulate the expression of genes involved in disease resistance and defense responses by mediating transcriptional or post-transcriptional gene silencing. Thus, these small RNAs play an important role in gene expression reprogramming in plant disease resistance and defense responses. This review focuses on the recent findings of plant endogenous small RNAs in antibacterial immunity. Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
effector-triggered immunity (ETI), which is mediated by resistance (R) proteins. R proteins recognize specific pathogen effectors, such as bacterial avirulence (avr) proteins, and rapidly turn on a series of defense responses to inhibit pathogen growth [1–4]. PTI, also referred to as ‘‘basal defense’’, is pathogen non-specific and less effective than ETI, the R gene-mediated resistance. Plant immune responses, including both PTI and ETI, are mediated by activation and repression of a large array of genes during infection. For example, over 2000 genes are differentially expressed in the disease resistance responses triggered by two NBS–LRR type of R genes, RPS2 and RPM1 [5]. How gene expression reprogramming is achieved, however, is largely unknown. Several recent studies suggest that endogenous small RNA-mediated gene silencing may serve as one important mechanism for gene expression reprogramming in plant immune responses. This review mainly discusses plant endogenous small RNAs in antibacterial immunity (Fig. 1 and Table 1).
Keywords: Endogenous siRNAs; Pathogen-regulated; lsiRNAs; miRNA; Plant immunity 2. Plant endogenous small RNAs
1. Multiple layers of plant immune responses Plants have evolved multiple layers of immune systems in response to various pathogen attacks [1,2]. The first layer of defense is so-called ‘‘non-host resistance’’, which enables plants to resist the vast majority of pathogens by either preventing pathogen penetration or by depriving the nutrients and other factors required for pathogen growth and multiplication. Once the bacterial or fungal pathogens gain access to the plant interior and get through the cell wall, they encounter another layer of defense that is triggered by the recognition of microbial or pathogen-associated molecular patterns (MAMPs or PAMPs), such as flagellin, by plant transmembrane pattern recognition receptors (PRRs). During the co-evolution of plant and pathogen, some successful pathogens have overcome this PAMPtriggered immunity (PTI) by delivering effectors into the plant cell to suppress the signal transduction of PTI. As part of the arms race between plants and pathogens, some plants have evolved another rapid and highly effective defense system –
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Endogenous small RNAs are now recognized as important regulators of gene expression in numerous cellular processes. These short non-coding small RNAs can be classified into microRNA (miRNA), short interfering RNA (siRNA), and PIWI-interacting RNA (piRNA) based on their distinct biogenesis pathways [6]. In plants, the biogenesis of small RNAs is especially complex, as reflected by the large numbers of enzymes involved. Arabidopsis has four type-III ribonuclease Dicer-like (DCL) proteins, six predicted RNA-dependent RNA polymerases (RDRs), and 10 predicted Argonautes (AGOs). Plants do not contain the PIWI subfamily of AGO proteins and no piRNAs are identified so far from plants. miRNAs are small (mostly 20–22-nt) RNAs generated from hairpin precursors mainly by DCL1. A few recently identified miRNAs with long hairpin precursors depend on DCL4. The biogenesis of miRNAs does not require RDRs or RNA polymerase IV (pol IV) components. In Arabidopsis, more than 200 miRNAs were reported, and many are important for plant development. Some miRNAs have been shown to be induced or repressed by various abiotic stresses and regulate gene expression in plant stress responses [7]. Plant miRNAs have high complementarity to their mRNA targets and were thought to predominantly induce post-transcriptional gene silencing by guiding mRNA cleavage. Translational inhibition,
0014-5793/$34.00 Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.06.053
[26] [26] [26]
[36]
Protoporphyrinogen oxidase These targets require experimental validation.
a
PPOXa AtlsiRNA-5
Induced by Pst (avrRpt2)
AtRAP IRT2 AT7SL-1a Induced by Pst (avrRpt2) Induced by Pst (avrRpt2) Induced by Pst (avrRpt2) and Pst (avrRpm1) AtlsiRNA-1 AtlsiRNA-2 AtlsiRNA-3
RPP4 confers resistance against Hyaloperonospora parasitica SNC1 contributes to the resistance against both Hyaloperonospora parasitica and Pst Contribute to both ETI and PTI Iron-responsive transporter A signal recognition particle gene, a RNA pol III-dependent RNA gene [26] RPP4 locus genes including SNC1 RPP4 locus associated siRNAs
[14] Negative regulator of RPS2 pathway (ETI) Induced by Pst (avrRpt2)
PPRL nat-siRNAATGB2 siRNAs
Auxin signaling and PTI Auxin signaling Auxin signaling Induced by biotic stresses Gene regulation Putative iron-chaperone protein TIR1, AFB2, AFB3 ARF6, ARF8 ARF10, ARF16, ARF17 Remorina Zinc finger homeobox genea Frataxin-related genea miR393 miR167 miR160 miR825 miRNAs
Induced by flg22 Induced by Pst (hrcC) Induced by Pst (hrcC) Suppressed by Pst (hrcC)
Targets Small RNAs
Table 1 List of plant endogenous small RNAs that may contribute to plant disease resistance and defense responses
however, was recently reported to be another widespread mode of action for many miRNAs in plants [8]. Endogenous siRNAs are generated from long doublestranded RNAs (dsRNAs) as a result of antisense transcription or the activity of RDRs. Millions of endogenous siRNAs have been identified from plants by deep sequencing. They are extremely diverse and are normally not conserved across different species. Plant endogenous siRNAs can induce transcriptional gene silencing via DNA methylation and/or histone modifications or cause post-transcriptional gene silencing via mRNA degradation and/or translation inhibition. Although siRNAs greatly outnumber miRNAs, the biological roles of many endogenous siRNAs have yet to be explored. The biogenesis of various siRNAs involves all four DCLs in Arabidopsis. DCL1 initiates the in-phase production of transacting siRNAs (ta-siRNAs) by producing miRNAs that guide the cleavage of ta-siRNA precursors [6]. DCL1 is also required for the bacterial-induced natural antisense transcripts-derived siRNA (nat-siRNA) [14] and the in-phase production of salt stress-induced 21-nt nat-siRNAs, which is initiated by a 24nt nat-siRNA produced by DCL2 [9]. DCL2 produces some viral siRNAs and substitutes for DCL3 or DCL4 when they are absent [6]. DCL3 generates 24-nt heterochromatin-associated or repeat-associated siRNAs that regulate heterochromatin formation and transcriptional gene silencing [6]. The inphase production of ta-siRNAs depends on DCL4 [6]. DCL4 also generates siRNAs from long hairpin RNAs in inverse repeat-induced PTGS. Furthermore, DCL4 serves as a primary antiviral sensor for production of virus-derived siRNAs and prevents viral exit from the vasculature [10]. In addition to DCLs, other components such as RDRs and AGOs have been implicated in siRNA accumulation. RDR2 and the RNA pol IV cooperate in generating heterochromatin-associated 24-nt siRNAs. These siRNAs are incorporated into AGO4 or AGO6 and induce transcriptional gene silencing by guiding DNA methylation and histone modifications at the homologous genomic loci [11–13]. Generation of ta-siRNAs and nat-siRNAs requires RDR6 and SUPPRESSOR OF GENE SILENCING 3 (SGS3) [6,9,14]. These findings indicate that plants have evolved multiple small RNA processing pathways with specific and overlapping functions.
Target functions
Fig. 1. Certain plant endogenous small RNAs (miRNAs and siRNAs) are up- or down-regulated by pathogen infection and target R genes or defense responses genes in plant immunity. These small RNAs contribute to gene expression reprogramming and fine-tuning in plant resistance and defense responses.
[15] [16] [16] [16]
H. Jin / FEBS Letters 582 (2008) 2679–2684
Reference
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H. Jin / FEBS Letters 582 (2008) 2679–2684
3. miRNAs and PAMP-triggered antibacterial resistance Arabidopsis miR393 was the first miRNA identified to play a role in plant antibacterial PTI by regulating the auxin signaling pathway [15]. miR393 was induced by a bacterial PAMP peptide flg22 at 20–60 min post-treatment. Expression of the miR393 targets, auxin receptor TIR1 (Transport Inhibitor Response 1) and two of its functional paralogs (AFB2 and AFB3), was decreased upon flg22 treatment. Constitutive overexpression of another TIR1 paralog that is not a target of miR393, AFB1, in a tirl-1 mutant background led to enhanced disease sensitivity to Pseudomonas syringae pv. tomato (Pst) DC3000, whereas miR393 overexpression lines restricted bacterial growth. Thus, the findings suggest that miR393 contributes to flg22-induced basal defense or PTI by targeting auxin receptor mRNAs and subsequently repressing auxin signaling in Arabidopsis (Fig. 2A). By using small RNA expression profiling on Arabidopsis leaves collected at 1 and 3 h post-inoculation of Pst DC3000 hrcC, Fahlgren et al. [16] identified three miRNAs, miR393, miR167, and miR160 that were highly induced after infection. Pst DC3000 hrcC strain has a mutation in the key effector delivery machine, the type III secretion system. This strain triggers PTI in plants and can no longer cause disease. Interest-
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ingly, all three miRNAs negatively regulate auxin signaling by either targeting auxin receptor genes or auxin response factors. Pathogen-induced small RNAs are likely to silence negative regulators of host resistance pathways. miR825 was downregulated by Pst DC3000 hrcC infection and has three predicted targets, including the Remorin gene, the zinc finger homeobox family gene, and a frataxin-related gene [16] (Fig. 2A). These predicted targets are likely up-regulated by the infection and presumably positively regulate plant defense responses, although their functions need to be confirmed by experimental data. These results indicate that host miRNAs play an important role in plant immunity. The importance of miRNA in host immunity has also been reported in animal systems. Several miRNAs, including miR150, miR155, and miR181a, are differentially expressed during the T-cell and B-cell differentiation and regulate their activation and sensitivity to antigens. The miRNAs help regulate the differentiation of haematopoieticcell lineage and the modulation of adaptive immune responses [17–20]. T-cell deficiencies were observed in Dicer conditional knockout mice [21]. Dicer ablation also affects B lymphocyte development by blocking pro-B to pre-B cell transition and interferes with antibody diversity [22]. Animal miRNAs also play a crucial role in innate immunity, the first line of defense mediated by recognition between Toll-like receptors (TLRs) and invading bacterial, viral, and other pathogens. For example, expression of miR-146a, miR132, and miR155 was largely induced by endotoxin lipopolysaccharide treatment, which mimicked bacterial infection [23]. Interestingly, miR146a can only be induced in a NF-kB dependent manner by the transmembrane TLRs that recognize bacterial constituents but not by the intracellular TLRs that mainly sense viral nucleic acids, whereas miR155 is induced by both bacterial and viral infection through TLR3-induced tumor-necrosis factor signaling [19,23]. By regulating TLR4 expression, let-7i also contributes to innate immune responses against the parasite Cryptosporidium parvum [24]. Thus, miRNA-mediated gene silencing is a general regulatory mechanism in host immunity in both plants and animals. Additional miRNA profiling in pathogen-infected plants is likely to identify more miRNAs that regulate plant immunity, including both PTI and ETI.
4. Endogenous siRNAs and bacterial effector-triggered resistance
Fig. 2. Identified miRNAs and endogenous siRNAs that play a role in antibacterial PTI (A) and ETI (B) in Arabidopsis [14–16,26]. The expression and putative function of miR167, miR160 and miR825 (A) were purely based on the deep sequencing data from Fahlgren et al. [16] and require further experimental validation. The putative function of AtlsiRNA-2, 3 and 5 (B) was based on Northern blot analysis and computational analysis and also requires further experimental validation.
The first example of a plant endogenous siRNA that regulates plant immunity is nat-siRNAATGB2 [14]. This nat-si RNA was identified from an expression study of Arabidopsis siRNAs from publicly available small RNA databases (ASRP and MPSS) that target pathogen-regulated genes from the AtGenExpress database [25]. nat-siRNAATGB2 is derived from the overlapping region of a pair of natural antisense transcripts, a Rab2-like small GTP-binding protein gene ATGB2 and a PPR (pentatricopeptide repeats) protein-like gene PPRL. It is specifically induced by Pst DC3000 carrying effector avrRpt2 but not other effectors. Induction of nat-siRNAATGB2 represses the expression of its antisense target PPRL, a negative regulator of RPS2-mediated ETI against Pst (avrRpt2) (Fig. 2B). Overexpression of PPRL delayed the hypersensitive response (HR), reduced cell death triggered by
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the recognition between RPS2 and Pst (avrRpt2), and enhanced the growth of Pst (avrRpt2). The biogenesis pathway of nat-siRNAATGB2 is unique. It depends on DCL1, HYL1, and HEN1, and also requires RDR6, SGS3, and RNA pol IV subunit NRPDla. Strikingly, the specific induction of nat-siRNAATGB2 by Pst (avrRpt2) depends on the cognate host resistance gene RPS2 and the NDR1 gene required for RPS2-specified resistance. This study demonstrated that a plant nat-siRNA is specifically induced by a bacterial effector and regulates ETI by down-regulating a negative regulator of the cognate R gene-mediated specific resistance pathway. To be cost effective, plant disease resistance responses need to be suppressed during normal conditions and to be rapidly turned on upon pathogen attack. Endogenous small RNA-mediated gene silencing may serve as one of the mechanisms for this rapid ‘‘off’’ and ‘‘on’’ regulation. In addition to the continuous study of small RNAs-targeting pathogen-regulated genes, our laboratory also conducted small RNA expression profiling via deep sequencing using small RNAs extracted from pathogen-infected Arabidopsis tissue to identify more pathogen-regulated plant endogenous small RNAs. Many small RNAs, including miRNAs and endogenous siRNAs, were induced or repressed by various pathogens, including bacteria and fungi (Jin et al., unpublished data), and these small RNAs may down- or up-regulate genes involved in plant disease resistance and defense responses (Figs. 1 and 2). Some of these small RNAs are unique to one specific pathogen, whereas others are relevant for plant basal defense responses and are regulated by multiple pathogens. During Northern blot expression validation of identified small RNAs, Katiyar-Agarwal et al. discovered a novel class of endogenous small RNAs, long siRNAs (lsiRNAs), which are 30–40 nt long and are induced by bacterial infection or by specific growth conditions [26]. Unlike the recently identified 25–31-nt animal piRNAs that are DICER-independent and require PIWI subfamily proteins for biogenesis, plant lsiRNAs depend on DCL1 and the AGO subfamily protein AGO7. Biogenesis of plant lsiRNAs also depends on HYL1, HEN1, HST1, RDR6, and Pol IV. Of the identified lsiRNAs, AtlsiRNA-1 is induced specifically by Pst (avrRpt2) and downregulates a putative RNA binding protein gene-AtRAP, possibly through a novel mode of action in plants—degradation of the target mRNA through decapping and XRN4-mediated 5 0 – 3 0 decay. Knockout mutation in AtRAP increases resistance to both virulent and avirulent Pst, which suggests that AtRAP may serve as a common component in basal defense but is controlled by AtlsiRNA-1 during the rapid response to avirulent factor avrRpt2. AtlsiRNA-2, -3, and -4 are modestly induced by mock treatment and infection by various Pst strains and target an iron-responsive transporter gene (IRT2), a signal recognition particle gene (AT7SL-1), and an anion transporter gene (ANTR2), respectively. AtlsiRNA-2 and 3 are induced to a higher level by Pst (avrRpt2). AtlsiRNA-5 is only weakly induced by Pst (avrRpt2) and targets the antisense gene protoporphyrinogen oxidase (PPOX) (Fig. 2B).
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ifications. The bacterial pathogen Pst DC3000 was shown to induce DNA hypomethylation in the absence of DNA replication at certain genomic loci, including some peri/centromeric repeats and retrotransposons, in Arabidopsis [27]. Both virulent Pst DC3000 and avirulent Pst DC3000 (avrRpt2) strains, but not the non-pathogenic strain Pst DC3000 hrpL, can induce chromatin alteration, such as decondensation of chromocenters, in the infected tissue. The authors speculated that Pst DC3000-induced DNA hypomethylation and heterochromatin decondensation may promote increased DNA recombination and genetic variability at some genomic loci, including centromeric regions harboring a group of resistance genes. This is a very interesting finding, and recently developed bisulfite sequencing using sequencing-by synthesis (SBS) technology will enable researchers to identify pathogen-regulated DNA methylation regions at a single-base resolution in a global level, as demonstrated in the elegant work by Lister et al. [28] and Cokus et al. [29]. The likelihood of small RNA involvement in this chromatin modification process upon pathogen challenge was indicated by the requirement of AGO4 in resistance to Pst infection in Arabidopsis [30]. Mutant ago4-2 was identified from a genetic screen and had enhanced susceptibility to the virulent Pst DC3000, to the avirulent Pst DC3000 carrying the effector avrRpml, as well as to a non-host strain P. syringae pv. phaseolicola, which is a pathogen of bean but not normally a pathogen of Arabidopsis. The extent of DNA cytosine methylation at CpNpG and CpHpH positions in various loci is clearly decreased in ago4 mutants. AGO4 associates with 24-nt siRNAs in a putative RNA-induced transcriptional silencing complex (RITS) to guide DNA methylation and histone modifications. The biogenesis of these chromatin-associated 24-nt siRNAs requires DCL3 and RDR2. However, mutations in DCL3 and RDR2 did not interfere with plant defense responses. Several DNA methyltransferases and chromatin-remodeling proteins, including Chromomethylase 3 (CMT3), Domains Rearranged Methyltransferase 1 and 2 (DRM1 and DRM2), and Defective in RNA-directed DNA methylation (DRD1), act with AGO4 in the RNA-dependent DNA Methylation (RdDM) pathway. No change of Pst growth rate was observed in drdl, cmt3 or the drml/drm2 double mutant as compared with wild type plants. This lack of phenotype may be due to the possible functional redundancy among various DCLs, RDRs, and methyltransferases, although at this stage we cannot rule out the possibility that AGO4 could contribute to disease resistance independently of the RdDM pathway. Disease susceptibility analysis on double or triple mutants of RDRs, DCLs, and methyltransferases, such as dcl2/3/4 and ddc (drm1-2 drm2-2 cmt3-11) triple mutants, will help to clarify this issue [31,32]. Bisulfite sequencing on the ago4 mutant may be able to determine the DNA methylation regions controlled by ago4 and may identify some R genes or resistance signaling genes controlled by RdDM.
6. NATs-associated endogenous siRNAs and beyond 5. Small RNA-mediated transcriptional gene silencing in plant immunity Chromatin-associated, 24-nt siRNAs induce transcriptional gene silencing by guiding DNA methylation and histone mod-
Interestingly, in addition to nat-siRNAs, several identified lsiRNAs are also derived from the overlapping region of NATs [26]. cis-NAT arrangement is a widespread phenomenon in both plant and animal genomes. About 7–30% of plant and animal genes are arranged as cis-NATs in spite of large intergenic
H. Jin / FEBS Letters 582 (2008) 2679–2684
regions, which suggests an evolutionary advantage of this genomic arrangement. Over 3000 Arabidopsis small RNAs from small RNA databases were found to match more than 60% of protein-coding NAT pairs [33], indicating that the small RNA-mediated gene regulation may serve as one of the regulatory mechanisms for the expression of at least a subgroup of NATs. Little conservation was observed on either overlapping NAT genes or nat-siRNAs among different species. Besides the overlapping gene regions, more than 30% of the Arabidopsis genome and about 60–80% of mammalian genomes can produce transcripts from both sense and antisense strands [34,35]. Antisense transcription provides perfect dsRNA precursors for siRNA production. Yi and Richards provided a nice example of siRNA generation from antisense transcription at the R gene recognition of the Hyaloperonospora parasitica (RPP) locus [36]. The Arabidopsis Columbia RPP4 locus (named RPP5 locus in Landsberg erecta ecotype) contains seven TIR–NBS–LRR class R genes, which are interspersed with two non-R genes and three related sequences. Two R genes in this locus, RPP4 and SNC1 (for Suppressor of nprl-1, constitutive 1), were shown to be functional and to confer resistance to the bacterium P. syringe and the oomycete H. parasitica. This cluster of R genes is coordinately regulated by transcription activation and siRNA-mediated RNA silencing. Endogenous siRNAs were detected at the RPP4 locus, including SNC1. SNC1 mRNA level is elevated in dcl4, ago1, and upf1 RNA silencing pathway mutants, indicating that these siRNAs function in the silencing of SNC1. Antisense transcripts were detected from the regions that produce these siRNAs, which suggests that these siRNAs may originate from sense and antisense RNA pairing.
7. Widespread occurrence of endogenous siRNAs in animal systems Until recently, no endogenous siRNAs (endo-siRNAs) were observed in animals lacking RDRPs, such as mammals and Drosophila. However, the RNAi machinery in Drosophila and mammals may not function only for antiviral defense by silencing exogenous viral RNAs. By using deep sequencing technologies, several laboratories discovered endogenous siRNAs (endo-siRNAs) that target transposons, intergenic regions, and mRNAs in mouse and Drosophila [37–42]. In Drosophila somatic S2 cells, 41% of the endo-siRNAs mapped to mRNAs do not map to transposons, suggesting a regulatory role of these endo-siRNAs in mRNA expression [37]. Furthermore, the mRNA-targeting endo-siRNAs were highly enriched (eg. over 10-fold in Drosophila) in the overlapping regions of cis-NATs or the regions that have antisense transcription [37–39,43]. Although the authors did not observe clear strand bias of these putative nat-siRNAs under the experimental conditions, these endo-siRNAs may also regulate gene expression of these cisNAT genes under specific conditions, as in plant systems. Thus, endogenous siRNA-mediated gene silencing is a general gene regulation mechanism in both plant and animal systems.
8. Conclusions Plants and animals employ small RNA-mediated gene silencing as an important mechanism for host immunity
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against bacterial, viral and fungal pathogens. We speculate that many endogenous small RNAs that have yet to be discovered have important functions in host immunity. By performing more small RNA profiling on pathogen-infected tissues using deep sequencing technologies, we expect to identify more small RNAs that are involved in host defense responses. These small RNAs can be up- or down-regulated in response to pathogen challenges and subsequently contribute to gene expression fine tuning and reprogramming by silencing negative regulators and inducing positive regulators of immune responses. Identification and functional analysis of the targets of these pathogen-regulated small RNAs would help discover new players in the host defense pathways and contribute to the elucidation of the molecular regulatory networks of host immune systems. Furthermore, the arms race between host and pathogens will never end. While hosts utilize silencing machinery for defense against pathogens, pathogens would likely develop countermeasures to fight against silencing. We speculate that bacterial and fungal pathogens may have evolved silencing suppressors, as in many RNA viruses. Acknowledgements: I would like to thank Jian-Kang Zhu, Shou-Wei Ding, and Xuemei Chen from University of California, Riverside for useful comments. Research in Jin Laboratory is supported by National Science Foundation Career Award MCB-0642843, University of California Discovery Grant Bio06-10566, and California Citrus Board Grants 5210-131 & 5210-132. I apologize for not citing some publications due to space limitations and referring to previous reviews for detailed information.
References [1] Jones, J.D.G. and Dangl, J.L. (2006) The plant immune system. Nature 444 (7117), 323–329. [2] Chisholm, S.T. et al. (2006) Host–microbe interactions: shaping the evolution of the plant immune response. Cell 124 (4), 803–814. [3] Dangl, J.L. and Jones, J.D.G. (2001) Plant pathogens and integrated defence responses to infection. Nature 411 (6839), 826–833. [4] Parker, J.E. et al. (2001) Genetic analysis of plant disease resistance pathways. Rice Biotechnology: Improving Yield, Stress Tolerance and Grain Quality, pp. 153–164. [5] Tao, Y. et al. (2003) Quantitative nature of Arabidopsis responses during compatible and incompatible interactions with the bacterial pathogen Pseudomonas syringae. Plant Cell 15 (2), 317–330. [6] Chapman, E.J. and Carrington, J.C. (2007) Specialization and evolution of endogenous small RNA pathways. Nat. Rev. Genet. 8 (11), 884–896. [7] Sunkar, R. et al. (2007) Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. Trends Plant Sci. 12 (7), 301–309. [8] Brodersen, P. et al. (2008) Widespread translational inhibition by plant miRNAs and siRNAs. Science 320 (5880), 1185–1190. [9] Borsani, O. et al. (2005) Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis. Cell 123, 1279–1291. [10] Deleris, A. et al. (2006) Hierarchical action and inhibition of plant Dicer-like proteins in antiviral defense. Science 313 (5783), 68–71. [11] Qi, Y.J. et al. (2006) Distinct catalytic and non-catalytic roles of ARGONAUTE4 in RNA-directed DNA methylation. Nature 443 (7114), 1008–1012. [12] Zilberman, D., Cao, X.F. and Jacobsen, S.E. (2003) ARGONAUTE4 control of locus-specific siRNA accumulation and DNA and histone methylation. Science 299 (5607), 716–719. [13] Zheng, X.W. et al. (2007) Role of Arabidopsis AGO6 in siRNA accumulation, DNA methylation and transcriptional gene silencing. EMBO J. 26 (6), 1691–1701.
2684 [14] Katiyar-Agarwal, S. et al. (2006) A pathogen-inducible endogenous siRNA in plant immunity. Proc. Natl. Acad. Sci. USA 103 (47), 18002–18007. [15] Navarro, L. et al. (2006) A plant miRNA contributes to antibacterial resistance by repressing auxin signaling. Science 312 (5772), 436–439. [16] Fahlgren, N. et al. (2007) High-throughput sequencing of Arabidopsis microRNAs: evidence for frequent birth and death of MIRNA genes. PLoS ONE 2, e219. [17] Xiao, C.C. et al. (2007) MiR-150 controls B cell differentiation by targeting the transcription factor c-myb. Cell 131 (1), 146–159. [18] Rodriguez, A. et al. (2007) Requirement of bic/microRNA-155 for normal immune function. Science 316 (5824), 608–611. [19] OÕConnell, R.M. et al. (2007) MicroRNA-155 is induced during the macrophage inflammatory response. Proc. Natl. Acad. Sci. USA 104 (5), 1604–1609. [20] Li, Q.J. et al. (2007) miR-181a is an intrinsic modulator of T cell sensitivity and selection. Cell 129 (1), 147–161. [21] Muljo, S.A. et al. (2005) Aberrant T cell differentiation in the absence of Dicer. J. Exp. Med. 202 (2), 261–269. [22] Koralov, S.B. et al. (2008) Dicer ablation affects antibody diversity and cell survival in the B lymphocyte lineage. Cell 132 (5), 860–874. [23] Taganov, K.D. et al. (2006) NF-kappa B-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. Proc. Natl. Acad. Sci. USA 103 (33), 12481–12486. [24] Chen, X.M. et al. (2007) A cellular Micro-RNA, let-7i, regulates toll-like receptor 4 expression and contributes to cholangiocyte immune responses against Cryptosporidium parvum infection. J. Biol. Chem. 282 (39), 28929–28938. [25] AtGenExpress.. [26] Katiyar-Agarwal, S. et al. (2007) A novel class of bacteriainduced small RNAs in Arabidopsis. Genes Dev. 21 (23), 3123– 3134. [27] Pavet, V. et al. (2006) Arabidopsis displays centromeric DNA hypomethylation and cytological alterations of heterochromatin upon attack by Pseudomonas syringae. Mol. Plant–Microbe Interact. 19 (6), 577–587. [28] Lister, R. et al. (2008) Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell 133 (3), 523–536.
H. Jin / FEBS Letters 582 (2008) 2679–2684 [29] Cokus, S.J. et al. (2008) Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452 (7184), 215–219. [30] Agorio, A. and Vera, P. (2007) ARGONAUTE4 is required for resistance to Pseudomonas syringae in Arabidopsis. Plant Cell 19 (11), 3778–3790. [31] Henderson, I.R. et al. (2006) Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning. Nat. Genet. 38 (6), 721–725. [32] Chan, S.W.L. et al. (2006) RNAi, DRD1, and histone methylation actively target developmentally important non-CG DNA methylation in Arabidopsis. PLoS Genet. 2, 791–797. [33] Jin, H.L. et al. (2008) Small RNAs and the regulation of cisnatural antisense transcripts in Arabidopsis. BMC Mol. Biol. 9 (6), doi:10.1186/1471-2199-9-6. [34] Yamada, K. et al. (2003) Empirical analysis of transcriptional activity in the Arabidopsis genome. Science 302 (5646), 842. [35] Katayama, S. et al. (2005) Antisense transcription in the mammalian transcriptome. Science 309 (5740), 1564–1566. [36] Yi, H. and Richards, E.J. (2007) A cluster of disease resistance genes in Arabidopsis is coordinately regulated by transcriptional activation and RNA silencing. Plant Cell 19 (9), 2929–2939. [37] Ghildiyal, M. et al. (2008) Endogenous siRNAs derived from transposons and mRNAs in Drosophila somatic cells. Science 320 (5879), 1077–1081. [38] Tarn, O.H. et al. (2008) Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes. Nature 453 (7194), 534-U8. [39] Watanabe, T. et al. (2008) Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature 453 (7194), 539-U9. [40] Kawamura, Y. et al. (2008) Drosophila endogenous small RNAs bind to Argonaute 2 in somatic cells. Nature 453 (7196), 793–797. [41] Okamura, K. et al. (2008) The Drosophila hairpin RNA pathway generates endogenous short interfering RNAs. Nature 453 (7196), 803–806. [42] Czech, B. et al. (2008) An endogenous small interfering RNA pathway in Drosophila. Nature 453 (7196), 798–802. [43] Okamura, K. et al. (2008) Two distinct mechanisms generate endogenous siRNAs from bidirectional transcription in Drosophila melanogaster. Nat. Struct. Mol. Biol. 15 (6), 581–590.
Minireview
Endogenous small RNAs and antibacterial immunity in plants Hailing Jin Department of Plant Pathology and Microbiology, Center for Plant Cell Biology and Institute for Integrative Genome Biology, University of California, Riverside, CA 92521, USA Received 10 June 2008; revised 26 June 2008; accepted 30 June 2008 Available online 11 July 2008 Edited by Shou-Wei Ding
Abstract Small RNAs are non-coding regulatory RNA molecules that control gene expression by mediating mRNA degradation, translational inhibition, or chromatin modification. Virusderived small RNAs induce silencing of viral RNAs and are essential for antiviral defense in both animal and plant systems. The role of host endogenous small RNAs on antibacterial immunity has only recently been recognized. Host disease resistance and defense responses are achieved by activation and repression of a large array of genes. Certain endogenous small RNAs in plants, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are induced or repressed in response to pathogen attack and subsequently regulate the expression of genes involved in disease resistance and defense responses by mediating transcriptional or post-transcriptional gene silencing. Thus, these small RNAs play an important role in gene expression reprogramming in plant disease resistance and defense responses. This review focuses on the recent findings of plant endogenous small RNAs in antibacterial immunity. Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
effector-triggered immunity (ETI), which is mediated by resistance (R) proteins. R proteins recognize specific pathogen effectors, such as bacterial avirulence (avr) proteins, and rapidly turn on a series of defense responses to inhibit pathogen growth [1–4]. PTI, also referred to as ‘‘basal defense’’, is pathogen non-specific and less effective than ETI, the R gene-mediated resistance. Plant immune responses, including both PTI and ETI, are mediated by activation and repression of a large array of genes during infection. For example, over 2000 genes are differentially expressed in the disease resistance responses triggered by two NBS–LRR type of R genes, RPS2 and RPM1 [5]. How gene expression reprogramming is achieved, however, is largely unknown. Several recent studies suggest that endogenous small RNA-mediated gene silencing may serve as one important mechanism for gene expression reprogramming in plant immune responses. This review mainly discusses plant endogenous small RNAs in antibacterial immunity (Fig. 1 and Table 1).
Keywords: Endogenous siRNAs; Pathogen-regulated; lsiRNAs; miRNA; Plant immunity 2. Plant endogenous small RNAs
1. Multiple layers of plant immune responses Plants have evolved multiple layers of immune systems in response to various pathogen attacks [1,2]. The first layer of defense is so-called ‘‘non-host resistance’’, which enables plants to resist the vast majority of pathogens by either preventing pathogen penetration or by depriving the nutrients and other factors required for pathogen growth and multiplication. Once the bacterial or fungal pathogens gain access to the plant interior and get through the cell wall, they encounter another layer of defense that is triggered by the recognition of microbial or pathogen-associated molecular patterns (MAMPs or PAMPs), such as flagellin, by plant transmembrane pattern recognition receptors (PRRs). During the co-evolution of plant and pathogen, some successful pathogens have overcome this PAMPtriggered immunity (PTI) by delivering effectors into the plant cell to suppress the signal transduction of PTI. As part of the arms race between plants and pathogens, some plants have evolved another rapid and highly effective defense system –
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Endogenous small RNAs are now recognized as important regulators of gene expression in numerous cellular processes. These short non-coding small RNAs can be classified into microRNA (miRNA), short interfering RNA (siRNA), and PIWI-interacting RNA (piRNA) based on their distinct biogenesis pathways [6]. In plants, the biogenesis of small RNAs is especially complex, as reflected by the large numbers of enzymes involved. Arabidopsis has four type-III ribonuclease Dicer-like (DCL) proteins, six predicted RNA-dependent RNA polymerases (RDRs), and 10 predicted Argonautes (AGOs). Plants do not contain the PIWI subfamily of AGO proteins and no piRNAs are identified so far from plants. miRNAs are small (mostly 20–22-nt) RNAs generated from hairpin precursors mainly by DCL1. A few recently identified miRNAs with long hairpin precursors depend on DCL4. The biogenesis of miRNAs does not require RDRs or RNA polymerase IV (pol IV) components. In Arabidopsis, more than 200 miRNAs were reported, and many are important for plant development. Some miRNAs have been shown to be induced or repressed by various abiotic stresses and regulate gene expression in plant stress responses [7]. Plant miRNAs have high complementarity to their mRNA targets and were thought to predominantly induce post-transcriptional gene silencing by guiding mRNA cleavage. Translational inhibition,
0014-5793/$34.00 Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.06.053
[26] [26] [26]
[36]
Protoporphyrinogen oxidase These targets require experimental validation.
a
PPOXa AtlsiRNA-5
Induced by Pst (avrRpt2)
AtRAP IRT2 AT7SL-1a Induced by Pst (avrRpt2) Induced by Pst (avrRpt2) Induced by Pst (avrRpt2) and Pst (avrRpm1) AtlsiRNA-1 AtlsiRNA-2 AtlsiRNA-3
RPP4 confers resistance against Hyaloperonospora parasitica SNC1 contributes to the resistance against both Hyaloperonospora parasitica and Pst Contribute to both ETI and PTI Iron-responsive transporter A signal recognition particle gene, a RNA pol III-dependent RNA gene [26] RPP4 locus genes including SNC1 RPP4 locus associated siRNAs
[14] Negative regulator of RPS2 pathway (ETI) Induced by Pst (avrRpt2)
PPRL nat-siRNAATGB2 siRNAs
Auxin signaling and PTI Auxin signaling Auxin signaling Induced by biotic stresses Gene regulation Putative iron-chaperone protein TIR1, AFB2, AFB3 ARF6, ARF8 ARF10, ARF16, ARF17 Remorina Zinc finger homeobox genea Frataxin-related genea miR393 miR167 miR160 miR825 miRNAs
Induced by flg22 Induced by Pst (hrcC) Induced by Pst (hrcC) Suppressed by Pst (hrcC)
Targets Small RNAs
Table 1 List of plant endogenous small RNAs that may contribute to plant disease resistance and defense responses
however, was recently reported to be another widespread mode of action for many miRNAs in plants [8]. Endogenous siRNAs are generated from long doublestranded RNAs (dsRNAs) as a result of antisense transcription or the activity of RDRs. Millions of endogenous siRNAs have been identified from plants by deep sequencing. They are extremely diverse and are normally not conserved across different species. Plant endogenous siRNAs can induce transcriptional gene silencing via DNA methylation and/or histone modifications or cause post-transcriptional gene silencing via mRNA degradation and/or translation inhibition. Although siRNAs greatly outnumber miRNAs, the biological roles of many endogenous siRNAs have yet to be explored. The biogenesis of various siRNAs involves all four DCLs in Arabidopsis. DCL1 initiates the in-phase production of transacting siRNAs (ta-siRNAs) by producing miRNAs that guide the cleavage of ta-siRNA precursors [6]. DCL1 is also required for the bacterial-induced natural antisense transcripts-derived siRNA (nat-siRNA) [14] and the in-phase production of salt stress-induced 21-nt nat-siRNAs, which is initiated by a 24nt nat-siRNA produced by DCL2 [9]. DCL2 produces some viral siRNAs and substitutes for DCL3 or DCL4 when they are absent [6]. DCL3 generates 24-nt heterochromatin-associated or repeat-associated siRNAs that regulate heterochromatin formation and transcriptional gene silencing [6]. The inphase production of ta-siRNAs depends on DCL4 [6]. DCL4 also generates siRNAs from long hairpin RNAs in inverse repeat-induced PTGS. Furthermore, DCL4 serves as a primary antiviral sensor for production of virus-derived siRNAs and prevents viral exit from the vasculature [10]. In addition to DCLs, other components such as RDRs and AGOs have been implicated in siRNA accumulation. RDR2 and the RNA pol IV cooperate in generating heterochromatin-associated 24-nt siRNAs. These siRNAs are incorporated into AGO4 or AGO6 and induce transcriptional gene silencing by guiding DNA methylation and histone modifications at the homologous genomic loci [11–13]. Generation of ta-siRNAs and nat-siRNAs requires RDR6 and SUPPRESSOR OF GENE SILENCING 3 (SGS3) [6,9,14]. These findings indicate that plants have evolved multiple small RNA processing pathways with specific and overlapping functions.
Target functions
Fig. 1. Certain plant endogenous small RNAs (miRNAs and siRNAs) are up- or down-regulated by pathogen infection and target R genes or defense responses genes in plant immunity. These small RNAs contribute to gene expression reprogramming and fine-tuning in plant resistance and defense responses.
[15] [16] [16] [16]
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Reference
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3. miRNAs and PAMP-triggered antibacterial resistance Arabidopsis miR393 was the first miRNA identified to play a role in plant antibacterial PTI by regulating the auxin signaling pathway [15]. miR393 was induced by a bacterial PAMP peptide flg22 at 20–60 min post-treatment. Expression of the miR393 targets, auxin receptor TIR1 (Transport Inhibitor Response 1) and two of its functional paralogs (AFB2 and AFB3), was decreased upon flg22 treatment. Constitutive overexpression of another TIR1 paralog that is not a target of miR393, AFB1, in a tirl-1 mutant background led to enhanced disease sensitivity to Pseudomonas syringae pv. tomato (Pst) DC3000, whereas miR393 overexpression lines restricted bacterial growth. Thus, the findings suggest that miR393 contributes to flg22-induced basal defense or PTI by targeting auxin receptor mRNAs and subsequently repressing auxin signaling in Arabidopsis (Fig. 2A). By using small RNA expression profiling on Arabidopsis leaves collected at 1 and 3 h post-inoculation of Pst DC3000 hrcC, Fahlgren et al. [16] identified three miRNAs, miR393, miR167, and miR160 that were highly induced after infection. Pst DC3000 hrcC strain has a mutation in the key effector delivery machine, the type III secretion system. This strain triggers PTI in plants and can no longer cause disease. Interest-
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ingly, all three miRNAs negatively regulate auxin signaling by either targeting auxin receptor genes or auxin response factors. Pathogen-induced small RNAs are likely to silence negative regulators of host resistance pathways. miR825 was downregulated by Pst DC3000 hrcC infection and has three predicted targets, including the Remorin gene, the zinc finger homeobox family gene, and a frataxin-related gene [16] (Fig. 2A). These predicted targets are likely up-regulated by the infection and presumably positively regulate plant defense responses, although their functions need to be confirmed by experimental data. These results indicate that host miRNAs play an important role in plant immunity. The importance of miRNA in host immunity has also been reported in animal systems. Several miRNAs, including miR150, miR155, and miR181a, are differentially expressed during the T-cell and B-cell differentiation and regulate their activation and sensitivity to antigens. The miRNAs help regulate the differentiation of haematopoieticcell lineage and the modulation of adaptive immune responses [17–20]. T-cell deficiencies were observed in Dicer conditional knockout mice [21]. Dicer ablation also affects B lymphocyte development by blocking pro-B to pre-B cell transition and interferes with antibody diversity [22]. Animal miRNAs also play a crucial role in innate immunity, the first line of defense mediated by recognition between Toll-like receptors (TLRs) and invading bacterial, viral, and other pathogens. For example, expression of miR-146a, miR132, and miR155 was largely induced by endotoxin lipopolysaccharide treatment, which mimicked bacterial infection [23]. Interestingly, miR146a can only be induced in a NF-kB dependent manner by the transmembrane TLRs that recognize bacterial constituents but not by the intracellular TLRs that mainly sense viral nucleic acids, whereas miR155 is induced by both bacterial and viral infection through TLR3-induced tumor-necrosis factor signaling [19,23]. By regulating TLR4 expression, let-7i also contributes to innate immune responses against the parasite Cryptosporidium parvum [24]. Thus, miRNA-mediated gene silencing is a general regulatory mechanism in host immunity in both plants and animals. Additional miRNA profiling in pathogen-infected plants is likely to identify more miRNAs that regulate plant immunity, including both PTI and ETI.
4. Endogenous siRNAs and bacterial effector-triggered resistance
Fig. 2. Identified miRNAs and endogenous siRNAs that play a role in antibacterial PTI (A) and ETI (B) in Arabidopsis [14–16,26]. The expression and putative function of miR167, miR160 and miR825 (A) were purely based on the deep sequencing data from Fahlgren et al. [16] and require further experimental validation. The putative function of AtlsiRNA-2, 3 and 5 (B) was based on Northern blot analysis and computational analysis and also requires further experimental validation.
The first example of a plant endogenous siRNA that regulates plant immunity is nat-siRNAATGB2 [14]. This nat-si RNA was identified from an expression study of Arabidopsis siRNAs from publicly available small RNA databases (ASRP and MPSS) that target pathogen-regulated genes from the AtGenExpress database [25]. nat-siRNAATGB2 is derived from the overlapping region of a pair of natural antisense transcripts, a Rab2-like small GTP-binding protein gene ATGB2 and a PPR (pentatricopeptide repeats) protein-like gene PPRL. It is specifically induced by Pst DC3000 carrying effector avrRpt2 but not other effectors. Induction of nat-siRNAATGB2 represses the expression of its antisense target PPRL, a negative regulator of RPS2-mediated ETI against Pst (avrRpt2) (Fig. 2B). Overexpression of PPRL delayed the hypersensitive response (HR), reduced cell death triggered by
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the recognition between RPS2 and Pst (avrRpt2), and enhanced the growth of Pst (avrRpt2). The biogenesis pathway of nat-siRNAATGB2 is unique. It depends on DCL1, HYL1, and HEN1, and also requires RDR6, SGS3, and RNA pol IV subunit NRPDla. Strikingly, the specific induction of nat-siRNAATGB2 by Pst (avrRpt2) depends on the cognate host resistance gene RPS2 and the NDR1 gene required for RPS2-specified resistance. This study demonstrated that a plant nat-siRNA is specifically induced by a bacterial effector and regulates ETI by down-regulating a negative regulator of the cognate R gene-mediated specific resistance pathway. To be cost effective, plant disease resistance responses need to be suppressed during normal conditions and to be rapidly turned on upon pathogen attack. Endogenous small RNA-mediated gene silencing may serve as one of the mechanisms for this rapid ‘‘off’’ and ‘‘on’’ regulation. In addition to the continuous study of small RNAs-targeting pathogen-regulated genes, our laboratory also conducted small RNA expression profiling via deep sequencing using small RNAs extracted from pathogen-infected Arabidopsis tissue to identify more pathogen-regulated plant endogenous small RNAs. Many small RNAs, including miRNAs and endogenous siRNAs, were induced or repressed by various pathogens, including bacteria and fungi (Jin et al., unpublished data), and these small RNAs may down- or up-regulate genes involved in plant disease resistance and defense responses (Figs. 1 and 2). Some of these small RNAs are unique to one specific pathogen, whereas others are relevant for plant basal defense responses and are regulated by multiple pathogens. During Northern blot expression validation of identified small RNAs, Katiyar-Agarwal et al. discovered a novel class of endogenous small RNAs, long siRNAs (lsiRNAs), which are 30–40 nt long and are induced by bacterial infection or by specific growth conditions [26]. Unlike the recently identified 25–31-nt animal piRNAs that are DICER-independent and require PIWI subfamily proteins for biogenesis, plant lsiRNAs depend on DCL1 and the AGO subfamily protein AGO7. Biogenesis of plant lsiRNAs also depends on HYL1, HEN1, HST1, RDR6, and Pol IV. Of the identified lsiRNAs, AtlsiRNA-1 is induced specifically by Pst (avrRpt2) and downregulates a putative RNA binding protein gene-AtRAP, possibly through a novel mode of action in plants—degradation of the target mRNA through decapping and XRN4-mediated 5 0 – 3 0 decay. Knockout mutation in AtRAP increases resistance to both virulent and avirulent Pst, which suggests that AtRAP may serve as a common component in basal defense but is controlled by AtlsiRNA-1 during the rapid response to avirulent factor avrRpt2. AtlsiRNA-2, -3, and -4 are modestly induced by mock treatment and infection by various Pst strains and target an iron-responsive transporter gene (IRT2), a signal recognition particle gene (AT7SL-1), and an anion transporter gene (ANTR2), respectively. AtlsiRNA-2 and 3 are induced to a higher level by Pst (avrRpt2). AtlsiRNA-5 is only weakly induced by Pst (avrRpt2) and targets the antisense gene protoporphyrinogen oxidase (PPOX) (Fig. 2B).
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ifications. The bacterial pathogen Pst DC3000 was shown to induce DNA hypomethylation in the absence of DNA replication at certain genomic loci, including some peri/centromeric repeats and retrotransposons, in Arabidopsis [27]. Both virulent Pst DC3000 and avirulent Pst DC3000 (avrRpt2) strains, but not the non-pathogenic strain Pst DC3000 hrpL, can induce chromatin alteration, such as decondensation of chromocenters, in the infected tissue. The authors speculated that Pst DC3000-induced DNA hypomethylation and heterochromatin decondensation may promote increased DNA recombination and genetic variability at some genomic loci, including centromeric regions harboring a group of resistance genes. This is a very interesting finding, and recently developed bisulfite sequencing using sequencing-by synthesis (SBS) technology will enable researchers to identify pathogen-regulated DNA methylation regions at a single-base resolution in a global level, as demonstrated in the elegant work by Lister et al. [28] and Cokus et al. [29]. The likelihood of small RNA involvement in this chromatin modification process upon pathogen challenge was indicated by the requirement of AGO4 in resistance to Pst infection in Arabidopsis [30]. Mutant ago4-2 was identified from a genetic screen and had enhanced susceptibility to the virulent Pst DC3000, to the avirulent Pst DC3000 carrying the effector avrRpml, as well as to a non-host strain P. syringae pv. phaseolicola, which is a pathogen of bean but not normally a pathogen of Arabidopsis. The extent of DNA cytosine methylation at CpNpG and CpHpH positions in various loci is clearly decreased in ago4 mutants. AGO4 associates with 24-nt siRNAs in a putative RNA-induced transcriptional silencing complex (RITS) to guide DNA methylation and histone modifications. The biogenesis of these chromatin-associated 24-nt siRNAs requires DCL3 and RDR2. However, mutations in DCL3 and RDR2 did not interfere with plant defense responses. Several DNA methyltransferases and chromatin-remodeling proteins, including Chromomethylase 3 (CMT3), Domains Rearranged Methyltransferase 1 and 2 (DRM1 and DRM2), and Defective in RNA-directed DNA methylation (DRD1), act with AGO4 in the RNA-dependent DNA Methylation (RdDM) pathway. No change of Pst growth rate was observed in drdl, cmt3 or the drml/drm2 double mutant as compared with wild type plants. This lack of phenotype may be due to the possible functional redundancy among various DCLs, RDRs, and methyltransferases, although at this stage we cannot rule out the possibility that AGO4 could contribute to disease resistance independently of the RdDM pathway. Disease susceptibility analysis on double or triple mutants of RDRs, DCLs, and methyltransferases, such as dcl2/3/4 and ddc (drm1-2 drm2-2 cmt3-11) triple mutants, will help to clarify this issue [31,32]. Bisulfite sequencing on the ago4 mutant may be able to determine the DNA methylation regions controlled by ago4 and may identify some R genes or resistance signaling genes controlled by RdDM.
6. NATs-associated endogenous siRNAs and beyond 5. Small RNA-mediated transcriptional gene silencing in plant immunity Chromatin-associated, 24-nt siRNAs induce transcriptional gene silencing by guiding DNA methylation and histone mod-
Interestingly, in addition to nat-siRNAs, several identified lsiRNAs are also derived from the overlapping region of NATs [26]. cis-NAT arrangement is a widespread phenomenon in both plant and animal genomes. About 7–30% of plant and animal genes are arranged as cis-NATs in spite of large intergenic
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regions, which suggests an evolutionary advantage of this genomic arrangement. Over 3000 Arabidopsis small RNAs from small RNA databases were found to match more than 60% of protein-coding NAT pairs [33], indicating that the small RNA-mediated gene regulation may serve as one of the regulatory mechanisms for the expression of at least a subgroup of NATs. Little conservation was observed on either overlapping NAT genes or nat-siRNAs among different species. Besides the overlapping gene regions, more than 30% of the Arabidopsis genome and about 60–80% of mammalian genomes can produce transcripts from both sense and antisense strands [34,35]. Antisense transcription provides perfect dsRNA precursors for siRNA production. Yi and Richards provided a nice example of siRNA generation from antisense transcription at the R gene recognition of the Hyaloperonospora parasitica (RPP) locus [36]. The Arabidopsis Columbia RPP4 locus (named RPP5 locus in Landsberg erecta ecotype) contains seven TIR–NBS–LRR class R genes, which are interspersed with two non-R genes and three related sequences. Two R genes in this locus, RPP4 and SNC1 (for Suppressor of nprl-1, constitutive 1), were shown to be functional and to confer resistance to the bacterium P. syringe and the oomycete H. parasitica. This cluster of R genes is coordinately regulated by transcription activation and siRNA-mediated RNA silencing. Endogenous siRNAs were detected at the RPP4 locus, including SNC1. SNC1 mRNA level is elevated in dcl4, ago1, and upf1 RNA silencing pathway mutants, indicating that these siRNAs function in the silencing of SNC1. Antisense transcripts were detected from the regions that produce these siRNAs, which suggests that these siRNAs may originate from sense and antisense RNA pairing.
7. Widespread occurrence of endogenous siRNAs in animal systems Until recently, no endogenous siRNAs (endo-siRNAs) were observed in animals lacking RDRPs, such as mammals and Drosophila. However, the RNAi machinery in Drosophila and mammals may not function only for antiviral defense by silencing exogenous viral RNAs. By using deep sequencing technologies, several laboratories discovered endogenous siRNAs (endo-siRNAs) that target transposons, intergenic regions, and mRNAs in mouse and Drosophila [37–42]. In Drosophila somatic S2 cells, 41% of the endo-siRNAs mapped to mRNAs do not map to transposons, suggesting a regulatory role of these endo-siRNAs in mRNA expression [37]. Furthermore, the mRNA-targeting endo-siRNAs were highly enriched (eg. over 10-fold in Drosophila) in the overlapping regions of cis-NATs or the regions that have antisense transcription [37–39,43]. Although the authors did not observe clear strand bias of these putative nat-siRNAs under the experimental conditions, these endo-siRNAs may also regulate gene expression of these cisNAT genes under specific conditions, as in plant systems. Thus, endogenous siRNA-mediated gene silencing is a general gene regulation mechanism in both plant and animal systems.
8. Conclusions Plants and animals employ small RNA-mediated gene silencing as an important mechanism for host immunity
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against bacterial, viral and fungal pathogens. We speculate that many endogenous small RNAs that have yet to be discovered have important functions in host immunity. By performing more small RNA profiling on pathogen-infected tissues using deep sequencing technologies, we expect to identify more small RNAs that are involved in host defense responses. These small RNAs can be up- or down-regulated in response to pathogen challenges and subsequently contribute to gene expression fine tuning and reprogramming by silencing negative regulators and inducing positive regulators of immune responses. Identification and functional analysis of the targets of these pathogen-regulated small RNAs would help discover new players in the host defense pathways and contribute to the elucidation of the molecular regulatory networks of host immune systems. Furthermore, the arms race between host and pathogens will never end. While hosts utilize silencing machinery for defense against pathogens, pathogens would likely develop countermeasures to fight against silencing. We speculate that bacterial and fungal pathogens may have evolved silencing suppressors, as in many RNA viruses. Acknowledgements: I would like to thank Jian-Kang Zhu, Shou-Wei Ding, and Xuemei Chen from University of California, Riverside for useful comments. Research in Jin Laboratory is supported by National Science Foundation Career Award MCB-0642843, University of California Discovery Grant Bio06-10566, and California Citrus Board Grants 5210-131 & 5210-132. I apologize for not citing some publications due to space limitations and referring to previous reviews for detailed information.
References [1] Jones, J.D.G. and Dangl, J.L. (2006) The plant immune system. Nature 444 (7117), 323–329. [2] Chisholm, S.T. et al. (2006) Host–microbe interactions: shaping the evolution of the plant immune response. Cell 124 (4), 803–814. [3] Dangl, J.L. and Jones, J.D.G. (2001) Plant pathogens and integrated defence responses to infection. Nature 411 (6839), 826–833. [4] Parker, J.E. et al. (2001) Genetic analysis of plant disease resistance pathways. Rice Biotechnology: Improving Yield, Stress Tolerance and Grain Quality, pp. 153–164. [5] Tao, Y. et al. (2003) Quantitative nature of Arabidopsis responses during compatible and incompatible interactions with the bacterial pathogen Pseudomonas syringae. Plant Cell 15 (2), 317–330. [6] Chapman, E.J. and Carrington, J.C. (2007) Specialization and evolution of endogenous small RNA pathways. Nat. Rev. Genet. 8 (11), 884–896. [7] Sunkar, R. et al. (2007) Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. Trends Plant Sci. 12 (7), 301–309. [8] Brodersen, P. et al. (2008) Widespread translational inhibition by plant miRNAs and siRNAs. Science 320 (5880), 1185–1190. [9] Borsani, O. et al. (2005) Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis. Cell 123, 1279–1291. [10] Deleris, A. et al. (2006) Hierarchical action and inhibition of plant Dicer-like proteins in antiviral defense. Science 313 (5783), 68–71. [11] Qi, Y.J. et al. (2006) Distinct catalytic and non-catalytic roles of ARGONAUTE4 in RNA-directed DNA methylation. Nature 443 (7114), 1008–1012. [12] Zilberman, D., Cao, X.F. and Jacobsen, S.E. (2003) ARGONAUTE4 control of locus-specific siRNA accumulation and DNA and histone methylation. Science 299 (5607), 716–719. [13] Zheng, X.W. et al. (2007) Role of Arabidopsis AGO6 in siRNA accumulation, DNA methylation and transcriptional gene silencing. EMBO J. 26 (6), 1691–1701.
2684 [14] Katiyar-Agarwal, S. et al. (2006) A pathogen-inducible endogenous siRNA in plant immunity. Proc. Natl. Acad. Sci. USA 103 (47), 18002–18007. [15] Navarro, L. et al. (2006) A plant miRNA contributes to antibacterial resistance by repressing auxin signaling. Science 312 (5772), 436–439. [16] Fahlgren, N. et al. (2007) High-throughput sequencing of Arabidopsis microRNAs: evidence for frequent birth and death of MIRNA genes. PLoS ONE 2, e219. [17] Xiao, C.C. et al. (2007) MiR-150 controls B cell differentiation by targeting the transcription factor c-myb. Cell 131 (1), 146–159. [18] Rodriguez, A. et al. (2007) Requirement of bic/microRNA-155 for normal immune function. Science 316 (5824), 608–611. [19] OÕConnell, R.M. et al. (2007) MicroRNA-155 is induced during the macrophage inflammatory response. Proc. Natl. Acad. Sci. USA 104 (5), 1604–1609. [20] Li, Q.J. et al. (2007) miR-181a is an intrinsic modulator of T cell sensitivity and selection. Cell 129 (1), 147–161. [21] Muljo, S.A. et al. (2005) Aberrant T cell differentiation in the absence of Dicer. J. Exp. Med. 202 (2), 261–269. [22] Koralov, S.B. et al. (2008) Dicer ablation affects antibody diversity and cell survival in the B lymphocyte lineage. Cell 132 (5), 860–874. [23] Taganov, K.D. et al. (2006) NF-kappa B-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. Proc. Natl. Acad. Sci. USA 103 (33), 12481–12486. [24] Chen, X.M. et al. (2007) A cellular Micro-RNA, let-7i, regulates toll-like receptor 4 expression and contributes to cholangiocyte immune responses against Cryptosporidium parvum infection. J. Biol. Chem. 282 (39), 28929–28938. [25] AtGenExpress.
H. Jin / FEBS Letters 582 (2008) 2679–2684 [29] Cokus, S.J. et al. (2008) Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452 (7184), 215–219. [30] Agorio, A. and Vera, P. (2007) ARGONAUTE4 is required for resistance to Pseudomonas syringae in Arabidopsis. Plant Cell 19 (11), 3778–3790. [31] Henderson, I.R. et al. (2006) Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning. Nat. Genet. 38 (6), 721–725. [32] Chan, S.W.L. et al. (2006) RNAi, DRD1, and histone methylation actively target developmentally important non-CG DNA methylation in Arabidopsis. PLoS Genet. 2, 791–797. [33] Jin, H.L. et al. (2008) Small RNAs and the regulation of cisnatural antisense transcripts in Arabidopsis. BMC Mol. Biol. 9 (6), doi:10.1186/1471-2199-9-6. [34] Yamada, K. et al. (2003) Empirical analysis of transcriptional activity in the Arabidopsis genome. Science 302 (5646), 842. [35] Katayama, S. et al. (2005) Antisense transcription in the mammalian transcriptome. Science 309 (5740), 1564–1566. [36] Yi, H. and Richards, E.J. (2007) A cluster of disease resistance genes in Arabidopsis is coordinately regulated by transcriptional activation and RNA silencing. Plant Cell 19 (9), 2929–2939. [37] Ghildiyal, M. et al. (2008) Endogenous siRNAs derived from transposons and mRNAs in Drosophila somatic cells. Science 320 (5879), 1077–1081. [38] Tarn, O.H. et al. (2008) Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes. Nature 453 (7194), 534-U8. [39] Watanabe, T. et al. (2008) Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature 453 (7194), 539-U9. [40] Kawamura, Y. et al. (2008) Drosophila endogenous small RNAs bind to Argonaute 2 in somatic cells. Nature 453 (7196), 793–797. [41] Okamura, K. et al. (2008) The Drosophila hairpin RNA pathway generates endogenous short interfering RNAs. Nature 453 (7196), 803–806. [42] Czech, B. et al. (2008) An endogenous small interfering RNA pathway in Drosophila. Nature 453 (7196), 798–802. [43] Okamura, K. et al. (2008) Two distinct mechanisms generate endogenous siRNAs from bidirectional transcription in Drosophila melanogaster. Nat. Struct. Mol. Biol. 15 (6), 581–590.