Genomics-guided discovery and structure identification of cyclic lipopeptides from the Bacillus siamensis JFL15.

In this research, a strain with broad-spectrum antimicrobial activities was isolated from the gastrointestinal tract of hairtail (Trichiurus haumela) and identified as Bacillus siamensis JFL15 through morphological, 16S rRNA, and average nucleotide identity analyses. The genome of B. siamensis JFL15 was sequenced, and three gene clusters involved in the biosynthesis of surfactin (srf), bacillibactin (dhb), and fengycin (fen) were predicted through antiSMASH analysis. The combined genomics-metabolics profiling of the strain revealed 20 active compounds, which belong to four main types of cyclic lipopeptides produced by Bacillus species: bacillibactin, iturin, fengycin, and surfactin. Among these lipopeptides, two high-purity antifungal components, namely, components b and c, were successfully identified as iturin A and bacillomycin F. The minimum inhibitory concentrations (MICs) of iturin A for Magnapothe grisea, Rhizoctorzia solani, and Colletotrichum gloeosporioides were 125.00, 62.50, and 125.00 µg/ml, respectively, whereas the MICs of bacillomycin F for these three organisms were 62.50, 31.25, and 62.50 µg/ml, respectively. The mechanism of bacillomycin F and iturin A against M. grisea was also investigated. Scanning electron microscopy (SEM) indicated that the surface of the hypha treated with iturin A or bacillomycin F became sunk, lumpy, and wrinkled. The diversity of the identified and predicted compounds from B. siamensis JFL15 suggested that this strain might be a promising biocontrol agent for an effective and environmentally friendly control of pathogenic microorganisms. To the best of our knowledge, this study is the first to describe cyclic lipopeptides purified and identified from B. siamensis.

Peroxisomal fission is induced during appressorium formation and is required for full virulence of the rice blast fungus.

Peroxisomes are involved in various metabolic processes and are important for virulence in different pathogenic fungi. How peroxisomes rapidly emerge in the appressorium during fungal infection is poorly understood. Here, we describe a gene, PEF1, which can regulate peroxisome formation in the appressorium by controlling peroxisomal fission, and is required for plant infection in the rice blast fungus Magnaporthe oryzae. Targeted deletion of PEF1 resulted in a reduction in virulence and a delay in penetration and invasive growth in host cells. PEF1 was particularly expressed during appressorial development, and its encoding protein was co-localized with peroxisomes during appressorial development. Compared with the massive vesicle-shaped peroxisomes formed in the wild-type appressorium, the Δpef1 mutant could only form stringy linked immature peroxisomes, suggesting that PEF1 was involved in peroxisomal fission during appressorium formation. We also found that the Δpef1 mutant could not utilize fatty acids efficiently, which can improve significantly the expression level of PEF1 and induce peroxisomal fission. As expected, the Δpef1 mutant showed reduced intracellular production of reactive oxygen species (ROS) during appressorium formation and induced ROS accumulation in host cells during infection. Taken together, PEF1-mediated peroxisomal fission is important for fungal infection by controlling the number of peroxisomes in the appressorium.

Appressorium-mediated penetration of Magnaporthe oryzae and Colletotrichum orbiculare into surface-cross-linked agar media.

Many phytopathogenic fungi form appressoria on some artificial substances. However, it is difficult to induce appressorium-mediated penetration into artificial substances. In the present study, novel artificial agar media were developed to investigate the in vitro penetration process of phytopathogenic fungi. The media contained sodium carboxymethyl cellulose or sodium alginate, and the surfaces were subjected to ionic cross-linking using trivalent metal ions. The hemibiotrophic phytopathogenic fungi, rice blast fungus and cucurbit anthracnose fungus, formed appressoria and penetrated into the surface cross-linked artificial agar media from the base of appressoria. These artificial media appeared to induce fungal infection behaviour that occurred on host plants.

Germination and infectivity of microconidia in the rice blast fungus Magnaporthe oryzae.

The rice blast fungus Magnaporthe oryzae is a model for studying fungal-plant interactions. Although it produces two types of spores (microconidia and macroconidia), previous infection studies have exclusively dealt with macroconidia. Germination of microconidia has not been reported, and their role in plant infection is not defined. Here we show that approximately 10% of microconidia germinate on plant surfaces, and that colonies derived from germinated microconidia are normal in growth and pathogenesis. In infection assays with rice and barley seedlings, microconidia fail to infect intact plants, but they can colonize and develop necrotic lesions on wounded leaves and stems. Microconidia also cause disease symptoms on inoculated spikelets in infection assays with barley and Brachypodium heads. Furthermore, microconidia are detected inside rice plants that developed blast lesions under laboratory or field conditions. Therefore, microconidia can germinate and are infectious, and may be an important factor in the rice blast cycle.

The natural product citral can cause significant damage to the hyphal cell walls of Magnaporthe grisea.

In order to find a natural alternative to the synthetic fungicides currently used against the devastating rice blast fungus, Magnaporthe grisea, this study explored the antifungal potential of citral and its mechanism of action. It was found that citral not only inhibited hyphal growth of M. grisea, but also caused a series of marked hyphal morphological and structural alterations. Specifically, citral was tested for antifungal activity against M. grisea in vitro and was found to significantly inhibit colony development and mycelial growth with IC50 and IC90 values of 40.71 and 203.75 µg/mL, respectively. Furthermore, citral reduced spore germination and germ tube length in a concentration-dependent manner. Following exposure to citral, the hyphal cell surface became wrinkled with folds and cell breakage that were observed under scanning electron microscopy (SEM). There was damage to hyphal cell walls and membrane structures, loss of villous-like material outside of the cell wall, thinning of the cell wall, and discontinuities formed in the cell membrane following treatment based on transmission electron microscopy (TEM). This increase in chitinase activity both supports the morphological changes seen in the hyphae, and also suggests a mechanism of action. In conclusion, citral has strong antifungal properties, and treatment with this compound is capable of causing significant damage to the hyphal cell walls of M. grisea.

PAF104, a synthetic peptide to control rice blast disease by blocking appressorium formation in Magnaporthe oryzae.

Magnaporthe oryzae is the most devastating pathogen of rice and the main cause of crop losses worldwide. The successful management of blast disease caused by this fungus is a clear necessity. The synthetic peptide PAF104 has been characterized by its inhibition of M. oryzae appressorium formation on hydrophobic surfaces. Growth and the ability of conidia to germinate was not affected by PAF104, indicating the lack of toxicity on fungal conidia. The addition of the cutin monomer 1,16-hexadecanediol does not interfere with the inhibitory effect of PAF104 on in vitro hydrophobic surfaces. On the other hand, inhibition of appressorium formation by PAF104 was nullified by the exogenous addition of cAMP. Our results suggest that PAF104 affects the Pmk1 pathway by repression of the gene expression of MoMSB2, which encodes a sensing surface protein, and the mitogen-activated protein/extracellular signal-regulated kinase kinase kinase MST11. The pathogenicity of M. oryzae was reduced after PAF104 treatment specifically blocking appressorium formation. Our results support PAF104 as a promising compound to control rice blast disease by blocking a specific target related to appressorium formation, a process essential for infection of rice leaves. Moreover, PAF104 is proposed as a lead compound to develop novel specific fungicides with improved properties.

Septin-mediated plant cell invasion by the rice blast fungus, Magnaporthe oryzae.

To cause rice blast disease, the fungus Magnaporthe oryzae develops a pressurized dome-shaped cell called an appressorium, which physically ruptures the leaf cuticle to gain entry to plant tissue. Here, we report that a toroidal F-actin network assembles in the appressorium by means of four septin guanosine triphosphatases, which polymerize into a dynamic, hetero-oligomeric ring. Septins scaffold F-actin, via the ezrin-radixin-moesin protein Tea1, and phosphatidylinositide interactions at the appressorium plasma membrane. The septin ring assembles in a Cdc42- and Chm1-dependent manner and forms a diffusion barrier to localize the inverse-bin-amphiphysin-RVS-domain protein Rvs167 and the Wiskott-Aldrich syndrome protein Las17 at the point of penetration. Septins thereby provide the cortical rigidity and membrane curvature necessary for protrusion of a rigid penetration peg to breach the leaf surface.

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.

Homeobox transcription factors are required for conidiation and appressorium development in the rice blast fungus Magnaporthe oryzae.

The appropriate development of conidia and appressoria is critical in the disease cycle of many fungal pathogens, including Magnaporthe oryzae. A total of eight genes (MoHOX1 to MoHOX8) encoding putative homeobox transcription factors (TFs) were identified from the M. oryzae genome. Knockout mutants for each MoHOX gene were obtained via homology-dependent gene replacement. Two mutants, DeltaMohox3 and DeltaMohox5, exhibited no difference to wild-type in growth, conidiation, conidium size, conidial germination, appressorium formation, and pathogenicity. However, the DeltaMohox1 showed a dramatic reduction in hyphal growth and increase in melanin pigmentation, compared to those in wild-type. DeltaMohox4 and DeltaMohox6 showed significantly reduced conidium size and hyphal growth, respectively. DeltaMohox8 formed normal appressoria, but failed in pathogenicity, probably due to defects in the development of penetration peg and invasive growth. It is most notable that asexual reproduction was completely abolished in DeltaMohox2, in which no conidia formed. DeltaMohox2 was still pathogenic through hypha-driven appressoria in a manner similar to that of the wild-type. However, DeltaMohox7 was unable to form appressoria either on conidial germ tubes, or at hyphal tips, being non-pathogenic. These factors indicate that M. oryzae is able to cause foliar disease via hyphal appressorium-mediated penetration, and MoHOX7 is mutually required to drive appressorium formation from hyphae and germ tubes. Transcriptional analyses suggest that the functioning of M. oryzae homeobox TFs is mediated through the regulation of gene expression and is affected by cAMP and Ca(2+) signaling and/or MAPK pathways. The divergent roles of this gene set may help reveal how the genome and regulatory pathways evolved within the rice blast pathogen and close relatives.

Autophagy-assisted glycogen catabolism regulates asexual differentiation in Magnaporthe oryzae.

Autophagy, a conserved pathway for bulk cellular degradation and recycling in eukaryotes, regulates proper turnover of organelles, membranes and certain proteins. Such regulated degradation is important for cell growth and development particularly during environmental stress conditions, which act as key inducers of autophagy. We found that autophagy and MoATG8 were significantly induced during asexual development in Magnaporthe oryzae. An RFP-tagged MoAtg8 showed specific localization and enrichment in aerial hyphae, conidiophores and conidia. We confirmed that loss of MoATG8 results in dramatically reduced ability to form conidia, the asexual spores that propagate rice-blast disease. Exogenous supply of glucose or sucrose significantly suppressed the conidiation defects in a MoATG8-deletion mutant. Comparative proteomics based identification and characterization of Gph1, a glycogen phosphorylase that catalyzes glycogen breakdown, indicated that autophagy-assisted glycogen homeostasis is likely important for proper aerial growth and conidiation in Magnaporthe. Loss of Gph1, or addition of G6P significantly restored conidiation in the Moatg8Delta mutant. Overproduction of Gph1 led to reduced conidiation in wild-type Magnaporthe strain. We propose that glycogen autophagy actively responds to and regulates carbon utilization required for cell growth and differentiation during asexual development in Magnaporthe.

Magnaporthe grisea cutinase2 mediates appressorium differentiation and host penetration and is required for full virulence.

The rice blast fungus Magnaporthe grisea infects its host by forming a specialized infection structure, the appressorium, on the plant leaf. The enormous turgor pressure generated within the appressorium drives the emerging penetration peg forcefully through the plant cuticle. Hitherto, the involvement of cutinase(s) in this process has remained unproven. We identified a specific M. grisea cutinase, CUT2, whose expression is dramatically upregulated during appressorium maturation and penetration. The cut2 mutant has reduced extracellular cutin-degrading and Ser esterase activity, when grown on cutin as the sole carbon source, compared with the wild-type strain. The cut2 mutant strain is severely less pathogenic than the wild type or complemented cut2/CUT2 strain on rice (Oryza sativa) and barley (Hordeum vulgare). It displays reduced conidiation and anomalous germling morphology, forming multiple elongated germ tubes and aberrant appressoria on inductive surfaces. We show that Cut2 mediates the formation of the penetration peg but does not play a role in spore or appressorium adhesion, or in appressorial turgor generation. Morphological and pathogenicity defects in the cut2 mutant are fully restored with exogenous application of synthetic cutin monomers, cAMP, 3-isobutyl-1-methylxanthine, and diacylglycerol (DAG). We propose that Cut2 is an upstream activator of cAMP/protein kinase A and DAG/protein kinase C signaling pathways that direct appressorium formation and infectious growth in M. grisea. Cut2 is therefore required for surface sensing leading to correct germling differentiation, penetration, and full virulence in this model fungus.

Emerging antifungal azoles and effects on Magnaporthe grisea.

Derivatives of pyrazolo[1,5-a][1, 3, 5]triazine-2,4-dione,pyrazolo[1,5-c][1, 3, 5]thiadiazine-2-one, pyrazolo[3,4-d][1, 3]thiazine-4-one, and pyrazolo[3,4-d][1, 3]thiazine-4-thione were screened for antifungal activity against the causal agent of rice blast disease, Magnaporthe grisea. The compounds were tested at doses ranging from 10 to 200mugml(-1), using the commercial fungicide tricyclazole as reference compound. All triazine derivatives inhibited the growth and pigmentation of the mycelia less effectively than tricyclazole. The thiadiazine derivatives proved to be more effective than their triazine counterparts, but only 4-(butylimino)-7-methylpyrazolo[1,5-c][1,3,5]thiadiazine-2-one (2h) and 4-(cyclohexylimino)-7-methylpyrazolo[1,5-c][1,3,5]thiadiazine-2-one (2j) were more effective than tricyclazole. Pyrazolo[3,4-d][1,3]thiazine-4-one derivatives were active only at the highest doses, whereas members of the pyrazolo[3,4-d][1,3]thiazine-4-thione series inhibited fungal growth at the lowest concentrations used, at which tricyclazole had no effect. A dose-dependent mechanism might be responsible for this effect, with lipophilicity as the governing factor. Within a given set, the presence of a cyclohexyl or an n-butyl group generally increased antifungal activity, with respect to both growth inhibition and cell de-pigmentation of the mycelium, suggesting that a higher lipophilicity might improve transport inside the cells. SEM and TEM of M. grisea hyphae showed that treatment with the most active substance (2h) caused significant ultrastructural effects, particularly on the endomembrane system, suggesting a mechanism of action similar to that of most azole fungicides. Dissimilarities were also observed, with no alterations of the cell wall evident. In conclusion, several compounds showed greater inhibition than tricyclazole, and therefore provide useful new chemistry for control of M. grisea infections.

Biomechanical model for appressorial design in Magnaporthe grisea.

The fungus Magnaporthe grisea, commonly referred to as the rice blast fungus, is responsible for destroying from 10% to 30% of the world's rice crop each year. The fungus attaches to the rice leaf and forms a dome-shaped structure, the appressorium, in which enormous pressures are generated that are used to blast a penetration peg through the rice cell walls and infect the plant. We develop a model of the appressorial design in terms of a bioelastic shell that can explain the shape of the appressorium, and its ability to maintain that shape under the enormous increases in turgor pressure that can occur during the penetration phase.

Live-cell imaging of tubulin in the filamentous fungus Magnaporthe grisea treated with anti-microtubule and anti-microfilament agents.

Microtubule dynamics were examined in live cells of the fungal plant pathogen Magnaporthe grisea transformed for constitutive expression of a fusion protein containing enhanced yellow-fluorescent protein and a Neurospora crassa benomyl-resistant allele of beta-tubulin. Transformants retained their ability to differentiate appressoria and cause disease but remained sensitive to benomyl. Linear microtubule arrays and low-level cytoplasmic fluorescence were observed in vegetative hyphae, conidia, germ tubes, and developing appressoria. Fluorescence within nuclei was conspicuously absent during interphase but increased rapidly at the onset of mitosis. Treatment with either benomyl or griseofulvin resulted in the appearance of prominent brightly fluorescent aggregates, including a large aggregate near the apex, with the concomitant disappearance of most cytoplasmic microtubules. Electron microscope imaging of treated cells indicated that the aggregates lacked any obvious profiles of intact microtubules. During these treatments, hyphal tip cells continued to elongate in a nonlinear and aerial fashion at a much slower rate than untreated cells. With subsequent removal of griseofulvin, distal aggregates disappeared rapidly but the apical aggregates persisted longer. Treatment with latrunculin A caused hyphal tip swelling without apparent effect on linear microtubule arrays. Simultaneous treatment with griseofulvin and latrunculin A resulted in depolymerization of microtubules and a cessation of growth, but near-apical fluorescent aggregates were not observed.

Independent genetic mechanisms mediate turgor generation and penetration peg formation during plant infection in the rice blast fungus.

The first barrier to infection encountered by foliar pathogens is the host cuticle. To traverse this obstacle, many fungi produce specialized infection cells called appressoria. MST12 is essential for appressorium-mediated penetration and infectious growth by the rice pathogen Magnaporthe grisea. In this study, we have characterized in detail the penetration defects of an mst12 deletion mutant. Appressoria formed by the mst12 mutant developed normal turgor pressure and ultrastructure but failed to form penetration pegs either on cellophane membranes or on plant epidermal cells. Deletion and site-directed mutagenesis analyses indicated that both the homeodomain and zinc finger domains, but not the middle region, of MST12 are essential for appressorial penetration and plant infection. The mst12 mutant appeared to be defective in microtubule reorganization associated with penetration peg formation. In mature appressoria, the mutant lacked vertical microtubules observed in the wild type. The mst12 mutant also failed to elicit localized host defence responses, including papilla formation and autofluorescence. Our data indicate that generation of appressorium turgor pressure and formation of the penetration peg are two independent processes. MST12 may play important roles in regulating penetration peg formation and directing the physical forces exerted by the appressorium turgor in mature appressoria.

Regulation of the MPG1 hydrophobin gene in the rice blast fungus Magnaporthe grisea.

The hydrophobin-encoding gene MPG1 of the rice blast fungus Magnaporthe grisea is highly expressed during the initial stages of host plant infection and targeted deletion of the gene results in a mutant strain that is reduced in virulence, conidiation, and appressorium formation. The green fluorescent protein-encoding allele sGFP was used as a reporter to investigate regulatory genes that control MPG1 expression. The MAP kinase-encoding gene PMK1 and the wide domain regulators of nitrogen source utilization, NPR1 and NUT1, were required for full expression of MPG1 in response to starvation stress. The CPKA gene, encoding the catalytic subunit of protein kinase A, was required for repression of MPG1 during growth in rich nutrient conditions. During appressorium morphogenesis, high-level MPG1 expression was found to require the CPKA and NPR1 genes. Expression of a destabilized GFP allele indicated that de novo MPG1 expression occurs during appressorium formation. Three regions of the MPG1 promoter were identified which are required for high-level expression of MPG1 during appressorium formation and are necessary for the biological activity of the MPG1 hydrophobin during spore formation and plant infection.

Reef coral fluorescent proteins for visualizing fungal pathogens.

The fluorescent proteins AmCyan, ZsGreen, ZsYellow, and AsRed, modified versions of proteins identified recently from several Anthozoa species of reef corals, were expressed for the first time in a heterologous system and used for imaging two different fungal plant pathogens. When driven by strong constitutive fungal promotors, expression of these reef coral fluorescent proteins yielded bright cytoplasmic fluorescence in Fusarium verticillioides and Magnaporthe grisea, and had no detectable effect on either growth rate or the ability to cause disease. Differential intracellular localization of the fluorescent proteins resulted in the discrimination of many subcellular organelles by confocal and multi-photon microscopy, and facilitated monitoring of such details as organelle dynamics and changes in the permeability of the nuclear envelope. AmCyan and ZsGreen were sufficiently excited at 855 and 880 nm, respectively, to allow for time-resolved in planta imaging by two-photon microscopy.

Live-cell imaging of endocytosis during conidial germination in the rice blast fungus, Magnaporthe grisea.

Although there is growing evidence that endocytosis is important in hyphal tip growth, it has not previously been shown to occur during fungal spore germination. We have analysed and characterized endocytosis during the germination of living conidia of the rice blast fungus, Magnaporthe grisea. Conidia treated with the endocytic markers Lucifer Yellow carbohydrazide, FITC-dextran, and FM4-64 were imaged by confocal microscopy. Internalization of these fluorescent marker dyes by conidia was blocked by chemical and temperature treatments that inhibit endocytosis, and the sequential staining of organelles by the membrane-selective dye FM4-64 was consistent with dye internalization by endocytosis. FM4-64 uptake occurred within 2-3 min of conidial hydration, more than 40 min before the emergence of the germ tube. The times at which each of the three conidial cells initiated dye internalization were different as were the rates of dye uptake by each cell. Using these techniques we have demonstrated for the first time that ungerminated and germinated spores of filamentous fungi undergo endocytosis. Furthermore, internalization of FITC-dextran and Lucifer Yellow carbohydrazide by germinating conidia provides the first direct evidence for fluid-phase endocytosis in a filamentous fungus. FM4-64 was internalized by both ungerminated conidia and conidial germlings on the rice leaf suggesting that endocytosis might play a significant role in spore germination and germ tube growth during the pre-penetration phase of infection.

The vacuole as central element of the lytic system and sink for lipid droplets in maturing appressoria of Magnaporthe grisea.

Histochemical and ultrastructural studies were carried out on a wild-type strain (Guy11) and a melanin-deficient mutant (buf1) of the rice-blast pathogen, Magnaporthe grisea (= Pyricularia oryzae), in order to investigate the destination of lipid storage reserves during appressorium development. Lipid droplets were abundant in conidia and were mobilised upon germination, accumulating in the appressorial hook which developed at the tip of each germ tube. Following the formation of a septum at the base of the nascent appressorium, one or a few closely appressed central vacuoles became established and were observed to enlarge in the course of appressorium maturation. On unyielding artificial surfaces such as glass or plastic, appressoria matured to completion within 36-48 h, by which time the enlarged vacuole filled most of the inside volume of the appressorium. Light and transmission electron microscopical observations revealed that the lipid droplets entered the vacuole by autophagocytosis and were degraded therein. Histochemical approaches confirmed the vacuole as the key lytic element in maturing appressoria. Endocytosis of a vital dye, Neutral Red, progressed via endosomes which migrated into the vacuole and lysed there, releasing their dye content into the vacuolar lumen. Furthermore, activity of the lysosomal marker enzyme, acid phosphomonoesterase, was strongly localised in the vacuole at all stages of appressorium maturation. It is therefore envisaged that vacuoles are involved in the degradation of lipid storage reserves which may act as sources of energy and/or osmotically active metabolites such as glycerol, which generate the very high turgor pressure known to be crucial for penetration of hard surfaces. On softer surfaces such as onion epidermis, appressoria of M. grisea were able to penetrate before degradation of lipid droplets had been completed.