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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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miR9863a regulates Mla1 mRNA and MLA1 protein in an uncoupled manner.(A) Schematic representation of Mla1-encoded domain structure and positions of amplicons for qRT-PCR quantification of Mla1 mRNA level. Amplicon 1, 2 or 3 positions at 5′, over or 3′ of the miR9863 cleavage site, respectively. (B) Quantification of Mla1 transcript and protein level at different time points post co-expression of Mla1-3HA with MIR9863a in N. benthamiana. The relative levels of three Mla1 amplicons were analyzed for samples of three time points, 16 and 24 and 36 hours-post-Agro-infiltration (hpai), by qRT-PCR quantification. For each amplicon, relative level was shown as fold-changes compared to the level at 16 hpai after first normalized to actin for each time point. The percentage above the bars indicates the remaining level of the amplicons relative to that of the EV control. Samples used for protein quantification were collected at 2 hrs after taking samples for mRNA quantification allowing sufficient time for protein translations. Western blots shown are exposed for more than 2 hrs. The asterisks indicate non-specific signals. All the experiments were performed at least twice with similar results.
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pgen-1004755-g007: miR9863a regulates Mla1 mRNA and MLA1 protein in an uncoupled manner.(A) Schematic representation of Mla1-encoded domain structure and positions of amplicons for qRT-PCR quantification of Mla1 mRNA level. Amplicon 1, 2 or 3 positions at 5′, over or 3′ of the miR9863 cleavage site, respectively. (B) Quantification of Mla1 transcript and protein level at different time points post co-expression of Mla1-3HA with MIR9863a in N. benthamiana. The relative levels of three Mla1 amplicons were analyzed for samples of three time points, 16 and 24 and 36 hours-post-Agro-infiltration (hpai), by qRT-PCR quantification. For each amplicon, relative level was shown as fold-changes compared to the level at 16 hpai after first normalized to actin for each time point. The percentage above the bars indicates the remaining level of the amplicons relative to that of the EV control. Samples used for protein quantification were collected at 2 hrs after taking samples for mRNA quantification allowing sufficient time for protein translations. Western blots shown are exposed for more than 2 hrs. The asterisks indicate non-specific signals. All the experiments were performed at least twice with similar results.

Mentions: Our data so far have showed that miR9863-directed Mla transcript cleavage and degradation play a role in the regulation of Mla. To examine whether translational inhibition directed by miR9863 may contribute to MLA suppression, again we co-expressed MIR9863a with Mla1 in N. benthamiana and then quantified Mla1 transcripts in parallel with MLA1 protein levels (Fig. 7). We monitored the level of three Mla1 amplicons (amplicon 1, Mla1407–783; amplicon 2, Mla11228–1582; amplicon 3 Mla12465–2621), positioned respectively upstream, over and downstream of the miR9863a cleavage site to reflect the levels of transcription, cleavage and cleavage-triggered decay of Mla1 transcripts (Fig. 7A) [84], at 16, 24, and 36 hours-post-Agro-infiltration (hpai). The corresponding MLA1 protein level was determined in samples collected 2 hrs later for each time point, allowing sufficient time for translation (Fig. 7B) [84]. At 16 hpai, we detected a basal level of the three Mla1 amplicons, but no accumulation of MLA1 protein (Fig. 7B, lanes 2 and 3). Later, at 24 hpai, we detected increased accumulation of Mla1 amplicons, as well as the presence of MLA1 protein in samples co-expressing the EV control (Fig. 7B, lane 4). However, the levels of the three Mla1 amplicons in samples co-expressing MIR9863a were 49–67% of that in EV control and MLA1 protein remained undetectable in the same samples (Fig. 7B, lane 5). At 36 hpai, Mla1 amplicons and MLA1 protein further increased in the EV control (Fig. 7B, lane 6), but the three Mla1 amplicons in MIR9863a-expressing samples were only 29–39% of the EV control (Fig. 7B, lane 7 vs. lane 6). It should be noted that these Mla1 transcript levels in the MIR9863a-expressing samples at 36 hpai are higher than those in the EV controls at 24 hpai (Fig. 7B, lane 7 vs. lane 4), yet MLA1 protein accumulation was not observed at 36 hpai, even though MLA1 protein was detected in the EV control at 24 hpai (Fig. 7B, lane 7 vs. lane 4). These findings suggest that in N. benthamiana Mla1 transcript levels have been uncoupled from accumulation of MLA1 protein in samples that Mla1 was co-expressed with miR9863, indicating apart from miR9863a-mediated transcript cleavage and decay, miR9863a-directed translational repression also plays a role in the regulation of Mla1 in this heterologous expression system.


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 regulates Mla1 mRNA and MLA1 protein in an uncoupled manner.(A) Schematic representation of Mla1-encoded domain structure and positions of amplicons for qRT-PCR quantification of Mla1 mRNA level. Amplicon 1, 2 or 3 positions at 5′, over or 3′ of the miR9863 cleavage site, respectively. (B) Quantification of Mla1 transcript and protein level at different time points post co-expression of Mla1-3HA with MIR9863a in N. benthamiana. The relative levels of three Mla1 amplicons were analyzed for samples of three time points, 16 and 24 and 36 hours-post-Agro-infiltration (hpai), by qRT-PCR quantification. For each amplicon, relative level was shown as fold-changes compared to the level at 16 hpai after first normalized to actin for each time point. The percentage above the bars indicates the remaining level of the amplicons relative to that of the EV control. Samples used for protein quantification were collected at 2 hrs after taking samples for mRNA quantification allowing sufficient time for protein translations. Western blots shown are exposed for more than 2 hrs. The asterisks indicate non-specific signals. All the experiments were performed at least twice with similar results.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4263374&req=5

pgen-1004755-g007: miR9863a regulates Mla1 mRNA and MLA1 protein in an uncoupled manner.(A) Schematic representation of Mla1-encoded domain structure and positions of amplicons for qRT-PCR quantification of Mla1 mRNA level. Amplicon 1, 2 or 3 positions at 5′, over or 3′ of the miR9863 cleavage site, respectively. (B) Quantification of Mla1 transcript and protein level at different time points post co-expression of Mla1-3HA with MIR9863a in N. benthamiana. The relative levels of three Mla1 amplicons were analyzed for samples of three time points, 16 and 24 and 36 hours-post-Agro-infiltration (hpai), by qRT-PCR quantification. For each amplicon, relative level was shown as fold-changes compared to the level at 16 hpai after first normalized to actin for each time point. The percentage above the bars indicates the remaining level of the amplicons relative to that of the EV control. Samples used for protein quantification were collected at 2 hrs after taking samples for mRNA quantification allowing sufficient time for protein translations. Western blots shown are exposed for more than 2 hrs. The asterisks indicate non-specific signals. All the experiments were performed at least twice with similar results.
Mentions: Our data so far have showed that miR9863-directed Mla transcript cleavage and degradation play a role in the regulation of Mla. To examine whether translational inhibition directed by miR9863 may contribute to MLA suppression, again we co-expressed MIR9863a with Mla1 in N. benthamiana and then quantified Mla1 transcripts in parallel with MLA1 protein levels (Fig. 7). We monitored the level of three Mla1 amplicons (amplicon 1, Mla1407–783; amplicon 2, Mla11228–1582; amplicon 3 Mla12465–2621), positioned respectively upstream, over and downstream of the miR9863a cleavage site to reflect the levels of transcription, cleavage and cleavage-triggered decay of Mla1 transcripts (Fig. 7A) [84], at 16, 24, and 36 hours-post-Agro-infiltration (hpai). The corresponding MLA1 protein level was determined in samples collected 2 hrs later for each time point, allowing sufficient time for translation (Fig. 7B) [84]. At 16 hpai, we detected a basal level of the three Mla1 amplicons, but no accumulation of MLA1 protein (Fig. 7B, lanes 2 and 3). Later, at 24 hpai, we detected increased accumulation of Mla1 amplicons, as well as the presence of MLA1 protein in samples co-expressing the EV control (Fig. 7B, lane 4). However, the levels of the three Mla1 amplicons in samples co-expressing MIR9863a were 49–67% of that in EV control and MLA1 protein remained undetectable in the same samples (Fig. 7B, lane 5). At 36 hpai, Mla1 amplicons and MLA1 protein further increased in the EV control (Fig. 7B, lane 6), but the three Mla1 amplicons in MIR9863a-expressing samples were only 29–39% of the EV control (Fig. 7B, lane 7 vs. lane 6). It should be noted that these Mla1 transcript levels in the MIR9863a-expressing samples at 36 hpai are higher than those in the EV controls at 24 hpai (Fig. 7B, lane 7 vs. lane 4), yet MLA1 protein accumulation was not observed at 36 hpai, even though MLA1 protein was detected in the EV control at 24 hpai (Fig. 7B, lane 7 vs. lane 4). These findings suggest that in N. benthamiana Mla1 transcript levels have been uncoupled from accumulation of MLA1 protein in samples that Mla1 was co-expressed with miR9863, indicating apart from miR9863a-mediated transcript cleavage and decay, miR9863a-directed translational repression also plays a role in the regulation of Mla1 in this heterologous expression system.

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