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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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Overexpression of MIR9863a and MIR9863b attenuates Mla1-triggered disease resistance and cell-death signaling.(A) Transient gene expression of MIR9863a, MIR9863c and MIR9863b in barley near-isogenic line containing Mla1 or Mla10. Empty vector or plasmids expressing indicated MIRNA precursors were delivered into leaf epidermal cells of indicated barley lines through particle bombardment, and spores of indicated avirulence Bgh isolates were inoculated 36 hrs post bombardment. Haustorial index shown are the percentage of cells that developed fungal haustoria in all transformed cells. (B) Transient gene expression assays in Golden promise (GP), a barley line susceptible to both Bgh K1 and Bgh A6. Indicated construct expressing MIR9863b was delivered alone or with Mla1-mYFP or Mla10-mYFP construct into GP leaf epidermal cells, and spores of Bgh K1 or Bgh A6 were inoculated at 4 hrs post bombardment. Error bars in (A) and (B) represent standard error (SE) from at least three replicates, and letters (a–c) above the bars represent groups with significant differences [p<0.05, Tukey's honest significant difference (HSD) test]. (C) to (D) Analysis of cell-death induction upon co-expressing Mla1 or Mla10 with MIR9863a and MIR9863c. The indicated constructs were expressed in N. benthamiana leaves by Agro-infiltration, and cell death was visualized by trypan blue staining at 48 hours-post-Agro-infiltration (hpai). Red circle indicates visible cell-death phenotype.
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pgen-1004755-g008: Overexpression of MIR9863a and MIR9863b attenuates Mla1-triggered disease resistance and cell-death signaling.(A) Transient gene expression of MIR9863a, MIR9863c and MIR9863b in barley near-isogenic line containing Mla1 or Mla10. Empty vector or plasmids expressing indicated MIRNA precursors were delivered into leaf epidermal cells of indicated barley lines through particle bombardment, and spores of indicated avirulence Bgh isolates were inoculated 36 hrs post bombardment. Haustorial index shown are the percentage of cells that developed fungal haustoria in all transformed cells. (B) Transient gene expression assays in Golden promise (GP), a barley line susceptible to both Bgh K1 and Bgh A6. Indicated construct expressing MIR9863b was delivered alone or with Mla1-mYFP or Mla10-mYFP construct into GP leaf epidermal cells, and spores of Bgh K1 or Bgh A6 were inoculated at 4 hrs post bombardment. Error bars in (A) and (B) represent standard error (SE) from at least three replicates, and letters (a–c) above the bars represent groups with significant differences [p<0.05, Tukey's honest significant difference (HSD) test]. (C) to (D) Analysis of cell-death induction upon co-expressing Mla1 or Mla10 with MIR9863a and MIR9863c. The indicated constructs were expressed in N. benthamiana leaves by Agro-infiltration, and cell death was visualized by trypan blue staining at 48 hours-post-Agro-infiltration (hpai). Red circle indicates visible cell-death phenotype.

Mentions: To investigate the role of miR9863 members in MLA-mediated disease resistance to Bgh, we used a well-established single-cell transient assay [77], [85]. Plasmid constructs of miRNA precursor are co-delivered with a β-glucuronidase (GUS) reporter into barley epidermal cells by particle bombardment, and then the haustorial index (HI%) is scored to assess the frequency of fungal haustoria in transformed cells upon fungal inoculation (Fig. 8A and 8B). First, we respectively delivered plasmids of EV, MIR9863a, MIR9863c, or MIR9863b into near-isogenic barley line P01 (Mla1) and then inoculated conidiospores of Bgh K1 (AVRa1) to activate MLA1-mediated immune responses (Fig. 8A, left panel). We observed a low HI% of ∼3% in leaves receiving EV control, likely due to MLA1-triggered immunity against Bgh K1 (Fig. 8A, bar 1), whereas in leaves expressing MIR9863a or MIR9863b, we observed significantly increased HI% to ∼22% or 15% (Fig. 8A, bars 2 and 4), indicating MLA1-triggered immunity was compromised by the expression of miR9863a or miR9863b.1/b.2. The HI% was similar in P01 leaves expressing MIR9863c to that in the EV control (Fig. 8A, bar 3), consistent with that miR9863c does not interact with the Mla1 allele (Fig. 2C). Further, plasmids of MIR9863a, MIR9863c and MIR9863b were respectively delivered into leaves of near-isogenic barley line P09 (Mla10) and inoculated with Bgh isolate A6 (AVRa10) (Fig. 8A, right panel), and we observed comparable HI% of ∼6% to 7% in leaves expressing EV or respective miR9863 precursors (Fig. 8A, bars 5 to 8), suggesting that expression of any miR9863 members had no effect on Mla10-mediated disease resistance to Bgh, consistent with that Mla members of group III are not miR9863 targets (Fig. 3B). Taken together, overexpression of miR9863a and miR9863b.1/b.2 specifically attenuates MLA1 but not MLA10-mediated disease resistance against Bgh in 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)

Overexpression of MIR9863a and MIR9863b attenuates Mla1-triggered disease resistance and cell-death signaling.(A) Transient gene expression of MIR9863a, MIR9863c and MIR9863b in barley near-isogenic line containing Mla1 or Mla10. Empty vector or plasmids expressing indicated MIRNA precursors were delivered into leaf epidermal cells of indicated barley lines through particle bombardment, and spores of indicated avirulence Bgh isolates were inoculated 36 hrs post bombardment. Haustorial index shown are the percentage of cells that developed fungal haustoria in all transformed cells. (B) Transient gene expression assays in Golden promise (GP), a barley line susceptible to both Bgh K1 and Bgh A6. Indicated construct expressing MIR9863b was delivered alone or with Mla1-mYFP or Mla10-mYFP construct into GP leaf epidermal cells, and spores of Bgh K1 or Bgh A6 were inoculated at 4 hrs post bombardment. Error bars in (A) and (B) represent standard error (SE) from at least three replicates, and letters (a–c) above the bars represent groups with significant differences [p<0.05, Tukey's honest significant difference (HSD) test]. (C) to (D) Analysis of cell-death induction upon co-expressing Mla1 or Mla10 with MIR9863a and MIR9863c. The indicated constructs were expressed in N. benthamiana leaves by Agro-infiltration, and cell death was visualized by trypan blue staining at 48 hours-post-Agro-infiltration (hpai). Red circle indicates visible cell-death phenotype.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004755-g008: Overexpression of MIR9863a and MIR9863b attenuates Mla1-triggered disease resistance and cell-death signaling.(A) Transient gene expression of MIR9863a, MIR9863c and MIR9863b in barley near-isogenic line containing Mla1 or Mla10. Empty vector or plasmids expressing indicated MIRNA precursors were delivered into leaf epidermal cells of indicated barley lines through particle bombardment, and spores of indicated avirulence Bgh isolates were inoculated 36 hrs post bombardment. Haustorial index shown are the percentage of cells that developed fungal haustoria in all transformed cells. (B) Transient gene expression assays in Golden promise (GP), a barley line susceptible to both Bgh K1 and Bgh A6. Indicated construct expressing MIR9863b was delivered alone or with Mla1-mYFP or Mla10-mYFP construct into GP leaf epidermal cells, and spores of Bgh K1 or Bgh A6 were inoculated at 4 hrs post bombardment. Error bars in (A) and (B) represent standard error (SE) from at least three replicates, and letters (a–c) above the bars represent groups with significant differences [p<0.05, Tukey's honest significant difference (HSD) test]. (C) to (D) Analysis of cell-death induction upon co-expressing Mla1 or Mla10 with MIR9863a and MIR9863c. The indicated constructs were expressed in N. benthamiana leaves by Agro-infiltration, and cell death was visualized by trypan blue staining at 48 hours-post-Agro-infiltration (hpai). Red circle indicates visible cell-death phenotype.
Mentions: To investigate the role of miR9863 members in MLA-mediated disease resistance to Bgh, we used a well-established single-cell transient assay [77], [85]. Plasmid constructs of miRNA precursor are co-delivered with a β-glucuronidase (GUS) reporter into barley epidermal cells by particle bombardment, and then the haustorial index (HI%) is scored to assess the frequency of fungal haustoria in transformed cells upon fungal inoculation (Fig. 8A and 8B). First, we respectively delivered plasmids of EV, MIR9863a, MIR9863c, or MIR9863b into near-isogenic barley line P01 (Mla1) and then inoculated conidiospores of Bgh K1 (AVRa1) to activate MLA1-mediated immune responses (Fig. 8A, left panel). We observed a low HI% of ∼3% in leaves receiving EV control, likely due to MLA1-triggered immunity against Bgh K1 (Fig. 8A, bar 1), whereas in leaves expressing MIR9863a or MIR9863b, we observed significantly increased HI% to ∼22% or 15% (Fig. 8A, bars 2 and 4), indicating MLA1-triggered immunity was compromised by the expression of miR9863a or miR9863b.1/b.2. The HI% was similar in P01 leaves expressing MIR9863c to that in the EV control (Fig. 8A, bar 3), consistent with that miR9863c does not interact with the Mla1 allele (Fig. 2C). Further, plasmids of MIR9863a, MIR9863c and MIR9863b were respectively delivered into leaves of near-isogenic barley line P09 (Mla10) and inoculated with Bgh isolate A6 (AVRa10) (Fig. 8A, right panel), and we observed comparable HI% of ∼6% to 7% in leaves expressing EV or respective miR9863 precursors (Fig. 8A, bars 5 to 8), suggesting that expression of any miR9863 members had no effect on Mla10-mediated disease resistance to Bgh, consistent with that Mla members of group III are not miR9863 targets (Fig. 3B). Taken together, overexpression of miR9863a and miR9863b.1/b.2 specifically attenuates MLA1 but not MLA10-mediated disease resistance against Bgh in 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
Related in: MedlinePlus