REL3介导的 TAS3 ta-siRNA合成途径调控百脉根根瘤形成的分子机理

The trans-acting RNAs ( ta-siRNAs) are a class of endogenous interfering RNAs(siRNAs) in plants, which are required for development of lateral organs, including leaf pattern, flower differentiation and lateral root growth. Legumes can develop two types of lateral organs from their primary roots, lateral roots and nitrogen-fixing nodules, the latter develop in response to symbiotic interactions with rhizobia. The formation of nitrogen-fixing nodules endows legumes the unique trait of nitrogen-fixation. REL3, a key component of TAS3 ta-siRNA biogenesis pathway in Lotus japonicus, was mutated and the corresponding mutant produces defects in compound leaf and flower. Analysis of symbiotic phenotypes showed that the rel3 mutant develops normal functional nitrogen-fixing nodules, but the nodule number is greatly reduced and the ratio of nodulation zone is significantly decreased in rel3. Moreover, rel3 showed altered responses to auxin and polar auxin inhibitors during nodulation. Also, rel3 has enhanced root-specific ethylene sensitivity, and can restore nodulation level of the wild type with ethylene inhibitors AVG and Ag+ treatment. Based on the current results, we intensively dissect the molecular mechanism on which the REL3 mediated TAS3 ta-siRNA synthetic pathway controls nodulation through integrating auxin and ethylene signaling by techniques including deep transcriptomics sequencing (RNA-seq)and yeast two-hybrization, etc. This proposal not only provides new insight into the symbiotic interaction but also contributes to better understanding of the regulatory mechanism of ta-siRNA.

许多研究证据表明ta-siRNA合成途径参与植物侧生器官叶、花和侧根发育的调控，然而其是否参与了豆科植物根部独特的侧生器官-共生根瘤的发育仍处于研究探索阶段。本项目利用豆科植物百脉根TAS3 ta-siRNA合成途径中的关键组分REL3的突变体探索ta-siRNA对于根瘤发育的调控作用。我们发现突变体rel3可形成正常功能的固氮根瘤，但结瘤数显著减少,结瘤区比率也明显降低。在结瘤过程中，rel3对生长素和生长素抑制剂的响应发生了改变。而且，rel3也显示出增强的根特异的乙烯敏感性响应，乙烯抑制剂AVG和Ag+处理可使rel3的结瘤数恢复至野生型水平。在这些研究基础上，本项目拟以转录组测序（RNA-seq)和酵母双杂交等技术解析TAS3 ta-siRNA途径整合生长素和乙烯信号从而协调控制结瘤过程的分子机理。该项目不仅为共生互作机理提供新的见解，也有助于完善对ta-siRNA调节机理的认识。

ta-siRNAs (trans-acting RNAs)是植物中一类重要的内源干扰RNAs，参与植物侧生器官叶、花和侧根发育的调控，但不清楚其是否参与共生根瘤的发育。本研究发现豆科植物百脉根TAS3 ta-siRNA合成途径中的关键组分REL3的突变体rel3可形成正常功能的固氮根瘤，但结瘤数显著减少,结瘤区比率也明显降低。在结瘤过程中，rel3对生长素和生长素抑制剂的响应发生了改变，也显示出根特异的乙烯敏感性增强的响应，乙烯抑制剂AVG和Ag+处理可使rel3的结瘤数恢复至野生型水平。基于RNA-seq转录组数据和qPCR检测证实TAS3 ta-siRNA负调节靶标基因ARF3a、ARF3b与ARF4的表达。突变体arf4-1的结瘤表型表明ARF4负调控结瘤过程，rel3与arf4-1双突体的结瘤结果，显示 arf4-1能部分互补rel3的根瘤数目减少表型。因此，从遗传上明确了REL3部分地通过TAS3 ta-siRNA对ARF4的沉默机制正调控结瘤过程。rel3和arf4-1突变体的结瘤过程对GA3处理分别表现不敏感和超敏感，表明REL3与ARF4分别正调控和负调控GA3对结瘤的抑制作用。酵母双杂交实验证实ARF4与DELLA1以及DELLA2之间存在相互作用，烟草叶片中共表达揭示ARF4与DELLA1分别正调控和负调控对方的积累。过表达GA失活形式的DELLA(della1△17与della2△18)的结瘤实验显示降低根瘤数，且della2△18过表达对GA3或PAC处理更敏感，表明过量积累DELLA蛋白能促进GA响应对结瘤的抑制作用。NAA处理和ARF4过表达与DR5::GUS的共表达实验，表明ARF4受到Auxin的诱导表达，并且其蛋白能够抑制Auxin响应。因此，ARF4通过自我负调节方式调节Auxin响应。总之，ARF4蛋白一方面可以通过自我负反馈方式抑制Auxin响应对结瘤的促进作用，另一方面可以通过促进DELLA蛋白的积累增强GA响应对结瘤的抑制作用。本研究第一次提出了 TAS3 ta-siRNA参与结瘤过程的证据，明确了保守的tasiR-ARF 途径在根瘤形成中的核心作用，指出TAS3 ta-siRNA通过整合生长素、乙烯和赤霉素信号协同控制根瘤菌侵染和根瘤器官起始。这些结果为共生互作机理提供新的见解，也促进了对ta-siRNA调节机理的认识。