Barley ROP-Interactive Partner-a organizes into RAC1- and MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN 1-dependent membrane domains.

BACKGROUND: Small ROP (also called RAC) GTPases are key factors in polar cell development and in interaction with the environment. ROP-Interactive Partner (RIP) proteins are predicted scaffold or ROP-effector proteins, which function downstream of activated GTP-loaded ROP proteins in establishing membrane heterogeneity and cellular organization. Grass ROP proteins function in cell polarity, resistance and susceptibility to fungal pathogens but grass RIP proteins are little understood. RESULTS: We found that the barley (Hordeum vulgare L.) RIPa protein can interact with barley ROPs in yeast. Fluorescent-tagged RIPa, when co-expressed with the constitutively activated ROP protein CA RAC1, accumulates at the cell periphery or plasma membrane. Additionally, RIPa, locates into membrane domains, which are laterally restricted by microtubules when co-expressed with RAC1 and MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN 1. Both structural integrity of MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN 1 and microtubule stability are key to maintenance of RIPa-labeled membrane domains. In this context, RIPa also accumulates at the interface of barley and invading hyphae of the powdery mildew fungus Blumeria graminis f.sp. hordei. CONCLUSIONS: Data suggest that barley RIPa interacts with barley ROPs and specifies RAC1 activity-associated membrane domains with potential signaling capacity. Lateral diffusion of this RAC1 signaling capacity is spatially restricted and the resulting membrane heterogeneity requires intact microtubules and MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN 1. Focal accumulation of RIPa at sites of fungal attack may indicate locally restricted ROP activity at sites of fungal invasion.

The Arabidopsis microtubule-associated protein MAP65-3 supports infection by filamentous biotrophic pathogens by down-regulating salicylic acid-dependent defenses.

The oomycete Hyaloperonospora arabidopsidis and the ascomycete Erysiphe cruciferarum are obligate biotrophic pathogens causing downy mildew and powdery mildew, respectively, on Arabidopsis. Upon infection, the filamentous pathogens induce the formation of intracellular bulbous structures called haustoria, which are required for the biotrophic lifestyle. We previously showed that the microtubule-associated protein AtMAP65-3 plays a critical role in organizing cytoskeleton microtubule arrays during mitosis and cytokinesis. This renders the protein essential for the development of giant cells, which are the feeding sites induced by root knot nematodes. Here, we show that AtMAP65-3 expression is also induced in leaves upon infection by the downy mildew oomycete and the powdery mildew fungus. Loss of AtMAP65-3 function in the map65-3 mutant dramatically reduced infection by both pathogens, predominantly at the stages of leaf penetration. Whole-transcriptome analysis showed an over-represented, constitutive activation of genes involved in salicylic acid (SA) biosynthesis, signaling, and defense execution in map65-3, whereas jasmonic acid (JA)-mediated signaling was down-regulated. Preventing SA synthesis and accumulation in map65-3 rescued plant susceptibility to pathogens, but not the developmental phenotype caused by cytoskeleton defaults. AtMAP65-3 thus has a dual role. It positively regulates cytokinesis, thus plant growth and development, and negatively interferes with plant defense against filamentous biotrophs. Our data suggest that downy mildew and powdery mildew stimulate AtMAP65-3 expression to down-regulate SA signaling for infection.

The Arabidopsis ROP-activated receptor-like cytoplasmic kinase RLCK VI_A3 is involved in control of basal resistance to powdery mildew and trichome branching.

KEY MESSAGE: The Arabidopsis receptor-like cytoplasmic kinase AtRLCK VI_A3 is activated by AtROPs and is involved in trichome branching and pathogen interaction. Receptor-like cytoplasmic kinases (RLCKs) belong to the large superfamily of receptor-like kinases, which are involved in a variety of cellular processes like plant growth, development and immune responses. Recent studies suggest that RLCKs of the VI_A subfamily are possible downstream effectors of the small monomeric G proteins of the plant-specific Rho family, called 'Rho of plants' (RAC/ROPs). Here, we describe Arabidopsis thaliana AtRLCK VI_A3 as a molecular interactor of AtROPs. In Arabidopsis epidermal cells, transient co-expression of plasma membrane located constitutively activated (CA) AtROP4 or CA AtROP6 resulting in the recruitment of green fluorescent protein-tagged AtRLCK VI_A3 to the cell periphery. Intrinsic kinase activity of AtRLCK VI_A3 was enhanced in the presence of CA AtROP6 in vitro and further suggested a functional interaction between the proteins. In the interaction of the biotrophic powdery mildew fungus Erysiphe cruciferarum (E. cruciferarum) and its host plant Arabidopsis, Atrlck VI_A3 mutant lines supported enhanced fungal reproduction. Furthermore Atrlck VI_A3 mutant lines showed slightly reduced size and an increase in trichome branch number compared to wild-type plants. In summary, our data suggest a role of the AtROP-regulated AtRLCK VI_A3 in basal resistance to E. cruciferarum as well as in plant growth and cellular differentiation during trichome morphogenesis. Results are discussed in the context of literature suggesting a function of RAC/ROPs in both resistance and susceptibility to pathogen infection.

A barley Engulfment and Motility domain containing protein modulates Rho GTPase activating protein HvMAGAP1 function in the barley powdery mildew interaction.

Engulfment and Motility (ELMO) proteins are involved in the regulation of small GTPase activity in eukaryotic organisms, but little is known about ELMO proteins in plants. We isolated the barley ELMO Domain Containing Protein, HvELMOD_C, in a yeast two hybrid screen for proteins interacting with HvMAGAP1 (Microtubule Associated ROP-GTPase Activating Protein 1). HvMAGAP1 is considered as an antagonist of barley RACB, a member of the RHO of plant (ROP) family GTPases, which functions as a susceptibility factor in the interaction of barley with the barley powdery mildew fungus Blumeria graminis f.sp. hordei. HvELMOD_C interacts with the central RHO-GAP domain of HvMAGAP1. Cytoplasmic HvELMOD_C translocates to microtubules on co-expression of HvMAGAP1 but not on co-expression of HvMAGAP1-R185G, a mutant of the catalytically active arginine R185 in the RHO-GAP domain. HvELMOD_C, when simultaneously expressed with HvMAGAP1, abolished the resistance-inducing effect of HvMAGAP1 to B. graminis f.sp. hordei. Therefore, HvELMOD_C might function as a new modulator of HvMAGAP1 and thus ROP activity in barley.

A barley ROP GTPase ACTIVATING PROTEIN associates with microtubules and regulates entry of the barley powdery mildew fungus into leaf epidermal cells.

Little is known about the function of host factors involved in disease susceptibility. The barley (Hordeum vulgare) ROP (RHO of plants) G-protein RACB is required for full susceptibility of the leaf epidermis to invasion by the biotrophic fungus Blumeria graminis f. sp hordei. Stable transgenic knockdown of RACB reduced the ability of barley to accommodate haustoria of B. graminis in intact epidermal leaf cells and to form hairs on the root epidermis, suggesting that RACB is a common element of root hair outgrowth and ingrowth of haustoria in leaf epidermal cells. We further identified a barley MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN (MAGAP1) interacting with RACB in yeast and in planta. Fluorescent MAGAP1 decorated cortical microtubules and was recruited by activated RACB to the cell periphery. Under fungal attack, MAGAP1-labeled microtubules built a polarized network at sites of successful defense. By contrast, microtubules loosened where the fungus succeeded in penetration. Genetic evidence suggests a function of MAGAP1 in limiting susceptibility to penetration by B. graminis. Additionally, MAGAP1 influenced the polar organization of cortical microtubules. These results add to our understanding of how intact plant cells accommodate fungal infection structures and suggest that RACB and MAGAP1 might be antagonistic players in cytoskeleton organization for fungal entry.

Barley ROP binding kinase1 is involved in microtubule organization and in basal penetration resistance to the barley powdery mildew fungus.

Certain plant receptor-like cytoplasmic kinases were reported to interact with small monomeric G-proteins of the RHO of plant (ROP; also called RAC) family in planta and to be activated by this interaction in vitro. We identified a barley (Hordeum vulgare) partial cDNA of a ROP binding protein kinase (HvRBK1) in yeast (Saccharomyces cerevisiae) two-hybrid screenings with barley HvROP bait proteins. Protein interaction of the constitutively activated (CA) barley HvROPs CA HvRACB and CA HvRAC1 with full-length HvRBK1 was verified in yeast and in planta. Green fluorescent protein-tagged HvRBK1 appears in the cytoplasm and nucleoplasm, but CA HvRACB or CA HvRAC1 can recruit green fluorescent protein-HvRBK1 to the cell periphery. Barley HvRBK1 is an active kinase in vitro, and activity is enhanced by CA HvRACB or GTP-loaded HvRAC1. Hence, HvRBK1 might act downstream of active HvROPs. Transient-induced gene silencing of barley HvRBK1 supported penetration by the parasitic fungus Blumeria graminis f. sp. hordei, suggesting a function of the protein in basal disease resistance. Transient knockdown of HvRBK1 also influenced the stability of cortical microtubules in barley epidermal cells. Hence, HvRBK1 might function in basal resistance to powdery mildew by influencing microtubule organization.

Fungal Development and Callose Deposition in Compatible and Incompatible Interactions in Melon Infected with Powdery Mildew.

Two post-haustorial resistance mechanisms (types I and II) against powdery mildew, caused by Podosphaera xanthii, have been described previously in melon according to the arresting of fungal development and the timing of hypersensitive response (HR) in host cells. In our work, host-pathogen interactions between races 1, 2, and 5 of Podosphaera and several melon accessions carrying different resistance genes, have been characterized by observing several parameters, such as the number of fungal penetration points with callose accumulation, the number of epidermal cells with callose accumulation in their cell walls, and the number of conidiophores developed. Influence of temperature was observed in some cases affecting the timing of fungal development arrest. According to our results, besides the compatible interaction, four different resistance behaviors in the plant-pathogen interaction have been observed herein: type I and II, as described previously, as well as an earlier and a later type II: IIa and IIb, respectively. Melon genotypes showing post-haustorial resistance mechanism types IIa and IIb against powdery mildew, seem to show different behavior according to temperature, affecting fungal development, mainly those genotypes carrying QTL of linkage group V for powdery mildew resistance, such as "TGR-1551".

Enemy at the gates: traffic at the plant cell pathogen interface.

The plant apoplast constitutes a space for early recognition of potentially harmful non-self. Basal pathogen recognition operates via dynamic sensing of conserved microbial patterns by pattern recognition receptors or of elicitor-active molecules released from plant cell walls during infection. Recognition elicits defence reactions depending on cellular export via SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex-mediated vesicle fusion or plasma membrane transporter activity. Lipid rafts appear also involved in focusing immunity-associated proteins to the site of pathogen contact. Simultaneously, pathogen effectors target recognition, apoplastic host proteins and transport for cell wall-associated defence. This microreview highlights most recent reports on the arms race for plant disease and immunity at the cell surface.

Transgenic suppression of cell death limits penetration success of the soybean rust fungus Phakopsora pachyrhizi into epidermal cells of barley.

The basidiomycete Phakopsora pachyrhizi (P. pachyrhizi) causes Asian soybean rust, one of the most devastating plant diseases on soybean. When inoculated on the nonhost barley P. pachyrhizi caused only very small necrotic spots, typical for an incompatible interaction, which involves a hypersensitive cell death reaction. A microscopic inspection of the interaction of barley with P. pachyrhizi revealed that the fungus germinated on barley and formed functional appressoria on epidermal cells. The fungus attempted to directly penetrate through periclinal cell walls but often failed, arrested in plant cell wall appositions that stained positively for callose. Penetration resistance depends on intact ROR1(REQUIRED FOR mlo-SPECIFIED RESISTANCE 1) and ROR2 genes of barley. If the fungus succeeded in penetration, epidermal cell death took place. Dead epidermal cells did not generally restrict fungal development but allowed for mesophyll invasion, which was followed by mesophyll cell death and fungal arrest. Transient or stable over expression of the barley cell death suppressor BAX inhibitor-1 reduced both epidermal cell death and fungal penetration success. Data suggest that P. pachyrhizi provokes a programmed cell death facilitating fungal entry into epidermal cells of barley.

Involvement of Arabidopsis thaliana endoplasmic reticulum KDEL-tailed cysteine endopeptidase 1 (AtCEP1) in powdery mildew-induced and AtCPR5-controlled cell death.

Programmed cell death (PCD) is a prerequisite for successful development and it limits the spread of biotrophic pathogens in a rapid hypersensitive response at the site of infection. KDEL-tailed cysteine endopeptidases (KDEL CysEP) are a subgroup of papain-type cysteine endopeptidases expressed in tissues undergoing PCD. In Arabidopsis, three KDEL CysEPs (AtCEP1, AtCEP2, and AtCEP3) are expressed. We have previously shown that AtCEP1 is a factor of basal resistance to powdery mildew caused by the biotrophic ascomycete Erysiphe cruciferarum, and is expressed in spatiotemporal association with the late fungal development on Arabidopsis leaves. The endoplasmic reticulum-localized proenzyme of AtCEP1 was further visualized at the haustorial complex encased with callose. The AtCPR5 gene (CONSTITUTIVE EXPRESSION OF PR GENES 5) is a regulator of expression of pathogenesis related genes. Loss of AtCPR5 leads to spontaneous expression of chlorotic lesions which was associated with enhanced expression of AtCEP1. We used the atcpr5-2 mutant plants and the atcep1 atcpr5-2 double mutants harboring a non-functional reporter (PCEP1::pre-pro-3xHA-EGFP-KDEL) for visualization of AtCEP1 promoter activity. We found the specific up-regulation of AtCEP1 in direct neighborhood of spreading leaf lesions thus likely representing cells undergoing PCD. Furthermore, we found a strong resistance of atcpr5 mutant plants against infection with E. cruciferarum. Loss of AtCEP1 had no obvious influence on the strong resistance of atcpr5-2 mutant plants against infection with E. cruciferarum. However, the area of necrotic leaf lesions associated with E. cruciferarum colonies was significantly larger in atcpr5-2 as compared to atcep1 atcpr5-2 double mutant plants. The presence of AtCEP1 thus contributes to AtCPR5-controlled PCD at the sites of powdery mildew infection.

A barley SKP1-like protein controls abundance of the susceptibility factor RACB and influences the interaction of barley with the barley powdery mildew fungus.

In an increasing number of plant-microbe interactions, it has become evident that the abundance of immunity-related proteins is controlled by the ubiquitin-26S proteasome system. In the interaction of barley with the biotrophic barley powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh), the RAC/ROP [RAT SARCOMA-related C3 botulinum toxin substrate/RAT SARCOMA HOMOLOGUE (RHO) of plants] guanosine triphosphatase (GTPase) HvRACB supports the fungus in a compatible interaction. By contrast, barley HvRBK1, a ROP-binding receptor-like cytoplasmic kinase that interacts with and can be activated by constitutively activated HvRACB, limits fungal infection success. We have identified a barley type II S-phase kinase 1-associated (SKP1)-like protein (HvSKP1-like) as a molecular interactor of HvRBK1. SKP1 proteins are subunits of the SKP1-cullin 1-F-box (SCF)-E3 ubiquitin ligase complex that acts in the specific recognition and ubiquitination of protein substrates for subsequent proteasomal degradation. Transient induced gene silencing of either HvSKP1-like or HvRBK1 increased protein abundance of constitutively activated HvRACB in barley epidermal cells, whereas abundance of dominant negative RACB only weakly increased. In addition, silencing of HvSKP1-like enhanced the susceptibility of barley to haustorium establishment by Bgh. In summary, our results suggest that HvSKP1-like, together with HvRBK1, controls the abundance of HvRACB and, at the same time, modulates the outcome of the barley-Bgh interaction. A possible feedback mechanism from RAC/ROP-activated HvRBK1 on the susceptibility factor HvRACB is discussed.

Endoplasmic reticulum KDEL-tailed cysteine endopeptidase 1 of Arabidopsis (AtCEP1) is involved in pathogen defense.

Programmed cell death (PCD) is a genetically determined process in all multicellular organisms. Plant PCD is effected by a unique group of papain-type cysteine endopeptidases (CysEP) with a C-terminal KDEL endoplasmic reticulum (ER) retention signal (KDEL CysEP). KDEL CysEPs can be stored as pro-enzymes in ER-derived endomembrane compartments and are released as mature CysEPs in the final stages of organelle disintegration. KDEL CysEPs accept a wide variety of amino acids at the active site, including the glycosylated hydroxyprolines of the extensins that form the basic scaffold of the cell wall. In Arabidopsis, three KDEL CysEPs (AtCEP1, AtCEP2, and AtCEP3) are expressed. Cell- and tissue-specific activities of these three genes suggest that KDEL CysEPs participate in the abscission of flower organs and in the collapse of tissues in the final stage of PCD as well as in developmental tissue remodeling. We observed that AtCEP1 is expressed in response to biotic stress stimuli in the leaf. atcep1 knockout mutants showed enhanced susceptibility to powdery mildew caused by the biotrophic ascomycete Erysiphe cruciferarum. A translational fusion protein of AtCEP1 with a three-fold hemaglutinin-tag and the green fluorescent protein under control of the endogenous AtCEP1 promoter (PCEP1::pre-pro-3xHA-EGFP-AtCEP1-KDEL) rescued the pathogenesis phenotype demonstrating the function of AtCEP1 in restriction of powdery mildew. The spatiotemporal AtCEP1-reporter expression during fungal infection together with microscopic inspection of the interaction phenotype suggested a function of AtCEP1 in controlling late stages of compatible interaction including late epidermal cell death. Additionally, expression of stress response genes appeared to be deregulated in the interaction of atcep1 mutants and E. cruciferarum. Possible functions of AtCEP1 in restricting parasitic success of the obligate biotrophic powdery mildew fungus are discussed.

ROPGAPs of Arabidopsis limit susceptibility to powdery mildew.

The barley ROP GTPase HvRACB is a susceptibility factor of barley to powdery mildew caused by the biotrophic fungus Blumeria graminis f.sp. hordei (Bgh). In a recent publication, we reported about a MICROTUBULE-ASSOCIATED ROP GTPASE-ACTIVATING PROTEIN 1 (HvMAGAP1) of barley. Transient-induced gene silencing or overexpression of HvMAGAP1 resulted in enhanced or reduced susceptibility to Bgh, respectively, indicating a possible HvRACB-antagonistic function of HvMAGAP1 in interaction with Bgh. HvMAGAP1 also influences the polarity of cortical microtubules in interaction with Bgh. In AtROPGAP1 and AtROPGAP4, Arabidopsis homologs of HvMAGAP1, knock-out T-DNA insertions enhanced susceptibility of Arabidopsis to the virulent powdery mildew fungus Erysiphe cruciferarum, indicating functions of ROPGAPs in pathogen interaction of monocots and dicots. Here we discuss the role of AtROPGAP1 and AtROPGAP4 in Arabidopsis pathogenesis of powdery mildew in some more detail.

RIP3 and AtKinesin-13A - a novel interaction linking Rho proteins of plants to microtubules.

RIP3 belongs to a group of recently identified proteins, classified as the ICR/RIP family whose members were described to interact with Rho proteins of plants (ROPs). Our in vivo and in vitro data demonstrate that RIP3 is a true ROP effector, interacting specifically with the active form of ROPs. We found that RIP3 has properties and cellular roles different from the previously described RIP family member ICR1/RIP1. We show that RIP3 is localized at microtubules and interacts with the kinesin-13 family member AtKinesin-13A, suggesting a role for RIP3 in microtubule reorganization and a possible function in ROP-regulated polar growth.