Data from: Tomato Sl3-MMP, a member of the Matrix metalloproteinase family, is required for disease resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000

Background: Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases. MMPs have been characterized in detail in mammals and shown to play key roles in many physiological and pathological processes. Although MMPs in some plant species have been identified, the function of MMPs in biotic stress responses remains elusive. Results: A total of five MMP genes were identified in tomato genome. qRT-PCR analysis revealed that expression of Sl-MMP genes was induced with distinct patterns by infection of Botrytis cinerea and Pseudomonas syringae pv. tomato (Pst) DC3000 and by treatment with defense-related hormones such as salicylic acid, jasmonic acid and ethylene precursor 1-amino cyclopropane-1-carboxylic acid. Virus-induced gene silencing (VIGS)-based knockdown of individual Sl-MMPs and disease assays indicated that silencing of Sl3-MMP resulted in reduced resistance to B. cinerea and Pst DC3000, whereas silencing of other four Sl-MMPs did not affect the disease resistance against these two pathogens. The Sl3-MMP-silenced tomato plants responded with increased accumulation of reactive oxygen species and alerted expression of defense genes after infection of B. cinerea. Transient expression of Sl3-MMP in leaves of Nicotiana benthamiana led to an enhanced resistance to B. cinerea and upregulated expression of defense-related genes. Biochemical assays revealed that the recombinant mature Sl3-MMP protein had proteolytic activities in vitro with distinct preferences for specificity of cleavage sites. The Sl3-MMP protein was targeted onto the plasma membrane of plant cells when transiently expressed in onion epidermal cells. Conclusion: VIGS-based knockdown of Sl3-MMP expression in tomato and gain-of-function transient expression of Sl3-MMP in N. benthamiana demonstrate that Sl3-MMP functions as a positive regulator of defense response against B. cinerea and Pst DC3000.

研究背景：基质金属蛋白酶（matrix metalloproteinases，MMPs）是一类锌依赖型内肽酶家族。该家族蛋白酶已在哺乳动物中得到详尽的功能表征，并被证实于诸多生理与病理过程中发挥关键调控作用。尽管已有部分植物物种中的MMPs被成功鉴定，但此类蛋白酶在生物胁迫响应中的具体功能仍尚不明确。实验结果：本研究在番茄基因组中共鉴定出5个MMP基因。实时定量聚合酶链反应（qRT-PCR）分析显示，灰葡萄孢（Botrytis cinerea）、丁香假单胞菌番茄致病变种（Pseudomonas syringae pv. tomato，Pst）DC3000侵染，以及水杨酸、茉莉酸、乙烯前体1-氨基环丙烷-1-羧酸（1-amino cyclopropane-1-carboxylic acid）等防卫相关激素处理，均可通过独特的表达模式诱导Sl-MMP基因的表达。基于病毒诱导基因沉默（virus-induced gene silencing，VIGS）的单个Sl-MMP基因敲降实验与抗病性测定结果表明，沉默Sl3-MMP会显著降低番茄对灰葡萄孢和Pst DC3000的抗性，而沉默其余4个Sl-MMP基因则不会对植株的这两种病原菌抗性产生影响。Sl3-MMP沉默的番茄植株在被灰葡萄孢侵染后，活性氧积累量显著升高，且防卫基因的表达模式发生改变。本氏烟草（Nicotiana benthamiana）叶片中瞬时过表达Sl3-MMP，可增强植株对灰葡萄孢的抗性，并上调防卫相关基因的表达水平。生化实验结果显示，重组成熟Sl3-MMP蛋白在体外具有蛋白水解活性，且对切割位点的特异性具有显著偏好性。在洋葱表皮细胞中瞬时表达Sl3-MMP蛋白时，该蛋白可被靶向定位于植物细胞的质膜。研究结论：通过VIGS技术敲降番茄中Sl3-MMP的表达，以及在本氏烟草中开展Sl3-MMP的功能获得性瞬时过表达实验，均证实Sl3-MMP是介导番茄对抗灰葡萄孢与Pst DC3000的防卫反应的正调控因子。