Protoplast transient expression is a powerful strategy for gene functional characterization, especially in biochemical mechanism studies. We herein developed a highly efficient transient expression system for apple protoplasts. The abilities of the Arabidopsis thaliana and Malus domestica ubiquitin-10 (AtUBQ10 and MdUBQ10) promoters to drive the expression of multiple genes were compared with that of the CaMV 35S promoter, and the results revealed that the AtUBQ10 and MdUBQ10 promoters were more efficient in apple protoplasts. With this system, we demonstrated that active AtMKK7ac could activate MAPK6/3/4 signaling cascades, which further regulated MdWRKY33 phosphorylation and stability in apple. Furthermore, the ligand-induced interaction between the immune receptor AtFLS2 and the coreceptor AtBAK1 was reconstituted in apple protoplasts. We also found that the stability of the bacterial effector AvrRpt2 was regulated by feedback involving auxin and the immune regulator RIN4. The system established herein will serve as a useful tool for the molecular and biochemical analyses of apple genes.

中文翻译：

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开发有效的启动子，驱动苹果原生质体中基因的有效表达

原生质体瞬时表达是基因功能表征的有力策略，特别是在生化机制研究中。我们在此开发了一种高效的苹果原生质体瞬时表达系统。的能力拟南芥和Malus domestica ubiquitin-10(AtUBQ10和MdUBQ10) 将驱动多个基因表达的启动子与CaMV 35S启动子，结果显示，AtUBQ10和MdUBQ10启动子在苹果原生质体中更有效。通过该系统，我们证明了活性 AtMKK7ac 可以激活 MAPK6/3/4 信号级联，从而进一步调节苹果中 MdWRKY33 的磷酸化和稳定性。此外，配体诱导的免疫受体 AtFLS2 和辅助受体 AtBAK1 之间的相互作用在苹果原生质体中被重建。我们还发现细菌效应器 AvrRpt2 的稳定性受到涉及生长素和免疫调节剂 RIN4 的反馈的调节。本文建立的系统将作为苹果基因分子和生化分析的有用工具。