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The miR9863 family regulates distinct Mla alleles in barley to attenuate NLR receptor-triggered disease resistance and cell-death signaling.

Liu J, Cheng X, Liu D, Xu W, Wise R, Shen QH - PLoS Genet. (2014)

Bottom Line: We show that miR9863 members guide the cleavage of Mla1 transcripts in barley, and block or reduce the accumulation of MLA1 protein in the heterologous Nicotiana benthamiana expression system.Overexpression of miR9863 members specifically attenuates MLA1, but not MLA10-triggered disease resistance and cell-death signaling.We propose a key role of the miR9863 family in dampening immune response signaling triggered by a group of MLA immune receptors in barley.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Plant Cell and Chromosome Engineering, Centre for Molecular Agrobiology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China; University of Chinese Academy of Sciences, Beijing, China.

ABSTRACT
Barley (Hordeum vulgare L.) Mla alleles encode coiled-coil (CC), nucleotide binding, leucine-rich repeat (NB-LRR) receptors that trigger isolate-specific immune responses against the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). How Mla or NB-LRR genes in grass species are regulated at post-transcriptional level is not clear. The microRNA family, miR9863, comprises four members that differentially regulate distinct Mla alleles in barley. We show that miR9863 members guide the cleavage of Mla1 transcripts in barley, and block or reduce the accumulation of MLA1 protein in the heterologous Nicotiana benthamiana expression system. Regulation specificity is determined by variation in a unique single-nucleotide-polymorphism (SNP) in mature miR9863 family members and two SNPs in the Mla miR9863-binding site that separates these alleles into three groups. Further, we demonstrate that 22-nt miR9863s trigger the biogenesis of 21-nt phased siRNAs (phasiRNAs) and together these sRNAs form a feed-forward regulation network for repressing the expression of group I Mla alleles. Overexpression of miR9863 members specifically attenuates MLA1, but not MLA10-triggered disease resistance and cell-death signaling. We propose a key role of the miR9863 family in dampening immune response signaling triggered by a group of MLA immune receptors in barley.

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22-nt miR9863s trigger the production of phasiRNAs with Mla1 transcripts.(A) Alignment of Mla1 sequence with 21-nt phased phasiRNAs. Raw reads of barley 21-nt phasiRNAs (y axis) obtained from deep sequencing were mapped to the sense strand (blue lines above the x axis) or antisense strand (red lines below the x axis) of the Mla1 sequence. Mla1 sequence adjacent to the miR9863 cleavage site was shown below the plot, and horizontal brackets below the sequence indicate 21-nt phasiRNAs, of which two most abundant ones (phasiRNAI and phasiRNAII) are indicated in the plot and in the sequence (in red). (B) Length of Mla1 phasiRNAs (x axis) was plotted to their raw reads value (y axis) obtained by sRNA deep sequencing in barley. (C) 5′-terminal nucleotide bias (x axis) of Mla1 phasiRNAs determined by raw reads (y axis) obtained by sRNA deep sequencing in barley. (D) Predicted secondary structure of precursors for expressing natural 22-nt miRNAs (MIR9863b, MIR9863a) and mutated 21-nt miRNAs (MIR9863b21 and MIR9863a21). (E) miR9863b.1/b.2 and miR9863a of 22-nt regulate Mla1 with higher efficiency than the mutated miRNAs of 21-nt. The mature miRNAs were detected by RNA gel blotting (panel a) with the mixture of [γ-32P]ATP labeled probes for miR9863a and miR9863b.1 (see supplemental Table 2 online), and 5S rRNA was employed as a loading control. MLA1 and actin protein levels were determined by immunoblotting (panel b), and rubisco is shown as loading control. (F) to (G) miR9863b.1/b.2 and miR9863a of 22-nt trigger the biogenesis of phasiRNAs with Mla1. Mla1 was co-expressed with EV or indicated miR9863 precursor in N. benthamiana, and the relative levels of phasiRNAI (F) and phasiRNAII (G) were measured by stem-loop quantitative RT-PCR (qRT-PCR) and normalized to U6 level. Letters above the bars (a-e) represent groups with significant differences [p<0.05, Tukey's honest significant difference (HSD) test].
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pgen-1004755-g006: 22-nt miR9863s trigger the production of phasiRNAs with Mla1 transcripts.(A) Alignment of Mla1 sequence with 21-nt phased phasiRNAs. Raw reads of barley 21-nt phasiRNAs (y axis) obtained from deep sequencing were mapped to the sense strand (blue lines above the x axis) or antisense strand (red lines below the x axis) of the Mla1 sequence. Mla1 sequence adjacent to the miR9863 cleavage site was shown below the plot, and horizontal brackets below the sequence indicate 21-nt phasiRNAs, of which two most abundant ones (phasiRNAI and phasiRNAII) are indicated in the plot and in the sequence (in red). (B) Length of Mla1 phasiRNAs (x axis) was plotted to their raw reads value (y axis) obtained by sRNA deep sequencing in barley. (C) 5′-terminal nucleotide bias (x axis) of Mla1 phasiRNAs determined by raw reads (y axis) obtained by sRNA deep sequencing in barley. (D) Predicted secondary structure of precursors for expressing natural 22-nt miRNAs (MIR9863b, MIR9863a) and mutated 21-nt miRNAs (MIR9863b21 and MIR9863a21). (E) miR9863b.1/b.2 and miR9863a of 22-nt regulate Mla1 with higher efficiency than the mutated miRNAs of 21-nt. The mature miRNAs were detected by RNA gel blotting (panel a) with the mixture of [γ-32P]ATP labeled probes for miR9863a and miR9863b.1 (see supplemental Table 2 online), and 5S rRNA was employed as a loading control. MLA1 and actin protein levels were determined by immunoblotting (panel b), and rubisco is shown as loading control. (F) to (G) miR9863b.1/b.2 and miR9863a of 22-nt trigger the biogenesis of phasiRNAs with Mla1. Mla1 was co-expressed with EV or indicated miR9863 precursor in N. benthamiana, and the relative levels of phasiRNAI (F) and phasiRNAII (G) were measured by stem-loop quantitative RT-PCR (qRT-PCR) and normalized to U6 level. Letters above the bars (a-e) represent groups with significant differences [p<0.05, Tukey's honest significant difference (HSD) test].

Mentions: Apart from many Bgh-responsive miRNA families in the sRNA libraries derived from infected barley leaf tissue (above, and see Materials and Methods), we identified additional sRNAs that perfectly match the Mla1 sense and antisense strand and are aligned downstream of the miR9863 target site (Fig. 6A, upper panel). These sRNAs are predominantly 21-nt in length (Fig. 6B) and biased for adenosine (A, ∼35%) and uridine (U, ∼20%) at the 5′-end position (Fig. 6C). Of the two abundant sRNAs at the 5′-proximal, one starts at 126 bp downstream of the miR9863a cleavage site (i.e., 1397 bp in Mla1), which corresponds to the 7th 21-nt register from the cleavage site; and the other starts at 504 bp downstream of the miR9863a cleavage site and corresponds to the 25th 21-nt register from the cleavage site (Fig. 6A, lower panel). These sRNAs are likely phasiRNAs [43] derived from the Mla1 transcripts, thus, for convenience of further analysis these two 21-nt register siRNAs are designated as phasiRNAI and phasiRNAII, respectively. To understand whether the biogenesis of these phasiRNAs are directly linked to miRNA action on its target, we co-expressed MIR9863b or MIR9863a with Mla1 in the heterologous N. benthamiana and then quantified the expression level of phasiRNAI and phasiRNAII (Fig. 6D, 6F and 6G). We found that phasiRNAI was significantly increased to ∼8 or 45 fold (Fig. 6F, bars 3 and 5), and phasiRNAII was increased to ∼2 to 2.5 fold (Fig. 6G, bars 3 and 5), compared to EV co-expressed with Mla1 (Fig. 6F and 6G, bar 2). These results indicate that miR9863b.1/b.2 and miR9863a trigger the biogenesis of phasiRNAs with Mla1 in N. benthamiana, and are consistent with our sRNA deep sequencing data obtained from barley.


The miR9863 family regulates distinct Mla alleles in barley to attenuate NLR receptor-triggered disease resistance and cell-death signaling.

Liu J, Cheng X, Liu D, Xu W, Wise R, Shen QH - PLoS Genet. (2014)

22-nt miR9863s trigger the production of phasiRNAs with Mla1 transcripts.(A) Alignment of Mla1 sequence with 21-nt phased phasiRNAs. Raw reads of barley 21-nt phasiRNAs (y axis) obtained from deep sequencing were mapped to the sense strand (blue lines above the x axis) or antisense strand (red lines below the x axis) of the Mla1 sequence. Mla1 sequence adjacent to the miR9863 cleavage site was shown below the plot, and horizontal brackets below the sequence indicate 21-nt phasiRNAs, of which two most abundant ones (phasiRNAI and phasiRNAII) are indicated in the plot and in the sequence (in red). (B) Length of Mla1 phasiRNAs (x axis) was plotted to their raw reads value (y axis) obtained by sRNA deep sequencing in barley. (C) 5′-terminal nucleotide bias (x axis) of Mla1 phasiRNAs determined by raw reads (y axis) obtained by sRNA deep sequencing in barley. (D) Predicted secondary structure of precursors for expressing natural 22-nt miRNAs (MIR9863b, MIR9863a) and mutated 21-nt miRNAs (MIR9863b21 and MIR9863a21). (E) miR9863b.1/b.2 and miR9863a of 22-nt regulate Mla1 with higher efficiency than the mutated miRNAs of 21-nt. The mature miRNAs were detected by RNA gel blotting (panel a) with the mixture of [γ-32P]ATP labeled probes for miR9863a and miR9863b.1 (see supplemental Table 2 online), and 5S rRNA was employed as a loading control. MLA1 and actin protein levels were determined by immunoblotting (panel b), and rubisco is shown as loading control. (F) to (G) miR9863b.1/b.2 and miR9863a of 22-nt trigger the biogenesis of phasiRNAs with Mla1. Mla1 was co-expressed with EV or indicated miR9863 precursor in N. benthamiana, and the relative levels of phasiRNAI (F) and phasiRNAII (G) were measured by stem-loop quantitative RT-PCR (qRT-PCR) and normalized to U6 level. Letters above the bars (a-e) represent groups with significant differences [p<0.05, Tukey's honest significant difference (HSD) test].
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Related In: Results  -  Collection

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pgen-1004755-g006: 22-nt miR9863s trigger the production of phasiRNAs with Mla1 transcripts.(A) Alignment of Mla1 sequence with 21-nt phased phasiRNAs. Raw reads of barley 21-nt phasiRNAs (y axis) obtained from deep sequencing were mapped to the sense strand (blue lines above the x axis) or antisense strand (red lines below the x axis) of the Mla1 sequence. Mla1 sequence adjacent to the miR9863 cleavage site was shown below the plot, and horizontal brackets below the sequence indicate 21-nt phasiRNAs, of which two most abundant ones (phasiRNAI and phasiRNAII) are indicated in the plot and in the sequence (in red). (B) Length of Mla1 phasiRNAs (x axis) was plotted to their raw reads value (y axis) obtained by sRNA deep sequencing in barley. (C) 5′-terminal nucleotide bias (x axis) of Mla1 phasiRNAs determined by raw reads (y axis) obtained by sRNA deep sequencing in barley. (D) Predicted secondary structure of precursors for expressing natural 22-nt miRNAs (MIR9863b, MIR9863a) and mutated 21-nt miRNAs (MIR9863b21 and MIR9863a21). (E) miR9863b.1/b.2 and miR9863a of 22-nt regulate Mla1 with higher efficiency than the mutated miRNAs of 21-nt. The mature miRNAs were detected by RNA gel blotting (panel a) with the mixture of [γ-32P]ATP labeled probes for miR9863a and miR9863b.1 (see supplemental Table 2 online), and 5S rRNA was employed as a loading control. MLA1 and actin protein levels were determined by immunoblotting (panel b), and rubisco is shown as loading control. (F) to (G) miR9863b.1/b.2 and miR9863a of 22-nt trigger the biogenesis of phasiRNAs with Mla1. Mla1 was co-expressed with EV or indicated miR9863 precursor in N. benthamiana, and the relative levels of phasiRNAI (F) and phasiRNAII (G) were measured by stem-loop quantitative RT-PCR (qRT-PCR) and normalized to U6 level. Letters above the bars (a-e) represent groups with significant differences [p<0.05, Tukey's honest significant difference (HSD) test].
Mentions: Apart from many Bgh-responsive miRNA families in the sRNA libraries derived from infected barley leaf tissue (above, and see Materials and Methods), we identified additional sRNAs that perfectly match the Mla1 sense and antisense strand and are aligned downstream of the miR9863 target site (Fig. 6A, upper panel). These sRNAs are predominantly 21-nt in length (Fig. 6B) and biased for adenosine (A, ∼35%) and uridine (U, ∼20%) at the 5′-end position (Fig. 6C). Of the two abundant sRNAs at the 5′-proximal, one starts at 126 bp downstream of the miR9863a cleavage site (i.e., 1397 bp in Mla1), which corresponds to the 7th 21-nt register from the cleavage site; and the other starts at 504 bp downstream of the miR9863a cleavage site and corresponds to the 25th 21-nt register from the cleavage site (Fig. 6A, lower panel). These sRNAs are likely phasiRNAs [43] derived from the Mla1 transcripts, thus, for convenience of further analysis these two 21-nt register siRNAs are designated as phasiRNAI and phasiRNAII, respectively. To understand whether the biogenesis of these phasiRNAs are directly linked to miRNA action on its target, we co-expressed MIR9863b or MIR9863a with Mla1 in the heterologous N. benthamiana and then quantified the expression level of phasiRNAI and phasiRNAII (Fig. 6D, 6F and 6G). We found that phasiRNAI was significantly increased to ∼8 or 45 fold (Fig. 6F, bars 3 and 5), and phasiRNAII was increased to ∼2 to 2.5 fold (Fig. 6G, bars 3 and 5), compared to EV co-expressed with Mla1 (Fig. 6F and 6G, bar 2). These results indicate that miR9863b.1/b.2 and miR9863a trigger the biogenesis of phasiRNAs with Mla1 in N. benthamiana, and are consistent with our sRNA deep sequencing data obtained from barley.

Bottom Line: We show that miR9863 members guide the cleavage of Mla1 transcripts in barley, and block or reduce the accumulation of MLA1 protein in the heterologous Nicotiana benthamiana expression system.Overexpression of miR9863 members specifically attenuates MLA1, but not MLA10-triggered disease resistance and cell-death signaling.We propose a key role of the miR9863 family in dampening immune response signaling triggered by a group of MLA immune receptors in barley.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Plant Cell and Chromosome Engineering, Centre for Molecular Agrobiology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China; University of Chinese Academy of Sciences, Beijing, China.

ABSTRACT
Barley (Hordeum vulgare L.) Mla alleles encode coiled-coil (CC), nucleotide binding, leucine-rich repeat (NB-LRR) receptors that trigger isolate-specific immune responses against the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). How Mla or NB-LRR genes in grass species are regulated at post-transcriptional level is not clear. The microRNA family, miR9863, comprises four members that differentially regulate distinct Mla alleles in barley. We show that miR9863 members guide the cleavage of Mla1 transcripts in barley, and block or reduce the accumulation of MLA1 protein in the heterologous Nicotiana benthamiana expression system. Regulation specificity is determined by variation in a unique single-nucleotide-polymorphism (SNP) in mature miR9863 family members and two SNPs in the Mla miR9863-binding site that separates these alleles into three groups. Further, we demonstrate that 22-nt miR9863s trigger the biogenesis of 21-nt phased siRNAs (phasiRNAs) and together these sRNAs form a feed-forward regulation network for repressing the expression of group I Mla alleles. Overexpression of miR9863 members specifically attenuates MLA1, but not MLA10-triggered disease resistance and cell-death signaling. We propose a key role of the miR9863 family in dampening immune response signaling triggered by a group of MLA immune receptors in barley.

Show MeSH