Determining the role of lipid droplet-associated triacylglycerol lipases under pathogen infection

脂滴（ld）是一种特殊的细胞器，由磷脂单层中的蛋白质组成，其周围主要是三酰基甘油（TAG）疏水核心。在植物中，它们不仅存在于种子和花粉中，也存在于营养组织中，它们在压力下积聚在那里。目前已知与ld相关的蛋白家族很少。关键的兴趣一直是三酰甘油脂肪酶参与TAG的降解，特别是在种子萌发过程中。在这个过程中的一个主要参与者是TAG脂肪酶SUGAR DEPENDENT 1，它对拟南芥幼苗的建立至关重要。然而，在拟南芥中还有许多脂肪酶的功能有待研究。我们一直在研究一个与ld相关的拟南芥TAG脂肪酶家族（已被烟草花粉管中的瞬时表达和拟南芥内在启动子下的部分稳定表达证实）。对于5个成员中的4个，我们能够显示tag降解活性。在生理水平上，OBL1对花粉管生长很重要，而OBL3似乎在病原体防御中起作用，因为突变体对丁香假单胞菌pt番茄DC3000 Delta avrPto/avrPtoB感染更敏感，这一作用可以通过在其原生启动子下重新引入OBL3来补充。OBL3的功能可能是释放脂肪酸，用于合成氧化脂类及其下游产物，因为氧化脂类可以作为植物alexine，而番茄中OBL1的同源物参与氧化脂类衍生挥发物的生成。此外，产生氧化脂的酶也与ld有关。我们希望进一步研究obl在病原体防御中的作用。obl3突变体将与野生型和两个互补系进行比较，以确定其对多种病原体的易感性。通过非靶向LC-MS（液相色谱-质谱联用）方法，记录了在丁香假单胞菌攻击期间，obl3突变体和oblQ突变体与野生型和两个互补系的代谢变化。然后，我们想通过测试obl下游合成的化合物对病原体生长的直接影响来证明它们作为植物alexine的作用。我们还想通过研究在OBL3启动子下表达与荧光团mVenus或GUS偶联的OBL3的细胞系，来证明OBL3在感染下改变其表达模式。最后，我们还想确定OBL3的交互伙伴。

Lipid droplets (LDs) are special organelles that consist of proteins in a phospholipid monolayer surrounding a hydrophobic core of mostly triacylglycerol (TAG). In plants, they occur not only in seeds and pollen but also in vegetative tissues, where they accumulate under stress. Only few protein families are known that associate with LDs. Of key interest have always been triacylglycerol lipases involved in the degradation of TAG especially during seed germination. One main player in this process is the TAG lipase SUGAR DEPENDENT 1, that is crucial for seedling establishment in Arabidopsis. However, there are many further lipases predicted in Arabidopsis whose function remains to be investigated. We have been working on a family of Arabidopsis TAG lipases that are associated with LDs (as confirmed by transient expression in tobacco pollen tubes and in part by stable expression under the intrinsic promoter in A. thaliana). For four of the five members we were able to show TAG-degradation activity.On the physiological level, OBL1 is important for pollen tube growth, while OBL3 appears to play a role in pathogen defense, as a mutant is more susceptible to Pseudomonas syringae pt tomato DC3000 Delta avrPto/avrPtoB infection, an effect that could be complemented by reintroducing OBL3 under its native promoter. The function of OBL3 could be to release fatty acids for the synthesis of oxylipins (oxidized fatty acids) and its downstream products, as oxylipins can act as phytoalexines, and as a homolog of OBL1 in tomato is involved in the generation of oxylipin-derived volatiles. Also, oxylipin-producing enzymes are associated with LDs.We want to further investigate the role of OBLs in pathogen defense. The obl3 mutant will be compared to the wild type and two complemented lines in respect to its susceptibility to a variety of pathogens.By an untargeted LC-MS (liquid chromatography coupled to mass spectrometry) approach, the metabolic changes during pathogen attack with Pseudomonas syringae will be recorded in the obl3 mutant and the oblQ mutant in comparison to the wild type and two complemented lines.We then want to show that the compounds synthesized downstream of the OBLs act as phytoalexines by testing them for direct effects on the growth of pathogens.We also want to demonstrate that OBL3 changes its expression pattern under infection by investigating lines expressing OBL3 coupled to the fluorophore mVenus, or GUS under the promoter of OBL3.At last, we also want to identify interaction partners of OBL3.