Barley RIC171 interacts with RACB in planta and supports entry of the powdery mildew fungus

RHO-like GTPases of plants (ROPs, also called RACs) are involved in plant development and interaction with the environment. The barley ROP protein RACB is involved in susceptibility to the fungal pathogen Blumeria graminis f.sp. hordei ( Bgh ) . By screening barley sequence databases for potential protein interactors of plant RHO-like proteins, we identified a ROP-interactive CRIB (CDC42/RAC interactive binding) motif containing protein of 171 amino acids (RIC171). The protein interacted with constitutively activated RACB in a targeted yeast two-hybrid assay. By use of split yellow fluorescing protein fusions, we demonstrated that RIC171 interacts with constitutively activated (CA) RACB-G15V but not with dominant negative RACB-T20N in planta . Transient overexpression of RIC171, similar to overexpression of CA RACB-G15V, rendered epidermal cells more susceptible to penetration by Bgh . In contrast, expression of a 46-amino-acid RIC171-CRIB peptide, which was sufficient to interact with CA RACB-G15V, had a dominant negative effect and reduced susceptibility to Bgh . A red fluorescing DsRED–RIC171 fusion protein colocalized with green fluorescing GFP–RACB-G15V at the cell periphery. Coexpression with CA RACB-G15V but not with RACB-T20N increased peripheral localization of DsRED–RIC171. Additionally, DsRED–RIC171 accumulated at sites of fungal attack, suggesting enhanced ROP activity at sites of attempted fungal penetration.     

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 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.     

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.     

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.     

Differences in early callose deposition during adapted and non-adapted powdery mildew infection of resistant Arabidopsis lines

The deposition of callose, a (1,3)-β-glucan cell wall polymer, can play an essential role in the defense response to invading pathogens. We could recently show that Arabidopsis thaliana lines with an overexpression of the callose synthase gene PMR4 gained complete penetration resistance to the adapted powdery mildew Golovinomyces cichoracearum and the non-adapted powdery mildew Blumeria graminis f. sp hordei. The penetration resistance is based on the transport of the callose synthase PMR4 to the site of attempted fungal penetration and the subsequent formation of enlarged callose deposits. The deposits differed in their total diameter comparing both types of powdery mildew infection. In this study, further characterization of these callose deposits revealed that size differences were especially pronounced in the core region of the deposits. This suggests that specific, pathogen-dependent factors exist, which might regulate callose synthase transport to the core region of forming deposits.     

Ultrastructural and Histochemical Studies on Mildew of Barley (Erysiphe graminis DC. f. sp. hordei Marchal)

Plants of the mildew susceptible barley cultivar Peruvian and the adult plant resistant cultivar Osiris were inoculated with Erysiphe graminis f. sp. hordei at the first and fifth leaf stages. Samples taken at 32 hr after inoculation were examined by electron microscopy to compare papillae associated either with penetration failure or with successful penetration of the fungus into the epidermal cell and haustorium formation. Four types of papillae with ultrastructural differences could, be classified. Although their definite association with fungal ingress or failure is not possible, our data suggest that papillae with larger, more compacted and amorphous or globular structures may be more effective as penetration barriers than others, with more or less uniform distribution of irregular, smaller electrondense structures.     

Constitutively activated barley ROPs modulate epidermal cell size, defense reactions and interactions with fungal leaf pathogens

RHO-like monomeric G-proteins of plants (ROPs, also called RACs), are involved in plant development and interaction with the environment. The barley (Hordeum vulgare) ROP protein HvRACB has been shown to be required for entry of the biotrophic powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh) into living host cells. To get a deeper insight into evolutionarily conserved functions of ROPs in cell polarity and pathogen responses, we stably expressed constitutively activated (CA) mutant variants of different barley ROPs (HvRACB, HvRAC1, HvRAC3) in barley. CA HvROPs induced epidermal cell expansion and/or abolished polarity in tip growing root hairs. All three CA HvROPs enhanced susceptibility of barley to penetration by Bgh whereas only CA HvRAC1 supported whole cell H₂O₂ production in non-penetrated cells. Despite increasing penetration by Bgh, CA HvRAC1 promoted callose deposition at sites of fungal attack and resistance to penetration by Magnaporthe oryzae. The data show an involvement of ROPs in polar growth processes of the monocot barley and in responses to fungal pathogens with different life style.     

The mlo resistance alleles to powdery mildew infection in barley trigger a developmentally controlled defence mimic phenotype

Recessive mlo resistance alleles of the Mlo locus in barley control a non race-specific resistance response to infection by the obligate biotrophic fungus Erysiphe graminis f.sp. hordei. All the mlo alleles analysed stop fungal growth at the same developmental stage within a subcellularly restricted, highly localized cell wall apposition directly beneath the site of abortive fungal penetration. We report that near-isogenic lines carrying the alleles mlo ₁, mlo ₃ or mlo ₅ undergo dramatic spontaneous formation of cell wall appositions, not only in the absence of the fungal pathogen but also in sterile grown plants. A comparative study of spontaneous and infection-triggered cell wall appositions reveals a high degree of similarity with respect to structure, chemical composition and distinct localization within plant tissue. We show that the rate of spontaneous apposition formation is dependent on the genetic background of the plant and that its onset is under developmental control. Furthermore, spontaneous formation of wall appositions is specifically triggered by mlo alleles, since it is unaffected in the presence of the race-specific resistance allele Mlg. We propose a model for the function of the Mlo locus that suggests that both Mlo and mlo alleles control qualitatively the same apposition-based resistance mechanism, which, in the presence of the wild-type Mlo allele, is merely less efficient to provide protection against the currently common races of E. graminis f.sp. hordei.     

Elektronenmikroskopische Untersuchungen des Gerstenmehltaus (Erysiphe graminis DC f. sp. hordei Marchal) nach Resistenzinduktion mit mikrobiellen Stoffwechselprodukten

Electron microscopical studies on Mildew of Barley (Erysiphe graminis DC f. sp. hordei Marchal) after induced Resistance with Products of Microbial Metabolism Ultrastructural studies of the infection progress of barley by Erysiphe graminis after induction of resistance with products of microbial metabolism showed that pathogen development was affected through inducer activated defence mechanisms. The formation of papillae-like structures and an accumulation of electron dense material below the host cell wall at the penetration site appeared to be associated with reduced penetration. The haustoria were also altered in the extrahaustorial membrane and electron dense material accumulated at the cell wall of haustorial body and neck, thus apparently impairing efficient functioning and allowing only limited growth of the pathogen.     

Medicarpin confers powdery mildew resistance in Medicago truncatula and activates the salicylic acid signalling pathway

Powdery mildew (PM) caused by the obligate biotrophic fungal pathogen Erysiphe pisi is an economically important disease of legumes. Legumes are rich in isoflavonoids, a class of secondary metabolites whose role in PM resistance is ambiguous. Here we show that the pterocarpan medicarpin accumulates at fungal infection sites, as analysed by fluorescein‐tagged medicarpin, and provides penetration and post‐penetration resistance against E. pisi in Medicago truncatula in part through the activation of the salicylic acid (SA) signalling pathway. Comparative gene expression and metabolite analyses revealed an early induction of isoflavonoid biosynthesis and accumulation of the defence phytohormones SA and jasmonic acid (JA) in the highly resistant M. truncatula genotype A17 but not in moderately susceptible R108 in response to PM infection. Pretreatment of R108 leaves with medicarpin increased SA levels, SA‐associated gene expression, and accumulation of hydrogen peroxide at PM infection sites, and reduced fungal penetration and colony formation. Strong parallels in the levels of medicarpin and SA, but not JA, were observed on medicarpin/SA treatment pre‐ or post‐PM infection. Collectively, our results suggest that medicarpin and SA may act in concert to restrict E. pisi growth, providing new insights into the metabolic and signalling pathways required for PM resistance in legumes.     

The conserved oligomeric Golgi complex is involved in penetration resistance of barley to the barley powdery mildew fungus

Membrane trafficking is vital to plant development and adaptation to the environment. It is suggested that post‐Golgi vesicles and multivesicular bodies are essential for plant defence against directly penetrating fungal parasites at the cell wall. However, the actual plant proteins involved in membrane transport for defence are largely unidentified. We applied a candidate gene approach and single cell transient‐induced gene silencing for the identification of membrane trafficking proteins of barley involved in the response to the fungal pathogen Blumeria graminis f.sp. hordei. This revealed potential components of vesicle tethering complexes [putative exocyst subunit HvEXO70F‐like and subunits of the conserved oligomeric Golgi (COG) complex] and Golgi membrane trafficking (COPIγ coatomer and HvYPT1‐like RAB GTPase) as essential for resistance to fungal penetration into the host cell.     

Lauric Acid Exhibits Antifungal Activity Against Plant Pathogenic Fungi

This study aimed at examining the effects of the saturated fatty acid lauric acid on mycelial growth of Rhizoctonia solani and Pythium ultimum and on infection of barley seedlings with Blumeria graminis f. sp. hordei. Mycelial growth of R. solani and P. ultimum in agar culture was significantly reduced by lauric acid at concentrations of 100 μm and above, while no fungal growth occurred in liquid culture at concentrations above 50 μm . Application of lauric acid at concentrations ranging from 250 to 1000 μm to barley leaves before or after inoculation with B. graminis f. sp. hordei led to significant reductions in infection. This study provides the first report of the activity of lauric acid against plant pathogenic fungi and indicates the need for investigation of its mechanism of action.     

Characterisation of the Nucleotide Exchange Factor ITSN1L: Evidence for a Kinetic Discrimination of GEF-Stimulated Nucleotide Release from Cdc42

Cdc42, a member of the Ras superfamily of small guanine nucleotide binding proteins, plays an important role in regulating the actin cytoskeleton, intracellular trafficking, and cell polarity. Its activation is controlled by guanine nucleotide exchange factors (GEFs), which stimulate the dissociation of bound guanosine-5′-diphosphate (GDP) to allow guanosine-5′-triphosphate (GTP) binding. Here, we investigate the exchange factor activity of the Dbl-homology domain containing constructs of the adaptor protein Intersectin1L (ITSN1L), which is a specific GEF for Cdc42. A detailed kinetic characterisation comparing ITSN1L-mediated nucleotide exchange on Cdc42 in its GTP- versus GDP-bound state reveals a kinetic discrimination for GEF-stimulated dissociation of GTP: The maximum acceleration of the intrinsic mGDP [2′/3′-O-(N-methyl-anthraniloyl)-GDP] release from Cdc42 by ITSN1L is accelerated at least 68,000-fold, whereas the exchange of mGTP [2′/3′-O-(N-methyl-anthraniloyl)-GTP] is stimulated only up to 6000-fold at the same GEF concentration. The selectivity in nucleotide exchange kinetics for GDP over GTP is even more pronounced when a Cdc42 mutant, F28L, is used, which is characterised by fast intrinsic dissociation of nucleotides. We furthermore show that both GTP and Mg²⁺ ions are required for the interaction with effectors. We suggest a novel model for selective nucleotide exchange residing on a conformational change of Cdc42 upon binding of GTP, which enables effector binding to the Cdc42 · GTP complex but, at the same time, excludes efficient modulation by the GEF. The higher exchange activity of ITSN1L towards the GDP-bound conformation of Cdc42 could represent an evolutionary adaptation of this GEF that ensures nucleotide exchange towards the formation of the signalling-active GTP-bound form of Cdc42 and avoids dissociation of the active complex.     

Infection of Brachypodium distachyon by Formae Speciales of Puccinia graminis: Early Infection Events and Host-Pathogen Incompatibility

Puccinia graminis causes stem rust, a serious disease of cereals and forage grasses. Important formae speciales of P. graminis and their typical hosts are P. graminis f. sp. tritici (Pg-tr) in wheat and barley, P. graminis f. sp. lolii (Pg-lo) in perennial ryegrass and tall fescue, and P. graminis f. sp. phlei-pratensis (Pg-pp) in timothy grass. Brachypodium distachyon is an emerging genetic model to study fungal disease resistance in cereals and temperate grasses. We characterized the P. graminis-Brachypodium pathosystem to evaluate its potential for investigating incompatibility and non-host resistance to P. graminis. Inoculation of eight Brachypodium inbred lines with Pg-tr, Pg-lo or Pg-pp resulted in sporulating lesions later accompanied by necrosis. Histological analysis of early infection events in one Brachypodium inbred line (Bd1-1) indicated that Pg-lo and Pg-pp were markedly more efficient than Pg-tr at establishing a biotrophic interaction. Formation of appressoria was completed (60–70% of germinated spores) by 12 h post-inoculation (hpi) under dark and wet conditions, and after 4 h of subsequent light exposure fungal penetration structures (penetration peg, substomatal vesicle and primary infection hyphae) had developed. Brachypodium Bd1-1 exhibited pre-haustorial resistance to Pg-tr, i.e. infection usually stopped at appressorial formation. By 68 hpi, only 0.3% and 0.7% of the Pg-tr urediniospores developed haustoria and colonies, respectively. In contrast, development of advanced infection structures by Pg-lo and Pg-pp was significantly more common; however, Brachypodium displayed post-haustorial resistance to these isolates. By 68 hpi the percentage of urediniospores that only develop a haustorium mother cell or haustorium in Pg-lo and Pg-pp reached 8% and 5%, respectively. The formation of colonies reached 14% and 13%, respectively. We conclude that Brachypodium is an apt grass model to study the molecular and genetic components of incompatiblity and non-host resistance to P. graminis.     

Cultivar-related differences in the distribution of cell-wall-bound thionins in compatible and incompatible interactions between barley and powdery mildew

Leaf-specific thionins of barley (Hordeum vulgare L.) have been identified as a novel class of cell-wall proteins toxic to plant-pathogenic fungi and possibly involved in the defence mechanism of plants. The distribution of these polypeptides has been studied in the host-pathogen system of barley and Erisyphe graminis DC.f.sp. hordei Marchal (powdery mildew). Immunogold-labelling of thionins in several barley cultivars indicates that resistance or susceptibility may be attributed to the presence or absence of thionins at the penetration site in walls and papillae of epidermal leaf cells.All of the leaf-specific thionin genes are confined to the distal end of the short arm of chromosome 6 of barley. None of the genes for cultivarspecific resistance to powdery mildew which have previously been mapped on barley chromosomes are found close to this locus.     

Formation of self-organizing functionally distinct Rho of plants domains involves a reduced mobile population

Rho family proteins are central to the regulation of cell polarity in eukaryotes. Rho of Plants-Guanyl nucleotide Exchange Factor (ROPGEF) can form self-organizing polar domains following co-expression with an Rho of Plants (ROP) and an ROP GTPase-Activating Protein (ROPGAP). Localization of ROPs in these domains has not been demonstrated, and the mechanisms underlying domain formation and function are not well understood. Here we show that six different ROPs form self-organizing domains when co-expressed with ROPGEF3 and GAP1 in Nicotiana benthamiana or Arabidopsis (Arabidopsis thaliana). Domain formation was associated with ROP–ROPGEF3 association, reduced ROP mobility, as revealed by time-lapse imaging and Fluorescence Recovery After Photobleaching beam size analysis, and was independent of Rho GTP Dissociation Inhibitor mediated recycling. The domain formation depended on the ROPs’ activation/inactivation cycles and interaction with anionic lipids via a C-terminal polybasic domain. Coexpression with the microtubule-associated protein ROP effector INTERACTOR OF CONSTITUTIVELY ACTIVE ROP 1 (ICR1) revealed differential function of the ROP domains in the ability to recruit ICR1. Taken together, the results reveal mechanisms underlying self-organizing ROP domain formation and function.

Plasma membrane self-organizing polarity domains of small GTP-binding proteins form upon their co-expression together with their activator and suppressor due to restriction of protein mobility.