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

Abstract

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 MLO͞calmodulin 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.

Myosins XI modulate host cellular responses and penetration resistance to fungal pathogens

“…All three PEN proteins are also recruited to attempted fungal penetration sites (6)(7)(8)(9)(10)(11). Intriguing findings show that the focal accumulation of PEN1 and PEN3 occurs outside the plasma membrane and within papillae or haustorial encasements (3,11,12).…”

“…However, it is important to note that Arabidopsis PEN1 is associated with the pathogen-induced PM microdomains and exhibits constitutive cycling between the PM and endosomes (6,9,43). This striking dynamic behavior may facilitate rapid retargeting of PEN1 to the pathogen infection site.…”

ArabidopsisPEN3/PDR8, an ATP Binding Cassette Transporter, Contributes to Nonhost Resistance to Inappropriate Pathogens That Enter by Direct Penetration

“…PEN1 (¼SYP121 syntaxin), the barley PEN1 homolog ROR2, and the barley MLO protein also localize in a similar pattern to the plasma membrane after powdery mildew infection (Assaad et al, 2004;Bhat et al, 2005). In addition, PEN2-GFP localizes to peroxisomes that accumulate near sites of invasion (Lipka et al, 2005).…”

Advances in Fluorescent Protein-Based Imaging for the Analysis of Plant Endomembranes

“…There are not yet many reports of FRET and FRET/FLIM analyses for studies in plant endomembranes. However, the technique has been successfully used to probe microdomains in the plasma membrane of cowpea (Vigna unguiculata) protoplasts (Vermeer et al, 2004) and barley (Hordeum vulgare) leaf epidermal cells during pathogen attack (Bhat et al, 2005) and to determine the orientation of the VHA-subunits of the head and peripheral stalk of the V-ATPase complex subunits in Arabidopsis protoplasts (Seidel et al, 2005). FRET/FLIM has been applied recently for the study of protein transport and targeting in plant endomembranes.…”