The miR9863 Family Regulates Distinct <i>Mla</i> Alleles in Barley to Attenuate NLR Receptor-Triggered Disease Resistance and Cell-Death Signaling

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.大麦（Hordeum vulgare L.）的Mla等位基因编码卷曲螺旋（coiled-coil, CC）、核苷酸结合富亮氨酸重复（nucleotide binding leucine-rich repeat, NB-LRR）受体，此类受体可触发针对禾本科布氏白粉菌大麦专化型（Blumeria graminis f. sp. hordei, Bgh）的小种特异性免疫应答。目前对于禾本科物种中Mla或NB-LRR基因的转录后调控机制尚不明确。 微小RNA（microRNA, miRNA）家族miR9863包含4个成员，可差异性调控大麦中不同的Mla等位基因。本研究证实，miR9863家族成员可介导大麦内Mla1转录本的剪切，并在异源本氏烟（Nicotiana benthamiana）表达体系中阻断或降低MLA1蛋白的积累。 调控特异性由成熟miR9863家族成员中独特的单核苷酸多态性（single-nucleotide-polymorphism, SNP）位点变异，以及Mla基因miR9863结合位点内的两个SNP位点共同决定，该变异将上述等位基因划分为3个类群。 进一步研究表明，长度为22核苷酸（nt）的miR9863可触发21 nt相位化小干扰RNA（phased siRNAs, phasiRNAs）的生物发生，此类小RNA共同构成前馈调控网络，以抑制第I类Mla等位基因的表达。 过表达miR9863家族成员可特异性减弱MLA1介导的抗病性与细胞死亡信号通路，但不影响MLA10触发的同类信号过程。 本研究提出，miR9863家族在抑制大麦中一类MLA免疫受体触发的免疫应答信号通路中发挥关键作用。