In the present study we combined activity based
In the present study, we combined activity-based protein profiling, biochemical and genetic approaches to identify the PLCPs involved in the sphingolipid pathway leading to plant PCD. By using Arabidopsis lrrk2 and leaves, we demonstrate that this sub-class of plant proteases is activated in response to PHS or FB1, respectively and we identify RD21 (responsive-to-desiccation-21) as one of the main PLCPs activated in response to sphingolipid treatment. Using a reverse genetic approach with two null mutants of RD21,we show that this protease acts as a negative regulator of FB1-induced cell death in Arabidopsis.
Material and methods
Discussion In plants, programmed cell death plays a fundamental role in development and defense responses. As an example, during necrotrophic pathogen-plant interactions, host death is induced enabling pathogen development . Interestingly, the necrotrophic fungus Fusarium moniliforme interferes with plant sphingolipid metabolism by secreting a mycotoxin called fumonisin B1 (FB1). This molecule, which is a structural analog of LCBs, inhibits the ceramide synthase and induces a concomitant increase in free intracellular levels of PHS and DHS, metabolites known to elicit a subsequent PCD in plants [6,7]. However, the LCB-mediated signaling pathway leading to PCD remains largely unknown so far. Nevertheless, previous studies have shown that different second messengers including intracellular Ca2+ ions and reactive oxygen species (ROS) or proteins such as MPK6, 14-3-3 s and the calcium-dependent protein kinase CPK3 are involved in this process [7,10,11]. In normal conditions, dimeric 14-3-3 proteins interact with CPK3 . In response to PHS, a cytosolic Ca2+ transient is induced and leads to CPK3 activation. Ca2+-activated CPK3 phosphorylates 14-3-3 s on a specific serine residue, which is located at the 14-3-3 dimer interface . Recent studies have shown that this phosphorylation event leads to plant 14-3-3 monomerization [29,30], that could disrupt the 14-3-3/CPK3 complex, leading to the release of CPK3 . Since RD21 is one of the major cysteine proteases in leaves of Arabidopsis thaliana and plays a role in plant immune responses [17,27], we speculated that it could play a role in FB1-induced cell death. RD21 belongs to the plant family of PLCPs . This protease is characterized by a unique C-terminal granulin domain that shares homology to granulin-containing growth factor in animals . The pre-pro-protease RD21 (51 kDa) is composed of five domains: a signal peptide, an autoinhibitory prodomain, the protease domain, a proline-rich domain and a granulin domain [21,22]. The activity of RD21 depends on two proteolytic steps. First, the autoinhibitory prodomain is removed leading to an active intermediary form iRD21 (40 kDa) and the granulin domain is then processed, resulting in active mature form mRD21 (30 kDa) [21,22,26]. These forms have been identified in our experiments in extracts of Arabidopsis leaf discs treated with FB1 (Fig. 2B). Recently, different contradicting studies have shown that RD21 plays a role in necrotrophic fungus-plant interaction. Shindo et al. (2012) have shown that leaves of Arabidopsis rd21 null mutants are more susceptible to Botrytis cinerea compared to WT plants, suggesting that RD21 is a positive regulator of plant resistance to Botrytis . By contrast, Lampl et al. (2013) showed that detached leaves of Arabidopsis mutant plants lacking RD21 are resistant to the two necrotrophic fungi Sclerotina sclerotiorum and Botrytis cinerea. Moreover, upon addition of oxalic acid, the major pathogenic effector of these two fungi, rd21 null mutants and RD21 overexpressing lines present a reduction and an increase in cell death, respectively, compared to WT plants. In this case, RD21 seems to play a pro-cell death function . These discrepancies may be due to the use of different Botrytis cinerea isolates and/or assays performed on whole plant  instead of detached leaves , as well as to differences in plant culture conditions. In our study, we show a clear FB1-susceptibility phenotype of the two rd21 mutants by using two different bioassays to quantify PCD revealing a similar behaviour than the one observed by Shindo et al. . Thus, both PCD tests demonstrate that RD21 is involved in FB1-induced signaling pathway and behaves as a negative regulator of cell death. We can speculate that RD21 activity could counterbalance the pro-death effect of vacuolar processing enzymes (VPEs), which are cysteine proteases involved in FB1-induced cell death .