A Critical Role of Lyst-Interacting Protein5, a Positive Regulator of Multivesicular Body Biogenesis,in Plant Responses to Heat and Salt Stresses

Multivesicular bodies (MVBs) are unique endosomes containing vesicles in the lumen and play critical roles in many cellular processes. We have recently shown that Arabidopsis (Arabidopsis thaliana) Lyst-Interacting Protein5 (LIP5), a positive regulator of the Suppressor of K⁺ Transport Growth Defect1 (SKD1) AAA ATPase in MVB biogenesis, is a critical target of the mitogen-activated protein kinases MPK3 and MPK6 and plays an important role in the plant immune system. In this study, we report that the LIP5-regulated MVB pathway also plays a critical role in plant responses to abiotic stresses. Disruption of LIP5 causes compromised tolerance to both heat and salt stresses. The critical role of LIP5 in plant tolerance to abiotic stresses is dependent on its ability to interact with Suppressor of K⁺ Transport Growth Defect1. When compared with wild-type plants, lip5 mutants accumulate increased levels of ubiquitinated protein aggregates and NaCl under heat and salt stresses, respectively. Further analysis using fluorescent dye and MVB markers reveals that abiotic stress increases the formation of endocytic vesicles and MVBs in a largely LIP5-dependent manner. LIP5 is also required for the salt-induced increase of intracellular reactive oxygen species, which have been implicated in signaling of salt stress responses. Basal levels of LIP5 phosphorylation by MPKs and the stability of LIP5 are elevated by salt stress, and mutation of MPK phosphorylation sites in LIP5 reduces the stability and compromises the ability to complement the lip5 salt-sensitive mutant phenotype. These results collectively indicate that the MVB pathway is positively regulated by pathogen/stress-responsive MPK3/6 through LIP5 phosphorylation and plays a critical role in broad plant responses to biotic and abiotic stresses.Multivesicular bodies (MVBs) are a subset of late endosomes that contain intraluminal vesicles generated when the limiting membrane of the endosome invaginates and buds into its own lumen. MVBs perform a variety of functions and, as a result, can have different compositions and morphologies. The most well-established role of MVBs in all eukaryotic cells is as a degradation route in the endocytic pathway that allows protein-containing intraluminal vesicles to be delivered into and degraded upon fusion with lysosomes or vacuoles (Reyes et al., 2011; Contento and Bassham, 2012). The route acts as a mechanism for removing damaged proteins as well as proteins that require down-regulation or clearing from the plasma membrane as part of a regulatory process. Those proteins retained in the limiting membrane of MVBs, on the other hand, can be delivered to the membrane of lysosomes or vacuoles or sorted back to the plasma membrane or other cellular compartments (Reyes et al., 2011; Contento and Bassham, 2012).The protein machinery involved in MVB biogenesis has been well studied in yeast and other eukaryotic organisms. A majority of proteins required for protein sorting into MVBs are components of three distinct protein complexes named ESCRT-I, ESCRT-II, and ESCRT-III (for Endosomal Sorting Complexes Required for Transport; Winter and Hauser, 2006). These complexes are recruited to endosomal membranes and function in sorting cargo and the formation of intraluminal vesicles. Ubiquitinated membrane proteins are first recognized by specific ubiquitin-binding proteins that also recruit ESCRT-I components from the cytoplasm. ESCRT-II and ESCRT-III complexes are then recruited and transiently assembled on the endosomal membrane for cargo sorting, concentration, and intraluminal vesicle formation. Unlike ESCRT-I and ESCRT-II, which are stable protein complexes, ESCRT-III proteins are monomers in the cytoplasm and only form complexes on the endosomal membrane. Disassembly of ESCRT-III, however, is not spontaneous but, rather, requires catalysis by the Vacuolar protein sorting 4p (Vps4p)/Suppressor of K⁺ Transport Growth Defect1 (SKD1) AAA ATPase together with its positive regulator Vacuolar protein sorting20-associated1 (Vta1)/LIP5 in an ATP-dependent reaction (Babst et al., 1998; Fujita et al., 2004; Scott et al., 2005; Azmi et al., 2006; Lottridge et al., 2006). In yeast and mammalian cells, both Vps4p/SKD1 and Vta1/LIP5 are critical players of MVB biogenesis (Yeo et al., 2003; Shiflett et al., 2004; Ward et al., 2005; Azmi et al., 2006).As sessile organisms, plants are constantly exposed to a wide range of biotic and abiotic stresses and, through evolution, have developed a battery of complicated adaptive mechanisms. Studies over the past decade have provided increasing evidence for the association of vesicle trafficking with plant responses to both biotic and abiotic stresses. Plant immune responses to biotic stresses consists of two interconnected branches: pattern-triggered immunity and effector-triggered immunity, which are conferred by pattern-recognition receptors and RESISTANCE (R) proteins, respectively. A number of pattern-recognition receptors, such as Arabidopsis (Arabidopsis thaliana) Flagellin-sensitive2 and R proteins, become associated with late endosomes/MVBs upon pattern and effector recognition, respectively, and there is strong evidence for a critical role of the association with vesicles in plant disease resistance (Choi et al., 2013; Spallek et al., 2013). In the penetration resistance of cereal plants against powdery mildew fungal pathogens, which is conferred by local cell wall appositions (papillae), electron and confocal microscopy detected trafficking molecules through late endosomes/MVBs for delivering defense-related materials to papillae, thereby executing a timely and localized defense response to invading pathogens (An et al., 2006a, 2006b; Meyer et al., 2009; Böhlenius et al., 2010; Nielsen et al., 2012). Similar relocalization of defense-related molecules, such as the PENTRATION RESISTANCE3 ATP-binding cassette transporter for cell surface defense in response to conserved pathogen elicitors, has also been observed in Arabidopsis (Underwood and Somerville, 2013). There is also evidence for a role of late endosomes/MVBs in plant abiotic stress responses (Jou et al., 2004, 2006; Ho et al., 2010; Xia et al., 2013). Generally speaking, however, while there is a large body of evidence for a critical role of general vesicle trafficking in plant stress responses, there has been only a limited number of studies that address specifically the roles and regulation of MVBs in plant responses to biotic and abiotic stresses. Studies on the role of MVBs in plant immune responses have been largely through microscopic analysis of the accumulation of the late endosomes in response to pathogen infection or elicitor treatment. Genetic analysis of the role of MVBs in plant stress responses has not been straightforward, because mutants for genes essential for MVB biogenesis are often lethal (Haas et al., 2007; Spitzer et al., 2009). While constitutive MVB biogenesis is known to be essential in many cellular processes, it remains to be determined whether there are specific pathogen/stress-responsive pathways for increased MVB biogenesis during plant stress responses.In Arabidopsis, disruption of the SKD1 gene is lethal, and expression of an ATPase-deficient mutant SKD1 causes alterations in the endosomal system and ultimately cell death (Haas et al., 2007). Arabidopsis LIP5 interacts strongly with SKD1 and increases in vitro the ATPase activity of SKD1 by 4- to 5-fold (Haas et al., 2007). However, disruption of LIP5 in Arabidopsis causes no major phenotypic alterations under normal growth conditions, indicating that the basal level of the SKD1 ATPase activity without stimulation by LIP5 is sufficient for plant growth and development (Haas et al., 2007). Recently, we reported the identification of Arabidopsis LIP5 as an interacting protein and a substrate of the pathogen-responsive mitogen-activated protein kinases (MAPKs) MPK6/MPK3 (Wang et al., 2014). Functional analysis with lip5 transfer DNA (T-DNA) insertion mutants indicates that LIP5 plays a critical role in pathogen-induced MVB trafficking and in basal resistance to Pseudomonas syringae strains (Wang et al., 2014). The critical role of LIP5 in the plant immune system is dependent on its ability to interact with SKD1. Further analysis reveals that LIP5 is expressed at low levels in healthy plants, but its protein levels can be substantially elevated through phosphorylation by the pathogen-responsive MPK cascade. Mutation of MPK phosphorylation sites in LIP5 does not affect its interaction with SKD1 but reduces its stability and, as a result, compromises its ability to complement the basal resistance of the lip5 mutants. These results provide genetic evidence for a critical role of induced MVB biogenesis in plant basal resistance and establish an important mechanism for the regulation of vesicle trafficking during plant-pathogen interactions (Wang et al., 2014).In this study, we report that the LIP5-regulated MVB pathway is also a critical cellular process during plant responses to abiotic stresses. Disruption of LIP5 causes compromised tolerance to both heat and salt stresses. The critical role of LIP5 in plant tolerance to abiotic stresses is again dependent on its ability to interact with SKD1. When compared with wild-type plants, lip5 mutants accumulate increased levels of ubiquitinated protein aggregates, suggesting a possible role of LIP5-regulated MVB trafficking as a critical route for the degradation of heat-damaged proteins. Compromised salt tolerance of the lip5 mutants was associated with an increased accumulation of cellular NaCl but reduced levels of cellular reactive oxygen species (ROS), which have been implicated in the signaling of salt stress responses (Kaye et al., 2011; Xie et al., 2011). The roles of LIP5 and its phosphorylation by MPK3/6 in plant responses to heat and salt stresses were also investigated. These results collectively provide important insights into the role and regulation of pathogen- and stress-responsive MVB biogenesis in broad plant stress responses.