Limits...
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.

Show MeSH
miR9863 family members target Mla1 transcripts in barley.(A) Alignment of Mla1 transcript with miR9863 family members from barley and wheat. Shown above the alignment are the MLA1 domain structure (CC, NB, ARC and LRR), the amino acid sequence of the miRNA target region and the neighboring RNBS-D motif (in red). hvu, Hordeum vulgare; tae, Triticum aestivum. (B) Predicted secondary structures of hvu-MIR9863a, hvu -MIR9863b and tae-MIR9863c precursors. Since hvu-MIR9863c could not be amplified from barley cultivars (i.e. Pallas and Golden promise), we used tae-MIR9863c for generating miR9863c in this study. The hvu-MIR9863b precursor generates both miR9863b.1 and miR9863b.2 that partially overlap with each other. (C) Ethidium bromide stained agarose gel of 5′ RACE products obtained from the barley line P01 (a Pallas near-isogenic line containing the Mla1 allele). 5′ RACE products were generated by using total RNAs isolated from P01, and ‘a’, ‘b’, and ‘c’ indicate three different products derived from different cleavage sites (see below). (D) Cleavage sites in the Mla1 transcript confirmed by the sequencing of 5′ RACE products. Arrows indicate the cleavage sites, ‘a’, ‘b’ and ‘c’, corresponding to three products shown in (C); numbers below the arrows show frequency of clones with matching 5′-RACE product from this site out of total clones confirmed by sequencing.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4263374&req=5

pgen-1004755-g001: miR9863 family members target Mla1 transcripts in barley.(A) Alignment of Mla1 transcript with miR9863 family members from barley and wheat. Shown above the alignment are the MLA1 domain structure (CC, NB, ARC and LRR), the amino acid sequence of the miRNA target region and the neighboring RNBS-D motif (in red). hvu, Hordeum vulgare; tae, Triticum aestivum. (B) Predicted secondary structures of hvu-MIR9863a, hvu -MIR9863b and tae-MIR9863c precursors. Since hvu-MIR9863c could not be amplified from barley cultivars (i.e. Pallas and Golden promise), we used tae-MIR9863c for generating miR9863c in this study. The hvu-MIR9863b precursor generates both miR9863b.1 and miR9863b.2 that partially overlap with each other. (C) Ethidium bromide stained agarose gel of 5′ RACE products obtained from the barley line P01 (a Pallas near-isogenic line containing the Mla1 allele). 5′ RACE products were generated by using total RNAs isolated from P01, and ‘a’, ‘b’, and ‘c’ indicate three different products derived from different cleavage sites (see below). (D) Cleavage sites in the Mla1 transcript confirmed by the sequencing of 5′ RACE products. Arrows indicate the cleavage sites, ‘a’, ‘b’ and ‘c’, corresponding to three products shown in (C); numbers below the arrows show frequency of clones with matching 5′-RACE product from this site out of total clones confirmed by sequencing.

Mentions: To investigate the role of miRNAs in barley (Hordeum vulgare L.) resistance to the powdery mildew pathogen, Bgh, we constructed small RNA (sRNA) libraries using samples derived from healthy and Bgh-infected leaves of the near-isogenic line P01 that harbors the Mla1 resistance allele [62]. Deep sequencing and data analyses of the sRNA libraries identified miRNAs that were either up- or down-regulated after Bgh infection. We searched in our sequence data for miRNAs potentially targeting Mla alleles and found that members of one miRNA family are complementary to the coding sequence for the region adjacent and with two-amino acids overlapping with the RNBS-D motif in Mla1 (Fig. 1A and 1D). Amino acid sequence alignment indicated that this potential miRNA target site is highly conserved among Mla alleles, as well as some R genes from wheat and related species (S1 Figure). The same miRNA family was also ascertained by an independent small RNA sequencing project [63] and described in previous small RNA deep sequencing studies in barley and bread wheat (Triticum aestivum L.). This family was originally designated in wheat as miR2009 with four members, i.e., miR2009a, miR2009b, miR2009c and miR2009d (S1 Table) [64]–[66]. However, the plant miR2009 was not formally included in miRBase20.0, while a sea urchin miRNA was already registered as miR-2009 [67]. Thus, the name of the wheat miR2009 was most likely assigned arbitrarily and thus bears the same name as that of the sea urchin miRNA. Therefore, we have renamed the plant miRNA family with its own unique designator in miRBase20.0, miR9863. We set out to search for barley and wheat EST sequences containing miR9863 members, and to BLAST wheat and barley genome databases. We found that miR9863a could be aligned to the wheat EST, CK193889, while miR9863c aligned to another wheat EST, DR736484 (S2A Figure). Interestingly, miR9863b and miR9863d overlap by 17 nucleotides, and cluster with miR9863a on the same wheat EST (i.e., CK193889) with 244 nt in-between (S2A Figure; S1 Table). In barley, miR9863b and miR9863d also align to one barley cDNA sequence, AK364228, overlapping by 17 nt (S2A Figure; S1 Table). Since miR9863b and miR9863d are two miRNAs that likely resulted from overlapping processing of the same locus, we designated these two miRNAs as miR9863b.1 and miR9863b.2 (Fig. 1A; S1 Table) [68]. The miRNA and miRNA* sequence and the flanking EST sequences together can form a typical hairpin structures, with the miRNA and miRNA* on the stem region (Fig. 1B; S2A Figure). Thus, these analyses indicate that the miR9863 family is present in the Triticeae, and members of this family could be potential regulators of Mla alleles.


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)

miR9863 family members target Mla1 transcripts in barley.(A) Alignment of Mla1 transcript with miR9863 family members from barley and wheat. Shown above the alignment are the MLA1 domain structure (CC, NB, ARC and LRR), the amino acid sequence of the miRNA target region and the neighboring RNBS-D motif (in red). hvu, Hordeum vulgare; tae, Triticum aestivum. (B) Predicted secondary structures of hvu-MIR9863a, hvu -MIR9863b and tae-MIR9863c precursors. Since hvu-MIR9863c could not be amplified from barley cultivars (i.e. Pallas and Golden promise), we used tae-MIR9863c for generating miR9863c in this study. The hvu-MIR9863b precursor generates both miR9863b.1 and miR9863b.2 that partially overlap with each other. (C) Ethidium bromide stained agarose gel of 5′ RACE products obtained from the barley line P01 (a Pallas near-isogenic line containing the Mla1 allele). 5′ RACE products were generated by using total RNAs isolated from P01, and ‘a’, ‘b’, and ‘c’ indicate three different products derived from different cleavage sites (see below). (D) Cleavage sites in the Mla1 transcript confirmed by the sequencing of 5′ RACE products. Arrows indicate the cleavage sites, ‘a’, ‘b’ and ‘c’, corresponding to three products shown in (C); numbers below the arrows show frequency of clones with matching 5′-RACE product from this site out of total clones confirmed by sequencing.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4263374&req=5

pgen-1004755-g001: miR9863 family members target Mla1 transcripts in barley.(A) Alignment of Mla1 transcript with miR9863 family members from barley and wheat. Shown above the alignment are the MLA1 domain structure (CC, NB, ARC and LRR), the amino acid sequence of the miRNA target region and the neighboring RNBS-D motif (in red). hvu, Hordeum vulgare; tae, Triticum aestivum. (B) Predicted secondary structures of hvu-MIR9863a, hvu -MIR9863b and tae-MIR9863c precursors. Since hvu-MIR9863c could not be amplified from barley cultivars (i.e. Pallas and Golden promise), we used tae-MIR9863c for generating miR9863c in this study. The hvu-MIR9863b precursor generates both miR9863b.1 and miR9863b.2 that partially overlap with each other. (C) Ethidium bromide stained agarose gel of 5′ RACE products obtained from the barley line P01 (a Pallas near-isogenic line containing the Mla1 allele). 5′ RACE products were generated by using total RNAs isolated from P01, and ‘a’, ‘b’, and ‘c’ indicate three different products derived from different cleavage sites (see below). (D) Cleavage sites in the Mla1 transcript confirmed by the sequencing of 5′ RACE products. Arrows indicate the cleavage sites, ‘a’, ‘b’ and ‘c’, corresponding to three products shown in (C); numbers below the arrows show frequency of clones with matching 5′-RACE product from this site out of total clones confirmed by sequencing.
Mentions: To investigate the role of miRNAs in barley (Hordeum vulgare L.) resistance to the powdery mildew pathogen, Bgh, we constructed small RNA (sRNA) libraries using samples derived from healthy and Bgh-infected leaves of the near-isogenic line P01 that harbors the Mla1 resistance allele [62]. Deep sequencing and data analyses of the sRNA libraries identified miRNAs that were either up- or down-regulated after Bgh infection. We searched in our sequence data for miRNAs potentially targeting Mla alleles and found that members of one miRNA family are complementary to the coding sequence for the region adjacent and with two-amino acids overlapping with the RNBS-D motif in Mla1 (Fig. 1A and 1D). Amino acid sequence alignment indicated that this potential miRNA target site is highly conserved among Mla alleles, as well as some R genes from wheat and related species (S1 Figure). The same miRNA family was also ascertained by an independent small RNA sequencing project [63] and described in previous small RNA deep sequencing studies in barley and bread wheat (Triticum aestivum L.). This family was originally designated in wheat as miR2009 with four members, i.e., miR2009a, miR2009b, miR2009c and miR2009d (S1 Table) [64]–[66]. However, the plant miR2009 was not formally included in miRBase20.0, while a sea urchin miRNA was already registered as miR-2009 [67]. Thus, the name of the wheat miR2009 was most likely assigned arbitrarily and thus bears the same name as that of the sea urchin miRNA. Therefore, we have renamed the plant miRNA family with its own unique designator in miRBase20.0, miR9863. We set out to search for barley and wheat EST sequences containing miR9863 members, and to BLAST wheat and barley genome databases. We found that miR9863a could be aligned to the wheat EST, CK193889, while miR9863c aligned to another wheat EST, DR736484 (S2A Figure). Interestingly, miR9863b and miR9863d overlap by 17 nucleotides, and cluster with miR9863a on the same wheat EST (i.e., CK193889) with 244 nt in-between (S2A Figure; S1 Table). In barley, miR9863b and miR9863d also align to one barley cDNA sequence, AK364228, overlapping by 17 nt (S2A Figure; S1 Table). Since miR9863b and miR9863d are two miRNAs that likely resulted from overlapping processing of the same locus, we designated these two miRNAs as miR9863b.1 and miR9863b.2 (Fig. 1A; S1 Table) [68]. The miRNA and miRNA* sequence and the flanking EST sequences together can form a typical hairpin structures, with the miRNA and miRNA* on the stem region (Fig. 1B; S2A Figure). Thus, these analyses indicate that the miR9863 family is present in the Triticeae, and members of this family could be potential regulators of Mla alleles.

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