During plant cell invasion, the oomycete Phytophthora infestans remains enveloped by host-derived membranes whose functional properties are poorly understood. P. infestans secretes a myriad of effector proteins through these interfaces for plant colonization. Recently we showed that the effector protein PexRD54 reprograms host-selective autophagy by antagonising antimicrobial-autophagy receptor Joka2/NBR1 for ATG8CL binding (Dagdas et al., 2016). Here, we show that during infection, ATG8CL/Joka2 labelled defense-related autophagosomes are diverted toward the perimicrobial host membrane to restrict pathogen growth. PexRD54 also localizes to autophagosomes across the perimicrobial membrane, consistent with the view that the pathogen remodels host-microbe interface by co-opting the host autophagy machinery. Furthermore, we show that the host-pathogen interface is a hotspot for autophagosome biogenesis. Notably, overexpression of the early autophagosome biogenesis protein ATG9 enhances plant immunity. Our results implicate selective autophagy in polarized immune responses of plants and point to more complex functions for autophagy than the widely known degradative roles.
Ichinose and Renier A. L. van der Hoorn INTRODUCTION: Immunogenic flagellin fragments are a signature of bacterial invasion in both plants and animals. Plants recognize flagellin fragments via FLS2, a model receptor kinase that is highly conserved amongst angiosperms. However, little is known about events upstream of flagellin perception by FLS2. The flagellin fragments recognized by FLS2 are buried in the flagellin polymer structure and require hydrolytic release before recognition can occur, yet the hydrolases releasing these elicitors remain to be identified. Uncovering their identity is a daunting task because the extracellular space of plants (the apoplast) is known to contain hundreds of uncharacterized glycosidases and proteases. RATIONALE:We reasoned that pathogenic bacteria would suppress plant hydrolases that are important for immunity. We therefore monitored the activity of apoplastic hydrolases using activitybased protein profiling (ABPP) using chemical probes that irreversibly label the active site of hydrolases in an activity-dependent manner. We applied this strategy to study the infection of the tobacco relative Nicotiana benthamiana with the model bacterial pathogens Pseudomonas syringae pathovars tomato DC3000 (PtoDC3000); tabaci (Pta6605); and syringae (PsyB728a). RESULTS:Glycosidase activity profiling of apoplastic fluids isolated from PtoDC3000-infected plants revealed that the activity of the β-galactosidase BGAL1 is suppressed in the apoplast during infection.BGAL1 suppression is caused by a heat-stable, basic, small inhibitor molecule that is produced by the bacteria under the control of the hrpR/S/L virulence regulators. Null mutants of N. benthamiana lacking BGAL1 generated by genome editing are more susceptible, demonstrating that BGAL1 contributes to immunity. When studying why BGAL1 is suppressed, we discovered that treatment of PtoDC3000 and Pta6605 with apoplastic fluids containing BGAL1 releases an elicitor that triggers the release of reactive oxygen species (ROS) in leaf discs, a signature immune response in plants. The released elicitor is flagellin-derived because the ROS burst requires both the FLS2 receptor in the plant and flagellinencoding fliC in the bacteria. The flagellin polymer of both PtoDC3000 and Pta6605 is O-glycosylated with a glycan consisting of several rhamnose residues and a terminal modified viosamine (mVio).Treatment of mutant Pta6605 bacteria carrying nonglycosylated flagellin triggers the ROS burst when treated with apoplastic fluids, even in the absence of BGAL1, demonstrating that BGAL1 requires
In plants, the highly conserved catabolic process of autophagy has long been known as a means of maintaining cellular homeostasis and coping with abiotic stress conditions. Accumulating evidence has linked autophagy to immunity against invading pathogens, regulating plant cell death, and antimicrobial defences. In turn, it appears that phytopathogens have evolved ways not only to evade autophagic clearance but also to modulate and co-opt autophagy for their own benefit. In this review, we summarize and discuss the emerging discoveries concerning how pathogens modulate both host and self-autophagy machineries to colonize their host plants, delving into the arms race that determines the fate of interorganismal interaction.
Eukaryotic cells deploy autophagy to eliminate invading microbes. In turn, pathogens have evolved effector proteins to counteract antimicrobial autophagy. How adapted pathogens co-opt autophagy for their own benefit is poorly understood. The Irish famine pathogen Phytophthora infestans secretes the effector protein PexRD54 that selectively activates an unknown plant autophagy pathway that antagonizes antimicrobial autophagy at the pathogen interface. Here, we show that PexRD54 induces autophagosome formation by bridging vesicles decorated by the small GTPase Rab8a with autophagic compartments labeled by the core autophagy protein ATG8CL. Rab8a is required for pathogen-triggered and starvation-induced but not antimicrobial autophagy, revealing specific trafficking pathways underpin selective autophagy. By subverting Rab8a-mediated vesicle trafficking, PexRD54 utilizes lipid droplets to facilitate biogenesis of autophagosomes diverted to pathogen feeding sites. Altogether, we show that PexRD54 mimics starvation-induced autophagy to subvert endomembrane trafficking at the host-pathogen interface, revealing how effectors bridge distinct host compartments to expedite colonization.
Bacterial bioluminescence is widely used to study the spatiotemporal dynamics of bacterial populations and gene expression in vivo at a population level but cannot easily be used to study bacterial activity at the level of individual cells. In this study, we describe the development of a new library of mini-Tn7-lux and lux::eyfp reporter constructs that provide a wide range of lux expression levels, and which combine the advantages of both bacterial bioluminescence and fluorescent proteins to bridge the gap between macro-and micro-scale imaging techniques. We demonstrate that a dual bioluminescencefluorescence approach using the lux operon and eYFP can be used to monitor bacterial movement in plants both macro-and microscopically and demonstrate that Pseudomonas syringae pv phaseolicola can colonize the leaf vascular system and systemically infect leaves of common bean (Phaseolus vulgaris). We also show that bacterial bioluminescence can be used to study the impact of plant immune responses on bacterial multiplication, viability and spread within plant tissues. The constructs and approach described in this study can be used to study the spatiotemporal dynamics of bacterial colonization and to link population dynamics and cellular interactions in a wide range of biological contexts.
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