Gavin E. Wakley

MPG1 Encodes a Fungal Hydrophobin Involved in Surface Interactions during Infection-Related Development of Magnaporthe grisea.

The rice blast fungus expresses a pathogenicity gene, MPG1, during appressorium formation, disease symptom development, and conidiation. The MPG1 gene sequence predicts a small protein belonging to a family of fungal proteins designated hydrophobins. Using random ascospore analysis and genetic complementation, we showed that MPG1 is necessary for infection-related development of Magnaporthe grisea on rice leaves and for full pathogenicity toward susceptible rice cultivars. The protein product of MPG1 appears to interact with hydrophobic surfaces, where it may act as a developmental sensor for appressorium formation. Ultrastructural studies revealed that MPG1 directs formation of a rodlet layer on conidia composed of interwoven ~5-nm rodlets, which contributes to their surface hydrophobicity. Using combined genetic and biochemical approaches, we identified a 15-kD secreted protein with characteristics that establish it as a class I hydrophobin. The protein is able to form detergent-insoluble high molecular mass complexes, is soluble in trifluoroacetic acid, and exhibits mobility shifts after treatment with performic acid. The production of this protein is directed by MPG1.

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

SummaryHistochemical and ultrastructural studies were carried out on a wild-type strain (Guyll) and a melanin-deficient mutant(büβ) 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 phospho-monoesterase, 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 ofM. grisea were able to penetrate before degradation of lipid droplets had been completed.

Complementation of the mpg1 mutant phenotype in Magnaporthe grisea reveals functional relationships between fungal hydrophobins.

The functional relationship between fungal hydrophobins was studied by complementation analysis of an mpg1− gene disruption mutant in Magnaporthe grisea. MPG1 encodes a hydrophobin required for full pathogenicity of the fungus, efficient elaboration of its infection structures and conidial rodlet protein production. Seven heterologous hydrophobin genes were selected which play distinct roles in conidiogenesis, fruit body development, aerial hyphae formation and infection structure elaboration in diverse fungal species. Each hydrophobin was introduced into an mpg1− mutant by transformation. Only one hydrophobin gene, SC1 from Schizophyllum commune, was able partially to complement mpg1− mutant phenotypes when regulated by its own promoter. In contrast, six of the transformants expressing hydrophobin genes controlled by the MPG1 promoter (SC1 and SC4 from S.commune, rodA and dewA from Aspergillus nidulans, EAS from Neurospora crassa and ssgA from Metarhizium anisopliae) could partially complement each of the diverse functions of MPG1. Complementation was always associated with partial restoration of a rodlet protein layer, characteristic of the particular hydrophobin being expressed, and with hydrophobin surface assembly during infection structure formation. This provides the first genetic evidence that diverse hydrophobin‐encoding genes encode functionally related proteins and suggests that, although very diverse in amino acid sequence, the hydrophobins constitute a closely related group of morphogenetic proteins.

Peroxisomal carnitine acetyl transferase is required for elaboration of penetration hyphae during plant infection by Magnaporthe grisea.

Magnaporthe grisea, the causal agent of rice blast disease, invades plant tissue due to the action of specialized infection structures called appressoria, which are used to breach the leaf cuticle and allow development of intracellular, infectious hyphae. In this report we demonstrate that peroxisomal carnitine acetyl transferase (CAT) activity is necessary for appressorium function, and in particular, for the elaboration of primary penetration hyphae. The major CAT activity in M. grisea is encoded by the PTH2 gene, which shows elevated expression in response to acetate and lipid, and is regulated by the cyclic AMP response pathway. Furthermore, a Pth2–GFP fusion protein colocalizes with a peroxisomal marker protein. Targeted deletion of PTH2, generated mutants that were completely non‐pathogenic, lacked CAT activity and were unable to utilize a range of lipid substrates. The impairment of appressorium function in Δpth2 was associated with a delay in lipid reserve mobilization from germ tubes into developing infection cells, and abnormal chitin distribution in infection structures. Addition of glucose to Δpth2 mutants partially restored the ability to cause rice blast disease and lipid reserve mobilization. Taken together, our findings provide evidence that Pth2 plays a role in the generation of acetyl CoA pools necessary for appressorium function and rapid elaboration of penetration hyphae during host infection.

A P-type ATPase required for rice blast disease and induction of host resistance

To cause diseases in plants, pathogenic microorganisms have evolved mechanisms to deliver proteins directly into plant cells, where they suppress plant defences and facilitate tissue invasion. How plant pathogenic fungi, which cause many of the worlds most serious plant diseases, deliver proteins during plant infection is currently unknown. Here we report the characterization of a P-type ATPase-encoding gene, MgAPT2, in the economically important rice blast pathogen Magnaporthe grisea, which is required for exocytosis during plant infection. Targeted gene replacement showed that MgAPT2 is required for both foliar and root infection by the fungus, and for the rapid induction of host defence responses in an incompatible reaction. ΔMgapt2 mutants are impaired in the secretion of a range of extracellular enzymes and accumulate abnormal Golgi-like cisternae. However, the loss of MgAPT2 does not significantly affect hyphal growth or sporulation, indicating that the establishment of rice blast disease involves the use of MgApt2-dependent exocytotic processes that operate during plant infection.

Production of a monoclonal antibody specific to the genus Trichoderma and closely related fungi, and its use to detect Trichoderma spp. in naturally infested composts.

Studies of the interactions between hyperparasitic fungi and their hosts are severely hampered by the absence of methods that allow the unambiguous identification of individual genera in complex environments that contain mixed populations of fungi, such as soil or compost. This study details the development of a monoclonal antibody (MF2) that allows the detection and recovery of Trichoderma spp. in naturally infested composts, and the visualization of hyperparasitic strains of Trichoderma during antagonistic interactions with their hosts. Murine monoclonal antibody MF2, of immunoglobulin class M (IgM), was raised against a protein epitope of a glycoprotein antigen(s) specific for species of the genus Trichoderma and for the closely related fungi Gliocladium viride, Hypomyces chrysospermus, Sphaerostilbella spp. and Hypocrea spp. MF2 did not react with antigens from Gliocladium catenulatum, Gliocladium roseum, Nectria ochroleuca and Clonostachys spp., nor with a range of unrelated soil- and compost-borne fungi. Extracellular production of the MF2 antigen was constitutive. Western-blotting analysis showed that MF2 bound to a ladder of proteins with apparent molecular masses in the range 35-200 kDa. Immunofluorescence studies showed that MF2 bound strongly to the cell walls of hyphae and phialides and the intercalary and terminal chlamydospores of Trichoderma spp., whereas immunogold electron microscopy revealed strong binding of MF2 to the cell walls and septa of hyphae and to the cell walls of phialoconidia. In immunofluorescence studies of dual cultures of Trichoderma and Rhizoctonia solani, only the cell walls of the hyperparasite, which coiled around the host, were stained by MF2. The specificity of MF2 enabled the development of a combined baiting-ELISA technique for the detection of Trichoderma spp. in naturally infested composts. The specificity of this technique was confirmed by phylogenetic analysis based on sequences of the ITS1-5.8S-ITS2 rRNA-encoding regions of the isolates.

Effects of dimethomorph on the morphology and ultrastructure of Phytophthora

Dimethomorph at a concentration of 1·0 μ m had profound effects on the morphology and ultrastructure of Phytophthora. The principal changes in ultrastructure were associated with extensive proliferation and aberrant deposition of cell-wall material. In actively growing regions of P. infestans, abnormal wall ingrowths (pegs) were laid down within 2 h exposure, some giving rise to false septa that occluded hyphae. The location of false septa in this species and in P. erythroseptica and P. cactorum coincided with the constrictions in hyphae that gave treated colonies a beaded morphology. Altered wall biogenesis was also manifested as thickening and in the development of more than one cell wall layer. Aberrant wall depositions contained ground and membranous cytoplasmic inclusions. Other ultrastructural changes induced by dimethomorph were the accumulation of fingerprint vacuoles, dense bodies and membrane debris (whorls). It is concluded that in the presence of dimethomorph there is loss of control of the biochemical processes involved in normal cell wall biogenesis.

Four conserved intramolecular disulphide linkages are required for secretion and cell wall localization of a hydrophobin during fungal morphogenesis

Hydrophobins are morphogenetic proteins produced by fungi during assembly of aerial hyphae, sporulation, mushroom development and pathogenesis. Eight cysteine residues are present in hydrophobins and form intramolecular disulphide bonds. Here, we show that expressing eight cysteine–alanine substitution alleles of the MPG1 hydrophobin gene from Magnaporthe grisea causes severe defects in development of aerial hyphae and spores. Immunolocalization revealed that Mpg1 hydrophobin variants, lacking intact disulphide bonds, retain the capacity to self‐assemble, but are not secreted to the cell surface. This provides the first genetic evidence that disulphide bridges in a hydrophobin are dispensable for aggregation, but essential for secretion.

Histochemical and Ultrastructural Characterization of Vacuoles and Spherosomes as Components of the Lytic System in Hyphae of the Fungus Botrytis cinerea

An integrated approach to acid phosphatase (EC 3.1.3.2) histochemistry by the azo-dye and lead-capture (‘Gomori’) methods in phosphate-starved hyphae of the fungus Botrytis cinerea revealed strikingly different patterns of localization of activity staining. Reaction product formed with the azo-dye method was found in numerous small organelles (<;0.5 µm diameter), which also accumulated the lipophilic dye Nile Red and mislocalized the formazan indicating mitochondrial succinate dehydrogenase activity. Such small organelles were stained only weakly and sporadically with the lead-capture method; instead, lead phosphate deposits were produced mainly in large vacuoles (up to 2.5 µm diam.), similar to those accumulating the vital dye Neutral Red. Additionally, acid phosphatase activity was detected in apical secretory vesicles with the lead-capture method but not with the azo-dye method. Ultrastructural studies by transmission electron microscopy confirmed the presence of large vacuoles which showed evidence of autophagic activity, and of small moderately osmiophilic organelles. The latter are considered to be spherosomes rather than lysosomes because of their weak reaction with the lead-capture method and their high lipid content. It is suggested that their apparently strong reaction with the azo-dye method is caused partly by false localization due to the lipophilic nature of the reaction product.

Septal Sealing in the Basidiomycete Coriolus versicolor

SUMMARY: The way in which the dolipore apparatus contains hyphal damage, and the process of septal sealing have been studied in Coriolus versicolor using combined light and electron microscopy. The technique used allows the structure of septa in adjacent damaged and undamaged hyphae to be compared. The results show that septal sealing, following damage, is a two stage process. The first is the instantaneous plugging of the pore channel with electron-dense material. The second, beginning several minutes later, involves the detachment of the septal apparatus present in the ruptured compartment and a re-modelling of the septal swelling on the other side of the wall to give a permanent seal. The parenthesomes play no part in the plugging response.