Date Published: July 10, 2014
Publisher: Public Library of Science
Author(s): Fei Wang, Yifen Shang, Baofang Fan, Jing-Quan Yu, Zhixiang Chen, Xin Li.
Multivesicular bodies (MVBs) play essential roles in many cellular processes. The MVB pathway requires reversible membrane association of the endosomal sorting complexes required for transports (ESCRTs) for sustained protein trafficking. Membrane dissociation of ESCRTs is catalyzed by the AAA ATPase SKD1, which is stimulated by LYST-INTERACTING PROTEIN 5 (LIP5). We report here that LIP5 is a target of pathogen-responsive mitogen-activated protein kinases (MPKs) and plays a critical role in plant basal resistance. Arabidopsis LIP5 interacts with MPK6 and MPK3 and is phosphorylated in vitro by activated MPK3 and MPK6 and in vivo upon expression of MPK3/6-activating NtMEK2DD and pathogen infection. Disruption of LIP5 has little effects on flg22-, salicylic acid-induced defense responses but compromises basal resistance to Pseudomonas syringae. The critical role of LIP5 in plant basal resistance is dependent on its ability to interact with SKD1. Mutation of MPK phosphorylation sites in LIP5 does not affect interaction with SKD1 but reduces the stability and compromises the ability to complement the lip5 mutant phenotypes. Using the membrane-selective FM1–43 dye and transmission electron microscopy, we demonstrated that pathogen infection increases formation of both intracellular MVBs and exosome-like paramural vesicles situated between the plasma membrane and the cell wall in a largely LIP5-dependent manner. These results indicate that the MVB pathway is positively regulated by pathogen-responsive MPK3/6 through LIP5 phosphorylation and plays a critical role in plant immune system likely through relocalization of defense-related molecules.
Endosomes traffic molecules from the plasma membrane to intracellular compartments and transport molecules from the biosynthetic apparatus to the sites of action , . Several different endosomes have been described based on biochemical composition, morphology, and function. Multivesicular bodies (MVBs) are late endosomes that contain intraluminal vesicles generated when the limiting membrane of the endosome invaginates and buds into its own lumen, thereby allowing cargo-containing intraluminal vesicles to be delivered into and degraded upon fusion with lysosomes or vacuoles , . Those proteins retained in the limiting membrane of MVBs, on the other hand, can be delivered to the membrane of lysosomes or vacuoles, or sort back to the plasma membrane or other cellular compartments , . Protein sorting into MVBs is highly regulated and is dependent on the action of three distinct protein complexes named ESCRT-I, II and III (Endosomal Sorting Complex Required for Transport) . Ubiquitinated membrane proteins are first recognized by ubiquitin-binding proteins such as the TOM1 families of proteins, which also recruit ESCRT-I components from the cytoplasm. ESCRT-II and ESCRT-III complexes then transiently assembly on the endosomal membrane for cargo sorting, concentration and vesicle formation. For sustained protein trafficking through the MVB pathway, it is necessary that the ESCRT complexes are dissociated and disassembled from the membrane and recycled back into the cytoplasm. The Vps4p/SKD1 AAA ATPase together with its positive regulator Vta1/LIP5 catalyzes the process of ESCRT disassembly in an ATP-dependent reaction , , , , . Studies in both yeast and mammalian cells indicate that both Vps4p/SKD1 and Vta1/LIP5 are critical players during MVB biogenesis , , , . In Arabidopsis, disruption of the SKD1 gene is lethal and expression of an ATPase-deficient version of SKD1 causes alterations in the endosomal system and ultimately cell death . Arabidopsis LIP5 interacts strongly with SKD1 and increases in vitro the ATPase activity of SKD1 by 4–5 fold . However, disruption of LIP5 in Arabidopsis causes no phenotypic alterations under normal growth conditions, indicating that the basal levels of the SKD1 ATPase activity are sufficient for plant growth and development .
The extensively characterized MPK3/MPK6 signaling cascade plays a crucial role in plant innate immunity . A number of Arabidopsis proteins including WRKY33, ACS2, ACS6 and ERF6 have been identified as substrates of MPK3/6 and functionally analyzed for their roles in plant defense against necrotrophic pathogens , , . Here, we report that LIP5, a positive regulator of the SKD1 AAA ATPase in MVB pathway, is another substrate of the pathogen-responsive MAPKs and plays an important role in plant basal resistance to the hemibiotrophic bacterial pathogen P. syringae. Through comprehensive genetic, molecular and biochemical analysis, we further demonstrated that the critical role of LIP5 in plant basal resistance is dependent on both its interaction with the SKD1 AAA ATPase in MVB biogenesis and its phosphorylation-enhanced stability. Our study provides genetic evidence for the critical role of MVB biogenesis in plant innate immunity and established an important mechanism for the regulation of vesicle trafficking during plant immune responses.