Molecular Biology of Pathogen-Induced Programmed Cell Death in Model Legume Medicago Truncatula

Plants have elaborated efficient mechanisms to survive in the changing environmental conditions, particularly during pathogen infection. The early plant response to the microbial pathogens is often accompanied by the induction of reactive oxygen species (ROS) and an oxidative burst which leads to rapid cell death in and around the initial infection site, a reaction known as the hypersensitive response (HR). Besides, the induction of a programmed cell death (PCD) in plants is assumed to be a common response to many different types of biotic stress. There is now compelling evidence that the mitochondrion integrates diverse cellular stress signals and initiates the death execution pathway in animals; on the flip-side a similar involvement for mitochondria in regulating PCD in plants has so far received very little attention. In this research study, we focused on the cellular responses in M. truncatula inoculated with zoospores from the oomycete A. euteiches, which is a severe root pathogen for legume crop plants. Using the model legume as a platform and A. euteiches to induce HR, mechanisms taking place in the plant cells as a response to pathogen infection particularly in the mitochondria, were studied via proteomic tools. The most crucial part of establishing an in vitro inoculation system was to ensure contact between cells and zoospores. It has been noticed under microscopic studies that zoospores are in contact with plant cells even under in vitro conditions. As expected, inoculated cells showed a clear reduction of viability and a reduction in mass as compared to the mock control. Notably, at 10 hpi & at 20 hpi cell viability went down to 72% and 39% respectively, while in the mock control cell viability only dropped to 88% and 70%. H₂O₂ oxidative burst measurement assays with A. euteiches zoospores at 0 h, 10 h, and 20 h induced moderate oxidative burst reactions. Maximal average values were 3.0 μM (0 h), 2.4 μM (10 h) and 1.8 μM (20 h) H₂O₂ production. Interestingly, double inoculation (at ‘0 h &10 h’ and at ‘0 h & 20 h’) with zoospores showed less than 1.0 μM H₂O₂ production. At 24 hpi, purification of mitochondria by density gradient centrifugation revealed an additional sub-fraction was positioned just below 40% of Percoll (the mitochondrial are normally are of 23-40% Percoll). Notably, super complex I+III₂ was observed absent while complex II, cyt c 1-1 & cyt c 1-2, dimeric complex III₂, complex IV, and porin protein complexes were less abundant in BN gels of the mitochondrial sub-fraction as compared to the gels of expected fractions. As expected, porin complexes (VDAC), complex II, complex III, cytochrome c 1, prohibitin complex V were highly abundant in the expected mitochondrial fraction in contrast to mock. In IEF gels, 13 protein subunits were of increased abundance at 20 hpi, 24 hpi, and 40 hpi, for example complex I, complex II, complex III, and proteins involved in amino acid degradation, and protein folding. In gel free analyses, 13 and 11 proteins were of increased abundance in the inoculated mitochondrial fraction at 24 h and at 40 h, respectively. There was similar pattern in protein abundance as observed in the BN gels and in the IEF gels.