A high-throughput procedure for the identification of genes contributing to plant defence mechanisms

Attempted host cell invasion by pathogenic fungi is known to trigger cell polarisation. Barley MLO is a member of a family of heptahelical membrane proteins unique to plants and is thought to regulate polarised SNARE protein-dependent secretory processes. Recessively inherited mutations in Mlo mediate resistance against all isolates (broad-spectrum resistance) of the biotrophic barley powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). It is assumed that the fungus utilizes MLO to suppress defence-associated secretion for successful host cell entry. In this study, a genetic screen was established based on gene silencing by double-stranded RNA interference (dsRNAi). This was accomplished by transient expression of inverted repeat DNAs in single epidermal barley cells. To identify novel components of MLO-modulated defence processes to Bgh, approximately 700 inverted repeat DNA constructs, derived from barley epidermal cDNAs, were generated and ballistically delivered into epidermal cells of both Mlo and mlo genotypes. Two dsRNAi constructs were identified permitting enhanced Bgh entry in a resistant mlo genotype. Both target the same isoform of an actin depolymerising factor, designated HvADF3. ADFs exist throughout all eukaryotic kingdoms and function in actin filament turnover (treadmilling), thereby contributing to actin cytoskeleton dynamics. Transient ectopic expression of HvADF3 in mlo epidermal cells also permitted enhanced Bgh invasion. Ectopic expression of HvADF3 or dsRNAi-mediated gene silencing resulted in the disappearance of phalloidin-stainable actin filaments, consistent with actin serving as substrate of ADFs. Closer inspection of the topology of the HvADF3 dsRNAi constructs revealed that the presumed silencing effect might be triggered by undesired overexpression. However, due to published pharmacological evidence implicating actin filament reorganisation in plant defence responses, the role of HvADF3 was further investigated. HvADF3 overexpression greatly reduced callose accumulation at pathogen entry sites, a common local stress response at the plant cell wall known to be sensitive to actin-depolymerising drugs. Replacements of an N-terminal serine residue, previously shown to be a phosphorylation target of ADF activity, to Ala or Asp enhanced and reduced Bgh entry rates, respectively, upon transient expression. Ectopic expression of six out of nine tested Arabidopsis ADF genes in barley phenocopied the effect of HvADF3 overexpression, demonstrating that multiple heterologous ADF isoforms can enhance pathogen entry. Although ectopic ADF expression impaired broad-spectrum resistance to Bgh, race-specific resistance (R) triggered by R genes Mlg, Mla1, or Mla6 was not affected. This is consistent with previous genetic data indicating separate pathways for broad-spectrum and R gene-mediated immune responses. Resistance to two tested inappropriate powdery mildews, Bg f sp tritici and Erysiphe pisi, that colonise in nature monocotyledonous wheat and dicotyledonous pea hosts respectively, was impaired upon HvADF3 overexpression. Interestingly, this impairment required the presence of Mlo, suggesting that both inappropriate powdery mildews potentially modulate barley defence reactions via MLO. To identify presumed common components of pathogen-triggered and developmentally controlled cell polarity, a range of Arabidopsis mutants with defects in leaf hair (trichome) development were tested for altered infection phenotypes to diverse powdery mildew fungi. Preliminary data indicate that the absence of an actin-related protein (ARP) 2/3 complex component, CROOKED, enhances pathogen entry of inappropriate powdery mildew species

Recruitment and interaction dynamics of plant penetration resistance components in a plasma membrane microdomain

Many fungal pathogens must enter plant cells for successful colonization. Barley mildew resistance locus o (Mlo) is required for host cell invasion upon attack by the ascomycete powdery mildew fungus, Blumeria graminis f.sp. hordei, and encodes the founder of a family of heptahelical integral membrane proteins unique to plants. Recessively inherited loss-of-function mutant alleles (mlo) result in effective penetration resistance to all isolates of the biotrophic parasite. We used noninvasive fluorescence-based imaging to show that fluorescently tagged MLO protein becomes redistributed in the plasma membrane (PM) and accumulates beneath fungal appressoria coincident with the initiation of pathogen entry into host cells. Polarized MLO accumulation occurs once upon attack and appears to be independent of actin cytoskeleton function. Likewise, barley ROR2 syntaxin, a genetically defined component of penetration resistance to B. graminis f.sp. hordei, and a subset of predicted PM-resident proteins become redistributed to fungal entry sites. We previously identified calmodulin, a cytoplasmic calcium sensor, as an interactor and positive regulator of MLO activity and demonstrate here by FRET microscopy an increase in MLO/calmodulin FRET around penetration sites coincident with successful host cell entry. Our data provide evidence for the formation of a pathogen-triggered PM microdomain that is reminiscent of membrane microdomains (lipid rafts) induced upon attempted entry of pathogenic bacteria in animal cells

Barley MLO Modulates Actin-Dependent and Actin-Independent Antifungal Defense Pathways at the Cell Periphery1[W][OA]

Cell polarization is a crucial process during plant development, as well as in plant-microbe interactions, and is frequently associated with extensive cytoskeletal rearrangements. In interactions of plants with inappropriate fungal pathogens (so-called non-host interactions), the actin cytoskeleton is thought to contribute to the establishment of effective barriers at the cell periphery against fungal ingress. Here, we impeded actin cytoskeleton function in various types of disease resistance using pharmacological inhibitors and genetic interference via ectopic expression of an actin-depolymerizing factor-encoding gene, ADF. We demonstrate that barley (Hordeum vulgare) epidermal cells require actin cytoskeleton function for basal defense to the appropriate powdery mildew pathogen Blumeria graminis f. sp. hordei and for mlo-mediated resistance at the cell wall, but not for several tested race-specific immune responses. Analysis of non-host resistance to two tested inappropriate powdery mildews, Erysiphe pisi and B. graminis f. sp. tritici, revealed the existence of actin-dependent and actin-independent resistance pathways acting at the cell periphery. These pathways act synergistically and appear to be under negative control by the plasma membrane-resident MLO protein

Conserved ERAD-Like Quality Control of a Plant Polytopic Membrane Protein

The endoplasmic reticulum (ER) of eukaryotic cells serves as a checkpoint tightly monitoring protein integrity and channeling malformed proteins into different rescue and degradation routes. The degradation of several ER lumenal and membrane-localized proteins is mediated by ER-associated protein degradation (ERAD) in yeast (Saccharomyces cerevisiae) and mammalian cells. To date, evidence for the existence of ERAD-like mechanisms in plants is indirect and based on heterologous or artificial substrate proteins. Here, we show that an allelic series of single amino acid substitution mutants of the plant-specific barley (Hordeum vulgare) seven-transmembrane domain mildew resistance o (MLO) protein generates substrates for a postinsertional quality control process in plant, yeast, and human cells, suggesting conservation of the underlying mechanism across kingdoms. Specific stabilization of mutant MLO proteins in yeast strains carrying defined defects in protein quality control demonstrates that MLO degradation is mediated by HRD pathway-dependent ERAD. In plants, individual aberrant MLO proteins exhibit markedly reduced half-lives, are polyubiquitinated, and can be stabilized through inhibition of proteasome activity. This and a dependence on homologs of the AAA ATPase CDC48/p97 to eliminate the aberrant variants strongly suggest that MLO proteins are endogenous substrates of an ERAD-related plant quality control mechanism