Miklis, Marco (2004) A high-throughput procedure for the identification of genes contributing to plant defence mechanisms. PhD thesis, Universität zu Köln.
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.
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