Nonhost resistance in Arabidopsis-Colletotrichum interactions acts at the cell periphery and requires actin filament function

Pathogenesis of nonadapted fungal pathogens is often terminated coincident with their attempted penetration into epidermal cells of nonhost plants. The genus Colletotrichum represents an economically important group of fungal plant pathogens that are amenable to molecular genetic analysis. Here, we investigated interactions between Arabidopsis and Colletotrichum to gain insights in plant and pathogen processes activating nonhost resistance responses. Three tested nonadapted Colletotrichum species differentiated melanized appressoria on Arabidopsis leaves but failed to form intracellular hyphae. Plant cells responded to Colletotrichum invasion attempts by the formation of PMR4/GSL5-dependent papillary callose. Appressorium differentiation and melanization were insufficient to trigger this localized plant cell response, but analysis of nonpathogenic C. lagenarium mutants implicates penetration-peg formation as the inductive cue. We show that Arabidopsis PEN1 syntaxin controls timely accumulation of papillary callose but is functionally dispensable for effective preinvasion (penetration) resistance in nonhost interactions. Consistent with this observation, green fluorescent protein-tagged PEN1 did not accumulate at sites of attempted penetration by either adapted or nonadapted Colletotrichum species, in contrast to the pronounced focal accumulations of PEN1 associated with entry of powdery mildews. We observed extensive reorganization of actin microfilaments leading to polar orientation of large actin bundles towards appressorial contact sites in interactions with the nonadapted Colletotrichum species. Pharmacological inhibition of actin filament function indicates a functional contribution of the actin cytoskeleton for both preinvasion resistance and papillary callose formation. Interestingly, the incidence of papilla formation at entry sites was greatly reduced in interactions with C. higginsianum isolates, indicating that this adapted pathogen may suppress preinvasion resistance at the cell periphery.     

Rapid and dynamic subcellular reorganization following mechanical stimulation of <Emphasis Type="Italic">Arabidopsis</Emphasis>epidermal cells mimics responses to fungal and oomycete attack

Background  

Plant cells respond to the presence of potential fungal or oomycete pathogens by mounting a basal defence response that involves aggregation of cytoplasm, reorganization of cytoskeletal, endomembrane and other cell components and development of cell wall appositions beneath the infection site. This response is induced by non-adapted, avirulent and virulent pathogens alike, and in the majority of cases achieves penetration resistance against the microorganism on the plant surface. To explore the nature of signals that trigger this subcellular response and to determine the timing of its induction, we have monitored the reorganization of GFP-tagged actin, microtubules, endoplasmic reticulum (ER) and peroxisomes inArabidopsis plants – after touching the epidermal surface with a microneedle.     

Simultaneous visualization of peroxisomes and cytoskeletal elements reveals actin and not microtubule-based peroxisome motility in plants

Peroxisomes were visualized in living plant cells using a yellow fluorescent protein tagged with a peroxisomal targeting signal consisting of the SKL motif. Simultaneous visualization of peroxisomes and microfilaments/microtubules was accomplished in onion (Allium cepa) epidermal cells transiently expressing the yellow fluorescent protein-peroxi construct, a green fluorescent protein-mTalin construct that labels filamentous-actin filaments, and a green fluorescent protein-microtubule-binding domain construct that labels microtubules. The covisualization of peroxisomes and cytoskeletal elements revealed that, contrary to the reports from animal cells, peroxisomes in plants appear to associate with actin filaments and not microtubules. That peroxisome movement is actin based was shown by pharmacological studies. For this analysis we used onion epidermal cells and various cell types of Arabidopsis including trichomes, root hairs, and root cortex cells exhibiting different modes of growth. In transient onion epidermis assay and in transgenic Arabidopsis plants, an interference with the actin cytoskeleton resulted in progressive loss of saltatory movement followed by the aggregation and a complete cessation of peroxisome motility within 30 min of drug application. Microtubule depolymerization or stabilization had no effect.     

Epidermal cells of a symbiosis-defective mutant of Lotus japonicus show altered cytoskeleton organisation in the presence of a mycorrhizal fungus

The influence of the mycorrhizal fungus Gigaspora margarita on cytoskeleton organisation in epidermal cells of Lotus japonicus roots was compared between plants of the wild type Gifu and the mutant Ljsym4-2, in which the fungus is confined to the epidermal cells. Immunofluorescence labelling of plant microtubules and microfilaments showed only limited alterations in the peripheral cytoskeleton of epidermal cells during early stages of fungal interaction with the wild type. Later, microtubules and microfilaments enveloped the growing hypha, while the host cell nucleus moved close to the fungus. In contrast, epidermal cells of the mutant responded with disorganisation and disassembly of microtubules and microfilaments before and during fungal penetration attempts. The fungus penetrated only as far as to epidermal cells, whose cytoplasm became devoid of tubulin and actin, suggesting cell death. The close relationship between host cytoskeleton organisation and compatibility with the fungus suggests that a functional Ljsym4 gene is necessary for correct reorganisation of the epidermal cell cytoskeleton in the presence of the fungus and for avoiding hypersensitivity-like reactions.     

RNA processing bodies, peroxisomes, Golgi bodies, mitochondria, and endoplasmic reticulum tubule junctions frequently pause at cortical microtubules

Organelle motility, essential for cellular function, is driven by the cytoskeleton. In plants, actin filaments sustain the long-distance transport of many types of organelles, and microtubules typically fine-tune the motile behavior. In shoot epidermal cells of Arabidopsis thaliana seedlings, we show here that a type of RNA granule, the RNA processing body (P-body), is transported by actin filaments and pauses at cortical microtubules. Interestingly, removal of microtubules does not change the frequency of P-body pausing. Similarly, we show that Golgi bodies, peroxisomes, and mitochondria all pause at microtubules, and again the frequency of pauses is not appreciably changed after microtubules are depolymerized. To understand the basis for pausing, we examined the endoplasmic reticulum (ER), whose overall architecture depends on actin filaments. By the dual observation of ER and microtubules, we find that stable junctions of tubular ER occur mainly at microtubules. Removal of microtubules reduces the number of stable ER tubule junctions, but those remaining are maintained without microtubules. The results indicate that pausing on microtubules is a common attribute of motile organelles but that microtubules are not required for pausing. We suggest that pausing on microtubules facilitates interactions between the ER and otherwise translocating organelles in the cell cortex.     

Visualization of peroxisomes in living plant cells reveals acto-myosin-dependent cytoplasmic streaming and peroxisome budding

Here we examine peroxisomes in living plant cells using transgenic Arabidopsis thaliana plants expressing the green fluorescent protein (GFP) fused to the peroxisome targeting signal 1 (PTS1). Using time-lapse laser scanning confocal microscopy we find that plant peroxisomes exhibit fast directional movement with peak velocities approaching 10 microm s(-1). Unlike mammalian peroxisomes which move on microtubules, plant peroxisome movement is dependent on actin microfilaments and myosin motors, since it is blocked by treatment with latrunculin B and butanedione monoxime, respectively. In contrast, microtubule-disrupting drugs have no effect on peroxisome streaming. Peroxisomes were further shown to associate with the actin cytoskeleton by the simultaneous visualization of actin filaments and peroxisomes in living cells using GFP-talin and GFP-PTS1 fusion proteins, respectively. In addition, peroxisome budding was observed, suggesting a possible mechanism of plant peroxisome proliferation. The strong signal associated with the GFP-PTS1 marker also allowed us to survey cytoplasmic streaming in different cell types. Peroxisome movement is most intense in elongated cells and those involved in long distance transport, suggesting that higher plants use cytoplasmic streaming to help transport vesicles and organelles over long distances.     

Cytoskeleton and cell wall function in penetration resistance

Plants successfully repel the vast majority of potential pathogens that arrive on their surface, with most microorganisms failing to breach the outer epidermal wall. Resistance to penetration at the epidermis is a key component of basal defence against disease and critically depends on fortification of the cell wall at the site of attempted penetration through the development of specialised cell wall appositions rich in antimicrobial compounds. Formation of cell wall appositions is achieved by rapid reorganisation of actin microfilaments, actin-dependent transport of secretory products to the infection site and local activation of callose synthesis. Plants are finely tuned to detect the presence of pathogens on their surface, perceiving both chemical and physical signals of pathogen origin. In the on-going evolution of interaction strategies, plants must continually monitor and out manoeuvre pathogen avoidance or suppression of plant defences in order to preserve the effectiveness of penetration resistance.     

An internal motor kinesin is associated with the Golgi apparatus and plays a role in trichome morphogenesis in Arabidopsis

Members of the kinesin superfamily are microtubule-based motor proteins that transport molecules/organelles along microtubules. We have identified similar internal motor kinesins, Kinesin-13A, from the cotton Gossypium hirsutum and Arabidopsis thaliana. Their motor domains share high degree of similarity with those of internal motor kinesins of animals and protists in the MCAK/Kinesin13 subfamily. However, no significant sequence similarities were detected in sequences outside the motor domain. In Arabidopsis plants carrying the T-DNA knockout kinesin-13a-1 and kinesin-13a-2 mutations at the Kinesin-13A locus, >70% leaf trichomes had four branches, whereas wild-type trichomes had three. Immunofluorescent results showed that AtKinesin-13A and GhKinesin-13A localized to entire Golgi stacks. In both wild-type and kinesin-13a mutant cells, the Golgi stacks were frequently associated with microtubules and with actin microfilaments. Aggregation/clustering of Golgi stacks was often observed in the kinesin-13a mutant trichomes and other epidermal cells. This suggested that the distribution of the Golgi apparatus in cell cortex might require microtubules and Kinesin-13A, and the organization of Golgi stacks could play a regulatory role in trichome morphogenesis. Our results also indicate that plant kinesins in the MCAK/Kinesin-13 subfamily have evolved to take on different tasks than their animal counterparts.     

Arabidopsis sterol endocytosis involves actin-mediated trafficking via ARA6-positive early endosomes

BACKGROUND: In contrast to the intense attention devoted to research on intracellular sterol trafficking in animal cells, knowledge about sterol transport in plant cells remains limited, and virtually nothing is known about plant endocytic sterol trafficking. Similar to animals, biosynthetic sterol transport occurs from the endoplasmic reticulum (ER) via the Golgi apparatus to the plasma membrane. The vesicle trafficking inhibitor brefeldin A (BFA) has been suggested to disrupt biosynthetic sterol transport at the Golgi level. RESULTS: Here, we report on early endocytic sterol trafficking in Arabidopsis root epidermal cells by introducing filipin as a tool for fluorescent sterol detection. Sterols can be internalized from the plasma membrane and localize to endosomes positive for the early endosomal Rab5 GTPase homolog ARA6 fused to green fluorescent protein (GFP) (ARA6-GFP). Early endocytic sterol transport is actin dependent and highly BFA sensitive. BFA causes coaccumulation of sterols, endocytic markers like ARA6-GFP, and PIN2, a polarly localized presumptive auxin transport protein, in early endosome agglomerations that can be distinguished from ER and Golgi. Sterol accumulation in such aggregates is enhanced in actin2 mutants, and the actin-depolymerizing drug cytochalasin D inhibits sterol redistribution from endosome aggregations. CONCLUSIONS: Early endocytic sterol trafficking involves transport via ARA6-positive early endosomes that, in contrast to animal cells, is actin dependent. Our results reveal sterol-enriched early endosomes as targets for BFA interference in plants. Early endocytic sterol trafficking and recycling of polar PIN2 protein share a common pathway, suggesting a connection between plant endocytic sterol transport and polar sorting events.     

Dynamic reorganization of microfilaments and microtubules is necessary for the expression of non-host resistance in barley coleoptile cells

To show the involvement of microfilaments and microtubules in non-host resistance of barley, partially dissected coleoptiles which had been inoculated with a non-pathogen, Erysiphe pisi, were treated with several actin and tubulin inhibitors. If the coleoptiles were not treated with any of the inhibitors, the non-pathogen always failed to penetrate the coleoptile cells. However, when coleoptiles were treated with actin or tubulin polymerization or depolymerization inhibitors, the non-pathogen was able to penetrate successfully and to form haustoria in coleoptile cells of a non-host plant, barley. Actin polymerization inhibitors, cytochalasins, were more effective in causing an increase in penetration efficiency of E. pisi than tubulin inhibitors. The effects of cytochalasins depended on the kind of cytochalasin; the strength of the actin depolymerizing activity correlated significantly with the efficiency of increasing the penetration of the non-pathogen. When both actin and tubulin inhibitors were added simultaneously, the polarization of defense-related responses, such as massive cytoplasmic aggregation, deposition of papillae and accumulation of autofluorescent compounds, at fungal penetration sites was suppressed. Actin inhibitors did not affect arrangement and stability of microtubules and vice versa, and a double treatment of coleoptile cells with both microfilament and microtubule inhibitors showed an additive effect in increasing the penetration efficiency of E. pisi. Furthermore, cytochalasin A treatment allowed other non-pathogens, Colletotrichum lagenarium and Alternaria alternata, to penetrate successfully into the non-host barley cells. These results strongly suggest that microfilaments and microtubules might play important roles in the expression of non-host resistance of barley.     

Actin Microfilaments are Required for the Expression of Nonhost Resistance in Higher Plants

We investigated the role of actin microfilaments in nonhostresistance of higher plants. Here we present several lines ofevidence to indicate that microfilaments are indeed involvedin blocking fungal penetration of nonhost plants. Erysiphe pisi,a pathogen of pea, normally fails to penetrate into nonhostplants such as barley, wheat, cucumber and tobacco. When tissuesof these nonhost plants were treated with cytochalasins, specificinhibitors of actin polymerization, this fungus became ableto penetrate and formed haustoria in epidermal cells of theseplants. Moreover, treatment of these plants with various kindsand concentrations of cytochalasins allowed several other non-pathogenicfungi, E.graminis hordei, E.graminis tritici, Sphaerotheca fuliginea,Colletotrichum graminicola, My-cosphaella pinodes, C. lagenarium,Altemaria kikuchiana and Corynespora melonis, to also penetratethe cells of these plants. The degree of microfilament depolymeriza-tionvaried depending on the kinds and concentrations of cytochalasinsapplied and we show that this is significantly correlated withthe penetration efficiency of C. graminicola. This indicatesthat the polymerized, filamentous state of actin is necessaryfor plants to block fungal penetration. These results stronglysuggest that actin microfilaments may play important roles inthe expression of nonhost resistance of higher plants. ¹Contribution no. 129 from the Laboratory of Plant Pathology,Mie University     

Actin cytoskeleton rearrangements during the gravitropic response of Arabidopsis roots

Gravitropic response is a plant growth response against changing its position relative to the gravity vector. In the present work we studied actin cytoskeleton rearrangements during Arabidopsis root gravitropic response. Two alternative approaches were used to visualize actin microfilaments: histochemical staining of fixed roots with rhodamine-phalloidin and live imaging of microfilaments in GFP-fABD2 transgenic plants. The curvature of actin microfilaments was shown to be increased within 30–60 min of gravistimulation, the fraction of axially oriented microfilaments decreased with a concomitant increase in the fraction of oblique and transversally oriented microfilaments. Methodological issues of actin cytoskeleton visualization in the study of Arabidopsis root gravitropic response, as well as the role of microfilaments at the stages of gravity perception, signal transduction and gravitropic bending formation are discussed. It is concluded that the actin cytoskeleton rearrangements observed are associated with the regulation of basic mechanisms of cell extension growth by which the gravitropic bending is formed.     

Actin Filaments in Mature Guard Cells Are Radially Distributed and Involved in Stomatal Movement

Stomatal movements, which regulate gas exchange in plants, involve pronounced changes in the shape and volume of the guard cell. To test whether the changes are regulated by actin filaments, we visualized microfilaments in mature guard cells and examined the effects of actin antagonists on stomatal movements. Immunolocalization on fixed cells and microinjection of fluorescein isothiocyanate-phalloidin into living guard cells of Commelina communis L. showed that cortical microfilaments were radially distributed, fanning out from the stomatal pore site, resembling the known pattern of microtubules. Treatment of epidermal peels with phalloidin prior to stabilizing microfilaments with m-maleimidobenzoyl N-hydroxysuccimimide caused dense packing of radial microfilaments and an accumulation of actin around many organelles. Both stomatal closing induced by abscisic acid and opening under light were inhibited. Treatment of guard cells with cytochalasin D abolished the radial pattern of microfilaments; generated sparse, poorly oriented arrays; and caused partial opening of dark-closed stomata. These results suggest that microfilaments participate in stomatal aperture regulation.     

ER-to-Golgi transport by COPII vesicles in Arabidopsis involves a ribosome-excluding scaffold that is transferred with the vesicles to the Golgi matrix

Plant Golgi stacks are mobile organelles that can travel along actin filaments. How COPII (coat complex II) vesicles are transferred from endoplasmic reticulum (ER) export sites to the moving Golgi stacks is not understood. We have examined COPII vesicle transfer in high-pressure frozen/freeze-substituted plant cells by electron tomography. Formation of each COPII vesicle is accompanied by the assembly of a ribosome-excluding scaffold layer that extends approximately 40 nm beyond the COPII coat. These COPII scaffolds can attach to the cis-side of the Golgi matrix, and the COPII vesicles are then transferred to the Golgi together with their scaffolds. When Atp115-GFP, a green fluorescent protein (GFP) fusion protein of an Arabidopsis thaliana homolog of the COPII vesicle-tethering factor p115, was expressed, the GFP localized to the COPII scaffold and to the cis-side of the Golgi matrix. Time-lapse imaging of Golgi stacks in live root meristem cells demonstrated that the Golgi stacks alternate between phases of fast, linear, saltatory movements (0.9-1.25 microm/s) and slower, wiggling motions (<0.4 microm/s). In root meristem cells, approximately 70% of the Golgi stacks were connected to an ER export site via a COPII scaffold, and these stacks possessed threefold more COPII vesicles than the Golgi not associated with the ER; in columella cells, only 15% of Golgi stacks were located in the vicinity of the ER. We postulate that the COPII scaffold first binds to and then fuses with the cis-side of the Golgi matrix, transferring its enclosed COPII vesicle to the cis-Golgi.     

Actin marker lines in grapevine reveal a gatekeeper function of guard cells

Resistance to abiotic and biotic stress is a central topic for sustainable agriculture, especially in grapevine, one of the field crops with the highest economic output per acreage. As early cellular factors for plant defense, actin microfilaments (AF) are of high relevance. We therefore generated a transgenic actin marker line for grapevine by expressing a fusion protein between green fluorescent protein and the second actin-binding domain of Arabidopsis (Arabidopsis thaliana) fimbrin, AtFIM1. Based on this first cytoskeletal-marker line in grapevine, the response of AFs to phytopathogenic microorganisms could be followed in vivo. Upon inoculation with fluorescently labeled strains of phytopathogenic bacteria, actin responses were confined to the guard cells. In contrast, upon contact with zoospores of Plasmopara viticola, not only the guard cells, but also epidermal pavement cells, where no zoospores had attached responded with the formation of a perinuclear actin basket. Our data support the hypothesis that guard cells act as pacemakers of defense, dominating the responses of the remaining epidermal cells.     

Stacks on tracks: the plant Golgi apparatus traffics on an actin/ER network †

We have visualized the relationship between the endoplasmic reticulum (ER) and Golgi in leaf cells of Nicotiana clevelandii by expression of two Golgi proteins fused to green fluorescent protein (GFP). A fusion of the trans -membrane domain (signal anchor sequence) of a rat sialyl transferase to GFP was targeted to the Golgi stacks. A second construct that expressed the Arabidopsis H/KDEL receptor homologue aERD2, fused to GFP, was targeted to both the Golgi apparatus and ER, allowing the relationship between these two organelles to be studied in living cells for the first time. The Golgi stacks were shown to move rapidly and extensively along the polygonal cortical ER network of leaf epidermal cells, without departing from the ER tubules. Co-localization of F-actin in the GFP-expressing cells revealed an underlying actin cytoskeleton that matched precisely the architecture of the ER network, while treatment of cells with the inhibitors cytochalasin D and N-ethylmaleimide revealed the dependency of Golgi movement on actin cables. These observations suggest that the leaf Golgi complex functions as a motile system of actin-directed stacks whose function is to pick up products from a relatively stationary ER system. Also, we demonstrate for the first time in vivo brefeldin A-induced retrograde transport of Golgi membrane protein to the ER.     

Mutations in actin-related proteins 2 and 3 affect cell shape development in Arabidopsis

ACTIN-RELATED PROTEINS 2 and 3 form the major subunits of the ARP2/3 complex, which is known as an important regulator of actin organization in diverse organisms. Here, we report that two genes, WURM and DISTORTED1, which are important for cell shape control in Arabidopsis, encode the plant ARP2 and ARP3 orthologs, respectively. Mutations in these genes result in misdirected expansion of various cell types: trichome expansion is randomized, pavement cells fail to produce lobes, hypocotyl cells curl out of the normal epidermal plane, and root hairs are sinuous. At the subcellular level, cell shape changes are linked to severe filamentous actin aggregation and compromised vacuole fusion. Because all seven subunits of the ARP2/3 complex are present in plants, our data indicate that this complex may play a pivotal role during plant cell morphogenesis.     

KATAMARI1/MURUS3 Is a novel golgi membrane protein that is required for endomembrane organization in Arabidopsis

In plant cells, unlike animal and yeast cells, endomembrane dynamics appear to depend more on actin filaments than on microtubules. However, the molecular mechanisms of endomembrane-actin filament interactions are unknown. In this study, we isolated and characterized an Arabidopsis thaliana mutant, katamari1 (kam1), which has a defect in the organization of endomembranes and actin filaments. The kam1 plants form abnormally large aggregates that consist of endoplasmic reticulum with actin filaments in the perinuclear region within the cells and are defective in normal cell elongation. Map-based cloning revealed that the KAM1 gene is allelic to the MUR3 gene. We demonstrate that the KAM1/MUR3 protein is a type II membrane protein composed of a short cytosolic N-terminal domain and a transmembrane domain followed by a large lumenal domain and is localized specifically on Golgi membranes. We further show that actin filaments interact with Golgi stacks via KAM1/MUR3 to maintain the proper organization of endomembranes. Our results provide functional evidence that KAM1/MUR3 is a novel component of the Golgi-mediated organization of actin functioning in proper endomembrane organization and cell elongation.     

Actin filaments play a critical role in vacuolar trafficking at the Golgi complex in plant cells

Actin filaments are thought to play an important role in intracellular trafficking in various eukaryotic cells. However, their involvement in intracellular trafficking in plant cells has not been clearly demonstrated. Here, we investigated the roles actin filaments play in intracellular trafficking in plant cells using latrunculin B (Lat B), an inhibitor of actin filament assembly, or actin mutants that disrupt actin filaments when overexpressed. Lat B and actin2 mutant overexpression inhibited the trafficking of two vacuolar reporter proteins, sporamin:green fluorescent protein (GFP) and Arabidopsis thaliana aleurain-like protein:GFP, to the central vacuole; instead, a punctate staining pattern was observed. Colocalization experiments with various marker proteins indicated that these punctate stains corresponded to the Golgi complex. The A. thaliana vacuolar sorting receptor VSR-At, which mainly localizes to the prevacuolar compartment, also accumulated at the Golgi complex in the presence of Lat B. However, Lat B had no effect on the endoplasmic reticulum (ER) to Golgi trafficking of sialyltransferase or retrograde Golgi to ER trafficking. Lat B also failed to influence the Golgi to plasma membrane trafficking of H+-ATPase:GFP or the secretion of invertase:GFP. Based on these observations, we propose that actin filaments play a critical role in the trafficking of proteins from the Golgi complex to the central vacuole.     

The receptor-like MLO protein and the RAC/ROP family G-protein RACB modulate actin reorganization in barley attacked by the biotrophic powdery mildew fungus Blumeria graminis f.sp. hordei

Cytoskeleton remodelling is a crucial process in determining the polarity of dividing and growing plant cells, as well as during interactions with the environment. Nothing is currently known about the proteins, which regulate actin remodelling during interactions with invading pathogens. The biotrophic powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh) invades susceptible barley (Hordeum vulgare L.) by penetrating epidermal cells, which remain intact during fungal development. In contrast, resistant host plants prevent infection by inhibiting penetration through apoplastic mechanisms, which require focusing defence reactions on the site of attack. We stained actin filaments in a susceptible Mlo-genotype and a near-isogenic race-non-specifically resistant barley mlo5-mutant genotype using fluorescence-labelled phalloidin after chemical fixation. This revealed that the actin cytoskeleton is differentially reorganized in susceptible and resistant hosts challenged by Bgh. Actin filaments were polarized towards the sites of attempted penetration in the resistant host, whereas when susceptible hosts were penetrated, a more subtle reorganization took place around fungal haustoria. Strong actin filament focusing towards sites of fungal attack was closely associated with successful prevention of penetration. Actin focusing was less frequent and seemingly delayed in susceptible wild-type barley expressing the susceptibility factor MLO. Additionally, single cell overexpression of a constitutively activated RAC/ROP G-protein, CA RACB, another potential host susceptibility factor and hypothetical actin cytoskeleton regulator, partly inhibited actin reorganization when under attack from Bgh, whereas knockdown of RACB promoted actin focusing. We conclude that RACB and, potentially, MLO are host proteins involved in the modulation of actin reorganization and cell polarity in the interaction of barley with Bgh.