miR477 acts as a negative regulator in tea plant immunity against Pseudopestalotiopsis infection by repressing the expression of its target gene PAL. MicroRNA (miRNA)-mediated post-transcriptional regulation plays a fundamental role in various plant physiological processes, including responses to pathogens. Our previous research revealed that miR477 might be involved in the tea plant–Pseudopestalotiopsis interaction (data not shown). In the present study, the accumulation of miR477 significantly decreased in tea plants during Pseudopestalotiopsis species infection. Using miRNA and degradome data sets, the targeting of phenylalanine ammonia-lyase (PAL) by miR477 was validated by 5′ RLM-RACE. GUS assay showed that the expression of PAL was post-transcriptionally regulated by miR477 and silenced by mRNA cleavage. A negative correlation between the expression of miR477 and PAL was found in tea plants infected by the pathogen. The transgenic lines overexpressing Csn-miR477 exhibited increased susceptibility to Pseudopestalotiopsis species, which was associated with reduced expression of PAL during infection. The degree of severity of the leaf lesions and the results of trypan blue staining showed that the plants overexpressing Csn-miR477 exhibited more severe damage upon pathogen infection than wild-type plants. In addition, more H2O2 and O2−, higher malondialdehyde (MDA) contents and less superoxide dismutase (SOD) and peroxidase (POD) activities were detected in the transgenic plants than in the wild-type plants after inoculation with Pseudopestalotiopsis species. Taken together, our results implied that Csn-miR477 might act as a negative regulator in pathogen-infected tea plants by inhibiting the expression of its target, PAL, and that Csn-miR477 is a candidate miRNA for improving the adaptation of tea plant to disease.

中文翻译：

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miR477靶向苯丙氨酸解氨酶基因并增强茶树（Camellia sinensis）在拟拟盘菌属物种感染期间对疾病的易感性


miR477 通过抑制其靶基因 PAL 的表达，在茶树对拟拟盘拟盘菌感染的免疫中发挥负调节作用。 MicroRNA (miRNA) 介导的转录后调控在各种植物生理过程中发挥着重要作用，包括对病原体的反应。我们之前的研究表明，miR477 可能参与茶树与拟盘拟盘菌的相互作用（数据未显示）。在本研究中，茶树中 miR477 的积累在拟拟盘菌感染期间显着减少。使用 miRNA 和降解组数据集，通过 5' RLM-RACE 验证了 miR477 对苯丙氨酸解氨酶 (PAL) 的靶向作用。 GUS 测定显示 PAL 的表达受到 miR477 的转录后调节，并通过 mRNA 切割沉默。在受病原菌感染的茶树中发现 miR477 和 PAL 的表达呈负相关。过表达 Csn-miR477 的转基因株系表现出对拟拟盘拟盘菌属物种的敏感性增加，这与感染期间 PAL 表达的减少有关。叶片病变的严重程度和台盼蓝染色结果表明，过表达Csn-miR477的植物在病原体感染时表现出比野生型植物更严重的损害。此外，与野生型植物相比，接种拟盘拟盘菌后，转基因植物中检测到更多的H2O2和O2−、更高的丙二醛（MDA）含量以及更低的超氧化物歧化酶（SOD）和过氧化物酶（POD）活性。 综上所述，我们的结果表明，Csn-miR477可能通过抑制其靶标PAL的表达，在病原菌感染的茶树中充当负调节因子，并且Csn-miR477是改善茶树对疾病的适应的候选miRNA 。