Department of Genetics, University of Valencia, Dr. Moliner 50, 46100, Burjassot, Valencia, Spain.
Plant Physiology Area, Biochemistry and Biotechnology Laboratory, Department CAMN, University Jaume I, 12071, Castellón, Spain.
Plant Cell Rep. 2018 Jan;37(1):167-176. doi: 10.1007/s00299-017-2219-8. Epub 2017 Oct 27.
SlyWRKY75: gene expression was induced in response to biotic stresses, especially in Botrytis cinerea-infected tomato plants, in which Sly-miR1127-3p is a putative SlyWRKY75 regulator and epigenetic marks were detected. WRKY75 transcription factor involved in Pi homeostasis was recently found also induced in defense against necrotrophic pathogens. In this study, we analyzed by RT-qPCR the expression of SlyWRKY75 gene in tomato plants in response to abiotic stresses (drought or heat) and biotic stresses (Colorado potato beetle larvae infestation, Pseudomonas syringae or Botrytis cinerea infection) being only differentially expressed following biotic stresses, especially upon B. cinerea infection (55-fold induction). JA and JA-Ile levels were significantly increased in tomato plants under biotic stresses compared with control plants, indicating that SlyWRKY75 might be a transcriptional regulator of the JA pathway. The contribution of miRNAs and epigenetic molecular mechanisms to the regulation of this gene in B. cinerea-infected tomato plants was explored. We identified a putative Sly-miR1127-3p miRNA predicted to bind the intronic region of the SlyWRKY75 genomic sequence. Sly-miR1127-3p miRNA was repressed in infected plants (0.4-fold) supporting that it might act as an epigenetic regulation factor of SlyWRKY75 gene expression rather than via the post-transcriptional mechanisms of canonical miRNAs. It has been proposed that certain miRNAs can mediate DNA methylation in the plant nucleus broadening miRNA functions with transcriptional gene silencing by targeting intron-containing pre-mRNAs. Histone modifications analysis by chromatin immunoprecipitation (ChIP) demonstrated the presence of the activator histone modification H3K4me3 on SlyWRKY75 transcription start site and gene body. The induction of this gene in response to B. cinerea correlates with the presence of an activator mark. Thus, miRNAs and chromatin modifications might cooperate as epigenetic factors to modulate SlyWRKY75 gene expression.
SlyWRKY75：基因表达受生物胁迫诱导，特别是在感染灰葡萄孢的番茄植株中，Sly-miR1127-3p 是 SlyWRKY75 的假定调节因子，并且检测到了表观遗传标记。WRKY75 转录因子参与磷稳态，最近也被发现参与对坏死型病原体的防御。在这项研究中，我们通过 RT-qPCR 分析了番茄植株在非生物胁迫（干旱或热胁迫）和生物胁迫（科罗拉多马铃薯甲虫幼虫侵袭、丁香假单胞菌或灰葡萄孢感染）下 SlyWRKY75 基因的表达，该基因仅在生物胁迫下差异表达，特别是在感染灰葡萄孢后（诱导 55 倍）。与对照植株相比，生物胁迫下番茄植株中的 JA 和 JA-Ile 水平显著升高，表明 SlyWRKY75 可能是 JA 途径的转录调节因子。探讨了 miRNA 和表观遗传分子机制对感染灰葡萄孢的番茄植株中该基因的调控作用。我们鉴定了一个推定的 Sly-miR1127-3p miRNA，该 miRNA 预测与 SlyWRKY75 基因组序列的内含子区域结合。在感染的植物中，Sly-miR1127-3p miRNA 被抑制（下调 0.4 倍），这表明它可能作为 SlyWRKY75 基因表达的表观遗传调控因子，而不是通过经典 miRNA 的转录后机制。已经提出某些 miRNA 可以介导植物细胞核中的 DNA 甲基化，通过靶向包含内含子的前体 mRNA 来扩大 miRNA 的功能并实现转录基因沉默。染色质免疫沉淀（ChIP）的组蛋白修饰分析表明，在 SlyWRKY75 转录起始位点和基因体上存在激活组蛋白修饰 H3K4me3。该基因在响应灰葡萄孢时的诱导与激活标记的存在相关。因此，miRNA 和染色质修饰可能作为表观遗传因子协同作用来调节 SlyWRKY75 基因表达。
