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
miR9863a and miR9863b.1/b.2 specifically regulate group I Mla alleles.(A) Mla alleles are classified into three groups according to the SNP haplotype in the miR9863 binding site. The two SNPs differ among Mla groups are highlighted. (B) Mla alleles of group I, but not group II and III, are regulated by miR9863a and miR9863b.1/b.2. Mla genes of group I (Mla28, Mla32), group II (Mla2, Mla6) and group III (Mla10, Mla12) were respectively co-expressed with either MIR9863a (upper panels) or MIR9863b (lower panels) in N. benthamiana as described in Fig. 2. Protein levels of MLA or actin were determined by immunoblotting with an anti-HA or anti-actin antibody; Rubisco was included as a loading control. The asterisks indicate non-specific signals.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4263374&req=5

pgen-1004755-g003: miR9863a and miR9863b.1/b.2 specifically regulate group I Mla alleles.(A) Mla alleles are classified into three groups according to the SNP haplotype in the miR9863 binding site. The two SNPs differ among Mla groups are highlighted. (B) Mla alleles of group I, but not group II and III, are regulated by miR9863a and miR9863b.1/b.2. Mla genes of group I (Mla28, Mla32), group II (Mla2, Mla6) and group III (Mla10, Mla12) were respectively co-expressed with either MIR9863a (upper panels) or MIR9863b (lower panels) in N. benthamiana as described in Fig. 2. Protein levels of MLA or actin were determined by immunoblotting with an anti-HA or anti-actin antibody; Rubisco was included as a loading control. The asterisks indicate non-specific signals.

Mentions: Cloned Mla alleles encode highly sequence related CNL-subtype NLR receptors [48], [59], [75]–[78]. Sequence alignment using 29 Mla short sequences covering only the miR9863 target site shows that these Mla sequences are almost identical except for 1 or 2 adjacent nucleotides (Fig. 3A). These two adjacent nucleotides were predicted to complement with the 2nd–3rd nt of miR9863a/b.2 or the 7th–8th nt of miR9863b.1, respectively. Based on these SNP haplotypes, 29 Mla alleles were classified into three groups, group I, II and III (Fig. 3A). To determine whether there are differences in miR9863 regulation on different Mla groups, we randomly selected from each group two Mla genes, i.e., Mla28 and Mla32 from group I, Mla2 and Mla6 from group II, and Mla10 and Mla12 from group III, and each of them was co-expressed with precursor MIR9863a or MIR9863b in N. benthamiana, respectively (Fig. 1B, Fig. 3B). Western analysis revealed that the accumulation of MLA28 and MLA32 (group I) was fully blocked by expression of MIR9863a and partially blocked by expression of MIR9863b (Fig. 3B, left panels). By contrast, the accumulation of MLA2 and MLA6 (group II, Fig. 3B middle panel), or MLA10 and MLA12 (group III, Fig. 3B right panel) were not affected by expression of MIR9863a or MIR9863b, compared to the EV control. Together, these data strongly suggest that only group I Mla members are regulated by miR9863a and miR9863b.1/b.2 in planta.


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)

miR9863a and miR9863b.1/b.2 specifically regulate group I Mla alleles.(A) Mla alleles are classified into three groups according to the SNP haplotype in the miR9863 binding site. The two SNPs differ among Mla groups are highlighted. (B) Mla alleles of group I, but not group II and III, are regulated by miR9863a and miR9863b.1/b.2. Mla genes of group I (Mla28, Mla32), group II (Mla2, Mla6) and group III (Mla10, Mla12) were respectively co-expressed with either MIR9863a (upper panels) or MIR9863b (lower panels) in N. benthamiana as described in Fig. 2. Protein levels of MLA or actin were determined by immunoblotting with an anti-HA or anti-actin antibody; Rubisco was included as a loading control. The asterisks indicate non-specific signals.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004755-g003: miR9863a and miR9863b.1/b.2 specifically regulate group I Mla alleles.(A) Mla alleles are classified into three groups according to the SNP haplotype in the miR9863 binding site. The two SNPs differ among Mla groups are highlighted. (B) Mla alleles of group I, but not group II and III, are regulated by miR9863a and miR9863b.1/b.2. Mla genes of group I (Mla28, Mla32), group II (Mla2, Mla6) and group III (Mla10, Mla12) were respectively co-expressed with either MIR9863a (upper panels) or MIR9863b (lower panels) in N. benthamiana as described in Fig. 2. Protein levels of MLA or actin were determined by immunoblotting with an anti-HA or anti-actin antibody; Rubisco was included as a loading control. The asterisks indicate non-specific signals.
Mentions: Cloned Mla alleles encode highly sequence related CNL-subtype NLR receptors [48], [59], [75]–[78]. Sequence alignment using 29 Mla short sequences covering only the miR9863 target site shows that these Mla sequences are almost identical except for 1 or 2 adjacent nucleotides (Fig. 3A). These two adjacent nucleotides were predicted to complement with the 2nd–3rd nt of miR9863a/b.2 or the 7th–8th nt of miR9863b.1, respectively. Based on these SNP haplotypes, 29 Mla alleles were classified into three groups, group I, II and III (Fig. 3A). To determine whether there are differences in miR9863 regulation on different Mla groups, we randomly selected from each group two Mla genes, i.e., Mla28 and Mla32 from group I, Mla2 and Mla6 from group II, and Mla10 and Mla12 from group III, and each of them was co-expressed with precursor MIR9863a or MIR9863b in N. benthamiana, respectively (Fig. 1B, Fig. 3B). Western analysis revealed that the accumulation of MLA28 and MLA32 (group I) was fully blocked by expression of MIR9863a and partially blocked by expression of MIR9863b (Fig. 3B, left panels). By contrast, the accumulation of MLA2 and MLA6 (group II, Fig. 3B middle panel), or MLA10 and MLA12 (group III, Fig. 3B right panel) were not affected by expression of MIR9863a or MIR9863b, compared to the EV control. Together, these data strongly suggest that only group I Mla members are regulated by miR9863a and miR9863b.1/b.2 in planta.

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