The Microrchidia (MORC) family proteins are important nuclear regulators in both animals and plants with critical roles in epigenetic gene silencing and genome stabilization. In the crop plant barley (Hordeum vulgare), seven MORC gene family members have been described. While barley HvMORC1 has been functionally characterized, very little information is available about other HvMORC paralogs. In this study, we elucidate the role of HvMORC6a and its potential interactors in regulating plant immunity via analysis of CRISPR/SpCas9-mediated single and double knockout (dKO) mutants, hvmorc1 (previously generated and characterized by our group), hvmorc6a, and hvmorc1/6a. For generation of hvmorc1/6a, we utilized two different strategies: (i) successive Agrobacterium-mediated transformation of homozygous single mutants, hvmorc1 and hvmorc6a, with the respective second construct, and (ii) simultaneous transformation with both hvmorc1 and hvmorc6a CRISPR/SpCas9 constructs. Total mutation efficiency in transformed homozygous single mutants ranged from 80 to 90%, while upon simultaneous transformation, SpCas9-induced mutation in both HvMORC1 and HvMORC6a genes was observed in 58% of T0 plants. Subsequent infection assays showed that HvMORC6a covers a key role in resistance to biotrophic (Blumeria graminis) and necrotrophic (Fusarium graminearum) plant pathogenic fungi, where the dKO hvmorc1/6a showed the strongest resistant phenotype. Consistent with this, the dKO showed highest levels of basal PR gene expression and derepression of TEs. Finally, we demonstrate that HvMORC1 and HvMORC6a form distinct nucleocytoplasmic homo-/heteromers with other HvMORCs and interact with components of the RNA-directed DNA methylation (RdDM) pathway, further substantiating that MORC proteins are involved in the regulation of TEs in barley.

中文翻译：

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CRISPR/SpCas9 介导的大麦 Microrchidia MORC1 和 MORC6a 双敲除揭示了它们在植物免疫、转录基因沉默和植物生长中的重要作用

Microrchidia (MORC) 家族蛋白是动物和植物中重要的核调节因子，在表观遗传基因沉默和基因组稳定中起关键作用。在农作物大麦（Hordeum vulgare）中，已经描述了七个MORC基因家族成员。虽然大麦HvMORC1已在功能上进行了表征，但有关其他HvMORC旁系同源物的信息很少。在这项研究中，我们通过分析 CRISPR/ Sp Cas9 介导的单敲除和双敲除 (dKO) 突变体hvmorc1（之前由我们小组生成和表征），阐明HvMORC6a及其潜在相互作用因子在调节植物免疫中的作用，hvmorc6a和hvmorc1/6a。对于hvmorc1/6a的生成，我们使用了两种不同的策略：（i）连续农杆菌介导的纯合单突变体hvmorc1和hvmorc6a的转化，以及各自的第二个构建体，以及（ii）同时转化hvmorc1和hvmorc6a CRISPR/ Sp Cas9 构建体。转化的纯合单突变体的总突变效率在 80% 到 90% 之间，而在同时转化时，Sp Cas9 诱导的HvMORC1和HvMORC6a突变在 58% 的 T0 植物中观察到基因。随后的感染检测表明，Hv MORC6a 在对生物营养性（禾本科）和坏死性营养性（禾谷镰刀菌）植物病原真菌的抗性中发挥了关键作用，其中 dKO hvmorc1 /6a表现出最强的抗性表型。与此一致，dKO 表现出最高水平的基础PR基因表达和TEs的去抑制。最后，我们证明Hv MORC1 和Hv MORC6a 与其他Hv形成不同的核质同源/异聚体MORCs 并与 RNA 指导的 DNA 甲基化 (RdDM) 途径的成分相互作用，进一步证实 MORC 蛋白参与调节大麦中的 TEs。