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

脂滴(LDS)是一种特殊的细胞器，由以三酰甘油(TAG)为主要疏水核心的磷脂单分子层中的蛋白质组成。在植物中，它们不仅存在于种子和花粉中，还存在于营养组织中，在胁迫下积累在营养组织中。目前已知的与腰椎间盘突出症相关的蛋白质家族很少。人们最感兴趣的一直是三酰甘油脂肪酶，它参与TAG的降解，特别是在种子萌发期间。在这一过程中的一个主要角色是标签脂肪酶糖依赖1，它对拟南芥的幼苗建立至关重要。然而，在拟南芥中预测到了许多进一步的脂肪酶，其功能仍有待研究。我们一直在研究与LDS相关的拟南芥标签脂肪酶家族(通过在烟草花粉管中的瞬时表达以及在A.thaliana固有启动子下的稳定表达证实了这一点)。在生理水平上，OBL1对花粉管的生长起重要作用，而OBL3似乎在病原菌防御中起作用，因为突变体对番茄DC3000 Delta avrPto/avrPtoB更敏感，这一效应可以通过在其天然启动子下重新导入OBL3来补充。OBL3的功能可能是释放脂肪酸，以合成氧化脂肪(氧化脂肪酸)及其下游产物，因为氧化脂肪可以作为植物防护素，并且作为番茄中OBL1的同系物，参与了氧化脂肪衍生挥发物的产生。此外，氧脂产生酶与LD有关。我们希望进一步研究OBLS在病原体防御中的作用。通过非靶向LC-MS(液-质联用)方法，我们将记录ob3突变体和obq突变体在病原菌攻击过程中的代谢变化，并与野生型和两个互补品系进行比较。我们想要通过测试OBL3下游合成的化合物对病原菌的生长的直接影响来证明OBL3作为植物外膜蛋白的作用。我们还想通过研究表达OBL3的与浮游生物连接的品系来证明OBL3在感染下改变了它的表达模式或OBL3启动者下的GUS。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.