Penetration Peg Research Articles

Ultrastructural analysis of responses of host and fungal cells during plant infection

Cellular responses in fungi and in susceptible or resistant hosts during fungus–plant interactions have been studied ultrastructurally to examine their role in pathogenicity. Pathogenicity is determined in some saprophytic fungi by various factors: the production of disease determinants such as the production of host-specific toxins (HSTs) or the extracellular matrix (ECM) by fungal infection structures and H2O2 generation from penetration pegs. Three different target sites for HSTs have been identified in host cells in many ultrastructural studies: plasma membranes, chloroplasts, and mitochondria. The mode of action of HSTs is characterized by the partial destruction of the target structures only in susceptible genotypes of host plants, with the result that the fungus can colonize the host. The infection structures of most fungal pathogen secrete ECM on plant surfaces during fungal differentiation, while the penetration pegs of some pathogens produce reactive oxygen species (ROS) in the cell walls and plasma membranes. The pathological roles of ECM and H2O2 generation are discussed here in light of ultrastructural evidence. Host and fungal characteristics in the incompatible interactions include the rapid formation of lignin in host epidermal cell walls, failure of penetration pegs to invade lignin-fortified pectin layers, the inhibition of subcuticular hyphal proliferation and the collapse of hyphae that have degraded cell walls within pectin layers of the host. Apoptosis-like host resistant mechanism is also discussed.

GcSTUA, an APSES Transcription Factor, Is Required for Generation of Appressorial Turgor Pressure and Full Pathogenicity of Glomerella cingulata

Glomerella cingulata, which infects a number of different hosts, gains entry to the plant tissue by means of an appressorium. Turgor pressure generated within the appressorium forces a penetration peg through the plant cuticle. A visible lesion forms as the fungus continues to grow within the host. A G. cingulata homolog (GcSTUA) of the genes encoding Asm1, Phd1, Sok2, Efg1, and StuA transcription factors in Magnaporthe grisea and other fungi was cloned and shown to be required for infection of intact apple fruit and penetration of onion epidermal cells. Mobilization of glycogen and triacylglycerol during formation of appressoria by the GcSTUA deletion mutant appeared normal and melanization of the maturing appressoria was also indistinguishable from that of the wild type. However, GcSTUA was essential for the generation of normal turgor pressure within the appressorium. As is the case for its homologs in other fungi, GcSTUA also was required for the formation of aerial hyphae, efficient conidiation, and the formation of perithecia (sexual reproductive structures).

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.

Attachment to and Penetration of Conventional and Silicone Hydrogel Contact Lenses by Fusarium solani and Ulocladium sp. In Vitro

To analyze the relative capacities of Fusarium solani and Ulocladium sp. to attach to and penetrate silicone hydrogel soft contact lenses. Representative silicone hydrogel (SH, siloxy complexes) and conventional [hydroxyethylmethylacrylate (HEMA)] soft contact lenses were exposed to suspensions of F. solani and Ulocladium sp. in vitro (10 conidia/mL in phosphate-buffered saline). The lenses were incubated with shaking at ambient temperatures and examined after rinsing in a multipurpose contact lens solution (MPS) by light and scanning electron microscopy. Isolates of both genera firmly attached to and penetrated both lens types, but Ulocladium sp. did so in greater density and more rapidly than F. solani. The extent of firm attachment and time needed for penetration into the lenses varied with strain and substratum, particularly with the isolates of F. solani. Morphologic characteristics (eg, penetration pegs, microcycle conidiation, and chlamydospores) of F. solani in the SH and HEMA lenses were similar to those observed in several lenses from patients with ReNu with MoistureLoc (RML)-associated Fusarium keratitis. To our knowledge, this is the first report that F. solani produces coiled penetration pegs in the matrices of SH hydrophilic soft contact lenses similar in morphology to those found in HEMA lenses. F. solani attaches firmly to SH lenses and rarely penetrates the lens matrix, but viable fungal propagules may remain on the lens after vigorous rinsing with MPS. Failure to use a manual cleaning-disinfection procedure may help to explain the increased incidence of Fusarium keratitis associated with contact lens wear.

Ultrastructural analysis of cell responses of host cells to pathogen infection

When plants come into contact with fungal pathogens and their disease chemical stressors such as toxins and elicitors, they exhibit a variety of complex cell responses against the disease stress. Some plants induce a series of resistant reactions to the stress, eventually resulting in survival by overcoming it, while some submit to fungal infection because of failure to establish defense reactions. Electron microscopy is one of a number of powerful techniques that can analyze cell functions with pathological significance by observing the responses of the stressed plant cells. This article describes the results of ultrastructural analysis on both the mode of action of Alternaria and Stemphylium host-specific toxins (HSTs) produced from some saprophytic pathogens in respective host cells to evaluate the pathological roles of HSTs. In addition, the aggressive roles of pathogen penetration pegs, lignin-induced resistant responses, and the apoptosis-like cell death mechanism by pathogen infection are also reported. Ultrastructural studies of plant diseases had been performed vigorously and extensively in the 1970s and 1980s in Japan, but they declined in number and in the 1990s they came to a standstill. This tendency is common in every field of natural science. Unfortunately, experts in electron microscopy with diverse and skillful techniques are now less common due to their gradual retirement from employment. The cytological posts have been insured by genome workers. The decline of electron microscopy is attributed to the difficulty of electron microscopy techniques and the rise of molecular biology. Molecular biology is excellent in analyzing the gene functions of pathogen-stressed host cells or differentiated pathogens during pathogenesis and the tech-

The anatomy of Santalum album (Sandalwood) haustoria

Structural attributes of Santalum album L. (Sandalwood) haustoria have been long overlooked in the literature. This is surprising since successful haustorial formation is key to the survival of individuals of this ecologically and economically important plant. We investigated the morphology of haustoria formed by S. album attached to one of its principal hosts Tithonia diversifolia (Hemsley) A. Gray. The bell-shaped mature haustoria were composed of a peripheral hyaline body and a centrally located penetration peg. The parasite penetration peg can penetrate the host by means of direct pressure and the secretion of cell-wall-degrading enzymes when forming a successful graft union. The latter mechanism is supported by this study as we observed no evidence of collapsed host cells as the result of parasite applied pressure. Upon reaching the xylem tissue of the host root, the penetration peg formed a thin ellipsoidal disc and the host–parasite interface was almost entirely composed of parenchymatous tissue. Luminal continuities were absent between the xylem conducting tissues of the partners, thus suggesting mass flow of solutes is unlikely to occur in this association. High densities of contact parenchyma were found at the host–parasite interface; thus it is probable that these are the principal structures formed by the parasite that facilitate the acquisition of host-derived xylem resources. This study therefore concludes that haustorial anatomy of S. album supports cross membrane (potentially selective) uptake of host-derived solutes as opposed to mass flow via vascular continuity.

Host invasion during rice‐blast disease requires carnitine‐dependent transport of peroxisomal acetyl‐CoA

In lower eukaryotes, beta-oxidation of fatty acids is restricted primarily to the peroxisomes and the resultant acetyl-CoA molecules (and the chain-shortened fatty acids) are transported via the cytosol into the mitochondria for further breakdown and usage. Using a loss-of-function mutation in the Magnaporthe grisea PEROXIN6 orthologue, we define an essential role for peroxisomal acetyl-CoA during the host invasion step of the rice-blast disease. We show that an Mgpex6Delta strain lacks functional peroxisomes and is incapable of beta-oxidation of long-chain fatty acids. The Mgpex6Delta mutant lacked appressorial melanin and host penetration, and was completely non-pathogenic. We further show that a peroxisome-associated carnitine acetyl-transferase (Crat1) activity is essential for such appressorial function in Magnaporthe. CRAT1-minus appressoria showed reduced melanization, but were surprisingly incapable of elaborating penetration pegs or infection hyphae. Exogenous addition of excess glucose during infection stage caused partial remediation of the pathogenicity defects in the crat1Delta strain. Moreover, Mgpex6Delta and crat1Delta mycelia showed increased sensitivity to Calcofluor white, suggesting that weakened cell wall biosynthesis in a glucose-deficient environment leads to appressorial dysfunction in these mutants. Interestingly, CRAT1 was itself essential for growth on acetate and long-chain fatty acids. Thus, carnitine-dependent metabolic activities associated with the peroxisomes, cooperatively facilitate the appressorial function of host invasion during rice-blast infections.

Fungal Pls1 tetraspanins as key factors of penetration into host plants: a role in re-establishing polarized growth in the appressorium?

The ability of plant pathogenic fungi to infect their host depends on successful penetration into plant tissues. This process often involves the differentiation of a specialized cell, the appressorium. Signalling pathways required for appressorium formation are conserved among fungi. However, the functions involved in appressorium maturation and penetration peg formation are still poorly understood. Recent studies have shown that Pls1 tetraspanins control an appressorial function required for penetration into host plants and are likely conserved among plant pathogenic fungi. Tetraspanins are small membrane proteins widely distributed among ascomycetes and basidiomycetes defining two distinct families; Pls1 tetraspanins are found in both ascomycetes and basidiomycetes and Tsp2 tetraspanins are specific to basidiomycetes. Both fungal tetraspanins families have similar secondary structures shared with animal tetraspanins. Pls1 tetraspanins are present as single genes in genomes of ascomycetes, allowing a unique opportunity to study their function in appressorium mediated penetration. Experimental evidence suggests that Pls1 tetraspanins are required for the formation of the penetration peg at the base of the appressorium, probably through re-establishing cell polarity.

植物の病原菌感染応答に関する電子顕微鏡学的解析

When plants come into contact with fungal pathogens and their disease chemical stressors such as toxins and elicitors, they exhibit a variety of complex cell responses against the disease stress. Some plants induce a series of resistant reactions to the stress, eventually resulting in survival by overcoming it, while some submit to fungal infection because of failure to establish defense reactions. Electron microscopy is one of a number of powerful techniques that can analyze cell functions with pathological significance by observing the responses of the stressed plant cells. This article describes the results of ultrastructural analysis on both the mode of action of Alternaria and Stemphylium host-specific toxins (HSTs) produced from some saprophytic pathogens in respective host cells to evaluate the pathological roles of HSTs. In addition, the aggressive roles of pathogen penetration pegs, lignin-induced resistant responses, and the apoptosis-like cell death mechanism by pathogen infection are also reported. Ultrastructural studies of plant diseases had been performed vigorously and extensively in the 1970s and 1980s in Japan, but they declined in number and in the 1990s they came to a standstill. This tendency is common in every field of natural science. Unfortunately, experts in electron microscopy with diverse and skillful techniques are now less common due to their gradual retirement from employment. The cytological posts have been insured by genome workers. The decline of electron microscopy is attributed to the difficulty of electron microscopy techniques and the rise of molecular biology. Molecular biology is excellent in analyzing the gene functions of pathogen-stressed host cells or differentiated pathogens during pathogenesis and the tech-

Arabidopsis thaliana subcellular responses to compatible Erysiphe cichoracearum infections

Subcellular events of Erysiphe cichoracearum infections of epidermal cells were visualized in living tissues of Arabidopsis plants carrying various green fluorescent protein (GFP)-tagged organelles via laser scanning confocal microscopy. Early in the infection sequence, cytoplasm and organelles moved towards penetration sites and accumulated near penetration pegs. Peroxisomes appeared to accumulate preferentially relative to the cytoplasm at penetration sites. Another early event, which preceded haustorium formation, was the aggregation of some GFP-tagged plasma membrane marker proteins into rings around penetration sites, which extended across cell-wall boundaries into neighboring cells. This feature localized to sites where papillae were deposited. The extrahaustorial membrane (EHM) encases the fungal feeding structure, the haustorium, separating it from the host cytoplasm. Eight plasma membrane markers were excluded from the EHM and remained in a collar-like formation around the haustorial neck. These observations support the suggestions that the EHM is a unique, specialized membrane and is different from the plasma membrane. Our results suggested two possibilities for the origin of the EHM: invagination of the plasma membrane coupled with membrane differentiation; or de novo synthesis of the EHM by targeted vesicle trafficking.

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.

Ultrastructure of the early stages of Colletotrichum acutatum infection of strawberry tissues

The early stages of the infection of attached leaves and petioles of strawberry (Fragaria ×ananassa Duch. 'Camarosa') by Colletotrichum acutatum Simmonds were studied using scanning and transmission electron microscopy. Pre-penetration events of these tissues were similar, but the production of secondary conidia (microcyclic conidiation) was detected only on leaves. At the ultrastructural level, different stages of maturation of appressoria were observed and described. In young appressoria, the cell wall was composed of two layers and the plasma membrane displayed a wavy appearance. In the following stage, the appressorium developed a third electron-transparent layer between the cell wall and the plasma membrane. This new electron-transparent material was especially visible in the region of the appressorium near the cuticle. The plasma membrane of this appressorium showed a smooth appearance. Afterwards, a penetration peg emerged through the pore penetrating the cuticle and reached the epidermal wall where it enlarged to form an intramural infection vesicle. Both structures of infection, the penetration peg and the intramural infection vesicle, produced during the early phases of infection of strawbery tissues by C. acutatum, have not been previously reported and confirm that its invasion strategy is that of a subcuticular intramural pathogen. Once the infection was well established, abundant subcuticular and intramural hyphae were produced on petioles, causing severe degradation of the host cell walls. Occasionally, the cuticle appeared disrupted in those regions where the host walls were very degraded and dilated. Differences between colonization of petioles and leaves were observed.Key words: Colletotrichum acutatum, Fragaria ×ananassa, infection vesicle, invasion strategy, penetration peg.

The tetraspanin gene ClPLS1 is essential for appressorium-mediated penetration of the fungal pathogen Colletotrichum lindemuthianum

Conservation of the molecular mechanisms controlling appressorium-mediated penetration during evolution was assessed through a functional study of the ClPLS1 gene from Colletotrichum lindemuthianum orthologous to the MgPLS1 from Magnaporthe grisea, involved in penetration peg development. These two plant-pathogenic Pyrenomycetes differentiate appressoria to penetrate into plant tissues. We showed that ClPLS1 is a functional homologue of MgPLS1 in M. grisea. Loss of ClPLS1 function had no effect on vegetative growth, conidiation or on appressorium differentiation and maturation. However, C lpls1::hph mutants are non-pathogenic on either intact or wounded bean leaves, as a result of a defect in the formation and/or positioning of the penetration pore and consequently in the formation of the penetration peg. These observations suggest that the fungal tetraspanins control a conserved appressorial function that could be required for the correct localization of the site where the penetration peg emerges.

The PEN1 Syntaxin Defines a Novel Cellular Compartment upon Fungal Attack and Is Required for the Timely Assembly of Papillae

Attack by the host powdery mildew Erysiphe cichoracearum usually results in successful penetration and rapid proliferation of the fungus on Arabidopsis. By contrast, the nonhost barley powdery mildew Blumeria graminis f. sp. hordei (Bgh) typically fails to penetrate Arabidopsis epidermal cells. In both instances the plant secretes cell wall appositions or papillae beneath the penetration peg of the fungus. Genetic screens for mutations that result in increased penetration of Bgh on Arabidopsis have recently identified the PEN1 syntaxin. Here we examine the role of PEN1 and of its closest homologue, SYP122, identified as a syntaxin whose expression is responsive to infection. pen1 syp122 double mutants are both dwarfed and necrotic, suggesting that the two syntaxins have overlapping functions. Although syp122-1 and the cell wall mur mutants have considerably more pronounced primary cell wall defects than pen1 mutants, these have relatively subtle or no effects on penetration resistance. Upon fungal attack, PEN1 appears to be actively recruited to papillae, and there is a 2-h delay in papillae formation in the pen1-1 mutant. We conclude that SYP122 may have a general function in secretion, including a role in cell wall deposition. By contrast, PEN1 appears to have a basal function in secretion and a specialized defense-related function, being required for the polarized secretion events that give rise to papilla formation.

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.

CDNAs generated from individual epidermal cells reveal that differential gene expression predicting subsequent resistance or susceptibility to rust fungal infection occurs prior to the fungus entering the cell lumen.

As the cowpea rust fungus penetrates the wall of a cowpea epidermal cell, resistant and susceptible plants exhibit different ultrastructural and cytochemical changes within the epidermal protoplast. To examine plant gene expression at this stage of infection, cytoplasm was extracted from individual inoculated or uninoculated epidermal cells before the fungal penetration peg reached the cell lumen. Initial differential colony hybridization screening of an expressed sequence tag library constructed from globally amplified cDNAs generated from the inoculated resistant cells resulted in 80 clones (out of 835) with a differential hybridization pattern. Further slot-blot screening and screening of the amplified cDNAs generated from inoculated or uninoculated, resistant or susceptible cells revealed 28 separate genes, mostly with no matching sequences in the databases, that were up-regulated in response to the growth of the fungus through the wall of resistant or susceptible cells. Five genes, including those coding for beta- and alpha-tubulin, were found to be down-regulated specifically in inoculated, susceptible cells, and five were specifically up-regulated in inoculated, resistant cells, including a PR-10 homolog and a phenylalanine ammonia-lyase gene. Probing the amplified cDNAs from each cell type for the expression of cell death-related genes revealed that an LLS1 homolog (vuLLS1), cloned from cowpea, was up-regulated by infection in both resistant and susceptible cells and that a homolog of HSR203J was differentially up-regulated in resistant cells. These data show that changes in gene expression predicting the subsequent expression of susceptibility or hypersensitive resistance to fungal infection occur prior to the fungus entering the cell lumen.

Confocal microscopy of Spitzenkörper dynamics during growth and differentiation of rust fungi.

The membrane-selective fluorescent dye FM4-64, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridium dibromide, was used to stain the apical vesicle cluster within the specialized Spitzenkörper of the germ tube of the rust fungi Uromyces vignae and Puccinia graminis f. sp. tritici grown on glass surfaces. The Spitzenkörper stained within 15 min following addition of the dye. Optical sectioning by confocal microscopy of stained hyphal tips showed that the Spitzenkörper was asymmetrically positioned close to the cell-substratum interface during germ tube growth. The Spitzenkörper showed variations in shape and positioning over short (5 s) time intervals. The movement to a new location in the hyphal dome was followed by new growth in that region, consistent with the view that the Spitzenkörper supplies secretory vesicles for germ tube growth. A pronounced Spitzenkörper disappeared at the onset of appressorium differentiation during swelling of the germ tube. However, a stained structure, similar in appearance to a Spitzenkörper, was again observed during the formation of the highly polarized penetration peg.

Digital Image Analysis of Internal Light Spots of Appressoria ofColletotrichum acutatum

ABSTRACT The initial penetration process of appressoria of Colletotrichum acutatum on almond leaves was studied using digital image analysis of light micrographs and scanning electron microscopy. For image analysis, a series of sequential, partially focused digital micrographs of appressoria was analyzed to generate a single, completely focused montage image with a continuous in-focus depth of field. In studies on the development of the internal light spot (ILS), we observed that 50.4% of the appressoria formed an ILS after leaves were inoculated and incubated for 12 h at 20 degrees C, and that this increased to 95.8% after 24 h. Comparative image analyses of appressoria with and without ILSs using depth relief mapping and line profile software options showed that the ILS had a depth relief that was below that of the leaf surface. Depth relief analysis in the ILS region during incubation revealed an increase in depth in this area of up to 1.8 mum in some of the appressoria. A comparative morphological study of the ILS in montage images and the penetration pore of appressoria in scanning electron micrographs showed similar shapes and dimensions of the two structures in the appressorium. Light micrographs of histological sections confirmed fungal penetration and internal vesicle formation in almond leaves within 36 h after inoculation and incubation at 20 degrees C. This study represents the first direct evidence that the ILS in appressoria corresponds to the penetration pore and the developing penetration peg using a rapid, digital image analysis technique.

Infection behavior of Alternaria alternata Japanese pear pathotype and localization of 1,3-β- d -glucan in compatible and incompatible interactions between the pathogen and host plants

The infection behavior of Alternaria alternata Japanese pear pathotype and host responses were examined cytologically and ultrastructurally in compatible and incompatible interactions between Japanese pear plants and the pathogen during the infection process. No differences in spore germination, appressorial formation, or cuticle penetration were detected between susceptible and resistant leaves inoculated with spores of the pathogen. On the other hand, infection hyphae emerged from penetration pegs and invaded the pectin layers of susceptible leaves but not those of resistant leaves. The results indicate that expression of resistance to the pathogen may be induced before the formation of infection hyphae in pear tissues. Plasma membrane modifications were observed ultrastructurally 3 h after inoculation only in susceptible leaf cells. Papillae were usually formed in epidermal cells underneath the appressoria in resistant leaves at the stage when the progress of the pegs was stopped 9 h after inoculation. Callose deposition was observed fluorescence-microscopically as papillae at the infection sites in abaxial epidermis of both cultivars. The immunoelectron microscopic results demonstrated that the components of the papillae and the extracellular polysaccharides that had accumulated in plasma membrane modification sites were compatible with 1,3-β-d-glucan. These results suggest that the glucan deposition results from host cuticle damage by fungal invasion and from plasma membrane damage by AK-toxin.

Microscopic detection of reactive oxygen species generation in the compatible and incompatible interactions of Alternaria alternata Japanese pear pathotype and host plants

Reactive oxygen species (ROS) generation was examined in the interaction of Alternaria alternata Japanese pear pathotype and host plants using three methods: nitro blue tetrazolium (NBT) method for microscopic detection of O2 −, diaminobenzidine (DAB) methods for microscopic detection of H2O2, and cerium chloride methods for ultrastructural detection of H2O2. ROS generation was detected by NBT and DAB methods at appressoria on leaves of susceptible cultivars and heat-shocked leaves of resistant cultivars but not in leaves of resistant cultivars. Ultrastructural detection by the cerium chloride method identified ROS generation at cell walls of appressoria and penetration pegs in susceptible, resistant leaves and heat-shocked leaves. These differences in the ultrastructural and microscopic data in resistant areas were due to the restriction of ROS generation in limited areas, the side facing the plant surface, of appressoria and penetration pegs. Therefore, ROS generation was apparently induced regardless of the resistance or susceptibility of the cultivar with the difference being in the volumes generated. After evaluating the pathological role of ROS generation in fungal structures, such generation was found to be associated with early penetration of cell walls in pear plants. Additionally, ROS generation in plants was also found in degrading pectin layers near infected hyphae and in plasma membrane modification sites in susceptible leaves but not in resistant leaves. ROS generation in susceptible leaves might be accompanied with plasma membrane damage, although the role of ROS generation in the pectin layers is not clear. ROS generation in both fungal and plant cells during their interaction was likely associated with the expression of susceptibility.