Plants encode numerous intracellular receptors known as nucleotide-binding leucine-rich repeat receptors (NLRs) that recognize pathogen-derived effectors or their activity to activate defenses. MiRNA regulate NLR genes in many species, often triggering the production of phasiRNAs. Most such examples involve genes encoding NLR carrying coiled-coil domains, although a few include genes encoding NLRs carrying a Toll/interleukin-1 domain (TNL). Here, we characterize the role of miR825-5p in Arabidopsis, using a combination of bioinformatics, transgenic plants with altered miRNA levels and/or reporters, small RNA and virulence assays. We demonstrate that miR825-5p downregulates TNL MIST1 by targeting for endonucleolytic cleavage the sequence coding for TIR2, a highly conserved amino acid motif, linked to a catalytic residue essential for immune function. MiR825-5p acts as a negative regulator of basal resistance against Pseudomonas syringae. MiR825-5p triggers the production from MIST1 of a large number of phased siRNAs that can mediate cleavage of both MIST1 and additional TNL gene transcripts, potentially acting as a regulatory hub. MiR825-5p is expressed in unchallenged leaves and transcriptionally downregulated in response to PAMPs. Our results show that miR825-5p, which is required for full expression of PTI, establishes a link between PAMP perception and expression of uncharacterized TNL genes.

Plants encode large numbers of intracellular immune receptors known as resistance (R) proteins or nucleotide-binding (NB) leucine-rich repeat (LRR) receptors (NLRs), involved in perception of pathogen-derived effectors and activation of immunity.Here, we report a two-tiered regulatory network mediated by microRNA and secondary phased small RNAs (phasiRNA) that targets the silencing of dozens of NLR genes encoding yet uncharacterized members of the Toll/interleukin-1 (TIR)-NBS-LRR (TNLs) subfamily in Arabidopsis. We show that miR825-5p downregulates expression of Arabidopsis AT5G38850 gene (renamed as microRNA-silenced TNL 1 or MIST1) by targeting the sequence coding for a highly conserved functional amino acid motif (TIR2) within the TIR domain of the receptor. Further, we show that MIST1 functions as a regulatory hub, since miRNA825-5p triggers RDR6-mediated processing of MIST1 transcripts, to generate trans-acting phasiRNAs that in turn target, a wide network of TNL genes for gene silencing. Regulation through MIST1 affects disease resistance against the model bacterial pathogen Pseudomonas syringae, since altered levels of miRNA825-5p lead to changes in Arabidopsis ability to establish basal defenses against this pathogen. MiR825-5p is expressed in unchallenged adult leaves and its production is down regulated in response to PAMPs such as bacterial flagellin but also fungal chitin.

Background: Small RNAs are sequence-dependent negative regulators of gene expression involved in many relevant plant processes such as development, genome stability, or stress response. Functional characterization of sRNAs in plants typically relies on the modification of the steady state levels of these molecules. State-of-the-art strategies to reduce plant sRNA levels include molecular tools such as Target Mimics (MIMs or TMs), Short Tandem Target Mimic (STTMs), or molecular SPONGES (SPs). Construction of these tools routinely involve many different molecular biology techniques, steps, and reagents rendering such processes expensive, time consuming, and difficult to implement, particularly high-throughput approaches. Results: We have developed a vector and a cloning strategy that significantly reduces the number of steps required for the generation of MIMs against any given small RNA (sRNA). Our pGREEN-based binary expression vector (pGREEN-DLM100) contains the IPS1 gene from A. thaliana bisected by a ccdB cassette that is itself flanked by restriction sites for a type IIS endonuclease. Using a single digestion plus a sticky-end ligation step, the ccdB cassette that functions as a negative (counter) selection system is replaced by a pair of 28 nt self-annealing primers that provide specificity against the selected target miRNA/siRNA. The method considerably reduces the number of steps and the time required to generate the construct, minimizes the errors derived from long-range PCRs, bypasses bottlenecks derived from subcloning steps, and eliminates the need for any additional cloning technics and reagents, overall saving time and reagents. Conclusions: Our streamlined system guarantees a low cost, fast and efficient cloning process that it can be easily implemented into high-throughput strategies, since the same digested plasmid can be used for any given sRNA. We believe this method represents a significant technical improvement on state-of-the-art methods to facilitate the characterization of functional aspects of sRNA biology.

Plant use different receptors to detect potential pathogens: membrane-anchored pattern-recognition receptors (PRRs) activated upon perception of pathogen-associated molecular patterns (PAMPs) that elicit pattern-triggered immunity (PTI), and intracellular nucleotide-binding leucine-rich repeats proteins (NLRs) proteins activated by detection of pathogen-derived effectors, triggering effector-triggered immunity (ETI). The interconnections between PTI and ETI responses are increasingly reported. Elevated NLR levels may cause autoimmunity with symptoms ranging from fitness-cost to developmental arrest, sometimes combined with run-away cell death, making accurate control of NLR dosage key for plant survival. Small RNA-mediated gene regulation has emerged as a major mechanism of control of NLR dosage. Twenty-two nucleotide microRNAs (miRNAs) with the unique ability to trigger secondary short interfering RNA (siRNA) production from target transcripts are particularly prevalent in NLR regulation. They enhance repression of the primary NLR-target, but also bring about repression of NLRs only complementary to secondary siRNAs. We summarize the current knowledge on miRNAs and siRNAs in the regulation of NLR expression with an emphasis on 22-nt miRNAs and propose that miRNA and siRNA regulation of NLR levels provides additional links between PTI and NLR defense pathways to increase plant responsiveness against a broad spectrum of pathogens and control an efficient deployment of defenses.