Immobilized Subpopulations of Leaf Epidermal Mitochondria Mediate PENETRATION2-Dependent Pathogen Entry Control in Arabidopsis

Abstract

The atypical myrosinase PENETRATION2 (PEN2) is required for broad-spectrum invasion resistance to filamentous plant pathogens. Previous localization studies suggested PEN2-GFP association with peroxisomes. Here, we show that PEN2 is a tail-anchored protein with dual-membrane targeting to peroxisomes and mitochondria and that PEN2 has the capacity to form homo-oligomer complexes. We demonstrate pathogen-induced recruitment and immobilization of mitochondrial subpopulations at sites of attempted fungal invasion and show that mitochondrial arrest is accompanied by peripheral accumulation of GFP-tagged PEN2. PEN2 substrate production by the cytochrome P450 monooxygenase CYP81F2 is localized to the surface of the endoplasmic reticulum, which focally reorganizes close to the immobilized mitochondria. Exclusive targeting of PEN2 to the outer membrane of mitochondria complements the pen2 mutant phenotype, corroborating the functional importance of the mitochondrial PEN2 protein subpool for controlled local production of PEN2 hydrolysis products at subcellular plant-microbe interaction domains. Moreover, live-cell imaging shows that mitochondria arrested at these domains exhibit a pathogen-induced redox imbalance, which may lead to the production of intracellular signals.

The Roles of Mitochondrial Reactive Oxygen Species in Cellular Signaling and Stress Response in Plants

“…AtOM66 overexpression plants show increased SA content and accelerated cell death rates and have altered pathogen resistance (Zhang et al, 2014a). The Arabidopsis defense protein PEN2 has recently been found to hyper-accumulate on the outer membrane of mitochondria that cluster around infection sites of leaf epidermal cells (Fuchs et al, 2015). Redox sensing revealed an oxidative shift in the matrix glutathione pool of mitochondria at the infection site in relation to other parts of the same cell or neighboring cells.…”

VPS9a Activates the Rab5 GTPase ARA7 to Confer Distinct Pre- and Postinvasive Plant Innate Immunity

“…One pathway, defined by the 1 b-glucoside hydrolase, PEN2, mediates synthesis and export of antimicrobial metabolites (Lipka et al, 2005;Stein et al, 2006;Clay et al, 2009;Bednarek et al, 2009). Here, the endoplasmic reticulum-resident P450 monooxygenase, CYP81F2, is thought to synthesize glucosinolate derivatives that are activated by PEN2 and finally secreted by the ATP binding cassette transporter protein, PEN3, thereby presenting potentially toxic compounds to the pathogen (Stein et al, 2006;Fuchs et al, 2016). Interestingly, loss of PEN2 and PEN3 also results in a substantial reduction of the hypersensitive response (HR) after recognition of the Pseudomonas syringae effectors, AvrRpm1 and AvrRps4, indicating that the role of the PEN2 pathway extends beyond that of preinvasive immunity (Johansson et al, 2014).…”

Techniques for the analysis of protein-protein interactions in vivo

“…Additionally, an interaction in CoIP could be due to the presence of a protein complex of multiple interactors, with an apparent binary interaction facilitated by known or unknown cofactors (Hall, 2005). This comes as a blessing in disguise, as it allows higher order protein complexes or ligand-induced dimerization to be dissected, a phenomenon that is neatly exploited when an interaction between two POIs is either induced by an exogenously applied factor (ligand, pathogen, and so on) or disrupted in a knockout line of the endogenous bridging factor (Chinchilla et al, 2007;Albert et al, 2015;Fuchs et al, 2016) Recently, two novel techniques have been developed that circumvent the limitations of traditional CoIP. Both developments, single-molecule pull down (Jain et al, 2011) and real-time single-molecule CoIP (Lee et al, 2013), use total internal reflection microscopy (Axelrod, 2001) to detect interactions on a coverslip.…”