Matrix metalloproteinases (MMPs) are evolutionary conserved metal-dependent endopeptidases widely present in animals and plants. Mammalian MMPs are well known as central regulators in a number of physiological and pathological processes such as tissue remodeling and cancer progression. Little is known about the detailed function and molecular mechanisms of MMPs in plants. Main focus of the present study was to analyze the potential involvement of Arabidopsis MMPs family in plant immune responses. Phylogenetic analysis using 44 MMPs from fourteen higher plants resulted in a clear classification of four subgroups, which are associated with certain plant species or functions. The expression profiles of five At-MMPs were examined in Col-0 plants during the interaction with distinct microbes, including the biotrophic fungus Golovinomyces orontii, necrotrophic fungus Botrytis cinerea, hemibiotrophic bacterium Pseudomonas syringae pv. tomato (Pst) DC3000 and the symbiotic mycorrhizal-like fungus Piriformospora indica. At2-MMP and At3-MMP were clearly up-regulated after infection with Pst DC3000 as well as B. cinerea. This indicates a potential involvement of MMPs in pathogen resistance. Arabidopsis T-DNA insertion mutant lines were tested for altered resistance. Mutation of At4-MMP and At5-MMP did not exhibit clear changes in pathogen resistance. At2-MMP mutants were identified to be more susceptible to B. cinerea infection. In contrast, At2-MMP overexpression lines 35S::MMP2 showed enhanced resistance to B. cinerea. Moreover, 35S::MMP2 exhibited early flowering compared with control plants. At3-MMP showed similar altered responses as At2-MMP based on mutants and overexpression analysis. Double mutants at2-mmp/at3-mmp were produced and showed significantly enhanced susceptibility to B. cinerea. These results confirmed the essential role of At2- and At3-MMP in the resistance towards B. cinerea. Bombardment-mediated transient transformation was used to clarify the subcellular localization of At2-MMP. Plasmolysis experiments demonstrated that a MMP2-GFP fusion protein was co-present in the plasma membrane and apoplastic space. In addition, recombinant proteins for Pro-MMP2 and mature MMP2 (Mat-MMP2) were produced and tested for proteolytic activity. As a result, Mat-MMP2 exhibited substantially higher activity than Pro-MMP2 against the substrate myelin basic protein (MBP). Activities of MMP2 can be inhibited by the metal chelator EDTA. Like most mammalian MMPs tested, the recombinant At2-MMP protein showed no direct antimicrobial activity towards B. cinerea conidia spore germination and hyphae growth. Expression pattern of At2-MMP and At3-MMP in Col-0 was compared in mutants which are compromised in SA (NahG, ics1, npr1-1, PAD3), JA (jar1, jin1) or ET (ein2-1) signalling after B. cinerea infection. The similar induction of At2-MMP and At3-MMP in all the mutants suggested that B. cinerea-induced expression of At2/3-MMP was likely independent of SA, JA and ET signalling. Reactive oxygen species (ROS) are early host responses to pathogen infection. To decipher the mode of actions for At2- and At3-MMP mediated immune responses, the ROS production in 35S::MMP2, at2-mmp and at3-mmp mutants was monitored after treatment of pathogen-associated molecular pattern molecules (PAMPs, flg22, elf18 and chitin) and a danger- associated molecular pattern peptide (DAMPs, Pep1). Intriguingly, the PAMP/DAMP-triggered ROS production was largely impaired in 35S:MMP2 plants. Wild-type level of ROS generation was observed in at2-mmp mutants. In contrast, flg22 and chitin-induced ROS generation was enhanced in at3-mmp mutants. Preliminary data also indicated that recombinant At2-MMP protein was capable of inducing ROS production in Col-0 plants. Taken together, At2-MMP and At3-MMP are playing essential roles in Arabidopsis immune responses likely through the modulation of ROS production. Future studies are suggested to focus on their physiological substrates, mode of actions and roles in resistance to other pathogens such as bacterial pathogens.
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