Dynamic reorganization of microtubules and microfilaments in flax cells during the resistance response to flax rust infection

The cytoskeleton in plant cells is a dynamic structure that can rapidly respond to extracellular stimuli. Alteration of the organization of microtubules and actin microfilaments was examined in mesophyll cells of flax, Linum usitatissimum L., during attempted infection by the flax rust fungus, Melampsora lini (Ehrenb.) Lev. Flax leaves that had been inoculated with either a compatible (yielding a susceptible reaction) or an incompatible (yielding a resistant reaction) strain of M. lini were embedded in butyl-methylmethacrylate resin; sections of this material were immunofluorescently labelled with anti-tubulin or anti-actin and examined using confocal laser scanning microscopy. In uninfected leaves, microtubules in the mesophyll cells formed a transverse array in the cell cortex. Microfilaments radiated through the cytoplasm from the nucleus. In an incompatible interaction, microtubules and microfilaments were extensively reorganized in mesophyll cells that were in contact with fungal infection hyphae or haustorial mother cells before penetration of the cell by the infection peg. After the initiation of haustorium development, microtubules disappeared from the infected cells, and growth of the haustoria ceased. In an incompatible interaction, hypersensitive cell death occurred in more than 70% of infected cells but occurred in less than 20% of cells in compatible interactions. After the infected cell had undergone hypersensitive cell death, the cytoskeleton in neighbouring cells became focused on the walls shared with the necrotic cell. In compatible interactions, reorganization of the cytoskeleton was either not observed at all or was observed much less frequently up to 48 h after inoculation.Abbreviations FITC fluorescein isothiocyanate - WGA wheatgerm agglutinin We thank Dr. G.J. Lawrence for providing valuable discussions and materials.     

Rapid induction of phenylalanine ammonia-lyase and chalcone synthase mRNAs during fungus infection of soybean (Glycine max L.) roots or elicitor treatment of soybean cell cultures at the onset of phytoalexin synthesis

The differential regulation of the activities and amounts of mRNAs for two enzymes involved in isoflavonoid phytoalexin biosynthesis in soybean was studied during the early stages after inoculation of primary roots with zoospores from either race 1 (incompatible, host resistant) or race 3 (compatible, host susceptible) of Phytophthora megasperma f.sp. glycinea, the causal fungus of root rot disease. In the incompatible interaction, cloned cDNAs were used to demonstrate that the amounts of phenylalanine ammonia-lyase and chalcone synthase mRNAs increased rapidly at the time of penetration of fungal germ tubes into epidermal cell layers (1–2 h after inoculation) concomitant with the onset of phytoalxxin accumulation; highest levels were reached after about 7 h. In the compatible interaction, only a slight early enhancement of mRNA levels was found and no further increase occurred until about 9 h after inoculation. The time course for changes in the activity of chalcone synthase mRNA also showed major differences between the incompatible and compatible interaction. The observed kinetics for the stimulation of mRNA expression related to phytoalexin synthesis in soybean roots lends further support to the hypothesis that phytoalexin production is an early defense response in the incompatible plant-fungus interaction. The kinetics for the enhancement of mRNA expression after treatment of soybean cell suspension cultures with a glucan elicitor derived from P. megasperma cell walls was similar to that measured during the early stages of the resistant response of soybean roots.Abbreviations cDNA copy DNA - CHS chalcone synthase - PAL phenylalanine ammonia-lyase     

A cytochrome P450 gene is differentially expressed in compatible and incompatible interactions between pepper (Capsicum annuum) and the anthracnose fungus, Colletotrichum gloeosporioides

The anthracnose fungus, Colletotrichum gloeosporioides, was previously shown to have an incompatible interaction with ripe-red fruit of pepper (Capsicum annuum). However, the fungus had a compatible interaction with unripe-mature-green fruit. Using mRNA differential display, we isolated and characterized a PepCYP gene expressed in the incompatible interaction. The PepCYP gene encodes a protein homologous to cytochrome P450 proteins containing a heme-binding domain. The expression level of PepCYP is higher in the incompatible interaction than in the compatible interaction, and then remains elevated in the incompatible interaction. In the compatible interaction, the expression of PepCYP is transient. The induction of PepCYP gene is up-regulated by wounding or jasmonic acid treatment during ripening. Analysis of PepCYP expression by in situ hybridization shows that the accumulation of PepCYP mRNA is localized in the epidermal cell layers, but not in the cortical cell layers. An examination of transverse sections of the fruits inoculated with the fungus shows that the fungus invades and colonizes the epidermal cell layers of the unripe fruit at 24 and 72 h after inoculation, respectively, but not those of the ripe fruit. These results suggest that the PepCYP gene product plays a role in the defense mechanism when the fungus invades and colonizes the epidermal cells of fruits in the incompatible interaction during the early fungal infection process.     

Chitinases of <Emphasis Type="Italic">Coffea arabica</Emphasis> genotypes resistant to orange rust <Emphasis Type="Italic">Hemileia vastatrix</Emphasis>

Two Coffea arabica — Hemileia vastatrix incompatible interactions (I₁: coffee cv. Caturra — rust race VI and I₂: coffee cv S4 Agaro — rust race II) and a compatible interaction (coffee cv. Caturra — rust race II) were compared in relation to the infection process and chitinase activity. In the two incompatible interactions the fungus ceased growth in the early infection stages, while in the compatible interaction no fungus growth inhibition was observed. A high constitutive level of chitinase activity was detected in the intercellular fluid of healthy leaves. Upon infection, chitinase isoforms were more abundant in incompatible interactions than in the compatible interaction. Immunodetection showed that class I chitinases are particularly relevant in the incompatible interactions and might participate in the defence response of the coffee plants.     

In situ localization of PR-1 mRNA and PR-1 protein in compatible and incompatible interactions of pepper stems withPhytophthora capsici

Summary In situ hybridization and immunogold labeling were performed to examine the temporal and spatial expression pattern of pathogenesis-related protein 1 (CABPR1) mRNA and PR-1 protein in pepper (Capsicum annuum L.) stem tissues infected by virulent and avirulent isolates ofPhytophthora capsici. CABPR1 mRNA accumulation was confirmed in the infected pepper stem tissue by Northern blot analysis and in situ hybridization. Northern blot analysis showed that the temporal expression ofCABPR1 mRNA varied greatly between compatible and incompatible interactions. An earlier expression of theCABPR1 gene, 6 h after inoculation, was observed in the incompatible interaction. In situ hybridization results revealed thatCABPR1 mRNA was expressed in the phloem areas of vascular bundles in infected pepper stem tissues, but especially strongly in the incompatible interaction. PR-1 protein was predominantly found in the intercellular spaces of pepper stem cells in the compatible and incompatible interactions 24 h after inoculation. Strikingly, the immunogold labeling was associated with fibrillar and electron-dense material localized in the intercellular space. Dense labeling of PR-1 protein was also seen at the interface of the pathogen and the host cell wall, whereas few gold particles were detected over the host cytoplasm. However, PR-1 protein was not detected over the fungal cell wall in either interaction.     

Chalara elegal-Infected Tobacco Roots: Ultrastructural Observations on in vitro Host-Fungus Relationships

Evidence, based on ultrastructural observations of stages involved in root infection oi Nicotiana tabacum cv. Xanthi n.c. in vitro by the black root rot fungus Chalara elegans, indicates that host cells from various layers react differently when challenged by the pathogenic fungus. All the host responses observed were associated with host cell wall modifications. Host reaction to fungal invasion occurring in the epidermal cells was limited to a disorganization of the cytoplasm. In the hypodermal cell layer, fibrillar cell wall outgrowths and wall thickenings were the earliest and the most obvious host reactions. In parenchymal cells, the host reacted by depositing papilla-like wall appositions directly adjacent to the infecting hyphae; with secondary infection of these cells, a densely staining material was laid down, mainly around the distal region of the infecting hyphae. In all these tissues, infection also led to disorganization of the host cytoplasm. Colonization of the endodermis did not lead to any rapid lethal modifications in either the host or the fungus, and a biotrophic-like state seemed to occur at this stage of the infection. No hyphal infection occurred in the central cylinder.     

Light and Electron Microscopy of the Compatible Interaction Between Arabidopsis and the Downy Mildew Pathogen Peronospora parasitica

In this study, we focused on compatible interactions between Peronospora parasitica isolate Emoy‐2 and wild‐type (Oy‐0) and mutant (Ws‐eds1) Arabidopsis thaliana accessions by using light and transmission electron microscopy (TEM). Light microscopy of compatible interactions revealed that conidia germinated and penetrated through the anticlinal cell walls of two epidermal cells. Rapid spreading of the hyphal growth with formation of numerous haustoria within the mesophyll cells was subsequently followed by profuse sporulation in the absence of host cell necrosis on both wild‐type and mutant accessions. TEM observations revealed that coenocytic intercellular hyphae ramified and spread intercellularly throughout the host tissue forming several haustoria in host mesophyll cells. Intracellular haustoria were lobed with the diameter of 6–7 μm. Each haustorium was connected to intercellular hyphae in the absence of apparent haustorial neck. The cytoplasm of the haustorium included the organelles characteristic of the pathogen. Callose‐like deposits were frequently observed at sites of penetration around the proximal region of the haustorial neck. Apart from a few callose ensheatments, no obvious response was observed in host cells following formation of haustoria. Most of mesophyll cells contained normal haustoria and the host cytoplasm displayed a high degree of structural integrity. Absence of host cell wall alteration and cell death in penetrated host cell of both accessions suggest that the pathogen exerts considerable control over basic cellular processes and in this respect, response to this biotroph oomycete differs considerably from responses to other pathogens such as necrotrophs.     

Components in the haustorial wall of the flax rust fungus,Melampsora lini, are labelled by three anti-calmodulin monoclonal antibodies

Summary Immunolocalisation studies, using flax leaf material infected with the flax rust fungus,Melampsora lini, and isolated haustorial complexes, have shown that three anti-calmodulin monoclonal antibodies bind to the haustorial wall of the fungus. The epitopes recognised by these antibodies are inserted into the wall during the early stages of haustorium development and remain in the wall throughout the life of the haustorium. The epitopes are present in both compatible and incompatible reactions and are oligosaccharide in nature. The results provide evidence for molecular differentiation within the haustorial complex ofM. lini.Abbreviations BMM butyl-methylmethacrylate - CaM calmodulin - FITC fluorescein isothiocyanate - MAb monoclonal antibody     

Powdery mildew (Podosphaera xanthii) resistance in melon is categorized into two types based on inhibition of the infection processes

Physiological races of powdery mildew (Podosphaera xanthii) cause different symptoms in eight melon lines. Infection by races 1, 2, and 5 was examined in different melon lines. After a compatible reaction, conidia germination, haustorium initiation from the germ tube, germ tube branching, and sporulation occurred within 12, 24, 48, and 120 h, respectively, and the conidia matured within 240 h. In contrast, type i and ii inhibition were identified through incompatible reactions. The germ tube and haustorium were initiated from conidia, but no germ tube branching occurred in the lines with type i resistance within 48-240 h. In type ii resistance, germ tube branching was observed within 120 h, but no sporulation was observed within 240 h. The number of fluorescing epidermal cells was higher within 24 h in type i, and within 48-120 h in type ii resistance lines than in susceptible lines. Callose accumulation around the haustorium was detected in type ii resistance lines within 48-120 h. This suggests that the rapid hypersensitive response (HR) within 24 h has an important role in the type i response, while HR and callose accumulation in the type ii response occur slowly between 48 and 120 h. Of the resistant lines, PMR 45 and WMR 29 showed a type i incompatible response; the PI 414723 response was entirely type ii; and PMR 5, PI 124112, and MR-1 showed different responses depending on the race. Therefore, the two types of incompatible responses were intermixed in the same germplasm.     

Ultrastructural comparison of incompatible and compatible interactions in the barley powdery mildew disease

Summary In the powdery mildew disease of barley,Erysiphe graminis f. sp.hordei forms an intimate relationship with compatible hosts, in which haustoria form in epidermal cells with no obvious detrimental effects on the host until late in the infection sequence. In incompatible interactions, by contrast, the deposition of papillae and localized host cell death have been correlated with the cessation of growth byE. g. hordei. With the advent of improved, low temperature methods of sample preparation, we felt that it was useful to reevaluate the structural details of interactions between barley andE. g. hordei by transmission electron microscopy. The haustoria that develop in susceptible barley lines appear highly metabolically active based on the occurrrence of abundant endoplasmic reticulum, Golgi-like cisternae, and vesicles. In comparison, haustoria found in the resistant barley line exhibited varying signs of degradation. A striking clearing of the matrix and loss of cristae were typical early changes in the haustorial mitochondria in incompatible interactions. The absence of distinct endoplasmic reticulum and Golgi-like cisternae, the formation of vacuoles, and the occurrence of a distended sheath were characteristic of intermediate stages of haustorial degeneration. At more advanced stages of degeneration, haustoria were dominated by large vacuoles containing membrane fragments. This process of degeneration was not observed in haustoria ofE. g. hordei developing in the susceptible barley line.Abbreviations b endoplasmic reticulum extension, blebbing - er endoplasmic reticulum - f fibrillar material - g Golgi-like structure - h haustorium - hb haustorial body - hcw haustorial cell wall - hcy haustorial cytoplasm - hf haustorial finger - hocw host cell wall - hocy host cytoplasm - 1 lipid-like droplet - m mitochondrion - mt microtubule - mve multivesicular body - n nucleus - p papilla - ph penetration site of an infection peg - pl plasma membrane - s sheath - sm extrahaustorial membrane - v vacuole - ve vesicle     

Subcellular localization of β-1,3-glucanase in Puccinia recondita f.sp. tritici-infected wheat leaves

An antiserum raised against the purified 33-kDa β-1,3-glucanase of wheat (Triticum aestivum L.) was employed to investigate the ultrastructural localization of the enzyme in wheat leaves infected with Puccinia recondita Rob. ex Desm. f.sp. tritici Eriks. and Henn. using a post-embedding immunogold labelling technique. In both compatible and incompatible interactions, β-1,3-glucanase was detected in the host plasmalemma and in the domain of the host cell wall near the plasmalemma of the mesophyll cells, but higher concentrations of the enzyme were detected in infected resistant wheat leaves than in infected susceptible ones. β-1,3-Glucanase was also found in the secondary thickening of xylem vessels and in the walls of guard cells, epidermal cells and phloem elements, while no labelling was observed in host organelles, viz. vacuoles, mitochondria, endoplasmic reticulum, Golgi bodies, nuclei and chloroplasts. A low concentration of the enzyme was detected on the intercellular hyphal wall and in the hyphal cytoplasm. In the compatible interaction, β-1,3-glucanase was demonstrated to accumulate predominantly in the haustorial wall and extrahaustorial matrix. In the incompatible interaction, strong labelling for β-1,3-glucanase was found in host cell wall appositions, in the extracellular matrix in the intercellular space, and in electron-dense structures of host origin which occurred in the incompatible interaction only. Received: 22 July 1997 / Accepted: 16 August 1997     

Epidermal derivatives as xylem elements and transfer cells: a study of the host-parasite interface in two species of Triphysaria (Scrophulariaceae)

Summary Haustoria ofTriphysaria pusilla andT. versicolor subsp.faucibarbata from a natural habitat were analysed by light and electron microscopy. The keel-shaped edge of the secondary haustorium generally splits the epidermis and cortex of the host root parallel to the root axis, and penetrates to the host vascular tissue. Anticlinally elongated epidermal cells of the haustorium constitute most of the host/parasite interface. Some of these epidermal cells are divided by oblique cell walls. Some of their oblique daughter cells as well as some undivided epidermal cells differentiate into xylem elements. Single epidermal cells occasionally intrude into the vascular tissue of the host and individual host cells can be invaded. The surface area of the plasmalemma in parasitic parenchymatous interface cells is increased by the differentiation of wall labyrinths characteristic of transfer cells and by the development of membrane-lined cytoplasmic tubules or flattened sacs which become embedded in the partly lignified interface cell-wall. Mycorrhizal fungal hyphae enter the xylem bridge in some haustoria. Implications of these observations for the function of the haustorium are discussed.     

Temporal and subcellular localization of PR-1 proteins in tomato stem tissues infected by virulent and avirulent isolates of Phytophthora capsici

Summary. Immunoblot analysis and immunogold labeling of PR-1 protein (pathogenesis-related protein 1) in tomato (Lycopersicon esculentum Mill.) were performed to examine the temporal and spatial expression patterns of PR-1 protein induced by Phytophthora capsici infection. Soluble proteins with molecular masses of 10, 17, 25, 27 and 75 kDa were induced and accumulated in P. capsici-infected stem tissues during the compatible and incompatible interactions. Western blot analysis revealed that expression of PR-1 protein (17 kDa), at 12 to 24 h after inoculation, occurred earlier in the incompatible than in the compatible interaction. Immunogold labeling of PR-1 proteins occurred over cell walls and cytoplasm of the host and the oomycete pathogen and at the interface between host and oomycete cell walls at 24 h after inoculation in the compatible interaction. In the incompatible interaction, numerous PR-1 proteins accumulated predominantly over oomycete cell walls and at the interface between host and oomycete cell walls. The quantity of PR-1 proteins deposited in both host and oomycete cells was much less in the compatible than the incompatible interaction. Healthy tomato stem tissue was nearly free of immunogold labeling of PR-1 proteins. Received October 9, 2001 Accepted January 18, 2002     

Involvement of reactive oxygen species in the response of resistant (hypersensitive) or susceptible cowpeas to the cowpea rust fungus

A previous study had indicated that scavengers of reactive oxygen species (ROS) delayed cell death (the hypersensitive response (HR)) triggered in epidermal cells of intact, resistant, cowpea ( Vigna unguiculata (L.) Walp) leaves by the monokaryotic stage of the cowpea rust fungus ( Uromyces vignae Barclay race 1). This HR had been monitored by cell autofluorescence, which occurs after protoplast collapse. In the present study, when cytoplasmic disorganization was used to monitor cell death more directly, ROS-scavengers, superoxide dismutase, catalase, horseradish peroxidase, and desferal-Mn(IV) had no effect on HR development. Cytological staining for superoxide or hydrogen peroxide generation also did not reveal the presence of ROS before or during the early stages of the HR, but did, as in the previous study, suggest a role in the autofluorescence and browning of invaded cells that occur following protoplast collapse. Staining of plant mitochondria with nitroblue tetrazolium, possibly attributable to increased dehydrogenase activity but not superoxide generation, occurred transiently around invasion hyphae (monokaryotic stage of the fungus) or haustoria (dikaryotic stage) of the fungus as they entered a cell in the susceptible or resistant cultivar. Around invasion hyphae in epidermal cells in resistant plants, this staining diminished as cytoplasmic streaming stopped, and gradually disappeared as cell death progressed. These data are consistent with other evidence that rust fungi initially negate non-specific defensive responses in both resistant and susceptible cells as part of the establishment of biotrophy. They also suggest that the HR in the cowpea–cowpea rust fungus pathosystem is not triggered by an oxidative burst.     

Temporal and spatial patterns of 1, 3-β-glucanase and chitinase induction in potato leaves infected by Phytophthora infestans

Infection of potato (Solarium tuberosum L.) leaves with the fungal pathogen Phytophthora infestans caused a similar, strong and coordinated induction of 1, 3-β-glucanases and chitinases in compatible (plant susceptible) and incompatible (plant resistant) interactions of two selected plant cultivars with appropriate races of the fungus. The temporal and spatial patterns of 1, 3-β-glucanase induction were studied in further detail by immunohistochemical and in-situ hybridization methods. Accumulation of the protein was preceded by progressive activation of the corresponding gene, commencing near infection sites and spreading rapidly throughout the whole infected leaf as well as to adjacent, non-infected leaves. Protein and mRNA distribution patterns were nearly identical in compatible and incompatible interactions. In comparison with 1, 3-β-glucanase mRNA, phenyl-alanine ammonia-lyase mRNA accumulated more rapidly and remained restricted to the vicinity of fungal infection sites, in addition to its constitutive occurrence in the vascular bundles. Even more rapid than any detectable mRNA induction was the accumulation of auto fluorescing material in plant cells immediately surrounding fungal structures, particularly and invariably in incompatible interactions and less frequently in compatible interactions. It is concluded that cultivar-race-specific resistance is established early in the interaction of potato leaves with P. infestans and hence the observed massive accumulation of 1, 3-β-glucanase and chitinase is presumably not involved in determining this specificity.     

The Differentiation of Infection Structures as a Result of Recognition Events between some Biotrophic Parasites and their Hosts

Most uredospores of rust fungi develop infection structures in a typical pattern so that they can infect the host plant. The function of these infection structures is divided into the following three phases:

• 1 In the recognition phase, the germ tube recognizes the cuticle and the stoma. This process may occur independently from the host plant since copies of the cuticle induce similar reactions of the fungus. During fungal growth on the epidermis, unspecific stress responses of the plant are triggered.
• 2 In the signal phase, the fungal substomatal vesicle and infection hypha(e) contact the host cells within the leaf parenchyma. A signal from the host induces further development of the fungus. Haustorium mother cell differentiation is effected and haustorium formation is initiated. At the same time, the fungus suppresses the synthesis of stress metabolites by the plant.
• 3 In the parasitic phase, the fungus penetrates the host cell and complex interactions between host and parasite begin. A highly specialized interface around the haustorium develops presumably in order to allow a more efficient nutrient transfer from host to parasite. Eventual defence reactions of the plant, generally on the race-cultivar level, fail to be evoked or are suppressed in compatible combinations.