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

Abstract

1Cell-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 1-3 . 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.Arabidopsis is immune to non-adapted powdery mildew fungi such as Blumeria graminis and Erysiphe pisi, which in nature colonize grass and pea species, respectively. This non-host resistance requires both pre-and post-invasive immune responses, which are under separate genetic control 2 . The former response engages PEN1 syntaxin, peroxisomal PEN2 b-glycosyl hydrolase and the plasmamembrane-resident PEN3 ABC transporter 1-3 . PEN2 and PEN3 act in the same pathway and are implicated in the cytoplasmic synthesis and transport of small antimicrobial compounds across the plasma membrane at attempted fungal entry sites, respectively 2,3 . PEN1 syntaxin acts in a second pathway and could, by analogy to known syntaxin functions in yeast and animals, either participate in vesicle fusion processes 4 or modulate ion-channel activity through interactions with plasma-membrane-resident ion channels 5 . Genetic studies defy mechanistic interpretations but suggest direct or indirect PEN1 repressor activity in defence responses that are dependent on salicylic acid, as well as an overlapping function with the closely related syntaxin of plant 122 (SYP122) 6 . Compared with largely resistant PEN1 wild type and severely defence-compromised pen1-1 null mutants, plants containing the pen1-3 allele allow intermediate B. graminis entry rates, indicating residual PEN1-3 resistance activity ( Supplementary Fig. 1a). In the deduced PEN1-3 protein, a glycine residue is substituted by a glutamate in the SNARE domain 1 (Supplementary Fig. 1b). Because this mutation affects a hydrophobic residue that is thought to stabilize interactions with ...

Requirement of Vesicle-Associated Membrane Protein 721 and 722 for Sustained Growth during Immune Responses in Arabidopsis

“…After growth for further two days to allow adaptation, the indicated amount of flg22 or MG132 was applied for the indicated time. For subcellular localization assays of VAMP722, transgenic plants expressing functional GFP-VAMP722 driven by its 5′ regulatory sequences (Kwon et al, 2008c) were used. To analyze the extent of growth inhibition by flg22, plants of the indicated genotypes were grown for one week, treated with 100 nM flg22 and allowed to grow five more days.…”

“…CYP81F2 is necessary for the generation of substrates of PEN2, which in turn initiates the metabolism of a group of tryptophan-derived compounds, known as indole glucosinolates, to release potential antimicrobial products by the PEN3 transporter into the extracellular space (Bednarek et al, 2009;Lipka et al, 2005;Stein et al, 2006). The other consists of the PM-anchored syntaxin PEN1, the PM-bound adaptor SNAP33, and the vesicle-associated membrane proteins (VAMPs) 721 and 722 that share 96% identical amino acid sequences (Collins et al, 2003;Kwon et al, 2008c;Pajonk et al, 2008). These proteins are proposed to operate in an exocytic plant defense pathway by forming ternary SNARE complexes (Kwon et al, 2008c).…”

“…The other consists of the PM-anchored syntaxin PEN1, the PM-bound adaptor SNAP33, and the vesicle-associated membrane proteins (VAMPs) 721 and 722 that share 96% identical amino acid sequences (Collins et al, 2003;Kwon et al, 2008c;Pajonk et al, 2008). These proteins are proposed to operate in an exocytic plant defense pathway by forming ternary SNARE complexes (Kwon et al, 2008c). We additionally showed that VAMP721/722 have a broader role in plant defense responses because their depletion in haplo-insufficient VAMP721 -/-VAMP722 +/-and VAMP721 +/-VAMP722 -/-genotypes leads to impaired defense to a virulent oomycete, Hyaloperonospora arabidopsidis, as well as to the non-adapted Erysiphe pisi and the host-adapted Golovinomyces orontii powdery mildew fungi (Kwon et al, 2008c).…”

“…These proteins are proposed to operate in an exocytic plant defense pathway by forming ternary SNARE complexes (Kwon et al, 2008c). We additionally showed that VAMP721/722 have a broader role in plant defense responses because their depletion in haplo-insufficient VAMP721 -/-VAMP722 +/-and VAMP721 +/-VAMP722 -/-genotypes leads to impaired defense to a virulent oomycete, Hyaloperonospora arabidopsidis, as well as to the non-adapted Erysiphe pisi and the host-adapted Golovinomyces orontii powdery mildew fungi (Kwon et al, 2008c). Since loss of PEN1 specifically allows enhanced plant cell entry of non-adapted powdery mildews (Collins et al, 2003), this points to the possible involvement of other syntaxins than PEN1 in defense responses against H. arabidopsidis and G. orontii.…”

“…Similarly, the VAMP721/722-cosilenced plants are dwarfed, and the snap33 and the vamp721 vamp722 double mutant genotypes are lethal at early seedling stage (Assaad et al, 2004;Heese et al, 2001;Kwon et al, 2008c;Zhang et al, 2007), suggesting that the PEN1-SNAP33-VAMP721/722 pathway also serves a role in constitutive secretion during plant growth, transporting materials required for cellular viability to the PM. We here describe a function of VAMP721/722 in sustained growth during immune responses in plants.…”