Regulation of plant immunity via small RNA-mediated control of NLR expression.

Plants 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 repeat proteins (NLRs) activated by detection of pathogen-derived effectors, activating effector-triggered immunity (ETI). The interconnections between PTI and ETI responses have been 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 miRNAs with the unique ability to trigger secondary 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 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.

miR825-5p targets the TIR-NBS-LRR gene MIST1 and down-regulates basal immunity against Pseudomonas syringae in Arabidopsis.

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. miRNAs regulate NLR genes in many species, often triggering the production of phased siRNAs (phasiRNAs). Most such examples involve genes encoding NLRs 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 RNAs, and virulence assays. We demonstrate that miR825-5p down-regulates the 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 phasiRNAs 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 down-regulated in response to pathogen-associated molecular patterns (PAMPs). Our results show that miR825-5p, which is required for full expression of PAMP-triggered immunity, establishes a link between PAMP perception and expression of uncharacterized TNL genes.

Protocol: low cost fast and efficient generation of molecular tools for small RNA analysis.

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.