Role of small RNAs in host-microbe interactions

Abstract

Plant defense responses against pathogens are mediated by activation and repression of a large array of genes. Host endogenous small RNAs are essential in this gene expression reprogramming process. Here, we discuss recent findings on pathogen-regulated host microRNAs (miRNAs) and small interfering RNAs (siRNAs) and their roles in plant-microbe interaction. We further introduce small RNA pathway components, including Dicer-like proteins (DCLs), double-stranded RNA (dsRNA) binding protein, RNA-dependent RNA polymerases (RDRs), small RNA methyltransferase HEN1, and Argonaute (AGO) proteins, that contribute to plant immune responses. The strategies that pathogens have evolved to suppress host small RNA pathways are also discussed. Collectively, host small RNAs and RNA silencing machinery constitute a critical layer of defense in regulating the interaction of pathogens with plants.

Figure 1

Endogenous small RNA pathways in Arabidopsis. (a) microRNA (miRNA) pathway. miRNAs are generated by transcription of noncoding miRNA genes by RNA Pol II. The primary miRNAs possess stem-loop structures that are acted upon by DCL1-HYL1-SE protein complex. DDL protein is known to be involved in the formation of precursor miRNA (pre-miRNA). The DCL1-HYL1 complex further processes pre-miRNA into 21-nucleotide (nt) miRNAs. The miRNA (miRNA:miRNA*) duplexes are then methylated at their 3’ ends by HEN1. These methylated miRNAs are transported into cytoplasm by an exportin homolog, HASTY (HST). The mature miRNA is incorporated into the RNA-induced silencing complex (RISC) containing AGO1 protein. The RISC is recruited to the target gene on the basis of sequence complementarity, and AGO1 represses gene expression by either mRNA degradation or translational repression. (b) trans-acting small interfering RNA (ta-siRNA) pathway. The process of TAS precursor is triggered by an miRNA-mediated cleavage. The resulting 5′ fragment (in case of TAS1a–c and TAS2) and 3′ fragment (in case of TAS3) act as templates for the formation of long double-stranded RNA (dsRNA) by concerted action of RDR6 and SGS3. These long dsRNAs are then recognized by the DCL4-DRB4 complex and cut into phased 21-nt small RNAs that undergo further methylation by HEN1. The ta-siRNAs are incorporated into a RISC containing AGO7 (in the case ofTAS3) or AGO1 (in the case of TAS1 and 2), which results in target cleavage. (c) natural antisense transcript-derived siRNA (nat-siRNA) pathway. Natural antisense transcripts produced by Pol II form dsRNA within their overlapping regions. The dsRNAs are processed by DCL1 and/or DCL2 into siRNAs that target antisense transcripts through an unidentified AGO protein containing RISC complex. RDR6-SGS3, together with Pol IV, forms an amplification loop to generate more nat-siRNAs, which reinforce the cleavage of antisense transcript. (d) heterochromatic siRNA (hc-siRNA) pathway. Transcription of heterochromatic regions, repeat regions or transposons by Pol II and/or Pol IV results in the formation of single-stranded RNA(ssRNA), which is converted into dsRNA by the action of RDR2. This dsRNA is processed into predominantly 24-nt long hc-siRNAs by DCL3. These 24-nt siRNAs associate with AGO4 (or AGO6, or AGO9) through an adaptor protein, KTF1, to form an RNA-directed DNA methylation (RdDM) effector complex that directly or indirectly recruits proteins involved in heterochromatin formation, including DRM2, DRD1, and DMS3, to the hc-siRNA target loci. (e) long siRNA (lsiRNA) pathway. lsiRNAs are generated by DCL1 from coding or noncoding genes, or overlapping regions of antisense transcription, or dsRNAs from the action of Pol IV and RDRs. lsiRNAs are methylated by HEN1 and repress the expression of target genes by guiding mRNA decapping mediated by DCP2 (decapping 2) and VCS (Varicose) and 5’–3’ RNA decay mediated by exoribonuclease XRN4.

Figure 2

Small RNAs regulate rhizobia-legume symbioses, resulting in nodule development for nitrogen fixation. Different steps in nodule formation are shown along with the miRNAs predicted to be involved at specific steps. (a) The interaction of nitrogen-starved plants with rhizobial bacteria results in the exchange of chemical signals as plants secrete flavonoids and bacteria produce lipochitooligosaccharides. (b) The recognition of signals results in the attachment of bacterial cells to root hairs. (c) Changes in ionic equilibrium lead to the deformation of root hairs and transcription of nodulation-specific genes. Curling of root hairs to engulf bacteria results in the formation of infection thread that transports bacteria deep into the root tissue followed by bacteroid development. (d) Within 2 to 3 weeks postinoculation, mature nitrogen-fixing nodules are formed. Superscripts i, ii, and iii represent the miRNAs identified by Subramanian et al. (90), Wang et al. (101), and Lelandais-Briére et al. (59), respectively. mtr, Medicago truncatula; gma, Glycine max.

Figure 3

Mechanism of action of bacterial suppressors of RNA silencing (BSRs) in plants. Different steps in small RNA pathways are suppressed by different effectors encoded by Pseudomonas syringae. FLS: flagellin receptor; TTSS: type III secretion system of bacterial pathogen; Pst: Pseudomonas syringae pv. tomato DC3000.

Figure 4

Immunity against pathogens is regulated by small RNAs in plants. In PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI) regulated by small RNAs, RNA silencing suppressors (VSRs and BSRs) repress the small RNA silencing pathway. PAMPs, pathogen-associated molecular patterns; PRRs, pattern recognition receptors; VSRs, viral suppressors of RNA silencing; BSRs, bacteria-encoded suppressors of RNA silencing.