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ANNUAL REVIEWS
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Small RNAs: A New Paradigm in Plant-Microbe Interactions Arne Weiberg, Ming Wang, Marschal Bellinger, and Hailing Jin Department of Plant Pathology and Microbiology, University of California, Riverside, California 92521; email: [email protected]
Annu. Rev. Phytopathol. 2014. 52:495–516
Keywords
The Annual Review of Phytopathology is online at phyto.annualreviews.org
small RNA, gene silencing, pathogen effector, transposable element, cross-kingdom RNAi
This article’s doi: 10.1146/annurev-phyto-102313-045933 c 2014 by Annual Reviews. Copyright All rights reserved
Abstract A never-ending arms race drives coevolution between pathogens and hosts. In plants, pathogen attacks invoke multiple layers of host immune responses. Many pathogens deliver effector proteins into host cells to suppress host immunity, and many plants have evolved resistance proteins to recognize effectors and trigger robust resistance. Here, we discuss findings on noncoding small RNAs (sRNAs) from plants and pathogens, which regulate host immunity and pathogen virulence. Recent discoveries have unveiled the role of noncoding sRNAs from eukaryotic pathogens and bacteria in pathogenicity in both plant and animal hosts. The discovery of fungal sRNAs that are delivered into host cells to suppress plant immunity added sRNAs to the list of pathogen effectors. Similar to protein effector genes, many of these sRNAs are generated from transposable element (TE) regions, which are likely to contribute to rapidly evolving virulence and host adaptation. We also discuss RNA silencing that occurs between organisms.
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INTRODUCTION
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Pathogen-associated molecular pattern (PAMP): conserved molecules common in pathogens, such as flagellin, peptidoglycan, and chitin PAMP-triggered immunity (PTI): plant receptor protein–based recognition of PAMPs that induces defense response Effector-triggered immunity (ETI): pathogen effector recognition by plant resistance proteins that induces defense response Noncoding RNAs: classes of RNA molecules that are not translated into proteins Dicer-like protein (DCL): RNA-binding type III endoribonuclease that processes doublestranded RNA molecules into mature miRNAs and siRNAs Argonaute (AGO): RNA-binding protein and central component of the RNA-induced gene-silencing complex Gene silencing: noncoding small RNAs mediate gene suppression via RNAi either transcriptionally by DNA methylation or posttranscriptionally by mRNA degradation or translational inhibition
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Pathogens are among the most specialized life-forms on Earth. The challenge of colonizing a living host cell comes from the evolutionary specificity that must exist for a pathogen to outmaneuver the host immune system. In plants, most of the microbial attackers are blocked by nonhost resistance and pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). Successful pathogens have evolved protein effectors to suppress PTI. To counter the pathogen protein effectors, plants have evolved resistance (R) genes, which encode R proteins that can recognize specific pathogen protein effectors directly or indirectly and induce a rapid and robust immune response, known as effector-triggered immunity (ETI). Pathogens then diversify the recognized effector gene or evolve new effector genes to diminish ETI, whereas plants subsequently evolve new R proteins to recognize the new effectors and again induce ETI, continuing the endless evolutionary arms race between pathogen and host (18, 53). Eukaryotic small RNAs (sRNAs) are short regulatory noncoding RNAs that induce silencing of target genes at transcriptional and posttranscriptional levels. The endoribonuclease Dicer or Dicer-like proteins (DCLs) process double-stranded RNAs (dsRNAs) or RNAs with hairpin structures, giving rise to mostly 20–30-nucleotide (nt)-long sRNAs, which are loaded into Argonaute (AGO) proteins to induce gene silencing of their complementary targets by guiding mRNA (messenger RNA) cleavage or degradation, translational inhibition, DNA methylation, and histone modification (5, 12). Prokaryotic regulatory noncoding sRNAs are strikingly different from those found in eukaryotes in terms of their length, structure characteristics, and functions. Bacterial noncoding sRNAs are heterogeneous in length (50–250 nts) and act through distinct RNA-binding protein complexes (133). Plant sRNAs and RNA interference (RNAi) pathway components are important regulatory players in plant immunity against viruses, bacteria, fungi, oomycetes, and pests (26, 51, 95, 106, 111). Recent studies also suggest that pathogen-derived sRNAs and RNAi machinery contribute to pathogen virulence (108, 109, 128). Phytopathologists have begun to study global sRNA expression profiles to identify infection-responsive sRNAs from plant hosts and pathogens, including bacteria, fungi, and oomycetes (35, 50, 75, 92, 100, 110, 122, 128, 135, 149). A vast majority of sRNAs in filamentous plant pathogens are transcribed from transposable element (TE) regions. Silencing of TEs by sRNAs is a ubiquitous phenomenon in all eukaryotes, including fungi (14, 17). This review discusses the role of sRNAs in plant-pathogen interactions, with an emphasis on the role of noncoding sRNAs from bacterial and eukaryotic plant pathogens in their pathogenicity. A brief introduction of sRNA-mediated gene silencing across different organisms is also included.
HOST ENDOGENOUS SRNAS AND RNAI PATHWAY COMPONENTS IN PLANT IMMUNITY Host sRNAs Many plant endogenous sRNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), play an important role in gene expression reprogramming and fine-tuning in host immune responses (51, 95, 106). Most miRNAs are 20–22 nts in length and are processed by DCL proteins from a single-stranded RNA (ssRNA) precursor with a hairpin structure. The model plant Arabidopsis genome encodes four DCLs and ten AGOs. Most Arabidopsis miRNAs are generated by DCL1 and loaded into AGO1 to induce mainly posttranscriptional gene silencing (PTGS). Plant endogenous siRNAs are numerous in quantity and are much more diverse than miRNAs in terms of their length and biogenesis pathways. siRNAs are processed from long dsRNA Weiberg et al.
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precursors by different DCLs and are loaded into different AGOs to guide silencing of their targets transcriptionally or posttranscriptionally (111). Trans-acting siRNAs (tasiRNAs), natural antisense transcript-derived siRNAs (nat-siRNAs), long siRNAs (lsiRNAs), and heterochromatic siRNAs (hcsiRNAs) are different siRNA subclasses present in plants (3, 61, 72, 95). Plant sRNAs are differentially expressed during pathogen attacks. Arabidopsis miR393 was the first miRNA discovered to be involved in plant immunity. It is induced by flg22, a PAMP, and targets auxin receptor TIR1 as well as its homologs and suppresses the auxin signaling pathway, thus contributing to PTI (85). Some conserved miRNAs, such as miR160 and miR167, are also induced by PAMPs and contribute to PTI (85, 144). However, other miRNAs, such as miR398a and miR773, are downregulated upon PAMP perception and bacterial infection to release target suppression and activate PTI (74). Plant sRNAs also play an important role in ETI. Arabidopsis nat-siRNAATGB2 was the first example of a plant endogenous siRNA that regulates R-genemediated immunity. It is highly induced by the bacterial pathogen Pseudomonas syringae pv. tomato (Pst), which carries an effector gene, avrRpt2, and contributes to resistance gene RPS2-mediated ETI by repressing a putative negative regulator of the RPS2 pathway (62). Other examples include Arabidopsis AtlsiRNA-1 and miR393∗ , both of which are induced by Pst (avrRpt2). miR393∗ is the complementary strand of the miR393 within the miRNA duplex. miRNA∗ s were once thought to be useless by-products, but we now know that some of them are functional. miR393∗ is loaded into AGO2 and regulates plant immunity by suppressing the MEMB12 gene that encodes a golgilocalized SNARE protein responsible for retrograde trafficking, which results in the increased exocytosis of antimicrobial pathogenesis-related (PR) proteins (145). AtlsiRNA-1 contributes to plant defense by targeting AtRAP, which encodes a putative RNA-binding protein that negatively regulates plant defense (60). Most plant R genes belong to the nucleotide-binding site (NBS)leucine-rich repeat (LRR) gene family, which is a hot spot for sRNA generation. A class of phased secondary siRNAs has been identified from the NBS-LRR type of R-gene regions in various plant species (69, 115, 142, 151). The biogenesis of these secondary siRNAs is initiated by cleavage of the NBS-LRR transcripts mediated by specific miRNA families, such as miR482 and miR2118 from both tomato (Solanum lycopersicum) and cotton (Gossypium hirsutum) and miR6019 and miR6020 from tobacco (Nicotiana benthamiana). These miRNAs and secondary siRNAs control the expression of NBS-LRR genes at a low level under normal conditions. However, this silencing effect is suppressed when plants are under attack from viral and bacterial pathogens, which leads to the upregulation of these R genes and triggers plant defense responses. Plant endogenous sRNAs that are responsive to fungal infection have also been identified from many plant species. In rice (Oryza sativa), sRNA profiling of the rice blast fungus Magnaporthe oryzae–challenged resistant and susceptible cultivars revealed a group of host endogenous miRNAs that contributes to gene regulation of defense responses (73). For example, miR160 and miR164 are induced, whereas miR396 is downregulated upon infection in a resistance cultivar but not in a susceptible one, suggesting their positive and negative roles in ETI, respectively. Furthermore, miR169, miR172, and miR398 are regulated in both susceptible and resistant cultivars, suggesting their regulatory role in basal responses (73). miR160 targets auxin responsive factor 16 (ARF16) and miR398 targets superoxide dismutase 2 (SOD2) and a copper chaperone of SOD2. All these genes are downregulated upon M. oryzae infection, which correlates well with induced miRNA levels (73). Overexpression of miR160 or miR398 in a susceptible rice cultivar leads to enhanced disease resistance toward M. oryzae, confirming that these two miRNAs are bona fide positive regulators of defense response against M. oryzae (73). In wheat (Triticum aestivum), sRNA profiling of a susceptible cultivar and a resistant cultivar upon infection with the powdery mildew fungus Blumeria graminis results in three categories of regulated miRNAs: (a) responsive only in the resistant cultivar, such as the upregulated miR2008 www.annualreviews.org • Small RNAs
RNA interference (RNAi): a biological process in which noncoding small RNA molecules block gene expression executed by protein members of the Argonaute class Plant immunity: innate system that protects plants against invading pathogens; based on two forms: preformed (constitutive) and pathogen-induced defense response Transposable element (TE): a genomic DNA unit that can undergo reposition, replication, and amplification within its host genome and introduce mutations MicroRNAs (miRNAs): small RNAs that are 20–22 nucleotide long and central in RNAi that are produced from a single-stranded RNA precursor with a hairpin structure by a Dicer-like protein and induce gene silencing of targets with imperfect matches Small interfering RNAs (siRNAs): small RNAs inducing RNAi that are produced from a longer doublestranded RNA precursor by a Dicer-like protein
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and miR2012 and the downregulated miR171; (b) responsive only in the susceptible cultivar, such as the induced miR393, miR444, miR827, miR2005, and miR2013 and the suppressed miR2001, miR2006, and miR2011; and (c) responsive in both cultivars, such as miR156, miR159, miR164, and miR396 (137). A distinct group of miRNAs, including miR167, miR171, miR444, miR408, and miR1138, are induced only in two susceptible wheat cultivars and only in the early stage after infection with the rust fungus Puccinia graminis f. sp. tritici (44). This group is not induced in the late stage of infection or in resistant cultivars. According to the miRNAs endogenous target genes, these miRNAs are likely to participate in early defense responses through regulating hormone signaling pathways, lignin synthesis, and protein biosynthesis. In eggplant (Solanum melongena), 33 Verticillium dahliae–responsive miRNAs were identified. For example, miR393 and miR399 are both highly downregulated after infection, allowing for the accumulation of their target gene auxin receptor, TIR1, and an E2 conjugating enzyme gene, PHO2. In general, TIR1 positively regulates plant defense against necrotrophic pathogens (76) and negatively regulates defense against biotrophic pathogens (85). miR399 is induced by phosphorus starvation in various plant species, and it downregulates PHO2, which is responsible for degrading two phosphate transporters, subsequently leading to enhanced phosphate uptake. Although miR393 is highly regulated by various pathogen infections (76, 85, 137), differential expression of miR399 in response to infection is not common, which may suggest a possible interaction between V. dahliae infection responses and phosphorus homeostasis. miR399 is highly induced in sweet orange Citrus sinensis that is Huanglongbing (HLB) positive (147). HLB, also named citrus greening, is caused by a phloem-limited bacterium within the genus Candidatus Liberibacter and is probably the most devastating citrus disease. Upregulation of miR399 led to the finding of 35% reduction of phosphorus content in HLB-positive trees. This finding links phosphorus deficiency to HLB disease symptomology (147). Application of phosphorus oxyanion solutions to HLB-infected trees reduced disease symptoms and increased fruit production. In cotton, 36 of its 300 NBS-LRR genes are potentially under the control of several members of the miR482 family. miR482, which targets the conserved P-loop sequences of NBS-LRR genes, is downregulated upon infection with V. dahliae. Correspondingly, expression of ten targeted NBS-LRR genes is derepressed. The cleavage of NBS-LRR transcripts by miR482 also triggers the generation of secondary siRNAs (151), which reinforces the silencing of these NBS-LRR genes. The tight repression of R genes by miR482 and secondary siRNAs under a noninfectious situation is turned off during infection by downregulation of miR482, which leads to R-gene activation. Similarly, altered miRNA expression patterns were observed in oomycete-infected plants. Three soybean (Glycine max) cultivars with different levels of resistance to the oomycete Phytophthora sojae were examined. miR403, which targets AGO protein genes, was downregulated in all three cultivars, suggesting a positive role of RNAi machinery in host defense against P. sojae. miR1510, along with family members of miR390 and miR1535, was downregulated. Expression of miRNAs negatively correlated with the expression of their predicted targets, indicating their regulatory role upon infection. Thus, it is clear that host endogenous sRNAs play a critical role in regulating and fine-tuning the expression of plant defense responsive genes and contribute to gene expression reprogramming in plant immune responses against a large array of pathogens, including bacteria, fungi, and oomycetes (43).
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Host RNAi Pathway Components Several plant RNAi pathway components have been shown to be important for antimicrobial immunity (111). In Arabidopsis, DCL1 and DCL4 are key components in miRNA and siRNA 498
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biosynthesis, respectively. The dcl1-9 mutant displays enhanced susceptibility to bacterial infection (86). Similarly, dcl1-7 is more susceptible to the fungal pathogen Botrytis cinerea, supporting the idea that RNAi is also involved in antifungal immunity (128). DCL4 is important for antiviral defense and is also partially involved in the accumulation of antibacterial AtlsiRNA-1 (60). The dcl4-2 mutant is more susceptible to the fungal pathogen V. dahliae (31). RNA-dependent RNA polymerases (RDRs or RdRPs) are responsible for generating dsRNAs using aberrant ssRNAs as templates, which are subsequently processed into sRNAs by DCLs. The Arabidopsis genome encodes six RDRs. RDR6 is involved in the biogenesis of secondary siRNAs, and the rdr6 mutant is more susceptible to bacterial as well as fungal infection (31, 62). Interestingly, suppressor of gene silencing 1 (sgs1), sgs2/rdr6, and sgs3 mutants showed enhanced susceptibility toward different pathogenic Verticillium spp. but did not show altered disease symptoms to other fungal pathogens, including B. cinerea, Alternaria brassicicola, and Plectosphaerella cucumerina (31). Although sgs1 has not been cloned, RDR6 (SGS2) and SGS3, which interact with each other, are required for secondary siRNA biogenesis and gene silencing, indicating a specific requirement for secondary siRNA-mediated function against vascular pathogens of the genus Verticillium. Among the ten Arabidopsis AGOs, AGO2 is highly induced during bacterial infection and is a key positive regulator of antibacterial defense. The Arabidopsis ago2 mutant is more susceptible to both virulent and avirulent strains of Pst. One of the most abundant sRNAs that is associated with AGO2 is miR393∗ , which suppresses a SNARE protein gene to promote secretion of antimicrobial peptides (145). miR393 also contributes to plant defense by loading into AGO1. Thus, this study provided an example of a pair of functional miRNA and miRNA∗ that function in the same cellular processes through two distinct AGOs. Similarly, miRNA∗ s are expressed differentially under different stress conditions in animal systems. They are also loaded into a different AGO from the one that associates with its miRNA counterpart (39, 42, 93). AGO1 plays a positive role in PTI through several associated miRNAs (74), e.g., miR160 and miR167, by targeting ARFs (auxin responsive factors) that positively regulate PAMP-triggered callose deposition, whereas AGO7 is partially involved in ETI but not PTI (74, 145). The Arabidopsis ago1-25 and ago1-27 mutants are compromised in flg22-triggered PTI and are more susceptible to bacterial pathogens (74). Interestingly, the ago1-27 mutant shows enhanced disease resistance against the fungal pathogens V. dahliae (31) and B. cinerea (128), indicating that AGO1 may possess an extra layer of regulation and play a delicate role in plant and fungal interaction, which is discussed in detail below. sRNAs regulate transcriptional gene silencing mainly through an RNA-dependent DNA methylation (RdDM) pathway (12, 65). Generally, a plant-specific RNA polymerase (Pol) IV generates transcripts that are replicated into dsRNAs by RDR2. The dsRNAs are processed by DCL3 and give rise to 24–30-nt hcsiRNAs that are loaded into AGO4/6/9 to induce DNA methylation and/or histone modifications. DNA cytosine methylation in plants occurs at CG, CHG, and CHH sequences (where H is A, C, or T), and is established by de novo methyltransferases DRM1 and DRM2 through the RdDM pathway. Methylation of CGs and CHGs is maintained by DNA methyltransferases MET1 and CMT3, respectively. Another plant-specific RNA polymerase, Pol V, also plays an important role in this pathway by generating scaffold RNAs that bring the hcsiRNAs to their target sites. The RdDM pathway is involved in regulating antibacterial and antifungal immunity (1, 28, 77, 141). Bacterial infection induces global hypomethylation, predominantly at centromeric and pericentromeric regions (96). Many Arabidopsis RdDM pathway mutants, including the drm1-2 drm2-2 cmt3-11 (ddc) triple mutant, nrpd1a (which encodes the largest subunit of Pol IV), rdr2, drd1, and the dcl2/3/4 triple mutant, show enhanced resistance to Pst DC3000 (28). Intriguingly, RdDM pathway mutants ago4-2, ago6-2, nrpd1b, and nrpd2a2b showed no obvious difference upon bacterial infection as compared with the wild type, although gene expression of AGO4, AGO6, NRPD2, NRPE5, NRPE7, www.annualreviews.org • Small RNAs
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and DRD1 is downregulated upon flg22 treatment (141). Furthermore, DNA glycosylase ROS1-mediated active demethylation is also involved in the activation process of antibacterial defense (141). These results indicate that antibacterial resistance pathways are under the control of the RdDM pathway. During bacterial infection, RdDM pathway genes are downregulated, and resistance pathway genes are demethylated and activated by loss of RdDM and/or by active demethylation, resulting in resistance. However, the Arabidopsis mutants ago4, drd1, rdr2, drm1drm2, and nrpd2 (encodes the second largest subunit of Pol IV and Pol V), but not nrpd1, showed enhanced disease susceptibility to necrotrophic fungal pathogens B. cinerea and P. cucumerina. Chromatin immunoprecipitation revealed that Pol V–dependent RdDM epigenetically controls salicylic acid–dependent defense responsive genes that are activated in nrpd2 and other RdDM mutants, which leads to enhanced susceptibility to jasmonic acid–dependent defense against B. cinerea and P. cucumerina (28, 77, 141). Intriguingly, the distribution of sRNAs also changes after infection with fungal pathogens, such as V. dahliae and the powdery mildew fungus Erysiphe graminis f. sp. tritici (137, 138), which causes reduced 21-nt sRNAs and increased 24-nt sRNAs (138). The 24-nt sRNAs are generated mainly from TEs and repeats, and mostly direct DNA methylation and/or histone modifications at their target sites, which further supports the role of the RdDM pathway in the regulation of defense responses against these pathogens.
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NONCODING SRNAS IN PATHOGENICITY Although host endogenous sRNAs have been extensively studied within plant-pathogen interactions, the importance of pathogen-derived noncoding sRNAs in host-pathogen interactions has been recognized only recently. Authentic regulatory sRNAs have been found in eukaryotic fungal and oomycete pathogens. Regulatory noncoding sRNAs with completely different structural features were also identified in bacteria. Their structures and functions are discussed here.
Noncoding sRNAs in Bacterial Virulence Bacterial noncoding sRNAs are completely different from the sRNAs found in eukaryotes because of the fact that prokaryotes do not possess DCL proteins. Instead, bacterial regulatory noncoding sRNAs are heterogeneous in length (50–300 nts) and regulate the stability and translation efficiency of target mRNAs through short and imperfect base-pairing (10–25 nts). They are often functionally associated with RNA-binding protein complexes. Although several high-throughput RNA-sequencing studies have identified potential sRNAs in phytopathogenic bacteria, including Agrobacterium tumefaciens (135), Pst (35), Xanthomonas campestris (50, 110), and Xanthomonas oryzae pv. oryzae (75), defined functions in pathogenesis remain scarce. Recently, an elegant study using genome-wide transcriptome analysis revealed that X. campestris pv. vesicatoria, the causal agent of bacterial spot disease in pepper (Capsicum annuum) and tomato, produces noncoding sRNAs. Some are under the control of HrpG and HrpX, two regulatory proteins of the type III secretion system, which is essential for bacterial pathogenesis (110). Gene deletion analysis demonstrated that the noncoding sRNAs sX12 and sX13 contribute to virulence (109). sX13 promotes the synthesis of HrpX and regulates expression of other proteins putatively involved in signal transduction, motility, transcriptional and posttranscriptional regulation, and virulence. Computational analysis has revealed that sX13-mediated repression of target mRNAs relies on the C-rich motifs in sX13 and the G-rich motifs in the target mRNAs. These structural features in sX13 are required for regulating virulence, and are conserved among distantly related bacterial pathogens. It is worth noting that although sX13 operates independently of Hfq (discussed in detail below), which encodes a global posttranscriptional regulator by
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binding to bacterial sRNAs, sX13 negatively affects the mRNA level of Hfq. Interestingly, structural similarities of sX13 have been found in the distantly related human pathogens Staphylococcus aureus and Helicobacter pylori (109), which suggests its functional importance. Bacterial noncoding sRNAs operate with RNA-binding protein complexes, the most prominent of which are the clustered regularly interspaced short palindromic repeat (CRISPR)CRISPR-associated (Cas) system (CRISPR-Cas) (49), the global regulatory protein Hfq, and the CsrA/RsmA RNA-binding protein (7, 118, 133). CRISPR-Cas is a unique prokaryotic adaptive immune system, similar to eukaryotic RNAi defense against genome-invading DNA and RNA elements, such as vectors and viruses (4, 10, 49). CRISPR loci are composed of contiguous, highly conserved repeat sequences and variable spacers. Spacers are acquired from the invading viral DNA or plasmid in a polar manner. Cas genes encode proteins that not only help to recognize and integrate foreign DNA to a new repeatspacer unit in the CRISPR locus but are also involved in the processing of small CRISPR RNAs (crRNAs) and the crRNA-guided suppression of the corresponding target DNA or RNA. The CRISPR-Cas systems of bacterial pathogens may also play critical roles in their pathogenesis (108). A small CRISPR-Cas-associated RNA from Francisella novicida guides the Cas9 protein to suppress an endogenous transcript encoding a lipoprotein involved in proinflammatory innate immune response. All CRISPR systems produce small crRNAs to recognize target DNA or RNA by short-seed base pairing with the inclusion of a PAM sequence. On the basis of the different actions of the RNA-binding proteins that execute CRISPR-mediated inhibition, CRISPR systems are divided into three classes, types I, II, and III. The type II CRISPR-Cas9 system provides a smart solution for sequence-specific cleavage at any designated genomic location and has a huge potential for biotechnological purposes in terms of targeted DNA suppression as well as in whole-genome editing applications in plants and animals (70, 79, 87, 112, 126). More than 40% of sequenced bacterial genomes, including many plant bacterial pathogens, such as some Xanthomonas and Pectobacteria strains, contain CRISPR-Cas systems (102). The feature of alternate conserved repeats with highly variable spacer elements makes CRISPR loci an optimal tool for pathogen subtyping and evolutionary research as well as a helpful tool in pathogen tracing (113). However, any direct link between the CRISPR-Cas system and plant bacterial pathogenesis has not yet been found. Hfq is a hexameric RNA-binding protein that acts as a global posttranscriptional regulator by binding to bacterial sRNAs to inhibit translation or promote degradation of target mRNAs (16, 125). Hfq protein is present in approximately 50% of all bacteria and is found in most plantpathogenic bacteria, including A. tumefaciens, Ralstonia solanacearum, Pectobacterium carotovorum, P. syringae, and Xanthomonas spp. (16). For example, in A. tumefaciens, Hfq binds to sRNA AbcR1, which controls expression of the ABC transporter component mRNA atu2422. The hfq mutant exhibits overproduction of several other ABC transporter components and shows ectopic phenotypes of delayed growth, altered cell morphology, reduced motility, and, most importantly, attenuated virulence (134). The CsrA/RsmA protein is a dimeric prokaryote-specific regulatory sRNA-binding protein that affects translation or the stability of target mRNAs (47). CsrA/RsmA protein genes are found in many pathogenic bacteria, including plant pathogens such as X. campestris and P. carotovorum. In P. carotovorum, RsmA is a global repressor that controls quorum sensing–dependent processes and participates in virulence as a negative regulator of the production of extracellular enzymes and affects components of the type III secretion system (hrpN, hrpL) (20). An rsmA deletion mutant in X. campestris pv. campestris exhibits reduced motility on agar plates, results in complete loss of virulence in the host plant Chinese radish (Raphanus sativus), and induces HR-mediated resistance in pepper plants (15).
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Table 1 The role of small RNAs in eukaryotic plant pathogens related to pathogenicity Pathogen
Disease
Features of sRNAs
Putative function
Phytophthora spp.
Late blight Sudden death
21 nts, DCL-dependent 25 or 26 nts, 32 nts, affected by AGO
Effector gene regulation TE regulation
Magnaporthe oryzae
Rice blast
18–23-nt TE loci 24 nts, DCL-, RDR-dependent 28–35-nt tRNA loci
TE regulation Stress response Appressoria formation
Botrytis cinerea
Gray mold rot
21 nts, DCL-dependent, LTR-TE loci
Suppression of host immunity
Sclerotinia sclerotiorum
White mold rot
20–24-nt milRNAs
Sclerotia development
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Abbreviations: AGO, Argonaute; DCL, Dicer-like protein; LTR, long terminal repeat; milRNAs, microRNA-like RNAs, nts, nucleotides; RDR, RNA-dependent RNA polymerase; TE, transposable element.
sRNAs in Plant Eukaryotic Pathogens
Long terminal repeat (LTR) retrotransposon: a type of retrotransposon with characteristic terminal sequence repeats that can sometimes occur in high copy numbers in host genomes
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sRNAs in eukaryotic plant pathogens have been characterized and are suggested to play a role in pathogenic development and virulence (Table 1). A global sRNA expression study in M. oryzae has classified sRNAs, some of which are associated with pathogenesis (92). sRNAs have been mapped to all kinds of genomic loci, but a large number of 18–23-nt sRNAs match to repetitive elements, including the long terminal repeat (LTR) retrotransposon MAGGY. A second group of 28–35-nt sRNAs is mapped to transfer RNA (tRNA) loci that are enriched in the appressoria, an infection-specific organ of this pathogen. A second study reports that 24 nts is the predominant size of sRNAs detected from M. oryzae under various physiological stress conditions and in planta during infection of rice (100). LTR-associated sRNA levels are increased during invasive growth. M. oryzae sRNAs regulate a subset of mRNAs posttranscriptionally, including an effector gene ACE1. ACE1 putatively codes for a hybrid between a polyketide synthase and a nonribosomal peptide synthetase, which likely functions in secondary metabolite production, and it is tightly controlled during initiation of appressorial penetration (8, 37). Similarly, sRNAs from the white mold fungus Sclerotinia sclerotiorum (149) and the entomopathogenic fungus Metarhizium anisopliae (150) show differential regulation during sclerotia production and conidiogenesis, respectively. These results support the notion that fungal endogenous sRNAs play a role in regulating virulence and developmental processes of fungal phytopathogens. The ascomycete B. cinerea is a destructive fungal plant pathogen that infects a broad spectrum of living plants, including almost all vegetables and fruits (121). It also causes serious postharvest losses (121). sRNA profiling of infected plant tissues has identified a set of B. cinerea sRNAs (Bc-sRNAs) that are predicted to target host genes in Arabidopsis and tomato, of which the majority are derived from LTR retrotransposon-like TEs (128). The Bc-siRNA targets, Arabidopsis mitogen-activated protein kinase genes MPK1 and MPK2, a cell wall–associated kinase (WAK), a peroxiredoxin (PRXIIF), and the tomato MPK-kinase kinase 4 (MAPKKK4), are suppressed upon B. cinerea infection. The suppression of these host target genes has also been observed in transgenic plants expressing Bc-sRNAs. Genetic analysis has demonstrated that these Bc-sRNA targets positively regulate plant immune responses against B. cinerea. Further analysis demonstrates that Bc-sRNAs silence the host genes by loading into a host AGO protein and utilizing the host gene-silencing machinery. In Arabidopsis, AGO1 is the predominant executer of miRNA-mediated PTGS and favors 21-nt miRNAs with a 5 terminal uracil (U) (82), and the majority of Bc-sRNAs that have predicted host targets are 21 nts in length and possess a 5 first nucleotide U. In addition, the Arabidopsis mutant ago1-27 exhibited reduced disease susceptibility to B. cinerea and infection was no longer able to suppress the expression of target mRNAs. By contrast, the Arabidopsis Weiberg et al.
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dcl1-7 mutant exhibited enhanced susceptibility toward B. cinerea infection, which suggests that utilizing host AGO1, rather than disrupting the plant miRNA biosynthesis pathway, is part of the fungal virulence strategy. This study was the first to show that sRNAs from a eukaryotic microbial pathogen act as effectors to suppress host immunity (6, 128) (Figure 1). This represents a naturally occurring cross-kingdom RNAi event (117). This sRNA-mediated virulence and the use of host gene-silencing machinery may be a more widely existing virulence mechanism in eukaryotic pathogens. For example, enhanced resistance in ago1 has also been observed during infection with V. dahliae (31). The oomycete Phytophthora infestans is the causal agent of late blight in potato and tomato. Although oomycetes and fungi look alike, they are phylogenetically very different. A global sRNA profiling of 19- to 40-nt sRNAs from P. infestans revealed three major size classes of sRNAs: 21 nts, 25 or 26 nts, and 30–33 nts (122). Numerous sRNAs are generated from TEs, predominantly LTR retrotransposons, and protein effector genes that include an RxLR amino acid motif and Crinkler (CRN). RxLR and CRN effectors are well-known virulence factors in pathogenic oomycetes. sRNAs from all size classes are mapped to TEs and RxLRs, whereas the sRNAs that are mapped to CRN genes predominantly belong to the 21-nt class that is mainly DCL1dependent. Long sRNAs (30–33 nts) are mapped mainly to TEs and are AGO dependent but DCL independent. Interestingly, differences in sRNA expression between two P. infestans strains exhibiting different virulence levels have been observed, further demonstrating a role for sRNA in P. infestans virulence. A negative correlation between the expression of sRNA and predicted targets, especially effector genes, supports a role of sRNAs in regulating effector gene expression and pathogen virulence. For example, PiAvr3a, an effector that suppresses plant programmed cell death for virulence by stabilizing host E3 ubiquitin ligase, is under the regulation of sRNAs (9). Six P. infestans miRNA candidates that have characteristics of plant and animal miRNAs were identified. Further characterization of sRNA populations from P. infestans, P. sojae, and Phytophthora ramorum identified two distinct classes of sRNAs: 21-nt miRNAs and siRNAs, and 25-nt siRNAs (33). The class of 25-nt siRNAs is predominantly mapped to TEs, whereas the class of 21-nt siRNAs is primarily derived from inverted repeats, conserved CRN effector genes, and type III fibronectin genes that are surrounded by TEs. A new family of miRNAs (MIR8788) is conserved among these three Phytophthora spp. and targets a family of amino acid/auxin permeases. The 25-nt siRNAs putatively regulate TE silencing, whereas the 21-nt sRNAs, including miRNAs, are proposed to regulate gene expression, including important virulence factors and effectors.
Cross-kingdom RNAi: a form of RNAi in which a gene-silencing trigger is produced in a donor organism but mediates gene silencing in an unrelated recipient organism
sRNA Biogenesis Proteins in Eukaryotic Plant Pathogens Most eukaryotic plant pathogen genomes encode DCL, AGO, and RDR proteins, the key components in the RNAi pathway. The filamentous saprophytic fungus Neurospora crassa was one of the first model organisms in which transgene-induced RNAi (called quelling) was discovered back in 1992 (103). Since then, genetic approaches, such as mutant screening (19), and numerous target gene disruption studies identified many RNAi pathway components and contributed much to the deciphering of the sophisticated architecture of RNAi pathways in fungi (14, 22, 71). Many RNAi-dependent phenomena in N. crassa and other fungal species have been studied. Quelling (38), the silencing of repeat-induced point (RIP) mutation (17), meiotic silencing of unpaired DNA (MSUD) (46, 114), DNA repair and homologous recombination (66, 146), sex-induced silencing (SIS) (127), and DNA methylation (41, 132) are examples of RNAi-dependent phenomena. However, it is not clear whether RNAi pathway components are directly required for pathogenicity in eukaryotic plant pathogens. www.annualreviews.org • Small RNAs
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The M. oryzae genome encodes two DCLs (MoDCL1 and MoDCL2), three putative AGOs (MoAGO1–MoAGO3), and three RDRs (MoRdRP1–MoRdRP3). MoDCL2 is required for transgene-induced gene silencing and sRNA-directed transposon silencing (57, 84). However, there are no reports of any RNAi mutant strains, including Mordrp, Modcl1, Modcl2, and Modcl1dcl2—although Modcl2 shows slightly reduced growth—that show obvious phenotypes
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associated with pathogenicity (57). In contrast, obvious defects in growth and sporulation have been observed in the zygomycete fruit rot pathogen Mucor circinellioides dcl1 and dcl2 mutants, respectively (24, 88). An ago1 mutant of M. circinellioides shows defects in asexual spore production (13). Similar defective growth and sporulation phenotypes were observed in nonpathogenic Trichoderma atroviride dcr-2, dcr-1dcr-2, and rdr-3 deletion mutants (11). B. cinerea encodes two DCL proteins. Although the dcl1 and dcl2 single mutants and the dcl1dcl2 double mutant exhibit growth retardation and reduced conidiation, only the dcl1dcl2 double mutant shows significantly reduced virulence on both Arabidopsis and tomato (128). This difference is mostly due to the levels of sRNA effectors, which are abolished in dcl1dcl2 but are not altered in the dcl1 and dcl2 single mutants. It is noteworthy that Saccharomyces cerevisiae and the basidiomycetous plant pathogen Ustilago maydis, but not its close relative Ustilago hordei, completely lost their essential RNAi pathway components (29, 64). P. infestans possesses possibly two DCLs (PiDCL1 and PiDCL2) (33), five putative AGOs (PiAGO1–PiAGO5) and one RDR (PiRDR). Transgene-induced silencing requires PiDCL1 and PiAGO1/PiAGO2 (123). RNAi components are likely to be involved in pathogenesis because mRNA levels of PiDCL1, PiAGO3, and PiAGO4 are highly induced at 24 hours post inoculation, although no obvious phenotypes in morphology, differentiation, or pathogenesis have been reported in RNAi mutants, including Piago1/2, Piago3, Piago4, Piago5, and Pidcl1 knockdown mutants, which is likely due to the low efficiency of RNAi in P. infestans or to possible functional redundancy of the homologous genes within the same family. The fungal RNAi pathways are very diverse; for example, miRNA pathways are conserved in plants and animals (21) but not in fungi. Instead, miRNA-like RNAs (milRNA) have been described in fungi (58, 63, 149, 150) and at least four different milRNA biogenesis pathways, including both DCL-dependent and DCL-independent pathways, are proposed in N. crassa (52, 67, 71). It is important to know whether similar sRNA biogenesis pathways exist in fungal and oomycete pathogens. Furthermore, DCLs seem to be functionally redundant in sRNA processing in most fungal species (67, 89, 128). Taken together, the possible existence of diverse sRNA biogenesis pathways in these organisms presents a challenge for scientists in the dissection of RNAi pathways and characterization of regulatory sRNAs in pathogenicity.
MicroRNA-like RNA (milRNAs): a class of small RNAs in fungi that share common features with microRNAs of higher eukaryotes but lack a common ancestor or functional conservation among different species
TRANSPOSON-ASSOCIATED SRNAS IN EUKARYOTIC PLANT PATHOGENS TEs are mobile genomic elements that drive genome evolution. TE transposition and replication are associated with genomic DNA rearrangements and mutations. Although temporal TE activity has beneficial effects in terms of adaptive evolution, it is obvious that such elements can be detrimental. In eukaryotes, a large number of sRNAs are generated from TE loci to suppress TE activity, which is essential for maintaining genome integrity. ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Figure 1 Some Botrytis cinerea small RNAs (Bc-sRNAs) are effectors that translocate into the host plant cell and hijack the host Argonaute (AGO) protein of the gene-silencing machinery to suppress plant immune response. (a) Upon B. cinerea pathogen recognition, host immune responses are activated, including cell wall–associated kinase (WAK), signaling components of mitogen-activated protein kinase (MAPK) cascade, and mitochondria-associated peroxiredoxin (PRXIIF) as well as other unidentified defense proteins (indicated by question mark). (b) B. cinerea generates sRNAs (blue), and some sRNA effectors (red ) are induced and translocate into the host plant cell during infection. Those sRNA effectors are predominantly transcribed from transposable elements (TEs). (c) Upon host cell entry, sRNA effectors are loaded into the plant AGO and target the mRNAs (messenger RNAs) of selective plant defense genes for silencing. (d ) Target silencing attenuates immune response and facilitates disease progression. www.annualreviews.org • Small RNAs
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Localization of Effector Genes and R Genes Within Regions Enriched with Transposable Elements is of Evolutionary Advantage
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As genome sequences became available for dozens of phytopathogens, scientists have started to realize that some of the most specialized host-adapted plant pathogens, such as the powdery mildew fungus Blumeria graminis (116) and P. infestans (45, 99), show large genome expansion by massive TE propagation. Genomic regions rich in TEs are normally poor in protein-coding genes, but TE-rich regions from these phytopathogens show a remarkable enrichment of virulence genes, such as protein effector genes (105, 107, 120, 140). In the fungus Fusarium oxysporum, these genome regions can be found on extra, linear-specific (LS) chromosomes that present low synteny rates to closely related Fusarium spp. (78, 101). Although LS chromosomes contain a low density of housekeeping genes, which may make LS chromosomes dispensable, they are linked to hypervirulence. Accordingly, it is evident that transposition of TEs shape effector gene repertoires (36, 55, 105, 107). Consistent with this notion, B. cinerea sRNA effectors are also derived from LTR-transposon regions. Although massive expansion of transposons has not occurred appreciably in the B. cinerea genome (2), population dynamics surveys and genetic marker analysis of B. cinerea species linked the presence of two TEs, the LTR retrotransposon Boty and Fot1-like element FLIPPER, to the virulent and host-adapted subpopulations named transposa isolates (40, 80). The presence of the two TEs in these transposa isolates in crop-producing areas suggests a functional role for these TEs in virulence and host adaptation of B. cinerea. Similarly, rice isolates of M. oryzae collected from geographically dispersed locations exhibit high copy numbers of the LTR retrotransposon MAGGY compared with non-rice isolates collected from other Gramineae (34). Botrytis FLIPPER elements are structurally similar to Fot1 and Pot2 TEs from pathogenic F. oxysporum and M. oryzae, respectively (68), further supporting their possible role in pathogenesis. Boty is an LTR retrotransposon element that is related to the gypsy family of retrotransposons (27). Boty-like elements often contain an antisense gene, brtn, in their 3 untranslated region of yet unknown function that is also present in Boty-like LTR retrotransposon elements in S. sclerotiorum (148), pointing to a potential functional role of brtn in fungi. The Boty and FLIPPER TEs are present in the virulent B. cinerea strain B05.10, in which a Boty-like LTR-retrotransposon element encodes the identified sRNA effectors that suppress host immunity genes (Figure 1). It is not surprising that sRNA effectors evolved from TEs, and this evolution ensures a high turnover frequency for natural selection (Figure 2a). TEs are also likely to shape plant R-gene evolution. Many R genes, in distinct plant species, are clustered within certain genomic loci and are often close to TEs and repeats, thus facilitating DNA mutation upon selection pressure. The TEs carry epigenetic information to control the expression of these R genes, as demonstrated for the Arabidopsis RPP5 and RPP7 R-gene loci (119, 139). R-gene clusters are also hot spots for sRNA generation. sRNAs play an essential role in regulating the epigenetic status of R-gene loci and fine-tuning the expression of R genes during infection (Figure 2b). TEs and sRNAs are indispensable regulators in the arms race between eukaryotic pathogens and hosts (130).
Transposon-Associated sRNAs Regulate and Fine-Tune the Expression of Effector Genes that Modulate Virulence Approximately 74% of the P. infestans genome consists of TEs (45). A remarkable number of P. infestans sRNAs are mapped to TEs, which is consistent with the large number of sRNAs from TE regions in plants and animals. These TE-derived sRNAs are likely to mediate silencing of TEs 506
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Figure 2 Transposon-associated small RNAs (sRNAs) in plant-microbe interactions. To maintain genome integrity, eukaryotic pathogens produce transposable element (TE)-associated sRNAs (blue bars) to silence TEs. (a) sRNA effectors (dark red bars), like those found in Botrytis cinerea, are transcribed from TEs and suppress host immunity-related genes. (b) Host plant resistance (R) genes are often clustered in genomic loci enriched with TEs. TEs manifest epigenetic control of R-gene expression by R-gene sRNAs ( green bars). (c) Similarly, pathogen protein effector genes are often found in clusters and interspersed with TEs. Protein effector gene–derived sRNAs (orange bars), as found in Phytophthora spp., regulate effector expression. (d ) In an epigenetic network, sRNAs are important regulators of pathogen protein effector genes and host plant R genes. TE transposition shapes the repertoires of effector genes in pathogens and R genes in plants. TEs are hot spots of sRNA production. In pathogens, sRNAs regulate expression of TEs and of TE-associated protein effector genes, and sRNA effectors are delivered into host cells to manipulate expression of host defense genes. In plants, sRNAs epigenetically control R-gene expression, which activates defense genes upon infection. It is possible that plants may also deliver plant RNA or protein molecules into pathogen cells. All these scenarios are likely to affect plant-pathogen interaction and contribute to host resistance, pathogen virulence, and host adaptation.
to maintain genome integrity. Many of the RxLR and CRN effectors are located in close vicinity (
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Further
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Small RNAs: A New Paradigm in Plant-Microbe Interactions Arne Weiberg, Ming Wang, Marschal Bellinger, and Hailing Jin Department of Plant Pathology and Microbiology, University of California, Riverside, California 92521; email: [email protected]
Annu. Rev. Phytopathol. 2014. 52:495–516
Keywords
The Annual Review of Phytopathology is online at phyto.annualreviews.org
small RNA, gene silencing, pathogen effector, transposable element, cross-kingdom RNAi
This article’s doi: 10.1146/annurev-phyto-102313-045933 c 2014 by Annual Reviews. Copyright All rights reserved
Abstract A never-ending arms race drives coevolution between pathogens and hosts. In plants, pathogen attacks invoke multiple layers of host immune responses. Many pathogens deliver effector proteins into host cells to suppress host immunity, and many plants have evolved resistance proteins to recognize effectors and trigger robust resistance. Here, we discuss findings on noncoding small RNAs (sRNAs) from plants and pathogens, which regulate host immunity and pathogen virulence. Recent discoveries have unveiled the role of noncoding sRNAs from eukaryotic pathogens and bacteria in pathogenicity in both plant and animal hosts. The discovery of fungal sRNAs that are delivered into host cells to suppress plant immunity added sRNAs to the list of pathogen effectors. Similar to protein effector genes, many of these sRNAs are generated from transposable element (TE) regions, which are likely to contribute to rapidly evolving virulence and host adaptation. We also discuss RNA silencing that occurs between organisms.
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INTRODUCTION
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Pathogen-associated molecular pattern (PAMP): conserved molecules common in pathogens, such as flagellin, peptidoglycan, and chitin PAMP-triggered immunity (PTI): plant receptor protein–based recognition of PAMPs that induces defense response Effector-triggered immunity (ETI): pathogen effector recognition by plant resistance proteins that induces defense response Noncoding RNAs: classes of RNA molecules that are not translated into proteins Dicer-like protein (DCL): RNA-binding type III endoribonuclease that processes doublestranded RNA molecules into mature miRNAs and siRNAs Argonaute (AGO): RNA-binding protein and central component of the RNA-induced gene-silencing complex Gene silencing: noncoding small RNAs mediate gene suppression via RNAi either transcriptionally by DNA methylation or posttranscriptionally by mRNA degradation or translational inhibition
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Pathogens are among the most specialized life-forms on Earth. The challenge of colonizing a living host cell comes from the evolutionary specificity that must exist for a pathogen to outmaneuver the host immune system. In plants, most of the microbial attackers are blocked by nonhost resistance and pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). Successful pathogens have evolved protein effectors to suppress PTI. To counter the pathogen protein effectors, plants have evolved resistance (R) genes, which encode R proteins that can recognize specific pathogen protein effectors directly or indirectly and induce a rapid and robust immune response, known as effector-triggered immunity (ETI). Pathogens then diversify the recognized effector gene or evolve new effector genes to diminish ETI, whereas plants subsequently evolve new R proteins to recognize the new effectors and again induce ETI, continuing the endless evolutionary arms race between pathogen and host (18, 53). Eukaryotic small RNAs (sRNAs) are short regulatory noncoding RNAs that induce silencing of target genes at transcriptional and posttranscriptional levels. The endoribonuclease Dicer or Dicer-like proteins (DCLs) process double-stranded RNAs (dsRNAs) or RNAs with hairpin structures, giving rise to mostly 20–30-nucleotide (nt)-long sRNAs, which are loaded into Argonaute (AGO) proteins to induce gene silencing of their complementary targets by guiding mRNA (messenger RNA) cleavage or degradation, translational inhibition, DNA methylation, and histone modification (5, 12). Prokaryotic regulatory noncoding sRNAs are strikingly different from those found in eukaryotes in terms of their length, structure characteristics, and functions. Bacterial noncoding sRNAs are heterogeneous in length (50–250 nts) and act through distinct RNA-binding protein complexes (133). Plant sRNAs and RNA interference (RNAi) pathway components are important regulatory players in plant immunity against viruses, bacteria, fungi, oomycetes, and pests (26, 51, 95, 106, 111). Recent studies also suggest that pathogen-derived sRNAs and RNAi machinery contribute to pathogen virulence (108, 109, 128). Phytopathologists have begun to study global sRNA expression profiles to identify infection-responsive sRNAs from plant hosts and pathogens, including bacteria, fungi, and oomycetes (35, 50, 75, 92, 100, 110, 122, 128, 135, 149). A vast majority of sRNAs in filamentous plant pathogens are transcribed from transposable element (TE) regions. Silencing of TEs by sRNAs is a ubiquitous phenomenon in all eukaryotes, including fungi (14, 17). This review discusses the role of sRNAs in plant-pathogen interactions, with an emphasis on the role of noncoding sRNAs from bacterial and eukaryotic plant pathogens in their pathogenicity. A brief introduction of sRNA-mediated gene silencing across different organisms is also included.
HOST ENDOGENOUS SRNAS AND RNAI PATHWAY COMPONENTS IN PLANT IMMUNITY Host sRNAs Many plant endogenous sRNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), play an important role in gene expression reprogramming and fine-tuning in host immune responses (51, 95, 106). Most miRNAs are 20–22 nts in length and are processed by DCL proteins from a single-stranded RNA (ssRNA) precursor with a hairpin structure. The model plant Arabidopsis genome encodes four DCLs and ten AGOs. Most Arabidopsis miRNAs are generated by DCL1 and loaded into AGO1 to induce mainly posttranscriptional gene silencing (PTGS). Plant endogenous siRNAs are numerous in quantity and are much more diverse than miRNAs in terms of their length and biogenesis pathways. siRNAs are processed from long dsRNA Weiberg et al.
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precursors by different DCLs and are loaded into different AGOs to guide silencing of their targets transcriptionally or posttranscriptionally (111). Trans-acting siRNAs (tasiRNAs), natural antisense transcript-derived siRNAs (nat-siRNAs), long siRNAs (lsiRNAs), and heterochromatic siRNAs (hcsiRNAs) are different siRNA subclasses present in plants (3, 61, 72, 95). Plant sRNAs are differentially expressed during pathogen attacks. Arabidopsis miR393 was the first miRNA discovered to be involved in plant immunity. It is induced by flg22, a PAMP, and targets auxin receptor TIR1 as well as its homologs and suppresses the auxin signaling pathway, thus contributing to PTI (85). Some conserved miRNAs, such as miR160 and miR167, are also induced by PAMPs and contribute to PTI (85, 144). However, other miRNAs, such as miR398a and miR773, are downregulated upon PAMP perception and bacterial infection to release target suppression and activate PTI (74). Plant sRNAs also play an important role in ETI. Arabidopsis nat-siRNAATGB2 was the first example of a plant endogenous siRNA that regulates R-genemediated immunity. It is highly induced by the bacterial pathogen Pseudomonas syringae pv. tomato (Pst), which carries an effector gene, avrRpt2, and contributes to resistance gene RPS2-mediated ETI by repressing a putative negative regulator of the RPS2 pathway (62). Other examples include Arabidopsis AtlsiRNA-1 and miR393∗ , both of which are induced by Pst (avrRpt2). miR393∗ is the complementary strand of the miR393 within the miRNA duplex. miRNA∗ s were once thought to be useless by-products, but we now know that some of them are functional. miR393∗ is loaded into AGO2 and regulates plant immunity by suppressing the MEMB12 gene that encodes a golgilocalized SNARE protein responsible for retrograde trafficking, which results in the increased exocytosis of antimicrobial pathogenesis-related (PR) proteins (145). AtlsiRNA-1 contributes to plant defense by targeting AtRAP, which encodes a putative RNA-binding protein that negatively regulates plant defense (60). Most plant R genes belong to the nucleotide-binding site (NBS)leucine-rich repeat (LRR) gene family, which is a hot spot for sRNA generation. A class of phased secondary siRNAs has been identified from the NBS-LRR type of R-gene regions in various plant species (69, 115, 142, 151). The biogenesis of these secondary siRNAs is initiated by cleavage of the NBS-LRR transcripts mediated by specific miRNA families, such as miR482 and miR2118 from both tomato (Solanum lycopersicum) and cotton (Gossypium hirsutum) and miR6019 and miR6020 from tobacco (Nicotiana benthamiana). These miRNAs and secondary siRNAs control the expression of NBS-LRR genes at a low level under normal conditions. However, this silencing effect is suppressed when plants are under attack from viral and bacterial pathogens, which leads to the upregulation of these R genes and triggers plant defense responses. Plant endogenous sRNAs that are responsive to fungal infection have also been identified from many plant species. In rice (Oryza sativa), sRNA profiling of the rice blast fungus Magnaporthe oryzae–challenged resistant and susceptible cultivars revealed a group of host endogenous miRNAs that contributes to gene regulation of defense responses (73). For example, miR160 and miR164 are induced, whereas miR396 is downregulated upon infection in a resistance cultivar but not in a susceptible one, suggesting their positive and negative roles in ETI, respectively. Furthermore, miR169, miR172, and miR398 are regulated in both susceptible and resistant cultivars, suggesting their regulatory role in basal responses (73). miR160 targets auxin responsive factor 16 (ARF16) and miR398 targets superoxide dismutase 2 (SOD2) and a copper chaperone of SOD2. All these genes are downregulated upon M. oryzae infection, which correlates well with induced miRNA levels (73). Overexpression of miR160 or miR398 in a susceptible rice cultivar leads to enhanced disease resistance toward M. oryzae, confirming that these two miRNAs are bona fide positive regulators of defense response against M. oryzae (73). In wheat (Triticum aestivum), sRNA profiling of a susceptible cultivar and a resistant cultivar upon infection with the powdery mildew fungus Blumeria graminis results in three categories of regulated miRNAs: (a) responsive only in the resistant cultivar, such as the upregulated miR2008 www.annualreviews.org • Small RNAs
RNA interference (RNAi): a biological process in which noncoding small RNA molecules block gene expression executed by protein members of the Argonaute class Plant immunity: innate system that protects plants against invading pathogens; based on two forms: preformed (constitutive) and pathogen-induced defense response Transposable element (TE): a genomic DNA unit that can undergo reposition, replication, and amplification within its host genome and introduce mutations MicroRNAs (miRNAs): small RNAs that are 20–22 nucleotide long and central in RNAi that are produced from a single-stranded RNA precursor with a hairpin structure by a Dicer-like protein and induce gene silencing of targets with imperfect matches Small interfering RNAs (siRNAs): small RNAs inducing RNAi that are produced from a longer doublestranded RNA precursor by a Dicer-like protein
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and miR2012 and the downregulated miR171; (b) responsive only in the susceptible cultivar, such as the induced miR393, miR444, miR827, miR2005, and miR2013 and the suppressed miR2001, miR2006, and miR2011; and (c) responsive in both cultivars, such as miR156, miR159, miR164, and miR396 (137). A distinct group of miRNAs, including miR167, miR171, miR444, miR408, and miR1138, are induced only in two susceptible wheat cultivars and only in the early stage after infection with the rust fungus Puccinia graminis f. sp. tritici (44). This group is not induced in the late stage of infection or in resistant cultivars. According to the miRNAs endogenous target genes, these miRNAs are likely to participate in early defense responses through regulating hormone signaling pathways, lignin synthesis, and protein biosynthesis. In eggplant (Solanum melongena), 33 Verticillium dahliae–responsive miRNAs were identified. For example, miR393 and miR399 are both highly downregulated after infection, allowing for the accumulation of their target gene auxin receptor, TIR1, and an E2 conjugating enzyme gene, PHO2. In general, TIR1 positively regulates plant defense against necrotrophic pathogens (76) and negatively regulates defense against biotrophic pathogens (85). miR399 is induced by phosphorus starvation in various plant species, and it downregulates PHO2, which is responsible for degrading two phosphate transporters, subsequently leading to enhanced phosphate uptake. Although miR393 is highly regulated by various pathogen infections (76, 85, 137), differential expression of miR399 in response to infection is not common, which may suggest a possible interaction between V. dahliae infection responses and phosphorus homeostasis. miR399 is highly induced in sweet orange Citrus sinensis that is Huanglongbing (HLB) positive (147). HLB, also named citrus greening, is caused by a phloem-limited bacterium within the genus Candidatus Liberibacter and is probably the most devastating citrus disease. Upregulation of miR399 led to the finding of 35% reduction of phosphorus content in HLB-positive trees. This finding links phosphorus deficiency to HLB disease symptomology (147). Application of phosphorus oxyanion solutions to HLB-infected trees reduced disease symptoms and increased fruit production. In cotton, 36 of its 300 NBS-LRR genes are potentially under the control of several members of the miR482 family. miR482, which targets the conserved P-loop sequences of NBS-LRR genes, is downregulated upon infection with V. dahliae. Correspondingly, expression of ten targeted NBS-LRR genes is derepressed. The cleavage of NBS-LRR transcripts by miR482 also triggers the generation of secondary siRNAs (151), which reinforces the silencing of these NBS-LRR genes. The tight repression of R genes by miR482 and secondary siRNAs under a noninfectious situation is turned off during infection by downregulation of miR482, which leads to R-gene activation. Similarly, altered miRNA expression patterns were observed in oomycete-infected plants. Three soybean (Glycine max) cultivars with different levels of resistance to the oomycete Phytophthora sojae were examined. miR403, which targets AGO protein genes, was downregulated in all three cultivars, suggesting a positive role of RNAi machinery in host defense against P. sojae. miR1510, along with family members of miR390 and miR1535, was downregulated. Expression of miRNAs negatively correlated with the expression of their predicted targets, indicating their regulatory role upon infection. Thus, it is clear that host endogenous sRNAs play a critical role in regulating and fine-tuning the expression of plant defense responsive genes and contribute to gene expression reprogramming in plant immune responses against a large array of pathogens, including bacteria, fungi, and oomycetes (43).
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Host RNAi Pathway Components Several plant RNAi pathway components have been shown to be important for antimicrobial immunity (111). In Arabidopsis, DCL1 and DCL4 are key components in miRNA and siRNA 498
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biosynthesis, respectively. The dcl1-9 mutant displays enhanced susceptibility to bacterial infection (86). Similarly, dcl1-7 is more susceptible to the fungal pathogen Botrytis cinerea, supporting the idea that RNAi is also involved in antifungal immunity (128). DCL4 is important for antiviral defense and is also partially involved in the accumulation of antibacterial AtlsiRNA-1 (60). The dcl4-2 mutant is more susceptible to the fungal pathogen V. dahliae (31). RNA-dependent RNA polymerases (RDRs or RdRPs) are responsible for generating dsRNAs using aberrant ssRNAs as templates, which are subsequently processed into sRNAs by DCLs. The Arabidopsis genome encodes six RDRs. RDR6 is involved in the biogenesis of secondary siRNAs, and the rdr6 mutant is more susceptible to bacterial as well as fungal infection (31, 62). Interestingly, suppressor of gene silencing 1 (sgs1), sgs2/rdr6, and sgs3 mutants showed enhanced susceptibility toward different pathogenic Verticillium spp. but did not show altered disease symptoms to other fungal pathogens, including B. cinerea, Alternaria brassicicola, and Plectosphaerella cucumerina (31). Although sgs1 has not been cloned, RDR6 (SGS2) and SGS3, which interact with each other, are required for secondary siRNA biogenesis and gene silencing, indicating a specific requirement for secondary siRNA-mediated function against vascular pathogens of the genus Verticillium. Among the ten Arabidopsis AGOs, AGO2 is highly induced during bacterial infection and is a key positive regulator of antibacterial defense. The Arabidopsis ago2 mutant is more susceptible to both virulent and avirulent strains of Pst. One of the most abundant sRNAs that is associated with AGO2 is miR393∗ , which suppresses a SNARE protein gene to promote secretion of antimicrobial peptides (145). miR393 also contributes to plant defense by loading into AGO1. Thus, this study provided an example of a pair of functional miRNA and miRNA∗ that function in the same cellular processes through two distinct AGOs. Similarly, miRNA∗ s are expressed differentially under different stress conditions in animal systems. They are also loaded into a different AGO from the one that associates with its miRNA counterpart (39, 42, 93). AGO1 plays a positive role in PTI through several associated miRNAs (74), e.g., miR160 and miR167, by targeting ARFs (auxin responsive factors) that positively regulate PAMP-triggered callose deposition, whereas AGO7 is partially involved in ETI but not PTI (74, 145). The Arabidopsis ago1-25 and ago1-27 mutants are compromised in flg22-triggered PTI and are more susceptible to bacterial pathogens (74). Interestingly, the ago1-27 mutant shows enhanced disease resistance against the fungal pathogens V. dahliae (31) and B. cinerea (128), indicating that AGO1 may possess an extra layer of regulation and play a delicate role in plant and fungal interaction, which is discussed in detail below. sRNAs regulate transcriptional gene silencing mainly through an RNA-dependent DNA methylation (RdDM) pathway (12, 65). Generally, a plant-specific RNA polymerase (Pol) IV generates transcripts that are replicated into dsRNAs by RDR2. The dsRNAs are processed by DCL3 and give rise to 24–30-nt hcsiRNAs that are loaded into AGO4/6/9 to induce DNA methylation and/or histone modifications. DNA cytosine methylation in plants occurs at CG, CHG, and CHH sequences (where H is A, C, or T), and is established by de novo methyltransferases DRM1 and DRM2 through the RdDM pathway. Methylation of CGs and CHGs is maintained by DNA methyltransferases MET1 and CMT3, respectively. Another plant-specific RNA polymerase, Pol V, also plays an important role in this pathway by generating scaffold RNAs that bring the hcsiRNAs to their target sites. The RdDM pathway is involved in regulating antibacterial and antifungal immunity (1, 28, 77, 141). Bacterial infection induces global hypomethylation, predominantly at centromeric and pericentromeric regions (96). Many Arabidopsis RdDM pathway mutants, including the drm1-2 drm2-2 cmt3-11 (ddc) triple mutant, nrpd1a (which encodes the largest subunit of Pol IV), rdr2, drd1, and the dcl2/3/4 triple mutant, show enhanced resistance to Pst DC3000 (28). Intriguingly, RdDM pathway mutants ago4-2, ago6-2, nrpd1b, and nrpd2a2b showed no obvious difference upon bacterial infection as compared with the wild type, although gene expression of AGO4, AGO6, NRPD2, NRPE5, NRPE7, www.annualreviews.org • Small RNAs
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and DRD1 is downregulated upon flg22 treatment (141). Furthermore, DNA glycosylase ROS1-mediated active demethylation is also involved in the activation process of antibacterial defense (141). These results indicate that antibacterial resistance pathways are under the control of the RdDM pathway. During bacterial infection, RdDM pathway genes are downregulated, and resistance pathway genes are demethylated and activated by loss of RdDM and/or by active demethylation, resulting in resistance. However, the Arabidopsis mutants ago4, drd1, rdr2, drm1drm2, and nrpd2 (encodes the second largest subunit of Pol IV and Pol V), but not nrpd1, showed enhanced disease susceptibility to necrotrophic fungal pathogens B. cinerea and P. cucumerina. Chromatin immunoprecipitation revealed that Pol V–dependent RdDM epigenetically controls salicylic acid–dependent defense responsive genes that are activated in nrpd2 and other RdDM mutants, which leads to enhanced susceptibility to jasmonic acid–dependent defense against B. cinerea and P. cucumerina (28, 77, 141). Intriguingly, the distribution of sRNAs also changes after infection with fungal pathogens, such as V. dahliae and the powdery mildew fungus Erysiphe graminis f. sp. tritici (137, 138), which causes reduced 21-nt sRNAs and increased 24-nt sRNAs (138). The 24-nt sRNAs are generated mainly from TEs and repeats, and mostly direct DNA methylation and/or histone modifications at their target sites, which further supports the role of the RdDM pathway in the regulation of defense responses against these pathogens.
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NONCODING SRNAS IN PATHOGENICITY Although host endogenous sRNAs have been extensively studied within plant-pathogen interactions, the importance of pathogen-derived noncoding sRNAs in host-pathogen interactions has been recognized only recently. Authentic regulatory sRNAs have been found in eukaryotic fungal and oomycete pathogens. Regulatory noncoding sRNAs with completely different structural features were also identified in bacteria. Their structures and functions are discussed here.
Noncoding sRNAs in Bacterial Virulence Bacterial noncoding sRNAs are completely different from the sRNAs found in eukaryotes because of the fact that prokaryotes do not possess DCL proteins. Instead, bacterial regulatory noncoding sRNAs are heterogeneous in length (50–300 nts) and regulate the stability and translation efficiency of target mRNAs through short and imperfect base-pairing (10–25 nts). They are often functionally associated with RNA-binding protein complexes. Although several high-throughput RNA-sequencing studies have identified potential sRNAs in phytopathogenic bacteria, including Agrobacterium tumefaciens (135), Pst (35), Xanthomonas campestris (50, 110), and Xanthomonas oryzae pv. oryzae (75), defined functions in pathogenesis remain scarce. Recently, an elegant study using genome-wide transcriptome analysis revealed that X. campestris pv. vesicatoria, the causal agent of bacterial spot disease in pepper (Capsicum annuum) and tomato, produces noncoding sRNAs. Some are under the control of HrpG and HrpX, two regulatory proteins of the type III secretion system, which is essential for bacterial pathogenesis (110). Gene deletion analysis demonstrated that the noncoding sRNAs sX12 and sX13 contribute to virulence (109). sX13 promotes the synthesis of HrpX and regulates expression of other proteins putatively involved in signal transduction, motility, transcriptional and posttranscriptional regulation, and virulence. Computational analysis has revealed that sX13-mediated repression of target mRNAs relies on the C-rich motifs in sX13 and the G-rich motifs in the target mRNAs. These structural features in sX13 are required for regulating virulence, and are conserved among distantly related bacterial pathogens. It is worth noting that although sX13 operates independently of Hfq (discussed in detail below), which encodes a global posttranscriptional regulator by
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binding to bacterial sRNAs, sX13 negatively affects the mRNA level of Hfq. Interestingly, structural similarities of sX13 have been found in the distantly related human pathogens Staphylococcus aureus and Helicobacter pylori (109), which suggests its functional importance. Bacterial noncoding sRNAs operate with RNA-binding protein complexes, the most prominent of which are the clustered regularly interspaced short palindromic repeat (CRISPR)CRISPR-associated (Cas) system (CRISPR-Cas) (49), the global regulatory protein Hfq, and the CsrA/RsmA RNA-binding protein (7, 118, 133). CRISPR-Cas is a unique prokaryotic adaptive immune system, similar to eukaryotic RNAi defense against genome-invading DNA and RNA elements, such as vectors and viruses (4, 10, 49). CRISPR loci are composed of contiguous, highly conserved repeat sequences and variable spacers. Spacers are acquired from the invading viral DNA or plasmid in a polar manner. Cas genes encode proteins that not only help to recognize and integrate foreign DNA to a new repeatspacer unit in the CRISPR locus but are also involved in the processing of small CRISPR RNAs (crRNAs) and the crRNA-guided suppression of the corresponding target DNA or RNA. The CRISPR-Cas systems of bacterial pathogens may also play critical roles in their pathogenesis (108). A small CRISPR-Cas-associated RNA from Francisella novicida guides the Cas9 protein to suppress an endogenous transcript encoding a lipoprotein involved in proinflammatory innate immune response. All CRISPR systems produce small crRNAs to recognize target DNA or RNA by short-seed base pairing with the inclusion of a PAM sequence. On the basis of the different actions of the RNA-binding proteins that execute CRISPR-mediated inhibition, CRISPR systems are divided into three classes, types I, II, and III. The type II CRISPR-Cas9 system provides a smart solution for sequence-specific cleavage at any designated genomic location and has a huge potential for biotechnological purposes in terms of targeted DNA suppression as well as in whole-genome editing applications in plants and animals (70, 79, 87, 112, 126). More than 40% of sequenced bacterial genomes, including many plant bacterial pathogens, such as some Xanthomonas and Pectobacteria strains, contain CRISPR-Cas systems (102). The feature of alternate conserved repeats with highly variable spacer elements makes CRISPR loci an optimal tool for pathogen subtyping and evolutionary research as well as a helpful tool in pathogen tracing (113). However, any direct link between the CRISPR-Cas system and plant bacterial pathogenesis has not yet been found. Hfq is a hexameric RNA-binding protein that acts as a global posttranscriptional regulator by binding to bacterial sRNAs to inhibit translation or promote degradation of target mRNAs (16, 125). Hfq protein is present in approximately 50% of all bacteria and is found in most plantpathogenic bacteria, including A. tumefaciens, Ralstonia solanacearum, Pectobacterium carotovorum, P. syringae, and Xanthomonas spp. (16). For example, in A. tumefaciens, Hfq binds to sRNA AbcR1, which controls expression of the ABC transporter component mRNA atu2422. The hfq mutant exhibits overproduction of several other ABC transporter components and shows ectopic phenotypes of delayed growth, altered cell morphology, reduced motility, and, most importantly, attenuated virulence (134). The CsrA/RsmA protein is a dimeric prokaryote-specific regulatory sRNA-binding protein that affects translation or the stability of target mRNAs (47). CsrA/RsmA protein genes are found in many pathogenic bacteria, including plant pathogens such as X. campestris and P. carotovorum. In P. carotovorum, RsmA is a global repressor that controls quorum sensing–dependent processes and participates in virulence as a negative regulator of the production of extracellular enzymes and affects components of the type III secretion system (hrpN, hrpL) (20). An rsmA deletion mutant in X. campestris pv. campestris exhibits reduced motility on agar plates, results in complete loss of virulence in the host plant Chinese radish (Raphanus sativus), and induces HR-mediated resistance in pepper plants (15).
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Table 1 The role of small RNAs in eukaryotic plant pathogens related to pathogenicity Pathogen
Disease
Features of sRNAs
Putative function
Phytophthora spp.
Late blight Sudden death
21 nts, DCL-dependent 25 or 26 nts, 32 nts, affected by AGO
Effector gene regulation TE regulation
Magnaporthe oryzae
Rice blast
18–23-nt TE loci 24 nts, DCL-, RDR-dependent 28–35-nt tRNA loci
TE regulation Stress response Appressoria formation
Botrytis cinerea
Gray mold rot
21 nts, DCL-dependent, LTR-TE loci
Suppression of host immunity
Sclerotinia sclerotiorum
White mold rot
20–24-nt milRNAs
Sclerotia development
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Abbreviations: AGO, Argonaute; DCL, Dicer-like protein; LTR, long terminal repeat; milRNAs, microRNA-like RNAs, nts, nucleotides; RDR, RNA-dependent RNA polymerase; TE, transposable element.
sRNAs in Plant Eukaryotic Pathogens
Long terminal repeat (LTR) retrotransposon: a type of retrotransposon with characteristic terminal sequence repeats that can sometimes occur in high copy numbers in host genomes
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sRNAs in eukaryotic plant pathogens have been characterized and are suggested to play a role in pathogenic development and virulence (Table 1). A global sRNA expression study in M. oryzae has classified sRNAs, some of which are associated with pathogenesis (92). sRNAs have been mapped to all kinds of genomic loci, but a large number of 18–23-nt sRNAs match to repetitive elements, including the long terminal repeat (LTR) retrotransposon MAGGY. A second group of 28–35-nt sRNAs is mapped to transfer RNA (tRNA) loci that are enriched in the appressoria, an infection-specific organ of this pathogen. A second study reports that 24 nts is the predominant size of sRNAs detected from M. oryzae under various physiological stress conditions and in planta during infection of rice (100). LTR-associated sRNA levels are increased during invasive growth. M. oryzae sRNAs regulate a subset of mRNAs posttranscriptionally, including an effector gene ACE1. ACE1 putatively codes for a hybrid between a polyketide synthase and a nonribosomal peptide synthetase, which likely functions in secondary metabolite production, and it is tightly controlled during initiation of appressorial penetration (8, 37). Similarly, sRNAs from the white mold fungus Sclerotinia sclerotiorum (149) and the entomopathogenic fungus Metarhizium anisopliae (150) show differential regulation during sclerotia production and conidiogenesis, respectively. These results support the notion that fungal endogenous sRNAs play a role in regulating virulence and developmental processes of fungal phytopathogens. The ascomycete B. cinerea is a destructive fungal plant pathogen that infects a broad spectrum of living plants, including almost all vegetables and fruits (121). It also causes serious postharvest losses (121). sRNA profiling of infected plant tissues has identified a set of B. cinerea sRNAs (Bc-sRNAs) that are predicted to target host genes in Arabidopsis and tomato, of which the majority are derived from LTR retrotransposon-like TEs (128). The Bc-siRNA targets, Arabidopsis mitogen-activated protein kinase genes MPK1 and MPK2, a cell wall–associated kinase (WAK), a peroxiredoxin (PRXIIF), and the tomato MPK-kinase kinase 4 (MAPKKK4), are suppressed upon B. cinerea infection. The suppression of these host target genes has also been observed in transgenic plants expressing Bc-sRNAs. Genetic analysis has demonstrated that these Bc-sRNA targets positively regulate plant immune responses against B. cinerea. Further analysis demonstrates that Bc-sRNAs silence the host genes by loading into a host AGO protein and utilizing the host gene-silencing machinery. In Arabidopsis, AGO1 is the predominant executer of miRNA-mediated PTGS and favors 21-nt miRNAs with a 5 terminal uracil (U) (82), and the majority of Bc-sRNAs that have predicted host targets are 21 nts in length and possess a 5 first nucleotide U. In addition, the Arabidopsis mutant ago1-27 exhibited reduced disease susceptibility to B. cinerea and infection was no longer able to suppress the expression of target mRNAs. By contrast, the Arabidopsis Weiberg et al.
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dcl1-7 mutant exhibited enhanced susceptibility toward B. cinerea infection, which suggests that utilizing host AGO1, rather than disrupting the plant miRNA biosynthesis pathway, is part of the fungal virulence strategy. This study was the first to show that sRNAs from a eukaryotic microbial pathogen act as effectors to suppress host immunity (6, 128) (Figure 1). This represents a naturally occurring cross-kingdom RNAi event (117). This sRNA-mediated virulence and the use of host gene-silencing machinery may be a more widely existing virulence mechanism in eukaryotic pathogens. For example, enhanced resistance in ago1 has also been observed during infection with V. dahliae (31). The oomycete Phytophthora infestans is the causal agent of late blight in potato and tomato. Although oomycetes and fungi look alike, they are phylogenetically very different. A global sRNA profiling of 19- to 40-nt sRNAs from P. infestans revealed three major size classes of sRNAs: 21 nts, 25 or 26 nts, and 30–33 nts (122). Numerous sRNAs are generated from TEs, predominantly LTR retrotransposons, and protein effector genes that include an RxLR amino acid motif and Crinkler (CRN). RxLR and CRN effectors are well-known virulence factors in pathogenic oomycetes. sRNAs from all size classes are mapped to TEs and RxLRs, whereas the sRNAs that are mapped to CRN genes predominantly belong to the 21-nt class that is mainly DCL1dependent. Long sRNAs (30–33 nts) are mapped mainly to TEs and are AGO dependent but DCL independent. Interestingly, differences in sRNA expression between two P. infestans strains exhibiting different virulence levels have been observed, further demonstrating a role for sRNA in P. infestans virulence. A negative correlation between the expression of sRNA and predicted targets, especially effector genes, supports a role of sRNAs in regulating effector gene expression and pathogen virulence. For example, PiAvr3a, an effector that suppresses plant programmed cell death for virulence by stabilizing host E3 ubiquitin ligase, is under the regulation of sRNAs (9). Six P. infestans miRNA candidates that have characteristics of plant and animal miRNAs were identified. Further characterization of sRNA populations from P. infestans, P. sojae, and Phytophthora ramorum identified two distinct classes of sRNAs: 21-nt miRNAs and siRNAs, and 25-nt siRNAs (33). The class of 25-nt siRNAs is predominantly mapped to TEs, whereas the class of 21-nt siRNAs is primarily derived from inverted repeats, conserved CRN effector genes, and type III fibronectin genes that are surrounded by TEs. A new family of miRNAs (MIR8788) is conserved among these three Phytophthora spp. and targets a family of amino acid/auxin permeases. The 25-nt siRNAs putatively regulate TE silencing, whereas the 21-nt sRNAs, including miRNAs, are proposed to regulate gene expression, including important virulence factors and effectors.
Cross-kingdom RNAi: a form of RNAi in which a gene-silencing trigger is produced in a donor organism but mediates gene silencing in an unrelated recipient organism
sRNA Biogenesis Proteins in Eukaryotic Plant Pathogens Most eukaryotic plant pathogen genomes encode DCL, AGO, and RDR proteins, the key components in the RNAi pathway. The filamentous saprophytic fungus Neurospora crassa was one of the first model organisms in which transgene-induced RNAi (called quelling) was discovered back in 1992 (103). Since then, genetic approaches, such as mutant screening (19), and numerous target gene disruption studies identified many RNAi pathway components and contributed much to the deciphering of the sophisticated architecture of RNAi pathways in fungi (14, 22, 71). Many RNAi-dependent phenomena in N. crassa and other fungal species have been studied. Quelling (38), the silencing of repeat-induced point (RIP) mutation (17), meiotic silencing of unpaired DNA (MSUD) (46, 114), DNA repair and homologous recombination (66, 146), sex-induced silencing (SIS) (127), and DNA methylation (41, 132) are examples of RNAi-dependent phenomena. However, it is not clear whether RNAi pathway components are directly required for pathogenicity in eukaryotic plant pathogens. www.annualreviews.org • Small RNAs
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The M. oryzae genome encodes two DCLs (MoDCL1 and MoDCL2), three putative AGOs (MoAGO1–MoAGO3), and three RDRs (MoRdRP1–MoRdRP3). MoDCL2 is required for transgene-induced gene silencing and sRNA-directed transposon silencing (57, 84). However, there are no reports of any RNAi mutant strains, including Mordrp, Modcl1, Modcl2, and Modcl1dcl2—although Modcl2 shows slightly reduced growth—that show obvious phenotypes
a
Activation of plant immune response Botrytis cinerea
sRNA effector sRNA
TE, etc.
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MAPKKK
WAK
AGO
MAPK
b
PRXIIF
Genomic DNA WRKY, etc.
AGO
?
Other defense proteins
Nucleus
Mitochondrion
Induction and translocation of fungal sRNA effectors TE, etc.
MAPKKK
WAK
AGO
AGO Immune response
AGO MAPKK
?
WRKY, etc.
MAPK
c
PRXIIF
AGO
Loading of sRNA effectors into plant AGO and degradation of mRNAs of defense genes TE, etc. WAK
AGO
MAPKKK
AGO
AGO
MAPKK mRNA
AGO PRXIIF
?
WRKY, etc.
MAPK
d
Immune response
Suppression of plant immunity and disease progression TE, etc. WAK
MAPKKK AGO
AGO
AGO
MAPKK MAPK
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AGO PRXIIF
WRKY, etc.
?
Induction Repression
Immune response
AGO MAPKK
Plant cell
AGO
Activation
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associated with pathogenicity (57). In contrast, obvious defects in growth and sporulation have been observed in the zygomycete fruit rot pathogen Mucor circinellioides dcl1 and dcl2 mutants, respectively (24, 88). An ago1 mutant of M. circinellioides shows defects in asexual spore production (13). Similar defective growth and sporulation phenotypes were observed in nonpathogenic Trichoderma atroviride dcr-2, dcr-1dcr-2, and rdr-3 deletion mutants (11). B. cinerea encodes two DCL proteins. Although the dcl1 and dcl2 single mutants and the dcl1dcl2 double mutant exhibit growth retardation and reduced conidiation, only the dcl1dcl2 double mutant shows significantly reduced virulence on both Arabidopsis and tomato (128). This difference is mostly due to the levels of sRNA effectors, which are abolished in dcl1dcl2 but are not altered in the dcl1 and dcl2 single mutants. It is noteworthy that Saccharomyces cerevisiae and the basidiomycetous plant pathogen Ustilago maydis, but not its close relative Ustilago hordei, completely lost their essential RNAi pathway components (29, 64). P. infestans possesses possibly two DCLs (PiDCL1 and PiDCL2) (33), five putative AGOs (PiAGO1–PiAGO5) and one RDR (PiRDR). Transgene-induced silencing requires PiDCL1 and PiAGO1/PiAGO2 (123). RNAi components are likely to be involved in pathogenesis because mRNA levels of PiDCL1, PiAGO3, and PiAGO4 are highly induced at 24 hours post inoculation, although no obvious phenotypes in morphology, differentiation, or pathogenesis have been reported in RNAi mutants, including Piago1/2, Piago3, Piago4, Piago5, and Pidcl1 knockdown mutants, which is likely due to the low efficiency of RNAi in P. infestans or to possible functional redundancy of the homologous genes within the same family. The fungal RNAi pathways are very diverse; for example, miRNA pathways are conserved in plants and animals (21) but not in fungi. Instead, miRNA-like RNAs (milRNA) have been described in fungi (58, 63, 149, 150) and at least four different milRNA biogenesis pathways, including both DCL-dependent and DCL-independent pathways, are proposed in N. crassa (52, 67, 71). It is important to know whether similar sRNA biogenesis pathways exist in fungal and oomycete pathogens. Furthermore, DCLs seem to be functionally redundant in sRNA processing in most fungal species (67, 89, 128). Taken together, the possible existence of diverse sRNA biogenesis pathways in these organisms presents a challenge for scientists in the dissection of RNAi pathways and characterization of regulatory sRNAs in pathogenicity.
MicroRNA-like RNA (milRNAs): a class of small RNAs in fungi that share common features with microRNAs of higher eukaryotes but lack a common ancestor or functional conservation among different species
TRANSPOSON-ASSOCIATED SRNAS IN EUKARYOTIC PLANT PATHOGENS TEs are mobile genomic elements that drive genome evolution. TE transposition and replication are associated with genomic DNA rearrangements and mutations. Although temporal TE activity has beneficial effects in terms of adaptive evolution, it is obvious that such elements can be detrimental. In eukaryotes, a large number of sRNAs are generated from TE loci to suppress TE activity, which is essential for maintaining genome integrity. ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Figure 1 Some Botrytis cinerea small RNAs (Bc-sRNAs) are effectors that translocate into the host plant cell and hijack the host Argonaute (AGO) protein of the gene-silencing machinery to suppress plant immune response. (a) Upon B. cinerea pathogen recognition, host immune responses are activated, including cell wall–associated kinase (WAK), signaling components of mitogen-activated protein kinase (MAPK) cascade, and mitochondria-associated peroxiredoxin (PRXIIF) as well as other unidentified defense proteins (indicated by question mark). (b) B. cinerea generates sRNAs (blue), and some sRNA effectors (red ) are induced and translocate into the host plant cell during infection. Those sRNA effectors are predominantly transcribed from transposable elements (TEs). (c) Upon host cell entry, sRNA effectors are loaded into the plant AGO and target the mRNAs (messenger RNAs) of selective plant defense genes for silencing. (d ) Target silencing attenuates immune response and facilitates disease progression. www.annualreviews.org • Small RNAs
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Localization of Effector Genes and R Genes Within Regions Enriched with Transposable Elements is of Evolutionary Advantage
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As genome sequences became available for dozens of phytopathogens, scientists have started to realize that some of the most specialized host-adapted plant pathogens, such as the powdery mildew fungus Blumeria graminis (116) and P. infestans (45, 99), show large genome expansion by massive TE propagation. Genomic regions rich in TEs are normally poor in protein-coding genes, but TE-rich regions from these phytopathogens show a remarkable enrichment of virulence genes, such as protein effector genes (105, 107, 120, 140). In the fungus Fusarium oxysporum, these genome regions can be found on extra, linear-specific (LS) chromosomes that present low synteny rates to closely related Fusarium spp. (78, 101). Although LS chromosomes contain a low density of housekeeping genes, which may make LS chromosomes dispensable, they are linked to hypervirulence. Accordingly, it is evident that transposition of TEs shape effector gene repertoires (36, 55, 105, 107). Consistent with this notion, B. cinerea sRNA effectors are also derived from LTR-transposon regions. Although massive expansion of transposons has not occurred appreciably in the B. cinerea genome (2), population dynamics surveys and genetic marker analysis of B. cinerea species linked the presence of two TEs, the LTR retrotransposon Boty and Fot1-like element FLIPPER, to the virulent and host-adapted subpopulations named transposa isolates (40, 80). The presence of the two TEs in these transposa isolates in crop-producing areas suggests a functional role for these TEs in virulence and host adaptation of B. cinerea. Similarly, rice isolates of M. oryzae collected from geographically dispersed locations exhibit high copy numbers of the LTR retrotransposon MAGGY compared with non-rice isolates collected from other Gramineae (34). Botrytis FLIPPER elements are structurally similar to Fot1 and Pot2 TEs from pathogenic F. oxysporum and M. oryzae, respectively (68), further supporting their possible role in pathogenesis. Boty is an LTR retrotransposon element that is related to the gypsy family of retrotransposons (27). Boty-like elements often contain an antisense gene, brtn, in their 3 untranslated region of yet unknown function that is also present in Boty-like LTR retrotransposon elements in S. sclerotiorum (148), pointing to a potential functional role of brtn in fungi. The Boty and FLIPPER TEs are present in the virulent B. cinerea strain B05.10, in which a Boty-like LTR-retrotransposon element encodes the identified sRNA effectors that suppress host immunity genes (Figure 1). It is not surprising that sRNA effectors evolved from TEs, and this evolution ensures a high turnover frequency for natural selection (Figure 2a). TEs are also likely to shape plant R-gene evolution. Many R genes, in distinct plant species, are clustered within certain genomic loci and are often close to TEs and repeats, thus facilitating DNA mutation upon selection pressure. The TEs carry epigenetic information to control the expression of these R genes, as demonstrated for the Arabidopsis RPP5 and RPP7 R-gene loci (119, 139). R-gene clusters are also hot spots for sRNA generation. sRNAs play an essential role in regulating the epigenetic status of R-gene loci and fine-tuning the expression of R genes during infection (Figure 2b). TEs and sRNAs are indispensable regulators in the arms race between eukaryotic pathogens and hosts (130).
Transposon-Associated sRNAs Regulate and Fine-Tune the Expression of Effector Genes that Modulate Virulence Approximately 74% of the P. infestans genome consists of TEs (45). A remarkable number of P. infestans sRNAs are mapped to TEs, which is consistent with the large number of sRNAs from TE regions in plants and animals. These TE-derived sRNAs are likely to mediate silencing of TEs 506
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d
TE
Pathogen cells TEs
b
R genes
TE
Induction
Protein effector genes
sRNAs
TE
Repression
Protein effectors
? Host proteins?
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c TE
Protein effector genes
?
Host defense genes
TE sRNAs
Types of sRNAs TE sRNAs sRNA effectors
R-gene sRNAs
R genes
TEs Plant cells
Protein effector gene sRNAs
Figure 2 Transposon-associated small RNAs (sRNAs) in plant-microbe interactions. To maintain genome integrity, eukaryotic pathogens produce transposable element (TE)-associated sRNAs (blue bars) to silence TEs. (a) sRNA effectors (dark red bars), like those found in Botrytis cinerea, are transcribed from TEs and suppress host immunity-related genes. (b) Host plant resistance (R) genes are often clustered in genomic loci enriched with TEs. TEs manifest epigenetic control of R-gene expression by R-gene sRNAs ( green bars). (c) Similarly, pathogen protein effector genes are often found in clusters and interspersed with TEs. Protein effector gene–derived sRNAs (orange bars), as found in Phytophthora spp., regulate effector expression. (d ) In an epigenetic network, sRNAs are important regulators of pathogen protein effector genes and host plant R genes. TE transposition shapes the repertoires of effector genes in pathogens and R genes in plants. TEs are hot spots of sRNA production. In pathogens, sRNAs regulate expression of TEs and of TE-associated protein effector genes, and sRNA effectors are delivered into host cells to manipulate expression of host defense genes. In plants, sRNAs epigenetically control R-gene expression, which activates defense genes upon infection. It is possible that plants may also deliver plant RNA or protein molecules into pathogen cells. All these scenarios are likely to affect plant-pathogen interaction and contribute to host resistance, pathogen virulence, and host adaptation.
to maintain genome integrity. Many of the RxLR and CRN effectors are located in close vicinity (
