MicroRNA regulation of plant innate immune receptors

Abstract

Plant genomes contain large numbers of cell surface leucine-rich repeat (LRR) and intracellular nucleotide binding (NB)-LRR immune receptors encoded by resistance (R) genes that recognize specific pathogen effectors and trigger resistance responses. The unregulated expression of NB-LRR genes can trigger autoimmunity in the absence of pathogen infection and inhibit plant growth. Despite the potential serious consequence on agricultural production, the mechanisms regulating R-gene expression are not well understood. We identified microRNA (miRNA) progenitor genes precursor transcripts, and two miRNAs [nta-miR6019 (22-nt) and nta-miR6020 (21-nt)] that guide cleavage of transcripts of the Toll and Interleukin-1 receptor-NB-LRR immune receptor N from tobacco that confers resistance to tobacco mosaic virus (TMV). We further showed that cleavage by nta-miR6019 triggers RNA-dependent RNA polymerase 6- and ribonuclease Dicer-like 4-dependent biogenesis of 21-nt secondary siRNAs "in phase" with the 22-nt miR6019 cleavage site. Furthermore, we found that processing of the 22-nt nta-miR6019 depended on an asymmetric bulge caused by mismatch in the nta-miR6019 precursor. Interestingly, coexpression of N with nta-miR6019 and nta-miR6020 resulted in attenuation of N-mediated resistance to TMV, indicating that these miRNAs have functional roles in NB-LRR regulation. Using a bioinformatics approach, we identified six additional 22-nt miRNA and two 21-nt miRNA families from three Solanaceae species-tobacco, tomato, and potato. We show that members of these miRNA families cleave transcripts of predicted functional R genes and trigger production of phased secondary 21-nt siRNAs. Our results demonstrate a conserved role for miRNAs and secondary siRNAs in NB-LRR/LRR immune receptor gene regulation and pathogen resistance in Solanaceae.

Fig. 1.

nta-miR6019 and nta-miR6020-directed N cleavage. (A) nta-miR6019a (bold red font), nta-miR6020a (dark red font), and N target sites (bold, black and shaded font) with N cleavage sites (Clv) indicated in base pairs (bp). N and N-homolog cleavage product sequences (black font), shown aligned with N. N (U15605) map, below with encoded protein domains are indicated as filled shaded rectangles: TIR, blue; NB, gray; LRR, green. The MiS1 transposon (alternative exon of N), filled orange triangle (14); the N miRNA target, red vertical line. (B) Predicted foldback structure of nta-premiR6019,6020a. (C) Ethidium bromide stained agarose gel of 5′ RACE products of RNA from N-CFP^T2T1 and N-CFP^t2t1 tobacco lines generated using N-CFP primers (Upper) and N-primers (Lower). (D) Maps of N-CFP^T2T1 and N-CFP^t2t1 transgenes with the N TIR domain, CFP gene, and miRNA target regions indicated as filled dark blue, cyan, and red rectangles, respectively. miRNA target sequences in N-CFP^T2T1 and N-CFP^t2t1 are shown in black (wild-type) and gray (mutated) bold font shown below maps. nta-miR6019 (red font) and nta-miR6020 (dark red font) shown above and below N sequences respectively. Number of nta-miR6020 cleavage products is indicated in dark red font and arrow.

Fig. 2.

N 21-nt secondary siRNAs are phased and require RDR6 and DCL4 for biogenesis. (A) Plot of secondary siRNAs (blue line) as the number of tobacco 21-nt sRNA raw reads (y axis) along the N gene (U15605 coordinates, x axis). A map of N (Fig. 1A) and N and nta-miR6019 sequences in black and red, respectively, are shown below. Horizontal brackets below N sequence indicate 21-nt siRNA phasing. nta-siRNAI and nta-siRNAII registers and coordinates are indicated in blue. (B) N siRNA length [nucleotide (nt), x axis] and number (raw reads, y axis) in wild-type tobacco sRNA library. (C) 5′-terminal nucleotide (x axis) of N 21-nt siRNAs (in raw reads, y axis). (D) Relative levels of nta-TAS3 siRNA2142, nta-siRNAI, and nta-siRNAII in TG34 (wt) and TG34::nta-amiR:RDR6 tobacco lines (rdr6), measured by quantitative miR-ID analysis. Data normalized to miRNA390 levels. Quantitative analysis was repeated with two biological replicas and three technical replicas for each. (E) Relative level of nta-TAS3 siRNA2142, nta-siRNAI, and nta-siRNAII in TG34 tobacco (wt) compared with levels in TG34::nta-RNAi:DCL2,DCL4 (dcl24). Data was normalized to miRNA390 levels in each library.

Fig. 3.

nta-miR6019 (22-nt) triggers N secondary siRNA biogenesis. (A) Map of N-CFP miRNA sensor (top) and sensor sequences (1–5) with N (black bold) shown aligned with nta-miR6019 (red) and nta-miR6020 (dark red). CFP is shown in cyan, and CFP-siRNA is indicated in bold cyan. Arrows indicate cleavage positions at targets T1, T2, and T3, and numbers indicate cloning frequency. (B) The relative levels of CFP-siRNA and tasiRNA2142 measured by quantitative miR-ID analysis in N. benthamiana samples coinfiltrated with indicated N-CFP miRNA sensors and 35S:nta-MIR6019,6020, normalized to levels of miRNA390. (C) Sequences and predicted secondary structures of wild-type (WT) nta-premiR6019 (red) and mutant nta-premiR6019 (AXC, A2C, C2A, and ACCA, dark red) constructions. (D) Northern blot hybridization of sRNAs isolated from N. benthamiana leaves coinfiltrated with indicated 35S:nta-MIR6019,6020 vectors and N-CFP^T2TE1 miRNA sensor. Hybridization probes, miR6019, CFP-siRNA, and miR171 (Table S1) are indicated.

Fig. 4.

Overexpression of nta-MIR6019,6020 attenuates N-mediated resistance to TMV. (A) A. tumefaciens expression vectors used in N. benthamiana coexpression assays, 35S:nta-fb6025, 35S:nta-MIR6019^AXC,6020, and 35S:nta-MIR6019,6020 are indicated as nta-fb6025.0, miR6019 AXC, and miR6019 WT, respectively. (B) Coinfiltrated leaves and control untreated leaves photographed under bright light at 6 d after infiltration. (C) Same leaves as in B, photographed under UV light. (D) Coinfiltrated leaves and control untreated leaves photographed under bright light at 7 d after infiltration. The number of plants of six plants tested that displayed representative phenotypes are indicated below each lane.