Appressorium-like Structures Research Articles

Spatiotemporal nature of Fusarium graminearum-wheat coleoptile interactions

Fusarium head blight (FHB) caused by the ascomycete fungus Fusarium graminearum can result in significant crop losses and render the crops harmful to human health due to contamination with mycotoxin. Although the pathogenesis of F. graminearum is widely investigated by molecular genetics approaches, detailed studies about its cellular and developmental processes at the initial stages of infection are very limited. We applied live-cell imaging approach to characterize the spatial and temporal development of growing hyphae and plant responses during F. graminearum and wheat coleoptile interactions. The present investigation demonstrates that F. graminearum uses two strategies to penetrate the host epidermal cells. The pathogen breaks through the host cell wall with appressoria-like structures derived from surficial hyphae, and also with the narrow pegs from thick and bulbous intracellular hyphae. Live cell imaging in the presence of the endocytic tracker FM4–64 showed that the plasma and intermembranes of the invaded wheat coleoptile cells were intact. Invasive hyphae exhibit branching, budding, pseudohyphae-like growth, cell-to-cell spreading ability, and were sealed within a plant membrane, indicating a biotrophic lifestyle of this fungus inside the invaded cells. Time-lapse imaging suggested that there were callose depositions at the plant cell walls in the form of continuous lines and also on the outer linings of the fungal invasive hyphae at colonization stage. In addition, our studies demonstrate that the activation of the toxisome-related gene (TRI4) requires external stimuli and is spatio-temporally modulated. Generally, this study provides new insights into the colonization strategies and host response features during F. graminearum-plant interactions. Further tracing of cellular details will significantly contribute to our understanding of molecular mechanisms of F. graminearum-host interactions.

Comparative expression analysis of Phytophthora sojae polysaccharide lyase family 3 (pectate lyase) genes during infection of the soybean Glycine max

Phytophthora sojae is an important plant pathogen affecting soybean crops worldwide. The specific mechanisms this pathogen uses to penetrate the host cell and initiate infection have not been fully elucidated. However, a greater understanding of the molecular mechanisms that facilitate these processes will be critical to the development of novel strategies to control this devastating agricultural pathogen. It is likely that, to initiate infection, P. sojae uses an appressorium-like structure to break through the plant cell wall. It is also presumed that the pathogen relies on enzymatic activity to weaken the cell wall. The polysaccharide lyase (PL) enzyme superfamily, particularly the PL family 3 (PL3), has been hypothesized to play an important role in the process of host cell penetration by contributing to the degradation of the structural polysaccharides of the cell wall. To investigate this hypothesis, we have scanned the revised version of the annotated P. sojae genome for the presence of putative PL3-coding genes and conducted an extensive sequence analysis of all gene models found. In addition, we have quantified the relative expression of each gene in P. sojae during infection of susceptible (Williams) and resistant (Williams 82) soybean cultivars over a 48 h time period. Twelve PL3-coding gene models were identified and initial results indicate that during infection of the Williams cultivar, several of these genes experience significant up-regulation during the first 48 h of infection. An increase in transcriptional activity is also observed during infection of Williams 82; however, this expression pattern is distinct from that observed in the Williams cultivar. These results provide evidence regarding the potential involvement of PL-coding genes in the early pathogenic processes of P. sojae and suggest that this pathogen expresses genes differentially, depending on whether infection is occurring on a resistant or a susceptible soybean cultivar.

Histopathology of infection and colonisation of Elsinoë ampelina on grapevine leaves

Elsinoe ampelina, the causal agent of grapevine anthracnose, infects all aboveground succulent parts of the plant, causing serious yield losses. There are no histopathological reports on this pathogen interaction with grapevine tissues. This study aimed at understanding the infection and colonisation processes of E. ampelina on Vitis labrusca cv. Niagara Rosada leaves by light, transmission and scanning microscopy. From one to five germ tubes per conidia were formed during conidia germination, predominantly one or two. Cuticle degradation was observed on plant surface underneath germ tubes and appressorium-like structures were rare. Monilioid and branched hyphae were observed 48 h post inoculation. Direct penetration occurred only on the adaxial leaf surface due to high trichome density on the abaxial leaf surface and conidia trapped on trichomes were frequently observed. The colonisation process was similar in the leaf blade, veins and petiole with the formation of inter- and intracellular hyphae. Phenolic compounds accumulated in infected areas; however, they did not prevent the colonisation progress. Tissues of foliar blade were totally changed by cell collapse and conidiogenous cells were observed on the surface of necrotic lesions. Ascus-like structures were observed within deeply unstructured tissues of leaf veins and necrotic areas of the petiole.

Synergistic deletion of RGS1 and COS1 may reduce the pathogenicity of Magnaporthe oryzae

Rice blast, caused by Magnaporthe oryzae, is a serious threat to global rice production. In recent years, many pathogenicity genes of M. oryzae have been identified, although most of their functions remain unknown. In this study, we report the synergistic deletion of RGS1 and COS1 that may reduce the pathogenicity of M. oryzae. The investigation involved comparing ΔMorgs1, ΔMocos1, and ΔMorgs1/ΔMocos1 mutants. The ΔMorgs1/ΔMocos1 mutant showed a weak reduction in vegetative growth, and the colonies displayed fewer and smoother aerial hyphae. The ΔMorgs1/ΔMocos1 mutant exhibited delayed appressorium-like structure formation and 'low pathogenicity' on detached rice seedling leaves when compared with ΔMocos1. Moreover, the melanin content of the single and double mutants was remarkably lower than that of the WT type. Thus, our results indicate that the synergy between RGS1 and COS1 may be crucial in the pathogenicity of M. oryzae.

A Small GTPase RHO2 Plays an Important Role in Pre-infection Development in the Rice Blast Pathogen Magnaporthe oryzae.

The rice blast pathogen Magnaporthe oryzae is a global threat to rice production. Here we characterized RHO2 gene (MGG_02457) that belongs to the Rho GTPase family, using a deletion mutant. This mutant ΔMorho2 exhibited no defects in conidiation and germination but developed only 6% of appressoria in response to a hydrophobic surface when compared to the wild-type progenitor. This result indicates that MoRHO2 plays a role in appressorium development. Furthermore, exogenous cAMP treatment on the mutant led to appressoria that exhibited abnormal morphology on both hydrophobic and hydrophilic surfaces. These outcomes suggested the involvement of MoRHO2 in cAMP-mediated appressorium development. ΔMorho2 mutation also delayed the development of appressorium-like structures (ALS) at hyphal tips on hydrophobic surface, which were also abnormally shaped. These results suggested that MoRHO2 is involved in morphological development of appressoria and ALS from conidia and hyphae, respectively. As expected, ΔMorho2 mutant was defective in plant penetration, but was still able to cause lesions, albeit at a reduced rate on wounded plants. These results implied that MoRHO2 plays a role in M. oryzae virulence as well.

Global insight into the distribution of velvet-like B protein in Cochliobolus species and implication in pathogenicity and fungicide resistance.

The Cochliobolus genus consist of over 55 species among which the 5 most devastating are Cochliobolus carbonum, Cochliobolus heterostrophus, Cochliobolus miyabeanus, Crocus sativus and Cochliobolus lunatus causing damages in sorghum, wheat, rice, maize, cassava and soybean estimated at over 10billion USD per annum worldwide. The dynamic pathogenicity of Cochliobolus species and the plethora of infected hosts is determined by the evolution of virulence determinants such as the velvet-like B protein (VelB). Nonetheless, the knowledge on the distribution of Cochliobolus VelB and its implication in pathogenicity and fungicide resistance are often lacking. By scanning through the annotated genomes of C. lunatus, C. heterostrophus, C. carbonum, C. victoriae, C. sativus and C. miyabeanus, it is revealed that the numbers of ortholog VelB and cognates vary. By using the phylogenetic approach, it is established that the diversification rates among velvet-domain-containing proteins for phytopathogenic Cochliobolus species could impact differently on their oxidant and fungicide resistance potentials, ability to form appressoria-like structures and infection pegs during infection. This study provides new insights into the pathogenicity evolution of Cochliobolus species at the VelB locus which is relevant for designing effective strategies for durable management of Cochliobolus diseases.

Anaeromyces contortus, sp. nov., a new anaerobic gut fungal species (Neocallimastigomycota) isolated from the feces of cow and goat

ABSTRACTTen different anaerobic gut fungal strains were isolated from fecal samples of cow and goat in Oklahoma, USA. The isolates displayed polycentric thalli, uniflagellated zoospores, and sausage-shaped hyphal constrictions, all characteristic of the genus Anaeromyces, and lobed or appressorium-like structures on the hyphae, previously reported in Anaeromyces elegans but not in A. mucronatus or A. robustus. Further, the strains exhibited highly coiled and entangled hyphae previously unreported in the Neocallimastigomycota, but resembling fungal traps previously observed in multiple nematophagous fungi. Scanning electron micrographs of isolates grown on switchgrass showed entrapment of plant material by the hyphal coils and the penetration of plant fibers at various sites by the appressorium-like structures. Molecular analysis based on sequences of both partial nuc rDNA ITS1 spacer region and the D1–D2 domains of the nuc28S rDNA confirmed the classification of all isolates in Anaeromyces and confirmed their phylogenetic distance from the available A. mucronatus and A. robustus sequences. On the basis of morphological and phylogenetic distinctions, we conclude that these strains represent a new Anaeromyces species, for which A. contortus is proposed.

Artificial Inoculation of Epichloë festucae into Lolium perenne, and Visualisation of Endophytic and Epiphyllous Fungal Growth.

Natural hosts for the fungal endophyte Epichloë festucae include Festuca rubra (fine fescue) and Festuca trachyphylla (hard fescue). Some strains also form stable associations with Lolium perenne (perennial ryegrass). L. perenne is a suitable host to study fungal endophyte-grass interactions, such as endophytic fungal growth within the plant and epiphyllous growth on the plant surface. Here we provide a detailed protocol based on work by, for artificial inoculation of E. festucae into L. perenne, and newly developed staining and visualization techniques for observing endophytic and epiphyllous hyphae and the expressorium, an appressorium-like structure used by the fungus to exit the plant. The staining method uses a combination of glucan binding aniline blue diammonium salt (AB) and chitin binding wheat germ agglutinin-conjugated Alexa Fluor®488 -(WGA-AF488). This protocol will be a useful tool to study Epichloë-grass interactions, particularly the comparison of different Epichloë-grass associations, various endophyte-host developmental stages, as well as the analysis of mutant Epichloë strains.

Disruption of actin motor function due to MoMyo5 mutation impairs host penetration and pathogenicity in Magnaporthe oryzae.

Actin motor myosin proteins are the driving forces behind the active transport of vesicles, and more than 20 classes of myosin have been found to contribute to a wide range of cellular processes, including endocytosis and exocytosis, autophagy, cytokinesis and the actin cytoskeleton. In Saccharomyces cerevisiae, class V myosin Myo2 (ScMyo2p) is important for the transport of distinct sets of cargo to regions of the cell along the cytoskeleton for polarized growth. To study whether myosins play a role in the formation or function of the appressorium (infectious structure) of the rice blast fungus Magnaporthe oryzae, we identified MoMyo5 as an orthologue of ScMyo2p and characterized its function. Targeted gene disruption revealed that MoMyo5 is required for intracellular transport and is essential for hyphal growth and asexual reproduction. Although the ΔMomyo5 mutant could form appressorium-like structures, the structures were unable to penetrate host cells and were therefore non-pathogenic. We further found that MoMyo5 moves dynamically from the cytoplasm to the hyphal tip, where it interacts with MoSec4, a Rab GTPase involved in secretory transport, hyphal growth and fungal pathogenicity. Our studies indicate that class V myosin and its translocation are tightly coupled with hyphal growth, asexual reproduction, appressorium function and pathogenicity in the rice blast fungus.

The glycogen synthase kinase MoGsk1, regulated by Mps1 MAP kinase, is required for fungal development and pathogenicity in Magnaporthe oryzae

Magnaporthe oryzae, the causal agent of blast disease, is one of the most destructive plant pathogens, causing significant yield losses on staple crops such as rice and wheat. The fungus infects plants with a specialized cell called an appressorium, whose development is tightly regulated by MAPK signaling pathways following the activation of upstream sensors in response to environmental stimuli. Here, we show the expression of the Glycogen synthase kinase 3 (GSK3) MoGSK1 in M. oryzae is regulated by Mps1 MAP kinase, particularly under the stressed conditions. Thus, MoGSK1 is functionally characterized in this study. MoGsk1 is functionally homologues to the Saccharomyces cerevisiae GSK3 homolog MCK1. Gene replacement of MoGSK1 caused significant delay in mycelial growth, complete loss of conidiation and inability to penetrate the host surface by mycelia-formed appressorium-like structures, consequently resulting in loss of pathogenicity. However, the developmental and pathogenic defects of Δmogsk1 are recovered via the heterologous expression of Fusarium graminearum GSK3 homolog gene FGK3, whose coding products also shows the similar cytoplasmic localization as MoGsk1 does in M. oryzae. By contrast, overexpression of MoGSK1 produced deformed appressoria in M. oryzae. In summary, our results suggest that MoGsk1, as a highly conservative signal modulator, dictates growth, conidiation and pathogenicity of M. oryzae.

MoCDC14 is important for septation during conidiation and appressorium formation in Magnaporthe oryzae.

As a typical foliar pathogen, appressorium formation and penetration are critical steps in the infection cycle of Magnaporthe oryzae. Because appressorium formation and penetration are closely co-regulated with the cell cycle, and Cdc14 phosphatases have an antagonistic relationship with cyclin-dependent kinases (CDKs) on proteins related to mitotic exit and cytokinesis, in this study, we functionally characterized the MoCDC14 gene in M. oryzae. The Mocdc14 deletion mutant showed significantly reduced growth rate and conidiation. It was also defective in septum formation and nuclear distribution. Septation was irregular in Mocdc14 hyphae and hyphal compartments became multi-nucleate. Mutant conidia often showed incomplete septa or lacked any septum. During appressorium formation, the septum delimiting appressoria from the rest of the germ tubes was often formed far away from the neck of the appressoria or not formed at all. Unlike the wild-type, some mutant appressoria had more than one nucleus at 24 h. In addition to appressoria, melanization occurred on parts of the germ tubes and conidia, depending on the irregular position of the appressorium-delimiting septum. The Mocdc14 mutant was also defective in glycogen degradation during appressorium formation and appressorial penetration of intact plant cells. Similar defects in septum formation, melanization and penetration were observed with appressorium-like structures formed at hyphal tips in the Mocdc14 mutant. Often a long fragment of mutant hyphae was melanized, together with the apical appressorium-like structures. These results indicate that MoCDC14 plays a critical role in septation, nuclear distribution and pathogenesis in M. oryzae, and correct septum formation during conidiogenesis and appressorium formation requires the MoCdc14 phosphatase.

Infection and symptom development by citrus scab pathogen Elsinoë fawcettii on leaves of satsuma mandarin

Elsinoe fawcettii, a fungal pathogen causing scab disease in citrus, is one of the destructive diseases having an adverse role in the fresh fruit market. We observed the infection behaviour of E. fawcettii on young leaves of satsuma mandarin daily and described the time course of symptom development with a focus on morphological changes during disease progress. The E. fawcettii isolate 16–1, belonging to Florida Broad Host Range (FBHR), was infectious and generated typical symptoms on adaxial as well as abaxial surfaces of the leaf. Of the conidia inoculated and adhered, only a few were able to germinate and create typical symptoms. Conidia extended their germ tubes from 1 day post-inoculation where a few possessed globose appressorium-like structures (APL) at their tip. The APL were found on both surfaces while symptoms could be produced without them. Dark regions were induced 1–2 days post-inoculation (dpi) under fluorescent microscopic observation. Cuticle and mesophyll cells including epidermis in the infected dark region were degraded, resulting in cellular necrosis. The adjacent cells of the necrotic region were enlarged and gradually covered leaving a grooved shaped necrotic center and appeared as a first visible symptom 3 dpi. Hyphae were extended through the necrotic epidermis and colonized intra- and inter-cellularly in mesophyll tissue. Also lamellated cell-wall was observed as a boundary between infected and non-infected regions in the lesion after typical symptoms were formed. The lamellation in the cell simultaneously occurred forming a compaction of host cells and a semi-circular arc of lammellated cells was formed on the boundary of the infected and non-infected regions of the lesion.

Structure-function analyses of the Pth11 receptor reveal an important role for CFEM motif and redox regulation in rice blast.

The interaction of Magnaporthe oryzae, the rice blast fungus, and rice begins when M.oryzae establishes contact with the host plant surface. On perception of appropriate surface signals, M.oryzae forms appressoria and initiates host invasion. Pth11, an important G-protein-coupled receptor necessary for appressorium formation in M.oryzae, contains seven transmembrane regions and a CFEM (common in several fungal extracellular membrane proteins) domain with the characteristic eight cysteine residues. We focused on gaining further insight into the role of the CFEM domain in the putative surface sensing/response function of Pth11. Increased/constitutive expression of CFEM resulted in precocious, albeit defective, appressoria formation in wild-type M.oryzae. The Pth11C63A/C65A mutant, probably with disrupted disulfide bonds in the CFEM, showed delayed appressorium formation and reduced virulence. Furthermore, the accumulation of reactive oxygen species (ROS) was found to be altered in the pth11Δ strain. Strikingly, antioxidant treatment induced appressorium formation in pth11Δ. The Gα subunit MagB and the mitogen-activated protein (MAP) kinase Pmk1 were required for the formation of antioxidant-induced appressoria. We conclude that the CFEM domain of Pth11 is required for proper development of the appressoria, appressoria-like structures and pathogenicity. Highly regulated ROS homeostasis is important for Pth11-mediated appressorium formation in M.oryzae.

Histopathological assessment of the infection of maize leaves by Fusarium graminearum, F. proliferatum, and F. verticillioides

Young maize plants were inoculated on unfolded mature leaves and on folded immature leaves with Fusarium graminearum, Fusarium proliferatum, and Fusarium verticillioides suspensions. Infection and symptom development of disease on these asymptomatic mature leaves and immature leaves were then documented. Subcuticular infection was found by the three Fusarium species on both symptomatic and symptomless leaves. The three Fusarium species penetrated the stomata of immature leaves by the formation of appressoria-like structures, infection cushions or by direct penetration. Infection by the three species of Fusarium via stomata is reported here for the first time. The superficial hyphae and re-emerging hyphae of the three species produced conidia. The macroconidia of F. graminearum produced secondary macroconidia and F. proliferatum formed microconidia inside the leaf tissues that sporulated through stomata and trichomes. The infection of maize leaves by the three species of Fusarium and their sporulation may contribute inoculum to cob and kernel infection.

Microscopic Aspects of Silicon-Mediated Rice Resistance to Leaf Scald.

This study investigated the effect of silicon (Si) on the potentiation of rice resistance against leaf scald at the microscopic level. Rice plants ('Primavera') were grown in a nutrient solution containing 0 (-Si) or 2 mM (+Si) Si. The foliar Si concentration of the +Si plants (3.6 dag/kg) increased in comparison with the -Si plants (0.3 dag/kg). An X-ray microanalysis revealed that the leaf tissue of +Si plants infected with Microdochium oryzae had higher peaks and deposition of insoluble Si than that of -Si plants. The high foliar Si concentration for the +Si plants reduced the expansion of leaf scald lesions. Scanning electron microscopy revealed that fungal hyphae and appressorium-like structures of M. oryzae were more abundant in the leaf surface of -Si plants relative to +Si plants. At both histopathological and ultrastructural levels, fungal hyphae grew abundantly into the leaf tissue of -Si plants. By contrast, rice cell walls were rarely degraded and fungal hyphae were often surrounded by amorphous granular material in the leaf tissue of +Si plants. Conidiophores emerged from stomata 36 h after fungal penetration, and conidia were noticed inside the leaf tissue of the -Si plants in great abundance. The collective results of the present study showed a high concentration and deposition of Si and a considerable deposition of phenolic-like compounds in the leaf tissue of +Si plants. These results indicate that the potentiation of the phenylpropanoid pathway in these plants supplied with Si was favorable for the increase in rice resistance to leaf scald.

Histopathological studies of the process of Phytophthora palmivora infection in oil palm

The predominant disease affecting oil palms in Colombia is bud rot, a decay of the growing leaf tissues that may eventually reach the meristem. Previous studies have suggested many biotic and abiotic causes, but Koch’s Postulates have recently confirmed Phytophthora palmivora as the primary pathogen of bud rot. In this paper, we describe the infection process in immature leaflets inoculated with zoospores of P. palmivora using light microscopy and scanning electron microscopy. Initially, zoospores were attracted primarily to leaf trichomes, where some developed appressorium-like structures and subsequently penetrated the tissue. Germinated zoospores were also observed penetrating the stomata or intercellular spaces. After penetration hyphae colonized the intercellular leaf spaces, followed by intracellular invasion, penetrating to the vascular bundles and then spreading throughout the host tissue. Between 12 and 24 h after inoculation, hyphae emerged from the surface of the leaflet through the stomata, trichomes and intercellular spaces. Within 36 and 48 h, sporangia, chlamydospores, and newly released zoospores were observed, starting a new cycle of secondary infections. This sequence was consistently observed in repeated experiments. We suggest that in the field collection of moisture at the base of emerging leaflets stimulates the release of zoospores and infection. Colonization is restricted once the leaflet emerges, leaving dormant chlamydospores and hyphae in small necrotic lesions. Successive rain events stimulate the formation of new generations of sporangia, zoospores and infections, which, if frequent enough, may completely colonize the bud area. Subsequent colonization of infected tissues by opportunistic bacteria and fungi increases the severity of bud rot symptoms. The information presented here provides an overview of histopathological aspects of the early infections process of P. palmivora in detached immature leaflets.

Cochliobolus lunatus colonizes potato by adopting different invasion strategies on cultivars: New insights on temperature dependent-virulence

Extreme temperature fluctuations affect the interaction dynamics of Cochliobolus lunatus through temperature-dependent virulence, virulence differentiation and induced-virulence which poses a major threat to global food security. The relationship between higher temperature and pathogenicity of C. lunatus on reported hosts are poorly understood. In this study, temperature stress was applied on C. lunatus to investigate the correlation among the different types of conidia. Additionally, a comparative dissection of the invasion process, infection structures and conidial germination pattern on four different Solanum tuberosum L. (potato) cultivars were performed. Based on microscopic examination, it was found that C. lunatus adopts different hyphae morphology and septation pattern at different temperature regimes and produce different types of conidia. The study showed that four-celled conidia are overproduced at elevated temperature (>30 °C) than one, two, three and five-celled conidia. Our finding revealed that C. lunatus conidia exhibit bipolar germination (>14.67%, P<0.05), unipolar germination (>35.33%, P<0.05), penetrate subcutaneously via epidermal anticlinal cell wall (>0.33%, P < 0.05) and differentially form appressoria-like structures during colonization of four different potato cultivars. Importantly, it is shown that unipolar germination and bipolar germination in C. lunatus are independently occurring phenomenon irrespective of the host. It is confirmed that C. lunatus adopt different but highly successful strategies on four different potato cultivars to incite brown-to-black leaf spot disease. Altogether, our data showed that increase in temperature enhances C. lunatus virulence on different potato cultivars irrespective of their inherent thermotolerant traits.

The Role of Mitogen-Activated Protein (MAP) Kinase Signaling Components in the Fungal Development, Stress Response and Virulence of the Fungal Cereal Pathogen Bipolaris sorokiniana.

Mitogen-activated protein kinases (MAPKs) have been demonstrated to be involved in fungal development, sexual reproduction, pathogenicity and/or virulence in many filamentous plant pathogenic fungi, but genes for MAPKs in the fungal cereal pathogen Bipolaris sorokiniana have not been characterized. In this study, orthologues of three MAPK genes (CsSLT2, CsHOG1 and CsFUS3) and one MAPK kinase kinase (MAPKKK) gene (CsSTE11) were identified in the whole genome sequence of the B. sorokiniana isolate ND90Pr, and knockout mutants were generated for each of them. The ∆Csfus3 and ∆Csste11 mutants were defective in conidiation and formation of appressoria-like structures, showed hypersensitivity to oxidative stress and lost pathogenicity on non-wounded leaves of barley cv. Bowman. When inoculated on wounded leaves of Bowman, the ∆Csfus3 and ∆Csste11 mutants were reduced in virulence compared to the wild type. No morphological changes were observed in the ∆Cshog1 mutants in comparison with the wild type; however, they were slightly reduced in growth under oxidative stress and were hypersensitive to hyperosmotic stress. The ∆Cshog1 mutants formed normal appressoria-like structures but were reduced in virulence when inoculated on Bowman leaves. The ∆Csslt2 mutants produced more vegetative hyphae, had lighter pigmentation, were more sensitive to cell wall degrading enzymes, and were reduced in virulence on Bowman leaves, although they formed normal appressoria like the wild type. Root infection assays indicated that the ∆Cshog1 and ∆Csslt2 mutants were able to infect barley roots while the ∆Csfus3 and ∆Csste11 failed to cause any symptoms. However, no significant difference in virulence was observed for ∆Cshog1 mutants while ∆Csslt2 mutants showed significantly reduced virulence on barley roots in comparison with the wild type. Our results indicated that all of these MAPK and MAPKKK genes are involved in the regulation of fungal development under normal and stress conditions and required for full virulence on barley plants.

MoTup1 is required for growth, conidiogenesis and pathogenicity of Magnaporthe oryzae.

The general transcriptional repressor Tup1 proteins play important regulatory roles in the growth and development of fungi. In this report, we characterized MoTup1, a protein homologous to Tup1 of Saccharomyces cerevisiae, from M. oryzae. Disruption of MoTUP1 resulted in severe mycelial growth reduction and a defect in conidiogenesis. We found that MoTup1 is required for the maintenance of cell wall integrity by regulating the expression of the genes involved in cell wall biosynthesis. Pathogenicity assays indicated that the ΔMotup1 mutants lost the ability to invade both rice and barley hosts. Moreover, observation of rice epidermis penetration showed that the hyphal tips of the mutants could still form appressorium-like structures, but were unable to invade host cells. Taken together, our results demonstrate that M. oryzae MoTup1 is an important regulatory factor in fungal growth, development and pathogenesis on hosts.

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.