Chian Kwon

Co-option of a default secretory pathway for plant immune responses

Cell-autonomous immunity is widespread in plant–fungus interactions and terminates fungal pathogenesis either at the cell surface or after pathogen entry. Although post-invasive resistance responses typically coincide with a self-contained cell death of plant cells undergoing attack by parasites, these cells survive pre-invasive defence. Mutational analysis in Arabidopsis identified PEN1 syntaxin as one component of two pre-invasive resistance pathways against ascomycete powdery mildew fungi. Here we show that plasma-membrane-resident PEN1 promiscuously forms SDS-resistant soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complexes together with the SNAP33 adaptor and a subset of vesicle-associated membrane proteins (VAMPs). PEN1-dependent disease resistance acts in vivo mainly through two functionally redundant VAMP72 subfamily members, VAMP721 and VAMP722. Unexpectedly, the same two VAMP proteins also operate redundantly in a default secretory pathway, suggesting dual functions in separate biological processes owing to evolutionary co-option of the default pathway for plant immunity. The disease resistance function of the secretory PEN1–SNAP33–VAMP721/722 complex and the pathogen-induced subcellular dynamics of its components are mechanistically reminiscent of immunological synapse formation in vertebrates, enabling execution of immune responses through focal secretion.

Secretory Pathways in Plant Immune Responses

Innate immune receptors in plants detect the presence of microbial pathogens and trigger defense responses to terminate or restrict pathogen growth. The molecular mechanisms of receptor-mediated nonself recognition and subsequent intracellular signaling pathways have received much attention in the

Not a peripheral issue: secretion in plant–microbe interactions

The sessile nature of flowering plants and their capacity to thrive in soil habitats likely resulted in specific adaptation mechanisms enabling co-existence with a vast diversity of air-borne and soil-borne microorganisms. Only a small fraction of these microbial encounters result in pathogenic or symbiotic interactions, whilst the majority appear to give rise to commensalistic or mutualistic associations. Considering the abundance of the latter associations the question arises whether plants evolved, besides the plant immune system, other dedicated mechanisms to communicate with and to host microbial communities in the phyllosphere and rhizosphere. We hypothesize that the constitutive and microbe-induced secretion of specialized plant-derived biomolecules creates a critical interface for all types of plant-microbe associations. Thus, the plant secretory machinery might serve an important role in establishing an extended phenotype with microbial life.

Activity determinants and functional specialization of Arabidopsis pen1 syntaxin in innate immunity

In eukaryotes, proteins of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family are believed to have a general role for the fusion of intracellular transport vesicles with acceptor membranes. Arabidopsis thaliana PEN1 syntaxin resides in the plasma membrane and was previously shown to act together with its partner SNAREs, the adaptor protein SNAP33, and endomembrane-anchored VAMP721/722 in the execution of secretory immune responses against powdery mildew fungi. We conducted a structure-function analysis of PEN1 and show that N-terminal phospho-mimicking and non-phosphorylatable variants neither affected binary nor ternary SNARE complex formation with cognate partners in vitro. However, expression of these syntaxin variants at native protein levels in a pen1 mutant background suggests that phosphorylation is required for full resistance activity in planta. All tested site-directed substitutions of SNARE domain or “linker region” residues reduced PEN1 defense activity. Two of the variants failed to form ternary complexes with the partner SNAREs in vitro, possibly explaining their diminished in planta activity. However, impaired pathogen defense in plants expressing a linker region variant is likely because of PEN1 destabilization. Although Arabidopsis PEN1 and SYP122 syntaxins share overlapping functions in plant growth and development, PEN1 activity in disease resistance is apparently the result of a complete functional specialization. Our findings are consistent with the hypothesis that PEN1 acts in plant defense through the formation of ternary SNARE complexes and point to the existence of unknown regulatory factors. Our data indirectly support structural inferences that the four-helical coiled coil bundle in ternary SNARE complexes is formed in a sequential order from the N- to C-terminal direction.

Les liaisons dangereuses: Immunological synapse formation in animals and plants

The immunological synapse in vertebrates describes a specialized junction between a T cell and a target cell, enabling execution of immune responses through focal secretion. Recent insights in the plant immune system suggest that plant cells assemble a pathogen-inducible machinery at the cell surface that shares several features with the immunological synapse. Apparent mechanistic commonalities include co-stimulatory non-self alarm signals as triggers, cell polarization driven by actin cytoskeleton remodeling, protein concentration into ring-shaped assemblies at the cell periphery and focal exocytosis mediated by soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins that are core factors for vesicle fusion. Although in plants, execution of immune responses by polar secretion seems to be a cell type-independent property, its confinement to T cells in the vertebrate immune system might reflect a greater division of labor.

Requirement of vesicle-associated membrane protein 721 and 722 for sustained growth during immune responses in Arabidopsis

Extracellular immune responses to ascomycete and oomycete pathogens in Arabidopsis are dependent on vesicle-associated secretion mediated by the SNARE proteins PEN1 syntaxin, SNAP33 and endomembrane-resident VAMP721/722. Continuous movement of functional GFP-VAMP722 to and from the plasma membrane in non-stimulated cells reflects the second proposed function of VAMP721/722 in constitutive secretion during plant growth and development. Application of the bacterium-derived elicitor flg22 stabilizes VAMP721/722 that are otherwise constitutively degraded via the 26S proteasome pathway. Depletion of VAMP721/722 levels by reducing VAMP721/722 gene dosage enhances flg22-induced seedling growth inhibition in spite of elevated VAMP721/722 abundance. We therefore propose that plants prioritize the deployment of the corresponding secretory pathway for defense over plant growth. Interstingly, VAMP721/722 specifically interact in vitro and in vivo with the plasma membrane syntaxin SYP132 that is required for plant growth and resistance to bacteria. This suggests that the plant growth/immunity-involved VAMP721/722 form SNARE complexes with multiple plasma membrane syntaxins to discharge cue-dependent cargo molecules.

Syntaxin of Plant Proteins SYP123 and SYP132 Mediate Root Hair Tip Growth in Arabidopsis thaliana

Root hairs are fast-growing tubular protrusions on root epidermal cells that play important roles in water and nutrient uptake in plants. The tip-focused polarized growth of root hairs is accomplished by the secretion of newly synthesized materials to the tip via the polarized membrane trafficking mechanism. Here, we report the function of two different types of plasma membrane (PM) Qa-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), SYP123 and SYP132, in the growth of root hair in Arabidopsis. We found that SYP123, but not SYP132, localizes in the tip region of root hairs by recycling between the brefeldin A (BFA)-sensitive endosomes and the PM of the expanding tip in an F-actin-dependent manner. The vesicle-associated membrane proteins VAMP721/722/724 also exhibited tip-focused localization in root hairs and formed ternary SNARE complexes with both SYP123 and SYP132. These results demonstrate that SYP123 and SYP132 act in a coordinated fashion to mediate tip-focused membrane trafficking for root hair tip growth.

Ready to fire: secretion in plant immunity

Effective recognition of pathogens and rapid execution of immune responses are essential for the survival of living organisms. Cell-autonomous immune responses of animal and plant cells rely on pattern recognition receptors that can distinguish self from non-self structures and that are able to activate a molecular execution machinery that ultimately terminates most pathogen attacks. Reminiscent of the situation in mammalian T cells, accumulating evidence points to a key role of vesicle trafficking and exocytosis in plant innate immunity. In this context, our recent finding that ternary soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complexes comprising PEN1, SNAP33 and VAMP721/722 function at pathogen entry sites is instrumental in understanding the execution of plant immune responses at the cell periphery. Our study further revealed unexpected overlapping functions of the same SNARE complexes in disease resistance and development. Here, we discuss the potential identity of cargo delivered through the PEN1-SNAP33-VAMP721/722-dependent secretory pathway and the necessity for a tight regulation of SNARE complex formation to avoid unintentional release of toxic load. Addendum to: Kwon C, Neu C, Pajonk S, Yun HS, Lipka U, Humphry M, Bau S, Straus M, Kwaaitaal M, Rampelt H, Kasmi FE, Jürgens G, Parker J, Panstruga R, Lipka V, Schulze-Lefert P. Co-option of a default secretory pathway for plant immune responses. Nature 2008; 451:835-40.

Plant exocytic secretion of toxic compounds for defense.

In contrast to animals, plants do not have a circulatory system as well as mobile immune cells that allow them to protect themselves against pathogens. Instead, plants exclusively depend on the innate immune system to defend against pathogens. As typically observed in the animal innate immunity, plant immune responses are composed of pathogen detection, defense signaling which includes transcriptional reprogramming, and secretion of antimicrobial compounds. Although knowledge on recognition and subsequent signaling of pathogen-derived molecules called elicitors is now expanding, the mechanisms of how these immune molecules are excreted are yet poorly understood. Therefore, current understandings of how plants secrete defense products especially via exocytosis will be discussed in this review.

Dual Effect of the Cubic Ag3PO4 Crystal on Pseudomonas syringae Growth and Plant Immunity

We previously found that the antibacterial activity of silver phosphate crystals on Escherichia coli depends on their structure. We here show that the cubic form of silver phosphate crystal (SPC) can also be applied to inhibit the growth of a plant-pathogenic Pseudomonas syringae bacterium. SPC pretreatment resulted in reduced in planta multiplication of P. syringae. Induced expression of a plant defense marker gene PR1 by SPC alone is suggestive of its additional plant immunity-stimulating activity. Since SPC can simultaneously inhibit P. syringae growth and induce plant defense responses, it might be used as a more effective plant disease-controlling agent.