Role of small RNAs in the interaction between Arabidopsis and Pseudomonas syringae

Abstract

Endogenous small RNAs (miRNAs and siRNAs) regulate gene expression in diverse biological processes. Research with the Arabidopsis-Pseudomonas syringae system has shown that small RNAs contribute to plant immunity by regulating the expression of their target genes. Plant immunity can be triggered by pathogen-associated molecular patterns (PAMPs) or effector proteins that are delivered into the host cell by the pathogen. Experimental evidence indicates that the miRNA pathway play a major role in PAMP-triggered immunity while some of the siRNA pathways appear to be more important in effector-triggered immunity. In addition, some P. syringae effector proteins appear to inhibit miRNA biogenesis or function to enhance bacterial virulence. These exciting findings illustrate a new battle ground for plant-pathogen interactions.

Keywords

miRNA / siRNA / PTI / ETI / effectors

Introduction

Based on mechanisms of pathogen recognition, plant immunity can be divided into pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) (Chisholm et al., 2006; Jones and Dangl, 2006). Pattern recognition receptors (PRRs) at plant plasma membrane detect conserved pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) to activate a battery of immune responses. PTI is thought to be the first layer of induced immunity that offers plants a highly effective protection against numerous potential pathogens (Zipfel et al., 2004; Li et al., 2005; Boller and He, 2009). Not surprisingly, pathogens must overcome this layer of immunity in order to adapt to their host plant species. In several bacterial pathogens, particularly the well-studied Pseudomonas syringae, this is achieved through the secretion of a repertoire of effector proteins directly into the plant cell to subvert the host immune system. To counteract, host plants have evolved resistance (R) proteins to recognize effectors to trigger even stronger immune responses, which often include hypersensitive response (HR) that may act to restrict pathogen progression.

Eukaryotic small RNAs of 20-40 nucleotides in length regulate the expression of their target genes in a sequence-specific manner. As such, they play crucial roles in virtually every aspect of biological processes including plant development, metabolism, and stresses. As expected, increasing evidence in recent years show that small RNAs also play an important role in plant immunity. Small RNAs include microRNAs (miRNAs) and various classes of small interfering RNAs (siRNAs) that differ in length, biogenesis, and function. In general, different classes of double-stranded small RNA precursors are cut by RNase-III ribonuclease activity possessed by specific Dicer-like (DCL) proteins to form mature small RNAs, and the mature small RNAs are incorporated into distinct Argonaute (AGO) proteins to form specific RNA-induced silencing complexes (RISC). RISC guides gene silencing at transcriptional level by DNA methylation or at post-transcriptional level by cleaving target mRNAs (Baulcombe, 2004; Chapman and Carrington, 2007). RISC formed by miRNA and AGO1 can also interact with polysomes to repress protein expression (Lanet et al., 2009), resulting in translational silencing. In plants, small RNAs globally regulate plant immunity by inhibiting target gene expression at transcriptional or post-transcriptional level (Mourrain et al., 2000; Morel et al., 2002; Katiyar-Agarwal et al., 2006, 2007). The study of small RNAs in plant immunity has been largely limited to the interaction between Arabidopsis and P. syringae. The biogenesis and involvement of various small RNAs in plant immunity have been reviewed comprehensively (Padmanabhan et al., 2009; Katiyar-Agarwal and Jin, 2010). This short review focuses on mechanistic implications brought about by the study of small RNAs in the context of PTI and ETI using Arabidopsis-P. syringae as a model plant-pathosystem.

PAMP-induced miRNAs contribute to PTI by suppressing auxin signaling

Although it is well known that RNAi plays a crucial role in plant resistance to viruses by directly targeting viral RNAs, a broader role of small RNAs in plant immunity was revealed only recently. Navarro et al. (2006) searched for evidence for a role of small RNAs in Arabidopsis immunity against bacterial pathogen by examining miRNA target gene transcript accumulation upon stimulation by bacterial flagellar peptide flg22, a well-established PAMP. The accumulation of TIR1, AFB2 and AFB3 transcripts, which encode auxin receptor F-box proteins, were found to be repressed by flg22. Because these transcripts are targets of miR393, the results suggest the involvement of miR393 in defenses against P. syringae. Indeed, subsequent experiments showed that miR393 was transcriptionally induced by flg22, and ectopic overexpression of miR393 led to increased resistance to a virulent strain of P. syringae. Conversely, overexpression of AFB1, a member of the TIR1/AFB family led to increased susceptibility to this bacterium, indicating that active auxin signaling renders plants more susceptible to P. syringae bacteria. Although AFB1 transcripts were not appreciably induced by flg22, these results nonetheless support that plants actively repress auxin signaling to combat P. syringae infection.

PAMP-induced expression of miR393 was independently confirmed by deep sequencing of Arabidopsis leaves pre-treated with flg22 or P. syringae pv. tomato DC3000 (hrcC^-), a nonpathogenic strain that is defective in type III secretion (Fahlgren et al., 2007; Li et al., 2010). Interestingly, these latter studies also identified two additional PAMP-induced miRNAs, miR160 and miR167 that target auxin-response factor (ARF) family members. ARFs directly regulate the transcription of auxin-response genes downstream of TIR/AFB. Consistent with the increased expression of miR160 in response to flg22 treatment, its target gene transcripts ARF10, ARF16, and ARF17 were all downregulated by flg22. The function of miR160 in defenses was further supported by transgenic plants overexpressing miR160a which showed reduced transcript levels for ARF16 and ARF17 and increased callose deposition in response to flg22 and hrcC^- bacterium, though the increased defenses did not result in elevated resistance to P. syringae bacteria (Li et al., 2010). Taken together, these results highlight an important role of auxin signaling pathway in Arabidopsis-P. syringae interactions.

PAMP-suppressed miRNAs negatively regulate PTI

In addition to upregulated miRNAs, some miRNAs are downregulated by bacteria. For example, miR398 is downregulated in plants challenged with incompatible strains of P. syringae (Jagadeeswaran et al., 2009). Consistent with this finding, the mRNA level of miR398 target gene CSD1 increases significantly correspondingly (Jagadeeswaran et al., 2009). Deep sequencing also identified miR398 and miR773 as significantly downregulated miRNAs within one hour of flg22 treatment. Transgenic overexpression of miR398b and miR773 greatly reduced mRNA level of their target genes and PAMP-induced callose deposition, rendering plants more susceptible to both virulent P. syringae strain and the nonpathogenic strain hrcC^- (Li et al., 2010). MiR398 targets cytochrome c oxidase (COX5b.1) and Cu/Zn superoxide dismutase enzymes (CSD1, CSD2) that convert superoxide anion to hydrogen peroxide (Mori and Schroeder, 2004), implying their positive role in PTI; whereas miR773 targets a DNA methyltransferase MET1. Although a role of MET1 in PTI remains to be tested, it has been reported that ArabidopsisMET1 and MET2 required for Agrobacterium-mediated transformation of Arabidopsis roots (Crane and Gelvin, 2007). Interestingly, the PAMP receptor EFR restricts Agrobacterium-mediated transient gene expression (Zipfel et al., 2006), indicating that PTI plays an important role in limiting Agrobacterium-mediated transformation. It is tempting to speculate that MET1- and MET2-mediated DNA methylation may play a positive role in PTI. Nevertheless, the identity of the target genes of miR398 suggests a role of stress response in PTI regulation. It will be important to determine if these target genes directly or indirectly regulate plant defenses and resistance to P. syringae.

Role of siRNAs in plant ETI

By examining Arabidopsis plants infected with P. syringae pv. tomato carrying avrRpt2, the Jin laboratory uncovered several unique small RNA species that play important roles in disease resistance. The Arabidopsis nat-siRNAATGB2, a natural antisense siRNA processed from the overlapping region of a natural antisense transcript (NATs) pair by DCL1 (Katiyar-Agarwal et al., 2006), is specifically induced by an avirulent P. syringae strain, Pst (avrRpt2), and suppresses the transcription of its antisense target gene PPRL, a pentatricopepetide protein-like gene. Overexpression of PPRL led to increased bacterial proliferation and delayed hypersensitive response (HR) when challenged with Pst (avrRpt2), but not other avirulent strains of bacteria in plants. Because AvrRpt2 specifically conditions resistance conferred by the resistance protein RPS2, the results indicate a specific role of PPRL and nat-siRNAATGB2 in RPS2 resistance. A novel class of long siRNAs (lsiRNAs) was reported in 2007. These 30-40 nucleotides long siRNAs were generated from natural antisense transcripts (NATs) pairs and induced by Pst (avrRpt2) or specific growth conditions, such as cell suspension culture (Katiyar-Agarwal et al., 2007). AtlsiRNA-1 degrades mRNA of its antisense gene AtRAP through decapping and XRN4-mediated 5′-3′ decay, which differs from other siRNA-mediated mRNA cleavage or DNA methylation. Reverse genetic study suggested that AtRAP negatively regulates plant resistance to bacteria (Padmanabhan et al., 2009). How PPRL and AtRAP affect RPS2 resistance is not understood.

Most recently, Zhang et al. (2011) showed that transcripts of AGO2 were dramatically induced by Pst (avrRpt2). Purification of AGO2-bound small RNAs led to the identification of several miRNA^*s which are considered passenger strand in the miRNA duplex. One of these miRNA^*s is miR393^*, which targets a gene encoding a Golgi-localized SNARE protein, MEMB12. Mutations in MEMB12 and AGO2 or overexpressing miR393^* result in increased secretion of pathogenesis-related proteins and enhanced disease resistance to Pst (avrRpt2) and a virulent strain of P. syringae, suggesting that MEMB12 negatively regulates PR protein secretion. MiR393^* regulates this process through translational inhibition of MEMB12. Interestingly, miR393^* is sorted primarily into AGO2 but not AGO1 whereas miRNA393 described above is sorted into AGO1 but not AGO2. These results also demonstrate a regulatory role of miRNA passenger strand, at least for miR393^*.

Regulation of PTI and ETI through distinct small RNA pathways

While it is clear that multiple classes of small RNAs regulate plant immunity against P. syringae bacteria, it is necessary to ask whether distinct small RNA pathways play differential roles in PTI and ETI. In general, different classes of small RNAs are loaded into distinct AGO proteins to carry out transcriptional or post-transcriptional gene silencing. For example, AGO1 and AGO10 mainly bind miRNAs, whereas AGO4 binds heterochromatin-siRNAs (hc-siRNA). AGO2 binds 21 nt small RNAs including miRNA^*s discussed above (Zhang et al., 2011). AGO7 binds TAS3 ta-siRNAs and miR390, and is also required for the biogenesis of AtlsiRNA (Katiyar-Agarwal et al., 2007). Thus analyses of various ago mutants in plant immune responses provide clues to the role of different small RNA pathways in disease resistance.

Among the 10 ArabidopsisAGO genes, three have been shown to contribute to resistance to P. syringae. Overall, AGO1 appears to play a positive role in PTI as indicated by the reduced callose deposition and PAMP-response gene expression, and a lack of PAMP-induced protection against virulent P. syringae in ago1 plants (Li et al., 2010). However, ago1 showed normal resistance to Pst (avrRpt2) (Li et al., unpublished results), suggesting that AGO1 may be specialized in PTI. This is consistent with a positive contribution of the miRNA pathway to PTI, in support of this notion, DCL1 and HEN1, which are required for the biogenesis of miRNAs, are also required for PTI (Navarro et al., 2008; Li et al., 2010). The miRNA pathway appears to impact late stage of PTI defenses such as defense gene expression and callose deposition. Early molecular events of PTI, such as MAPK activation and transient oxidative burst, are not affected in ago1 and dcl1 mutants. These results demonstrate that miRNA pathway positively regulates PTI to against bacterial attack. However, the role of siRNAs in PTI is not clear, and deeper bioinformatics and functional mechanism assay will help us to pick out new and interesting siRNAs. It should be pointed out that not all miRNAs play a positive role in PTI, as miR398 and miR773 play negative role in PTI (Li et al., 2010).

Unlike ago1, ago7 plants show increased susceptibility to Pst (avrRpt2) but not to hrcC^- bacteria (Katiyar-Agarwal et al., 2007; Li et al., 2010). Furthermore the PTI responses in ago7 plants are normal as measured by PAMP-induced callose deposition and gene expression (Li et al., unpublished results), indicating that AGO7 plays a specific role in AvrRpt2-induced ETI, which is consistent with a specific role of nat-siRNAATGB2 and AtlsiRNA-1 in Pst (avrRpt2) resistance discussed above. ago2 mutant plants are greatly reduced in resistance to Pst (avrRpt2), and ago2ago7 double mutants show additive effects compared to ago2 and ago7 single mutants (Zhang et al., 2011). It is not known, however, if AGO2 plays a general role in ETI specified by multiple resistance proteins. It also remains to be tested if ago2 is compromised in PTI defenses.

AGO4 is known to associate with siRNA to direct DNA methylation (RNA-directed DNA methylation, RdDM) and histone modifications (Zilberman et al., 2003; 2004). ago4-1 and ago4-2 mutant plants show enhanced disease susceptibility to virulent and avirulent P. syringae strains and nonadapted strains such as P. syringae pv. tabaci (Agorio and Vera, 2007), indicating that AGO4 may be required for both PTI and ETI. This possibility remains to be tested rigorously. DNA methylation assay showed that the methylation extent at CpNpG and CpHpH positions were decreased in both mutants. However, the mutations in other components in RdDM pathway, such as RDR2, DCL3, DRD1, CMT3, DRM1, and DRM2, failed to show increased susceptibility to P. syringae. It is not known if any of the AGO4-associated small RNAs play a role in resistance to P. syringae. It is also possible that AGO4 positively regulates resistance to bacteria independently of RdDM pathway. Alternatively, the lack of phenotype in these RdDM component mutants may reflect functional redundancy among family members of these components.

Some bacterial effectors are capable of modulating miRNA/siRNA pathways

P. syringae effector proteins are known to actively suppress host immunity. This is achieved mainly through direct targeting of host components that are key to immune signaling. Some of the target proteins include receptor kinases, MAP kinases, RIN4 that are required for sensing PAMPs or early steps of signal transduction (Zhang et al., 2007, 2010; Göhre et al., 2008; Xiang et al., 2008; Gimenez-Ibanez et al., 2009; Cui et al., 2010; Wang et al., 2010; Wilton et al., 2010; Liu et al., 2011). A recent report expanded the list to include an enzyme for phytoalexin biosynthesis (Zhou et al., 2011). By using Agrobacterium-mediated transient-expression assay, Navarro et al. (2008) identified several P. syringae effectors including AvrPto, AvrPtoB, and HopT1-1 that were capable of inhibiting the biogenesis or function of miRNA. For example, AvrPtoB downregulates pri-miR393 transcription, suggesting that AvrPtoB is likely to suppress miRNA generation. AvrPto suppresses mature miR393 accumulation without affecting pri-miR393 level. The third effector HopT1-1 appears to enhance the translation of miR834 target gene COP1-interacting protein 4 by interfering AGO1 function within RISC.

Surprisingly, a recent report suggested that several P. syringae effectors positively regulate siRNA-mediated gene silence (Sarris et al., 2011). Transient expression of HopAB1, HopX1, HopF2 and, to a lesser extent, AvrPtoB, in a transgenic GFP Nicotiana benthamiana line enhanced post transcriptional GFP gene silencing, and this enhancement could be blocked by two well-known viral silencing suppressors P38 and P19. HopAB1 is highly homologous to AvrPtoB that was reported to block miRNA biogenesis (Navarro et al., 2008). HopAB1 and AvrPtoB contain a C-terminal E3 ligase domain, and this domain appears to be required and sufficient for the enhancement of silencing (Sarris et al., 2011). Moreover, transient expression of HopAB1 and HopX1 can induce the accumulation of nat-siRNAATGB2 and AtlsiRNA-1 within the efr-1 background independent of avr-R interaction and hypersensitive response (Sarris et al., 2011). Although these effectors enhance siRNAs in the absence of visible hypersensitive response, it cannot be excluded that they are recognized by weak R genes which indirectly stimulate siRNA pathway. It will be interesting to test if these effectors can also induce siRNA production in wild type background so as to understand whether the induction of siRNAs by these effectors is associated with pattern recognition.

While it is intriguing that both miRNA and siRNA pathways are affected by certain effectors, a number of questions remain unanswered. While the effector-mediated inhibition of miRNA biogenesis is consistent with a role of these effectors in virulence (Navarro et al., 2008), it is not clear how the siRNA pathway enhancement by effectors would impact the outcome of host-bacterial pathogen interaction. In addition, it is not clear how AvrPtoB differentially modulate miRNA and siRNA pathways. In most cases, the observed alterations of miRNA/siRNA were made by overexpression of effectors, which may or may not occur when these effectors are naturally delivered into the plant cell by P. syringae bacterium. Furthermore, it cannot be ruled out that the alterations of miRNA/siRNA pathways are caused indirectly by these effectors as a consequence of resistance or virulence. Future investigation is needed to elucidate the molecular basis by which these effectors impacting miRNA and siRNA pathways.

Conclusions

Endogenous small RNA pathways are important players regulating both PTI and ETI during P. syringae infection. Over a few years, we have learned that small RNAs can regulate plant immunity through the regulation of auxin signaling, stress responses and PR protein secretion. Continued characterization of additional pathogen-responsive small RNAs is likely to yield new insight in disease resistance mechanisms. We can expect that similar findings will be made in other plant-pathogen systems. Indeed, He et al. (2008) reported that turnip mosaic virus (TuMV) infection induces two Brassica miRNAs bra-miR158 and bra-miR1885 in Brassica. Furthermore, several miRNA families in loblolly pine were regulated by endemic rust fungus Cronartium quercuum f. sp. fusiforme, which caused fusiform rust disease (cankers) in pines (Lu et al., 2007). A genome-wide study showed that more than 6% rice genes are predicted to be cis-NATs (Zhou et al., 2009), and the transcription of which are regulated by abiotic and biotic stresses (Zhou et al., 2010; Yan et al., 2011). With high throughput sequencing and bio-informatics, more pathogen-responsive small RNAs will surely be identified. The characterization of these small RNA target genes will help us to find new components in plant immunity signaling pathways.

It remains to be seen whether any of the P. syringae effectors directly target small RNA pathways and whether other pathogens also carry inhibitors of small RNA pathways. Given the importance of various small RNAs in plant immunity, it is tempting to speculate that regulation of small RNA biogenesis and function can be an emerging battle ground between plants and pathogenic microbes.

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