The powdery mildew resistance protein RPW8.2 is carried on VAMP721/722 vesicles to the extrahaustorial membrane of haustorial complexes

Abstract

Plants employ multiple cell-autonomous defense mechanisms to impede pathogenesis of microbial intruders. Previously we identified an exocytosis defense mechanism in Arabidopsis against pathogenic powdery mildew fungi. This pre-invasive defense mechanism depends on the formation of ternary protein complexes consisting of the plasma membrane-localized PEN1 syntaxin, the adaptor protein SNAP33 and closely sequence-related vesicle-resident VAMP721 or VAMP722 proteins. The Arabidopsis thaliana resistance to powdery mildew 8.2 protein (RPW8.2) confers disease resistance against powdery mildews upon fungal entry into host cells and is specifically targeted to the extrahaustorial membrane (EHM), which envelops the haustorial complex of the fungus. However, the secretory machinery involved in trafficking RPW8.2 to the EHM is unknown. Here we report that RPW8.2 is transiently located on VAMP721/722 vesicles, and later incorporated into the EHM of mature haustoria. Resistance activity of RPW8.2 against the powdery mildew Golovinomyces orontii is greatly diminished in the absence of VAMP721 but only slightly so in the absence of VAMP722. Consistent with this result, trafficking of RPW8.2 to the EHM is delayed in the absence of VAMP721. These findings implicate VAMP721/722 vesicles as key components of the secretory machinery for carrying RPW8.2 to the plant-fungal interface. Quantitative fluorescence recovery after photobleaching suggests that vesicle-mediated trafficking of RPW8.2-yellow fluorescent protein (YFP) to the EHM occurs transiently during early haustorial development and that lateral diffusion of RPW8.2-YFP within the EHM exceeds vesicle-mediated replenishment of RPW8.2-YFP in mature haustoria. Our findings imply the engagement of VAMP721/722 in a bifurcated trafficking pathway for pre-invasive defense at the cell periphery and post-invasive defense at the EHM.

Homologues of the RPW8 Resistance Protein Are Localized to the Extrahaustorial Membrane that Is Likely Synthesized De Novo

“…8B). In line with this inference, an exocytosis pathway defined by a SNARE complex defined by Vamp721/Vamp722 and SNAP33 has recently been shown to be required for efficient targeting of RPW8.2 vesicles to the EHM (Kim et al, 2014). Third, consistent with the above results, we also found that YFP-HR3's localization to the papilla is BFA sensitive, whereas EHM-targeting of YFP-HR3 (and RPW8.2-RFP) is BFA insensitive (Fig.…”

“…We recently revealed that RPW8.2 [also RPW8.1 when expressed in the haustorium-invaded cells (Ma et al, 2014)] is specifically targeted to the EHM where it enhances formation of the haustorial encasement and promotes onsite H 2 O 2 accumulation and/or triggers hypersensitive response . Specific targeting of RPW8.2 to the EHM requires two basic residue-enriched motifs in RPW8.2 and engages an EHM-oriented specific trafficking pathway (Wang et al, 2013;Kim et al, 2014). Thus, the EHM is the subcellular locale for launching RPW8.2-mediated, haustorium-targeted defenses.…”

Mutant Allele-Specific Uncoupling of PENETRATION3 Functions Reveals Engagement of the ATP-Binding Cassette Transporter in Distinct Tryptophan Metabolic Pathways

“…Genetic screens for impaired extracellular defense in nonhost resistance to the nonadapted powdery mildew pathogens also identified SYNTAXIN RELATED PROTEIN1 (SYR1), also known as SYP121/PEN1 and PEN2, which encode a plasma membrane-resident syntaxin and a myrosinase, respectively (Leyman et al, 1999;Collins et al, 2003;Lipka et al, 2005;Stein et al, 2006). PEN2 and PEN3 act in the same pathway for extracellular defense, whereas PEN1 functions in a parallel secretory defense pathway (Collins et al, 2003;Kwon et al, 2008;Kim et al, 2014). PEN2 function has been assigned to a glucosinolate metabolic pathway, which comprises biosynthesis of indole glucosinolates (IGs), pathogen-inducible redirection of this biosynthesis through the CYTO-CHROME P450 81F2 (CYP81F2) monooxygenase to 4-methoxyindol-3-ylmethylglucosinolate (4MI3G), and 4MI3G activation by PEN2 myrosinase through deglucosylation (Bednarek et al, 2009).…”

Co-regulation of theGlycine maxsoluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE)-containing regulon occurs during defense to a root pathogen

“…SYP121 forms a complex on the PM in association with two vesicle-associated membrane proteins (VAMPs), VAMP721 and VAMP722 (Collins et al 2003;Kwon et al 2008). VAMP721 and VAMP722 exhibit homology to the rat (Rattus norvegicus) synaptobrevin (SYB) and S. cerevisiae Ykt6p and Sec22p (Baumert et al 1989;Dujon et al 1994;Søgaard et al 1994;McNew et al 1997;Sanderfoot et al 2000;Collins et al 2003;Lipka et al 2007;Kwon et al 2008;Kim et al 2014). Therefore, 4 VAMP protein classes exist (Lipka et al 2007).…”

“…Therefore, 4 VAMP protein classes exist (Lipka et al 2007). SYP121 also functions with the 33 kilodalton (kD) soluble N-ethylmaleimide-sensitive factor (NSF) adaptor protein (SNAP33), related to the mouse (Mus musculus) SNAP-25 and S. cerevisiae Sec9p (Oyler et al 1989;Collins et al 2003;Kwon et al 2008;Kim et al 2014). PEN1 functions in the formation of a membranous defense apparatus called a cell wall apposition (Aist 1976;Collins et al 2003).…”