Effects of latrunculin B on the actin cytoskeleton and hyphal growth in Phytophthora infestans

The actin cytoskeleton is conserved in all eukaryotes, but its functions vary among different organisms. In oomycetes, the function of the actin cytoskeleton has received relatively little attention. We have performed a bioinformatics study and show that oomycete actin genes fall within a distinct clade that is divergent from plant, fungal and vertebrate actin genes. To obtain a better understanding of the functions of the actin cytoskeleton in hyphal growth of oomycetes, we studied the actin organization in Phytophthora infestans hyphae and the consequences of treatment with the actin depolymerising drug latrunculin B (latB). This revealed that latB treatment causes a concentration dependent inhibition of colony expansion and aberrant hyphal growth. The most obvious aberrations observed upon treatment with 0.1 μM latB were increased hyphal branching and irregular tube diameters whereas at higher concentrations latB (0.5 and 1 μM) tips of expanding hyphae changed into balloon-like shapes. This aberrant growth correlated with changes in the organization of the actin cytoskeleton. In untreated hyphae, staining with fluorescently tagged phalloidin revealed two populations of actin filaments: long, axially oriented actin filament cables and cortical actin filament plaques. Two hyphal subtypes were recognized, one containing only plaques and the other containing both cables and plaques. In the latter, some hyphae had an apical zone without actin filament plaques. Upon latB treatment, the proportion of hyphae without actin filament cables increased and there were more hyphae with a short apical zone without actin filament plaques. In general, actin filament plaques were more resilient against actin depolymerisation than actin filament cables. Besides disturbing hyphal growth and actin organization, actin depolymerisation also affected the positioning of nuclei. In the presence of latB, the distance between nuclei and the hyphal tip decreased, suggesting that the actin cytoskeleton plays a role in preventing the movement of nuclei towards the hyphal tip.     

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.     

Actin‐bundling protein fimbrin regulates pathogenicity via organizing F‐actin dynamics during appressorium development in Colletotrichum gloeosporioides

Anthracnose caused by Colletotrichum gloeosporioides leads to serious economic loss to rubber tree yield and other tropical crops. The appressorium, a specialized dome‐shaped infection structure, plays a crucial role in the pathogenesis of C. gloeosporioides. However, the mechanism of how actin cytoskeleton dynamics regulate appressorium formation and penetration remains poorly defined in C. gloeosporioides. In this study, an actin cross‐linking protein fimbrin homologue (CgFim1) was identified in C. gloeosporioides, and the knockout of CgFim1 led to impairment in vegetative growth, conidiation, and pathogenicity. We then investigated the roles of CgFim1 in the dynamic organization of the actin cytoskeleton. We observed that actin patches and cables localized at the apical and subapical regions of the hyphal tip, and showed a disc‐to‐ring dynamic around the pore during appressorium development. CgFim1 showed a similar distribution pattern to the actin cytoskeleton. Moreover, knockout of CgFim1 affected the polarity of the actin cytoskeleton in the hyphal tip and disrupted the actin dynamics and ring structure formation in the appressorium, which prevented polar growth and appressorium development. The CgFim1 mutant also interfered with the septin structure formation. This caused defects in pore wall overlay formation, pore contraction, and the extension of the penetration peg. These results reveal the mechanism by which CgFim1 regulates the growth and pathogenicity of C. gloeosporioides by organizing the actin cytoskeleton.     

Plasma-mediated enhancement of enzyme secretion in Aspergillus oryzae

Technical bottlenecks in protein production and secretion often limit the efficient and robust industrial use of microbial enzymes. The potential of non-thermal atmospheric pressure plasma to overcome these technical barriers was examined. Spores of the fermenting fungus Aspergillus oryzae (A. oryzae) were submerged in potato dextrose broth (PDB) (5 × 10⁶ per ml) and treated with micro dielectric barrier discharge plasma at an input voltage of 1.2 kV and current of 50 to 63 mA using nitrogen as the feed gas. The specific activity of α-amylase in the broth was increased by 7.4 to 9.3% after 24 and 48 h of plasma treatment. Long-lived species, such as NO₂⁻ and NO₃⁻, generated in PDB after plasma treatment may have contributed to the elevated secretion of α-amylase. Observations after 24 h of plasma treatment also included increased accumulation of vesicles at the hyphal tip, hyphal membrane depolarization and higher intracellular Ca²⁺ levels. These results suggest that long-lived nitrogen species generated in PDB after plasma treatment can enhance the secretion of α-amylase from fungal hyphae by depolarizing the cell membrane and activating Ca²⁺ influx into hyphal cells, eventually leading to the accumulation of secretory vesicles near the hyphal tips.     

Rice ubiquitin-conjugating enzyme OsUBC26 is essential for immunity to the blast fungus Magnaporthe oryzae

The functions of ubiquitin-conjugating enzymes (E2) in plant immunity are not well understood. In this study, OsUBC26, a rice ubiquitin-conjugating enzyme, was characterized in the defence against Magnaporthe oryzae. The expression of OsUBC26 was induced by M. oryzae inoculation and methyl jasmonate treatment. Both RNA interference lines and CRISPR/Cas9 null mutants of OsUBC26 reduced rice resistance to M. oryzae. WRKY45 was down-regulated in OsUBC26 null mutants. In vitro E2 activity assay indicated that OsUBC26 is an active ubiquitin-conjugating enzyme. Yeast two-hybrid assays using OsUBC26 as bait identified the RING-type E3 ligase UCIP2 as an interacting protein. Coimmunoprecipitation assays confirmed the interaction between OsUBC26 and UCIP2. The CRISPR/Cas9 mutants of UCIP2 also showed compromised resistance to M. oryzae. Yeast two-hybrid screening using UCIP2 as bait revealed that APIP6 is a binding partner of UCIP2. Moreover, OsUBC26 working with APIP6 ubiquitinateds AvrPiz-t, an avirulence effector of M. oryzae, and OsUBC26 null mutation impaired the proteasome degradation of AvrPiz-t in rice cells. In summary, OsUBC26 plays important roles in rice disease resistance by regulating WRKY45 expression and working with E3 ligases such as APIP6 to counteract the effector protein AvrPiz-t from M. oryzae.     

Does Hyphal‐Tip Ensure the Same Allelic Composition at SSR Loci as Monosporic Isolates of Sclerotinia sclerotiorum?

The proper characterization of individual is a basic stage in population genetic studies. In Sclerotinia sclerotiorum, genetic uniformity of an individual can be obtained by isolation of single ascospore; however, hyphal‐tip isolates are commonly used in genetic studies. The aim of this study was to assess whether hyphal‐tip isolates of S. sclerotiorum can be used as surrogate of monoascosporic (monosporic) isolates. Twenty‐eight isolates of S. sclerotiorum were collected from common bean plants with white mold symptoms and were purified by hyphal‐tip or single ascospore. The correspondence between hyphal‐tip and monosporic isolates was assessed through the allelic composition at 10 microsatellite (SSR) loci of the isolates obtained by both methods. For the SSR loci comprised of dinucleotide repeats in 92% of the cases, the difference (di) between the amplicon size values for hyphal‐tip and monosporic isolates was no more than one base pair. For the loci comprised of tetra or pentanucleotide repeats in 89% of the cases, di was no more than one base pair. The same allelic profile was found for hyphal‐tip or single ascospore isolates of S. sclerotiorum. When monosporic isolates cannot be easily obtained, hyphal‐tip can safeguard the genotypic identity of S. sclerotiorum isolates.     

Identification of Sternbin and Naringenin as Detoxified Metabolites from the Rice Flavanone Phytoalexin Sakuranetin by Pyricularia oryzae

Sakuranetin ( 1 ) is a flavanone phytoalexin that has been reported to play an important role in disease resistance in rice plants. The rice blast fungus Pyricularia oryzae (syn. Magnaporthe oryzae) has been reported to metabolize 1 to lower its antifungal activity. Here, two flavanones, sternbin ( 2 ) and naringenin ( 3 ), were identified as metabolites of 1 in P. oryzae suspension culture by liquid chromatography tandem mass spectrometry (LC/MS/MS). The inhibition of 1 , 2 , and 3 on P. oryzae mycelial growth were 45%, 19%, and 19%, respectively, at a concentration of 100 μm . Thus, 2 and 3 are detoxified metabolites of 1 by P. oryzae.     

Steady-state force–velocity relation of ATP-dependent sliding between slime mold myosin, arranged on paramyosin filaments, and algal cell actin cables

To investigate characteristics of ATP-dependent sliding of a non-muscle cell myosin, obtained from a cellular slime mold Dictyostelium discoideum, on actin filament, we prepared hybrid thick filaments, in which Dictyostelium myosin was regularly arranged around paramyosin filaments obtained from a molluscan smooth muscle. A single to a few hybrid filaments were attached to a polystyrene bead (diameter, 4.5 μm; specific gravity, 1.5), and the filaments were made to slide on actin filament arrays (actin cables) in the internodal cell of an alga Chara corallina, mounted on the rotor of a centrifuge microscope. The filament-attached bead was observed to move with a constant velocity under a constant external load for many seconds. The steady-state force–velocity relation of Dictyostelium myosin sliding on actin cables was hyperbolic in shape except for large loads ≤0.7–0.8 P₀, being qualitatively similar to that of skeletal muscle fibres, despite a considerable variation in the number of myosin molecules interacting with actin cables. Comparison of the P–V curves between Dictyostelium myosin and muscle myosins sliding on actin cables suggests that the time of attachment to actin in a single attachment–detachment cycle is much longer in Dictyostelium myosin than in muscle myosins.     

Actin dynamics in Phytophthora infestans; rapidly reorganizing cables and immobile,long‐lived plaques

The actin cytoskeleton is a dynamic but well‐organized intracellular framework that is essential for proper functioning of eukaryotic cells. Here, we use the actin binding peptide Lifeact to investigate the in vivo actin cytoskeleton dynamics in the oomycete plant pathogen Phytophthora infestans. Lifeact–eGFP labelled thick and thin actin bundles and actin filament plaques allowing visualization of actin dynamics. All actin structures in the hyphae were cortically localized. In growing hyphae actin filament cables were axially oriented in the sub‐apical region whereas in the extreme apex in growing hyphae, waves of fine F‐actin polymerization were observed. Upon growth termination, actin filament plaques appeared in the hyphal tip. The distance between a hyphal tip and the first actin filament plaque correlated strongly with hyphal growth velocity. The actin filament plaques were nearly immobile with average lifetimes exceeding 1 h, relatively long when compared to the lifetime of actin patches known in other eukaryotes. Plaque assembly required ～30 s while disassembly was accomplished in ～10 s. Remarkably, plaque disassembly was not accompanied with internalization and the formation of endocytic vesicles. These findings suggest that the functions of actin plaques in oomycetes differ from those of actin patches present in other organisms.     

CDK phosphorylates the polarisome scaffold Spa2 to maintain its localization at the site of cell growth

Polarisome is a protein complex that plays an important role in polarized growth in fungi by assembling actin cables towards the site of cell growth. For proper morphogenesis, the polarisome must localize to the right place at the right time. However, the mechanisms that control polarisome localization remain poorly understood. In this study, using the polymorphic fungus Candida albicans as a model, we have discovered that the cyclin‐dependent kinase (CDK) Cdc28 phosphorylates the polarisome scaffold protein Spa2 to govern polarisome localization during both yeast and hyphal growth. In a yeast cell cycle, Cdc28‐Clb2 phosphorylates Spa2 and controls the timing of polarisome translocation from the bud tip to the bud neck. And during hyphal development, Cdc28‐Clb2 and the hyphal‐specific Cdc28‐Hgc1 cooperate to enhance Spa2 phosphorylation to maintain the polarisome at the hyphal tip. Blocking the CDK phosphorylation causes premature tip‐to‐neck translocation of Spa2 during yeast growth and inappropriate septal localization of Spa2 in hyphae and abnormal hyphal morphology under certain inducing conditions. Together, our results generate new insights into the mechanisms by which fungi regulate polarisome localization in the control of polarized growth.     

On‐site secretory vesicle delivery drives filamentous growth in the fungal pathogen Candida albicans

Candida albicans is an opportunistic fungal pathogen that colonises the skin as well as genital and intestinal mucosa of most healthy individuals. The ability of C. albicans to switch between different morphological states, for example, from an ellipsoid yeast form to a highly polarised, hyphal form, contributes to its success as a pathogen. In highly polarised tip‐growing cells such as neurons, pollen tubes, and filamentous fungi, delivery of membrane and cargo to the filament apex is achieved by long‐range delivery of secretory vesicles tethered to motors moving along cytoskeletal cables that extend towards the growing tip. To investigate whether such a mechanism is also critical for C. albicans filamentous growth, we studied the dynamics and organisation of the C. albicans secretory pathway using live cell imaging and three‐dimensional electron microscopy. We demonstrate that the secretory pathway is organised in distinct domains, including endoplasmic reticulum membrane sheets that extend along the length of the hyphal filament, a sub‐apical zone exhibiting distinct membrane structures and dynamics and a Spitzenkörper comprised of uniformly sized secretory vesicles. Our results indicate that the organisation of the secretory pathway in C. albicans likely facilitates short‐range “on‐site” secretory vesicle delivery, in contrast to filamentous fungi and many highly polarised cells.     

Induction of defense responses in tomato against Pseudomonas syringae pv. tomato by regulating the stress ethylene level with Methylobacterium oryzae CBMB20 containing 1-aminocyclopropane-1-carboxylate deaminase

This study aimed to examine the induction of defense responses in tomato elicited by Methylobacterium oryzae CBMB20 as a consequence of reduced stress ethylene level possibly through its ACC deaminase activity. Significantly increased activities of pathogenesis-related (PR) proteins and defense enzymes such as β-1,3-glucanase, phenylalanine ammonia-lyase, peroxidase and polyphenol oxidase were noted in M. oryzae CBMB20 pretreated and challenged with Pseudomonas syringae pv. tomato (Pst) compared to either control or M. oryzae-treated tomato plants in both growth chamber and greenhouse conditions. Increased PR proteins and defense enzyme activities were correlated with the reduction of stress ethylene level. M. oryzae CBMB20 reduced the stress ethylene level about 27% and 55% when challenged with Pst, in growth chamber and greenhouse on day 7 respectively and the effect was comparable to that of the chemical ethylene biosynthesis inhibitor AVG, L-α-(2-aminoethoxyvinyl)-glycine hydrochloride. As a consequence of reduced stress ethylene level and its effect on defense response in crop plants, the disease severity was reduced 26% in M. oryzae CBMB20-treated plants challenged with pathogen. Therefore, inoculation of M. oryzae CBMB20 would induce the defense enzymes and contribute to the enhanced resistance of tomato plants against the pathogen Pst.     

Actin cable distribution and dynamics arising from cross-linking,motor pulling,and filament turnover

The growth of fission yeast relies on the polymerization of actin filaments nucleated by formin For3p, which localizes at tip cortical sites. These actin filaments bundle to form actin cables that span the cell and guide the movement of vesicles toward the cell tips. A big challenge is to develop a quantitative understanding of these cellular actin structures. We used computer simulations to study the spatial and dynamical properties of actin cables. We simulated individual actin filaments as semiflexible polymers in three dimensions composed of beads connected with springs. Polymerization out of For3p cortical sites, bundling by cross-linkers, pulling by type V myosin, and severing by cofilin are simulated as growth, cross-linking, pulling, and turnover of the semiflexible polymers. With the foregoing mechanisms, the model generates actin cable structures and dynamics similar to those observed in live-cell experiments. Our simulations reproduce the particular actin cable structures in myoVΔ cells and predict the effect of increased myosin V pulling. Increasing cross-linking parameters generates thicker actin cables. It also leads to antiparallel and parallel phases with straight or curved cables, consistent with observations of cells overexpressing α-actinin. Finally, the model predicts that clustering of formins at cell tips promotes actin cable formation.     

Endocytic protein Pal1 regulates appressorium formation and is required for full virulence of Magnaporthe oryzae

Endocytosis plays key roles during infection of plant-pathogenic fungi, but its regulatory mechanisms are still largely unknown. Here, we identified a putative endocytosis-related gene, PAL1, which was highly expressed in appressorium of Magnaporthe oryzae, and was found to be important for appressorium formation and maturation. Deletion of PAL1 significantly reduced the virulence of M. oryzae due to defects in appressorial penetration and invasive growth in host cells. The Pal1 protein interacted and colocalized with the endocytosis protein Sla1, suggesting it is involved in endocytosis. The Δpal1 mutant was significantly reduced in appressorium formation, which was recovered by adding exogenous cAMP and 3-isobutyl-1-methylxanthine (IBMX). Moreover, the phosphorylation level of Pmk1 in Δpal1 was also reduced, suggesting Pal1 functions upstream of both the cAMP and Pmk1 signalling pathways. As a consequence, the utilization of glycogen and lipid, appressorial autophagy, actin ring formation, localization of septin proteins, as well as turgor accumulation were all affected in the Δpal1 mutant. Taken together, Pal1 regulates cAMP and the Pmk1 signalling pathway for appressorium formation and maturation to facilitate infection of M. oryzae.     

2,6‐Dimethoxy‐1,4‐Benzoquinone Enhances Resistance Against the Rice Blast Fungus Magnaporthe oryzae

We investigated the effect of 2,6‐dimethoxy‐1,4‐benzoquinone (DMBQ) on induced resistance to Magnaporthe oryzae in rice. DMBQ concentrations greater than 50 μg/ml inhibited spore germination and appressorium formation in M. oryzae. When rice leaves pretreated with 10 μg/ml DMBQ, which did not show antifungal activity against spore germination and appressorium formation of M. oryzae, were inoculated with M. oryzae spores 5 days after DMBQ pretreatment, blast lesion formation was inhibited compared with control leaves pretreated with distilled water. In addition, infection‐inhibiting activity against M. oryzae was significantly enhanced in rice leaf sheaths pretreated with 10 μg/ml DMBQ. H₂O₂ generation was observed in rice leaves pretreated with DMBQ, and PAL, POX, CHS and PR10a were significantly expressed in these leaves. These results suggested that DMBQ can protect rice from blast disease caused by M. oryzae.     

Septum-directed secretion in the filamentous fungus Aspergillus oryzae

Although exocytosis in fungal cells takes place at hyphal tips, there also seems a line of circumstantial evidence suggesting the occurrence of exocytosis at other sites of cells, such as septa. To investigate whether exocytosis takes place at fungal septa, we monitored dynamics of EGFP‐fused α‐amylase (AmyB–EGFP), the representative secretory enzyme of the filamentous fungus Aspergillus oryzae. We found that AmyB–EGFP accumulates in Spitzenkörper at hyphal tips as well as septal periplasm between the plasma membrane and cell walls. The septal accumulation of AmyB–EGFP was a rapid process, and required microtubules but not F‐actin. Thus, this process is independent of exocytosis at hyphal tips that requires both microtubules and F‐actin. In addition, fluorescence recovery after photobleaching (FRAP) analysis of EGFP‐fused AoSnc1 revealed that secretory vesicles constitutively fuse with the septal plasma membrane. These results demonstrated that exocytosis takes place at septa in addition to hyphal tips. Analysis of two plasma membrane transporters, AoUapC and AoGap1, revealed that they preferentially accumulate at septa and the lateral plasma membrane with no clear accumulation at apical Spitzenkörper, suggesting that non‐tip directed exocytosis is important for delivery of these proteins.