Genetic analysis of a mutation on appressorium formation in Magnaporthe grisea

The rice blast fungus, Magnaporthe grisea, forms a dome-shaped and darkly pigmented infection structure, an appressorium, to penetrate its host. Differentiation and maturation of appressoria are critical steps for successful infection. A spontaneous developmental mutant (MG01) defective in appressorium formation was found in this fungus. The mutant did not form appressoria either on inductive hydrophobic surfaces or on rice leaves. The addition of cyclic AMP or 1,16-hexadecanediol was not effective in inducing appressorium formation in this mutant. This mutant did not cause lesions on rice when inoculated with conidial suspension by spraying or injecting into the leaf sheath. Genetic analysis of the mutant indicated that the phenotype is under single gene control, designated APP5. Crosses with previously described appressorium defective mutants (app1⁻ and app3⁻) of Magnaporthe grisea suggested that the mutations are at different loci. Bulked segregant analysis was employed to obtain DNA markers linked to the APP5 locus.     

cAMP介导的梨果表皮物化信号对链格孢侵染的调控

采用药理学方法,用cAMP抑制剂阿托品（atropine）处理链格孢Alternaria alternata孢子悬浮液,通过体外试验分析cAMP信号级联通路在链格孢响应梨果皮蜡质疏水性、化学组分和外源乙烯利等刺激后启动孢子萌发、附着胞形成的调控作用,并通过体内试验研究其对链格孢致病性的调控。结果表明,高疏水性表面和梨果蜡涂膜表面及1μmol/L的乙烯利均可显著促进链格孢的孢子萌发和附着胞形成。cAMP信号级联通路抑制剂atropine处理后显著抑制了表皮疏水性、蜡质和外源乙烯介导的链格孢的孢子萌发和附着胞形成,其中抑制剂处理后4h,链格孢在疏水性、果蜡涂膜表面和乙烯等处理中附着胞形成率分别较对照降低了75.3%、63.7%和74.3%,同时抑制剂处理还可抑制损伤接种链格孢早酥梨黑斑病的扩展。外源cAMP可以部分恢复抑制剂的作用,外源cAMP+atropine处理后4h,在高疏水性（108°）和果蜡涂膜表面,链格孢附着胞形成率为抑制剂atropine单独处理的2.4倍和1.6倍,表明cAMP信号级联通路可通过调控侵染结构的形成而影响链格孢对梨果表皮物理化学信号的识别和应答。     

Generality of the prerequisite of conidium attachment to a hydrophobic substratum as a signal for germination among Phyllosticta species

It has been shown that conidia of Phyllosticta ampelicida require attachment to a substratum to initiate germination. Furthermore this attachment occurs only on hydrophobic surfaces. This study was initiated to ascertain the breadth of this phenomenon among other species of the genus Phyllosticta. We tested 23 isolates of Phyllosticta representing at least 14 named species. These isolates were collected from North America, Asia and Africa. For 22 of the 23 isolates tested spore attachment occurred at a rate of 60-100% on hydrophobic polystyrene but at 0-5% on hydrophilic polystyrene. The one exception to the preference for a hydrophobic substratum for attachment was an unnamed species of Phyllosticta from Rhus glauca that attached less than 10% on either surface. A similar response was observed when assaying germination and appressorium formation for 17 isolates. Germination and appressorium formation for these isolates proceeded on hydrophobic polystyrene but not on nutrient agar, which is hydrophilic. In five of the tested isolates germination was high on both hydrophobic polystyrene and hydrophilic nutrient media. The isolate from Rhus glauca did not germinate appreciably on either surface. Taken together these results suggest that the requirement for conidium contact/attachment to trigger germination is pervasive to the genus Phyllosticta.     

cAMP Regulates Infection Structure Formation in the Plant Pathogenic Fungus Magnaporthe grisea

Magnaporthe grisea, the causal agent of rice blast, is one of the most destructive fungal pathogens of rice throughout the world. Infection of rice by M. grisea requires the formation of an appressorium, a darkly pigmented, dome-shaped structure. The germ tube tip differentiates into an appressorium following germination of conidia on a leaf surface. When conidia germinate on growth medium or other noninductive surfaces, the emerging germ tube does not differentiate and continues to grow vegetatively. Little is known about the endogenous or exogenous signals controlling the developmental process of infection structure formation. We show here that a hydrophobic surface was sufficient for the induction of the appressorium. Furthermore, we demonstrate that the addition of cAMP, its analogs (8-bromo cAMP and N6-monobutyryl cAMP), or 3-isobutyl-1-methylxanthine (an inhibitor of phosphodiesterase) to germinating conidia or to vegetative hyphae induced appressorium formation on noninductive surfaces. The identification of cAMP as a mediator of infection structure formation provides a clue to the regulation of this developmental process. Elucidation of the mechanism involved is not only of biological interest but may also provide the basis for new disease control strategies.     

Signal transduction leading to appressorium formation in germinating conidia of Magnaporthe grisea: effects of second messengers diacylglycerols, ceramides and sphingomyelin

The inhibition of appressorium formation in germinating conidiospores of Magnaporthe grisea on an inductive surface by glisoprenin A could be reversed in a competitive manner by 1,2-dioctanoylglycerol, a known activator of protein kinase C. Dioctanoylglycerol and linoleic acid methyl ester induced appressorium formation on a noninductive surface. The effect of ceramides as inducers was heavily dependent on the fatty acid in the molecule. Ceramide IIIa containing linoleic acid was a good inducer whereas others had only weak effects. Sphingomyelin, however, inhibited appressorium formation induced by 1,16-hexadecanediol and to a lesser extent after induction with 8-(4-chlorophenylthio)-adenosine-3′,5′-monophosphate. The results are in agreement with our previous findings that in appressorium formation two signal transducing pathways are involved.     

Mucin Msb2 cooperates with the transmembrane protein Sho1 in various plant surface signal sensing and pathogenic processes in the poplar anthracnose fungus Colletotrichum gloeosporioides

Colletotrichum gloeosporioides is a hemibiotrophic ascomycete fungus that causes anthracnose on numerous plants worldwide and forms a specialized infection structure known as an appressorium in response to various plant surface signals. However, the associated mechanism of host surface signal recognition remains unclear. In the present study, three putative sensors, namely the mucin Msb2, the membrane sensor protein Sho1, and the G‐protein‐coupled receptor Pth11, were identified and characterized. The results showed that CgMsb2 plays a major role in the recognition of various host surface signals; deletion of CgMsb2 resulted in significant defects in appressorium formation, appressorium penetration, cellophane membrane penetration, and pathogenicity. CgSho1 plays a minor role and together with CgMsb2 cooperatively regulates host signal recognition, cellophane membrane penetration, and pathogenicity; deletion of CgSho1 resulted in an expansion defect of infection hyphae. Deletion of CgPth11 in wildtype, ΔCgMsb2, and ΔCgSho1 strains only resulted in a slight defect in appressorium formation at the early stage, and CgPth11 was dispensable for penetration and pathogenicity. However, exogenous cAMP failed to restore the defect of appressorium formation in ΔCgPth11 at the early stage. CgMsb2 contributed to the phosphorylation of the mitogen‐activated protein kinase CgMk1, which is essential for infection‐associated functions, while CgSho1 was unable to activate CgMk1 alone but rather cooperated with CgMsb2 to activate CgMk1. These data suggest that CgMsb2 contributes to the activation of CgMk1 and has overlapping functions with CgSho1 in plant surface sensing, appressorium formation, and pathogenicity.     

Physical-chemical plant-derived signals induce differentiation in Ustilago maydis

Ustilago maydis is able to initiate pathogenic development after fusion of two haploid cells with different mating type. On the maize leaf surface, the resulting dikaryon switches to filamentous growth, differentiates appressoria and penetrates the host. Here, we report on the plant signals required for filament formation and appressorium development in U. maydis. In vitro , hydroxy-fatty acids stimulate filament formation via the induction of pheromone genes and this signal can be bypassed by genetically activating the downstream MAP kinase module. Hydrophobicity also induces filaments and these resemble the dikaryotic filaments formed on the plant surface. With the help of a marker gene that is specifically expressed in the tip cell of those hyphae that have formed an appressorium, hydrophobicity is shown to be essential for appressorium development in vitro . Hydroxy-fatty acids or a cutin monomer mixture isolated from maize leaves have a stimulatory role when a hydrophobic surface is provided. Our results suggest that the early phase of communication between U. maydis and its host plant is governed by two different stimuli.     

Different Chitin Synthase Genes Are Required for Various Developmental and Plant Infection Processes in the Rice Blast Fungus Magnaporthe oryzae

Chitin is a major component of fungal cell wall and is synthesized by chitin synthases (Chs). Plant pathogenic fungi normally have multiple chitin synthase genes. To determine their roles in development and pathogenesis, we functionally characterized all seven CHS genes in Magnaporthe oryzae. Three of them, CHS1, CHS6, and CHS7, were found to be important for plant infection. While the chs6 mutant was non-pathogenic, the chs1 and chs7 mutants were significantly reduced in virulence. CHS1 plays a specific role in conidiogenesis, an essential step for natural infection cycle. Most of chs1 conidia had no septum and spore tip mucilage. The chs6 mutant was reduced in hyphal growth and conidiation. It failed to penetrate and grow invasively in plant cells. The two MMD-containing chitin synthase genes, CHS5 and CHS6, have a similar expression pattern. Although deletion of CHS5 had no detectable phenotype, the chs5 chs6 double mutant had more severe defects than the chs6 mutant, indicating that they may have overlapping functions in maintaining polarized growth in vegetative and invasive hyphae. Unlike the other CHS genes, CHS7 has a unique function in appressorium formation. Although it was blocked in appressorium formation by germ tubes on artificial hydrophobic surfaces, the chs7 mutant still produced melanized appressoria by hyphal tips or on plant surfaces, indicating that chitin synthase genes have distinct impacts on appressorium formation by hyphal tip and germ tube. The chs7 mutant also was defective in appressorium penetration and invasive growth. Overall, our results indicate that individual CHS genes play diverse roles in hyphal growth, conidiogenesis, appressorium development, and pathogenesis in M. oryzae, and provided potential new leads in the control of this devastating pathogen by targeting specific chitin synthases.     

Multiple plant surface signals are sensed by different mechanisms in the rice blast fungus for appressorium formation

Surface recognition and penetration are among the most critical plant infection processes in foliar pathogens. In Magnaporthe oryzae, the Pmk1 MAP kinase regulates appressorium formation and penetration. Its orthologs also are known to be required for various plant infection processes in other phytopathogenic fungi. Although a number of upstream components of this important pathway have been characterized, the upstream sensors for surface signals have not been well characterized. Pmk1 is orthologous to Kss1 in yeast that functions downstream from Msb2 and Sho1 for filamentous growth. Because of the conserved nature of the Pmk1 and Kss1 pathways and reduced expression of MoMSB2 in the pmk1 mutant, in this study we functionally characterized the MoMSB2 and MoSHO1 genes. Whereas the Momsb2 mutant was significantly reduced in appressorium formation and virulence, the Mosho1 mutant was only slightly reduced. The Mosho1 Momsb2 double mutant rarely formed appressoria on artificial hydrophobic surfaces, had a reduced Pmk1 phosphorylation level, and was nonresponsive to cutin monomers. However, it still formed appressoria and caused rare, restricted lesions on rice leaves. On artificial hydrophilic surfaces, leaf surface waxes and primary alcohols-but not paraffin waxes and alkanes- stimulated appressorium formation in the Mosho1 Momsb2 mutant, but more efficiently in the Momsb2 mutant. Furthermore, expression of a dominant active MST7 allele partially suppressed the defects of the Momsb2 mutant. These results indicate that, besides surface hydrophobicity and cutin monomers, primary alcohols, a major component of epicuticular leaf waxes in grasses, are recognized by M. oryzae as signals for appressorium formation. Our data also suggest that MoMsb2 and MoSho1 may have overlapping functions in recognizing various surface signals for Pmk1 activation and appressorium formation. While MoMsb2 is critical for sensing surface hydrophobicity and cutin monomers, MoSho1 may play a more important role in recognizing rice leaf waxes.     

Screening of a synthetic peptide combinatorial library to identify inhibitors of the appressorium formation in Magnaporthe oryzae

The rice blast disease caused by Magnaporthe oryzae is one of the most devastating diseases of cultivated rice. One of the most important stages in the infective cycle of M. oryzae is the formation of the dome-shaped structure called appressorium. The purpose of the present study was to identify novel peptides to control the rice blast disease by blocking the appressorium formation through screening of a synthetic peptide combinatorial library. As result of the screening, a set of 29 putative bioactive peptides were identified, synthesized and assayed in comparison with the previously identified peptide PAF104. The peptides MgAPI24, MgAPI40 and MgAPI47 showed improved inhibitory activity on the M. oryzae appressorium formation. Our data show that these peptides have a differential effect on two developmental structures: appressoria and appressorium-like structures. Antimicrobial assays against M. oryzae and other non-target microorganisms showed a weak or no toxicity of these peptides, demonstrating their specific activity blocking the appressorium formation. Therefore, the outcome of this research would be useful in the development of novel target-oriented peptides to use in plant protection.     

Cutinase and hydrophobin interplay: A herald for pathogenesis?

Surface-penetrating phytopathogenic fungi frequently form appressoria. These are specialised infection structures pivotal to fungal ingress into the host. Recently, we demonstrated that one member of a family of cutinases in Magnaporthe grisea is involved in surface sensing, mediating appressorium differentiation and penetration peg formation and hence facilitates host penetration. Cutinase2 serves as an upstream activator of cAMP/PKA and DAG/PKC signalling cascades and is essential for full virulence. Here, we speculate on the role of rice blast hydrophobins as surface interactors facilitating fungal cutinase activity.Key words: rice blast fungus, appressorium, cutinase, hydrophobin, penetration, surface sensing, signalling     

Electron microscopic observation of cell wall structure during appressorium formation in Colletotrichum lagenarium

Summary The formation of cell walls during the appressorium formation inColletotrichum lagenarium was observed by electron microscope on the materials prepared by replicas and sectioning. The outer layer of conidia cell walls ruptured at the time of germination and the inner layer bulged out to form a germ tube. The germ tubes and primordia of appressoria had smooth surface and were consisted of one-layered cell wall. However, as the appressorium matured, the electron dense materials appeared on the outer surface of the cell wall which grew into granules. These granules are believed to form the outer layer of appressoria. The under side of the appressorium in contact with the glass surface showed a round pore.Contribution No. 191.     

Involvement of extracellular matrix and integrin-like proteins on conidial adhesion and appressorium differentiation in Magnaporthe oryzae

Conidial adhesion and appressorium formation of Magnaporthe oryzae on the rice surface are important early events in the infection process. As an initiative step to understand the mechanisms underlying these cellular processes at a biochemical level, the effect of a human fibronectin antibody (HFA) and RGD peptides on conidial adhesion and appressorium formation was evaluated. HFA inhibited conidial adhesion and appressorium formation in a dosage-dependent manner. RGD peptides also inhibited these cellular events. Conidial adhesion and appressorium formation inhibited by RGD peptides were restored by chemicals involved in the cyclic AMP-dependent signaling pathway. These results suggest that extracellular matrix proteins might be involved in conidial adhesion and appressorium formation through integrin-like receptor mediation and modulation of cAMP-dependent signaling in the cells.     

The Late Endosomal HOPS Complex Anchors Active G-Protein Signaling Essential for Pathogenesis in Magnaporthe oryzae

In Magnaporthe oryzae, the causal ascomycete of the devastating rice blast disease, the conidial germ tube tip must sense and respond to a wide array of requisite cues from the host in order to switch from polarized to isotropic growth, ultimately forming the dome-shaped infection cell known as the appressorium. Although the role for G-protein mediated Cyclic AMP signaling in appressorium formation was first identified almost two decades ago, little is known about the spatio-temporal dynamics of the cascade and how the signal is transmitted through the intracellular network during cell growth and morphogenesis. In this study, we demonstrate that the late endosomal compartments, comprising of a PI3P-rich (Phosphatidylinositol 3-phosphate) highly dynamic tubulo-vesicular network, scaffold active MagA/Gα_S, Rgs1 (a GAP for MagA), Adenylate cyclase and Pth11 (a non-canonical GPCR) in the likely absence of AKAP-like anchors during early pathogenic development in M. oryzae. Loss of HOPS component Vps39 and consequently the late endosomal function caused a disruption of adenylate cyclase localization, cAMP signaling and appressorium formation. Remarkably, exogenous cAMP rescued the appressorium formation defects associated with VPS39 deletion in M. oryzae. We propose that sequestration of key G-protein signaling components on dynamic late endosomes and/or endolysosomes, provides an effective molecular means to compartmentalize and control the spatio-temporal activation and rapid downregulation (likely via vacuolar degradation) of cAMP signaling amidst changing cellular geometry during pathogenic development in M. oryzae.     

Spatial Uncoupling of Mitosis and Cytokinesis during Appressorium-Mediated Plant Infection by the Rice Blast Fungus Magnaporthe oryzae

To infect plants, many pathogenic fungi develop specialized infection structures called appressoria. Here, we report that appressorium development in the rice blast fungus Magnaporthe oryzae involves an unusual cell division, in which nuclear division is spatially uncoupled from the site of cytokinesis and septum formation. The position of the appressorium septum is defined prior to mitosis by formation of a heteromeric septin ring complex, which was visualized by spatial localization of Septin4:green fluorescent protein (GFP) and Septin5:GFP fusion proteins. Mitosis in the fungal germ tube is followed by long-distance nuclear migration and rapid formation of an actomyosin contractile ring in the neck of the developing appressorium, at a position previously marked by the septin complex. By contrast, mutants impaired in appressorium development, such as Δpmk1 and ΔcpkA regulatory mutants, undergo coupled mitosis and cytokinesis within the germ tube. Perturbation of the spatial control of septation, by conditional mutation of the SEPTATION-ASSOCIATED1 gene of M. oryzae, prevented the fungus from causing rice blast disease. Overexpression of SEP1 did not affect septation during appressorium formation, but instead led to decoupling of nuclear division and cytokinesis in nongerminated conidial cells. When considered together, these results indicate that SEP1 is essential for determining the position and frequency of cell division sites in M. oryzae and demonstrate that differentiation of appressoria requires a cytokinetic event that is distinct from cell divisions within hyphae.     

Deformation of Stomatal Guard Cell Lips and Microfabricated Artificial Topographies during Appressorium Formation by Uromyces

Terhune, B. T., Bojko, R. J., and Hoch, H. C. 1993. Deformation of stomatal guard cell lips and microfabricated artificial topographies during appressorium formation by Uromyces. Experimental Mycology 17, 70-78. The inductive signal, stomatal guard cell lips or 0.5-μm-high plastic ridges, for appressorium formation in urediospore germlings of Uromyces appendiculatus was examined for signs of physical deformation during the course of appressorium development. The normally erect stomatal guard cell lips were usually observed prostrate at most stages of appressorium development; and, there were no persistent or significant indentations into the fungal cell that might have been caused by the topographical features. To further evaluate the events that occurred at the lip-appressorium interface in situ, polycarbonate and/or polystyrene ridges (0.5 μm high and 0.25 μm wide), mimicking stomatal guard cell lips of Phaseolus vulgaris, were cast on specifically microfabricated silicon templates. These artificial lips induced appressoria and became deformed approximately 30 min after initial contact by the germ tube apex as recorded and observed with time-lapsed video light microscopy. The collapsed nature of the ridges was further evaluated by both transmission and scanning electron microscopy. These results suggest that mechanical forces imposed by a combination of cell turgor pressure and adhesion of the appressorium to the substrate were responsible for deformation of the inductive topography.