A pathogen-responsive gene cluster for the production of highly modified fatty acids in tomato

In response to biotic stress, plants produce suites of highly modified fatty acids that bear unusual chemical functionality. Despite their chemical complexity and proposed roles in pathogen defense, little is known about the biosynthesis of these decorated fatty acids in plants. Falcarindiol is a prototypical acetylenic lipid present in carrot, tomato, and celery that inhibits growth of fungi and human cancer cell lines. Using a combination of untargeted metabolomics and RNA sequencing, we discovered a biosynthetic gene cluster in tomato (Solanum lycopersicum) that is required for the production of falcarindiol in response to an adapted fungal pathogen, Cladosporium fulvum. By reconstituting the initial biosynthetic steps in a heterologous host (Nicotiana benthamiana) and generating transgenic pathway mutants in tomato, we demonstrate a direct role for three genes in the cluster in falcarindiol biosynthesis. This work reveals a mechanism by which plants sculpt their lipid pool in response to pathogens, and provides critical insight into the complex biochemistry of alkynyl lipid production. Tomato (Solanum lycopersicum VF36, 4.5 week-old plants) were treated with a panel of fungal elicitors via leaf infiltration: chitin (Ct, 0.5 mg/ml), Malassezia restricta (Mz, OD600 = 0.5), Cladosporium fulvum (Cf, OD600 = 1.0), and a panel of bacterial elicitors: flg22 (Fg, 1 μM), Staphylococcus epidermidis (Sc, OD600 = 1.0), Propionibacterium acnes (Pb, OD600 = 1.0) and Xanthomonas euvesicatoria (Xe, OD600 = 0.2), and water (mock, control). After pathogen inoculation, leaves (one leaf from three individual plants per treatment, n=3) were collected at 12, 24, and 48 hours post-inoculation using razor, flash frozen by liquid nitrogen, and stored at -80 °C for later use in gene profiling and RNA-Sequencing.

面对生物胁迫时，植物会合成一系列带有特殊化学官能团的高度修饰脂肪酸。尽管这类修饰脂肪酸化学结构复杂，且被认为在病原菌防御中发挥作用，但学界对植物体内这类带修饰的脂肪酸的生物合成机制仍知之甚少。
镰叶芹醇（falcarindiol）是一类典型的炔烃类脂质，存在于胡萝卜、番茄和芹菜中，可抑制真菌及人类癌细胞系的增殖。本研究结合非靶向代谢组学与RNA测序技术，在番茄（Solanum lycopersicum）中发现了一个生物合成基因簇，该基因簇对于番茄在适应型真菌病原菌黄枝孢菌（Cladosporium fulvum）侵染时合成镰叶芹醇是必需的。
通过在异源宿主本氏烟（Nicotiana benthamiana）中重构该通路的初始生物合成步骤，并在番茄中构建该通路的转基因突变体，本研究证实了该基因簇中的三个基因直接参与镰叶芹醇的生物合成。
本研究揭示了植物响应病原菌侵染时重塑自身脂质组的机制，并为炔烃类脂质合成的复杂生化过程提供了关键见解。
本研究采用叶片注射法，对4.5周龄的番茄（Solanum lycopersicum VF36）植株施加一系列激发子处理：包括真菌激发子：几丁质（chitin，Ct，0.5 mg/ml）、限制性马拉色菌（Malassezia restricta，Mz，OD600=0.5）、黄枝孢菌（Cladosporium fulvum，Cf，OD600=1.0）；以及细菌激发子：鞭毛蛋白22肽（flg22，Fg，1 μM）、表皮葡萄球菌（Staphylococcus epidermidis，Sc，OD600=1.0）、痤疮丙酸杆菌（Propionibacterium acnes，Pb，OD600=1.0）、蚀纹黄单胞菌（Xanthomonas euvesicatoria，Xe，OD600=0.2），并设置无菌水处理组作为空白对照（mock，对照）。
病原菌接种后，分别于接种后12、24、48小时采集叶片（每个处理组取3株独立植株的1片叶片，n=3），使用无菌手术刀切割样品，经液氮快速冷冻后置于-80℃冰箱保存，用于后续基因表达分析与RNA测序实验。