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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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SNP variations in Mla alleles dictate miR9863 specificity.(A) Alignment of miR9863 target site of WT and mutated Mla alleles. SNPs in bold font and color-coded represent Mla type of group I (black), II (magenta) or III (red). Nucleotide substitutions TC1278GC or TC1278GA were introduced into Mla1; GC1278TC into Mla2 and Mla6, and GA1278TC into Mla10 and Mla12. (B) to (D) Determination of miR9863a regulation specificity on WT or mutated Mla alleles from different groups. Using transient gene expression assays in N. benthamiana, Hvu-MIR9863a was respectively co-expressed with group I Mla1 alleles (B), or group II Mla2 and Mla6 alleles (C), or group III Mla10 and Mla12 alleles (D). The protein accumulation levels of indicated Mla isoforms and actin were determined by immunoblotting. The asterisks indicate non-specific signals.
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pgen-1004755-g004: SNP variations in Mla alleles dictate miR9863 specificity.(A) Alignment of miR9863 target site of WT and mutated Mla alleles. SNPs in bold font and color-coded represent Mla type of group I (black), II (magenta) or III (red). Nucleotide substitutions TC1278GC or TC1278GA were introduced into Mla1; GC1278TC into Mla2 and Mla6, and GA1278TC into Mla10 and Mla12. (B) to (D) Determination of miR9863a regulation specificity on WT or mutated Mla alleles from different groups. Using transient gene expression assays in N. benthamiana, Hvu-MIR9863a was respectively co-expressed with group I Mla1 alleles (B), or group II Mla2 and Mla6 alleles (C), or group III Mla10 and Mla12 alleles (D). The protein accumulation levels of indicated Mla isoforms and actin were determined by immunoblotting. The asterisks indicate non-specific signals.

Mentions: To further verify the above finding that 1–2 SNPs in the Mla allele miR9863-binding site dictate miRNA specificity, we conducted targeted mutagenesis at these nt positions for each individual Mla from groups I, II, or III (Fig. 4A). First, we replaced the ‘TC’ (nt at 1278 and 1279) in Mla1 (from group I) with ‘GC’ or ‘GA’ to mimic the SNP haplotype of group II or III Mla, and reciprocally we replaced ‘GC’ in Mla2 and Mla6, as well as ‘GA’ in Mla10 and Mla12 to ‘TC’ to mimic the SNP haplotype of group I Mla (Fig. 4A). Upon co-expression of MIR9863a with WT Mla1 or two Mla1 variants (Mla1-TC1278GC, Mla1-TC1278GA), we could not detect by Western analysis any WT MLA1 accumulation, but the levels of two MLA1 variants are comparable to that in the EV control (Fig. 4B), suggesting loss of miR9863a effects on mutated Mla1 genes. In contrast, when MIR9863a was co-expressed with WT Mla2 and Mla6, or the respective Mla variants (Fig. 4C), as well as with WT Mla10 and Mla12, or their variants (Fig. 4D), we detected the expression of the WT MLAs similar to EV control, but no accumulation was detected for all mutant variants, compared to the EV control (Fig. 4C and 4D). This indicates a gain of function for miR9863a for Mla variant genes that mimic the group I Mla SNP haplotype. We obtained similar gain of function results for MIR9863b (S7 Figure). These results suggest that miR9863 targeting specificity on Mla alleles is largely determined by the two SNP compositions in the miR9863-binding site of each Mla.


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)

SNP variations in Mla alleles dictate miR9863 specificity.(A) Alignment of miR9863 target site of WT and mutated Mla alleles. SNPs in bold font and color-coded represent Mla type of group I (black), II (magenta) or III (red). Nucleotide substitutions TC1278GC or TC1278GA were introduced into Mla1; GC1278TC into Mla2 and Mla6, and GA1278TC into Mla10 and Mla12. (B) to (D) Determination of miR9863a regulation specificity on WT or mutated Mla alleles from different groups. Using transient gene expression assays in N. benthamiana, Hvu-MIR9863a was respectively co-expressed with group I Mla1 alleles (B), or group II Mla2 and Mla6 alleles (C), or group III Mla10 and Mla12 alleles (D). The protein accumulation levels of indicated Mla isoforms and actin were determined by immunoblotting. The asterisks indicate non-specific signals.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4263374&req=5

pgen-1004755-g004: SNP variations in Mla alleles dictate miR9863 specificity.(A) Alignment of miR9863 target site of WT and mutated Mla alleles. SNPs in bold font and color-coded represent Mla type of group I (black), II (magenta) or III (red). Nucleotide substitutions TC1278GC or TC1278GA were introduced into Mla1; GC1278TC into Mla2 and Mla6, and GA1278TC into Mla10 and Mla12. (B) to (D) Determination of miR9863a regulation specificity on WT or mutated Mla alleles from different groups. Using transient gene expression assays in N. benthamiana, Hvu-MIR9863a was respectively co-expressed with group I Mla1 alleles (B), or group II Mla2 and Mla6 alleles (C), or group III Mla10 and Mla12 alleles (D). The protein accumulation levels of indicated Mla isoforms and actin were determined by immunoblotting. The asterisks indicate non-specific signals.
Mentions: To further verify the above finding that 1–2 SNPs in the Mla allele miR9863-binding site dictate miRNA specificity, we conducted targeted mutagenesis at these nt positions for each individual Mla from groups I, II, or III (Fig. 4A). First, we replaced the ‘TC’ (nt at 1278 and 1279) in Mla1 (from group I) with ‘GC’ or ‘GA’ to mimic the SNP haplotype of group II or III Mla, and reciprocally we replaced ‘GC’ in Mla2 and Mla6, as well as ‘GA’ in Mla10 and Mla12 to ‘TC’ to mimic the SNP haplotype of group I Mla (Fig. 4A). Upon co-expression of MIR9863a with WT Mla1 or two Mla1 variants (Mla1-TC1278GC, Mla1-TC1278GA), we could not detect by Western analysis any WT MLA1 accumulation, but the levels of two MLA1 variants are comparable to that in the EV control (Fig. 4B), suggesting loss of miR9863a effects on mutated Mla1 genes. In contrast, when MIR9863a was co-expressed with WT Mla2 and Mla6, or the respective Mla variants (Fig. 4C), as well as with WT Mla10 and Mla12, or their variants (Fig. 4D), we detected the expression of the WT MLAs similar to EV control, but no accumulation was detected for all mutant variants, compared to the EV control (Fig. 4C and 4D). This indicates a gain of function for miR9863a for Mla variant genes that mimic the group I Mla SNP haplotype. We obtained similar gain of function results for MIR9863b (S7 Figure). These results suggest that miR9863 targeting specificity on Mla alleles is largely determined by the two SNP compositions in the miR9863-binding site of each Mla.

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