Biotrophic Plant Pathogens Research Articles

Terrific protein traffic: the mystery of effector protein delivery by filamentous plant pathogens.

Many biotrophic fungal and oomycete plant pathogens deliver effector proteins directly into host cells during infection. Recent advances are revealing the extensive effector repertoires of these pathogens and are beginning to shed light on how they manipulate host cells to establish a parasitic relationship. Surprisingly, oomycete effectors seem to share a common uptake system with those from the human malaria pathogen. The current explosion of information is opening new research avenues in molecular plant pathology and is providing new opportunities to limit the impact of plant disease on food production.

Evidence for the importance of enzymatic digestion of epidermal walls during subepidermal sporulation and pustule opening in white blister rusts (Albuginaceae)

Albugo candida, A. ipomoeae-panduratae, Pustula tragopogonis, Wilsoniana bliti and W. portulacae are widespread obligate biotrophic plant pathogens causing white blister diseases on a variety of flowering plants. Their subepidermal mode of sporulation is unique amongst Oomycetes and leads to blister-like structures on their hosts similar to those produced by true rusts ( Uredinales). Unlike in true rusts, sporangia are colourless and produced in chains; the first formed, primary sporangium, differing in size and morphology from subsequent secondary sporangia. According to current interpretations of pustule development the rising pressure of the growing chains of sporangia tear off the epidermal layer from the mesophyll and, in the end, ruptures the epidermis to release the sporangia. This is not convincing considering the rigidity of the epidermal layer and the fact that thin-walled mesophyll cells show no signs of pressure endurance. Our detailed light-, scanning electron-, and transmission electron microscopic observations provide evidence that pustule development and opening are regulated and delicate processes that involve directed enzymatic dissection of host tissue cell walls. The process starts when intercellular hyphae separate the epidermal layer from the parenchyma, forming a cavity in which sporulation takes place. Then thick-walled sporogenous hyphae with club-shaped but thin-walled tips develop and produce sporangia in basipetal succession from the apices of the sporogenous hyphae. The short-living primary sporangia attach tightly to the inner cell walls of the epidermal layer and undergo dramatic cytological changes during pustule maturation, including vacuolisation and development of numerous electron-dense vesicles that might deliver cell wall degrading enzymes. In ripe pustules, the disintegration of areas of epidermal cells leads to the opening of the pustules and to the release of the secondary sporangia. Also the comparison of samples prepared for scanning electron microscopy with fresh pustules, as well as the comparison of the inner epidermal layers detached by the pathogens and detached by force supports our conclusion that delicate enzymatic activity and not force are involved in pustule development and opening by these highly sophisticated pathogens.

Plasmopara halstedii

<i>Plasmopara halstedii</i>

A cultivation independent, PCR-based protocol for the direct identification of plant pathogens in infected plant material

A protocol involving PCR, shot-gun cloning and sequencing was developed as a pre-diagnostic screening tool working directly on disease symptoms. The method was used to show the presence of biotrophic and non-biotrophic eukaryotic plant pathogens in leaves and fruits.

Selective Elicitation of the Phytoalexin Rutalexin in Rutabaga and Turnip Roots by a Biotrophic Plant Pathogen

Stress responses of roots of rutabaga ( Brassica napus ssp. napobrassica) and turnip ( B. rapa ssp. rapa) were analyzed. Phytoalexin production due to biotic elicitation by the biotroph Albugo candida and abiotic elicitation using UV irradiation was quantified by HPLC. The phytoalexin rutalexin was produced in substantially higher amounts in rutabaga and turnip roots inoculated with A. candida than in UV irradiated roots. By contrast, production of the phytoalexins brassinin in rutabaga and cyclobrassinin in turnip was higher in UV irradiated roots than in roots inoculated with A. candida. Overall, the results suggest that A. candida is able to redirect the phytoalexin biosynthetic pathway towards rutalexin, a phytoalexin substantially less inhibitory than either brassinin or cyclobrassinin.

The NADPH oxidase Cpnox1 is required for full pathogenicity of the ergot fungus Claviceps purpurea

The role of reactive oxygen species (ROS) in interactions between phytopathogenic fungi and their hosts is well established. An oxidative burst mainly caused by superoxide formation by membrane-associated NADPH oxidases is an essential element of plant defence reactions. Apart from primary effects, ROS play a major role as a second messenger in host response. Recently, NADPH oxidase (nox)-encoding genes have been identified in filamentous fungi. Functional analyses have shown that these fungal enzymes are involved in sexual differentiation, and there is growing evidence that they also affect developmental programmes involved in fungus-plant interactions. Here we show that in the biotrophic plant pathogen Claviceps purpurea deletion of the cpnox1 gene, probably encoding an NADPH oxidase, has impact on germination of conidia and pathogenicity: Deltacpnox1 mutants can penetrate the host epidermis, but they are impaired in colonization of the plant ovarian tissue. In the few cases where macroscopic signs of infection (honeydew) appear, they are extremely delayed and fully developed sclerotia have never been observed. C. purpurea Nox1 is important for the interaction with its host, probably by directly affecting pathogenic differentiation of the fungus.

Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis

Ustilago maydis is a ubiquitous pathogen of maize and a well-established model organism for the study of plant-microbe interactions. This basidiomycete fungus does not use aggressive virulence strategies to kill its host. U. maydis belongs to the group of biotrophic parasites (the smuts) that depend on living tissue for proliferation and development. Here we report the genome sequence for a member of this economically important group of biotrophic fungi. The 20.5-million-base U. maydis genome assembly contains 6,902 predicted protein-encoding genes and lacks pathogenicity signatures found in the genomes of aggressive pathogenic fungi, for example a battery of cell-wall-degrading enzymes. However, we detected unexpected genomic features responsible for the pathogenicity of this organism. Specifically, we found 12 clusters of genes encoding small secreted proteins with unknown function. A significant fraction of these genes exists in small gene families. Expression analysis showed that most of the genes contained in these clusters are regulated together and induced in infected tissue. Deletion of individual clusters altered the virulence of U. maydis in five cases, ranging from a complete lack of symptoms to hypervirulence. Despite years of research into the mechanism of pathogenicity in U. maydis, no 'true' virulence factors had been previously identified. Thus, the discovery of the secreted protein gene clusters and the functional demonstration of their decisive role in the infection process illuminate previously unknown mechanisms of pathogenicity operating in biotrophic fungi. Genomic analysis is, similarly, likely to open up new avenues for the discovery of virulence determinants in other pathogens.

Host range expansion in a powdery mildew fungus (Golovinomyces sp.) infecting Arabidopsis thaliana: Torenia fournieri as a new host

Since 2003, Torenia fournieri plants grown for experimental purposes were repeatedly infected by powdery mildew in a laboratory in Hungary. Based on morphological characteristics, the pathogen belonged to the mitosporic genus Oidium subgen. Reticuloidium, the anamorph stage of Golovinomyces. The rDNA ITS sequence was identical to that of two other powdery mildew fungi, infecting Arabidopsis and Veronica, respectively, in different parts of the world. According to a previous phylogenetic analysis of ITS and 28S rDNA sequences, those two powdery mildews belong to a recently evolved group of Golovinomyces characterized by multiple host range expansions during their evolution. Both the ITS sequence and the morphological data indicate that the powdery mildew anamorph infecting Torenia also belongs to this group. It is likely that the powdery mildew infections of the experimental T. fournieri plants, native to south-east Asia, were the result of a very recent host range expansion of a polyphagous Golovinomyces because (i) T. fournieri is absent from our region, except as an experimental plant grown in the laboratory, (ii) the powdery mildew fungus infecting this exotic plant belongs to a group of Golovinomyces where host range expansion is a frequent evolutionary scenario, (iii) cross-inoculation tests showed that this pathogen is also able to infect other plant species, notably A. thaliana and tobacco, and (iv) no Golovinomyces species are known to infect T. fournieri anywhere in the world. Although host range expansion has often been proposed as a common evolutionary process in the Erysiphales, and also in other biotrophic plant pathogens, this has not been clearly demonstrated in any case studies so far. To our knowledge, this is the first convincing case of a host range expansion event in the Erysiphales.

Survey and expression analysis of five new chitin synthase genes in the biotrophic rust fungus Puccinia graminis

We have isolated and characterised the first set of chitin synthase genes from a rust fungus, a large group of economically highly important, obligately biotrophic plant pathogens. Puccinia graminis was used as a model organism for the rust fungi which are not well investigated on the molecular level today. One of the major structural components of most fungal cell walls is the chitin polymer which is synthesised by a family of enzymes called chitin synthases. In P. graminis, we have isolated five new chitin synthase genes from four different classes, chsII, chsIIIa, chsIIIb, chsIV, and chsV. The genes contain a high number of introns, unusual for other known fungal chitin synthases. The dinucleic stage of the fungus seems to contain two slightly different genes or alleles for four isoforms. One isoform, chsIIIa, seems to be expressed only in the youngest stages of fungal growth. Analysis of the derived proteins shows that together with other basidiomycete CHS, the pgtCHS form separate subgroups in the phylogenetic tree. This set of five rust chitin synthase genes, with some unusual features compared to known fungal chitin synthases, allows new insights into chitin synthase classification, and may help in the development of novel functional fungicides.

Spatially Resolved Sulphur K-edge XANES Spectroscopy for in situ Characterization of the Fungus-plant Interaction Puccinia triticina and Wheat Leaves

An innovative application of X-ray absorption near edge structure (XANES) spectroscopy for the characterization of interactions of biotrophic plant pathogens with their hosts as exemplified by Puccinia triticina colonizing wheat leaves is described. Spatially resolved, synchrotron radiation-based XANES spectroscopy was used for the detection of changes in sulphur metabolism induced by leaf rust infections. A significant accumulation of sulphate occurred at the site of the sporulating urediniosori of P. triticina. Compared with non-infected leaf areas, minor changes in the spectra were observed for the non-visibly colonized tissue neighbouring the rust sori. As the spectra for isolated urediniospores and the healthy leaf areas did not match the spectra of the urediniosori, a significant impact of the biotrophic pathogen on sulphur metabolism of wheat has been demonstrated. Spatially resolved XANES spectroscopy will extend the range of qualitative and quantitative methods for in situ investigations of host–pathogen interactions, thus contributing to enlarge our knowledge about the metabolism of diseased plants.

Characterization of a novel NADP(+)-dependent D-arabitol dehydrogenase from the plant pathogen Uromyces fabae.

We have identified and characterized a novel NADP(+)-dependent D-arabitol dehydrogenase and the corresponding gene from the rust fungus Uromyces fabae, a biotrophic plant pathogen on broad bean (Vicia faba). The new enzyme was termed ARD1p (D-arabitol dehydrogenase 1). It recognizes D-arabitol and mannitol as substrates in the forward reaction, and D-xylulose, D-ribulose and D-fructose as substrates in the reverse reaction. Co-factor specificity was restricted to NADP(H). Kinetic data for the major substrates and co-factors are presented. A detailed analysis of the organization and expression pattern of the ARD1 gene are also given. Immunocytological data indicate a localization of the gene product predominantly in haustoria, the feeding structures of these fungi. Analyses of metabolite levels during pathogenesis indicate that the D-arabitol concentration rises dramatically as infection progresses, and D-arabitol was shown in an in vitro system to be capable of quenching reactive oxygen species involved in host plant defence reactions. ARD1p may therefore play an important role in carbohydrate metabolism and in establishing and/or maintaining the biotrophic interaction in U. fabae.

Possible roles for mannitol and mannitol dehydrogenase in the biotrophic plant pathogen Uromyces fabae.

Levels of the C6-polyol mannitol were observed to rise dramatically in the biotrophic interaction of the rust fungus Uromyces fabae and its host plant Vicia faba. Mannitol was found in millimolar concentrations in extracts and apoplastic fluids of infected leaves and also in extracts of spores. We suggest that this polyol might have at least a dual function: first, as a carbohydrate storage compound, and second, as a scavenger of reactive oxygen species. Mannitol accumulation is accompanied by high expression of a mannitol dehydrogenase (MAD1) in haustoria. While MAD1 transcripts were detected in haustoria only, immunolocalization studies show that the gene product is also present in spores. Kinetic and thermodynamic analyses of the MAD1p catalyzed reactions indicate that the enzyme might be responsible for the production of mannitol in haustoria and for the utilization of mannitol in spores. Since V. faba is normally unable to synthesize or utilize polyols, the multipurpose usage of mannitol seems an ideal strategy for the fungal pathogen.

Mutualistic interaction between a weevil and a rust fungus, two parasites of the weed Cirsium arvense

We present a mutualism between a stem-boring weevil, Apion onopordi Kirby (Coleoptera: Apionidae), and a rust fungus, Puccinia punctiformis (Str.) Röhl. (Uredinales), both parasites of the creeping thistle, Cirsium arvense (L.) Scop. (Asteraceae). Females, but not males, of A. onopordi induced systemic rust infections of thistle shoots in the season after they were attacked by the weevil, indicating that insect oviposition is a crucial stage in pathogen transmission. Adult weevils emerged from systemically infected thistle shoots were heavier than weevils from healthy C. arvense shoots. Heavier females had a higher fecundity and laid larger eggs. The weevil preferred to deposit eggs in systemically rust-infected over healthy thistle shoots, which seemed to be a sub-optimal host. This is to our knowledge the first report of a mutualistic interaction between an herbivorous insect and a biotrophic plant pathogen. The mechanism responsible for the advantage of rust-infected shoots for A. onopordi causes a different outcome in other thistle herbivores, and therefore can not be explained by a general enhancement of nutritional quality in rust-infected tissue. This mutualism likely has evolved from a competitive relationship. Unlike other thistle herbivores A. onopordi seems to be better suited as mutualist for P. punctiformis because of its small impact on the host plant and its feeding niche on plant parts not directly associated with pathogen reproduction.

Infection structures of biotrophic and hemibiotrophic fungal plant pathogens

Summary Biotrophic plant pathogenic fungi are one of the major causes of crop losses. The infection processes they exhibit are typified by infected host plant cells remaining alive for several days. This requires the development of specialized infection structures such as haustoria which are produced by obligate biotrophs, and intracellular hyphae which are produced by many hemibiotrophs. These infection hyphae are surrounded by the host plant plasma membrane, and in the case of haustoria the extrahaustorial membrane differs biochemically and structurally from the normal membrane. An interfacial matrix separates haustoria and intracellular hyphae from the invaginated membrane and this seems to be characteristic of biotrophic interactions. There is clear evidence for molecular differentiation of the haustorial plasma membrane in powdery mildews and rusts in comparison with the other fungal membranes. Relatively few pathogenicity genes related to biotrophy, and the switch from biotrophy to necrotrophy in hemibiotrophs, have been identified.

Apoptosis, programmed cell death and the hypersensitive response

Apoptosis is typically a morphologically identifiable form of programmed cell death in mammals that is regulated by genes with homologues in other animal Phyla. Although both plants and fungal plant pathogens exhibit forms of developmental programmed cell death, demonstrated morphological or genetic homologies with mammalian apoptosis are still generally lacking. Because of its ubiquity and the involvement of signal transduction pathways in its induction, a strong case is developing that the hypersensitive response is a specific form of plant programmed cell death evolved as a defense against microbial parasites. Data suggest that separate signalling pathways may lead to the cell death and the defense gene activation that characterize this response and that parasite-specific resistance genes represent only one of many types of genes involved in response regulation. However, despite some biochemical similarities between the hypersensitive response, forms of developmental programmed cell death in plants, and animal apoptosis, no unique and consistent markers for the hypersensitive response (or plant programmed cell death in general) have yet been found. Whether any of these forms of plant cell death should be called apoptosis depends on how the term is defined. Assuming the hypersensitive response is a form of programmed cell death and is the ‘default state’ upon pathogen entry into a cell, it seems likely that intracellular biotrophic plant pathogens resemble some animal viruses in being able to suppress this response in susceptible hosts.

New CJD strain

In contrast to most biotrophic plant pathogens, smut fungi belonging to the family of Ustilaginaceae are facultative parasites that have a yeastlike, saprophytic phase contrasting the filamentous, parasitic growth during plant infection. This dimorphic lifestyle allows easy culturing, reverse genetic modifications and cell biological analyses in the saprophytic stage; impact on virulence or pathogenicity can then been monitored easily in infection assays. Such excellent preconditions made the maize smut fungus Ustilago maydis the most advanced model organism for fungal biotrophs. A recent key step in Ustilago research was the high-quality sequencing and manual annotation of its genome, which identified a wealth of novel virulence genes that are arranged in physical gene clusters. In next steps, the genomes of the maize head smut Sporisorium reilianum f. sp. zeae and the barley covered smut Ustilago hordei have been sequenced. These closely related pathogens exhibit significant differences regarding genome organization and gene equipment, likely reflecting different infection styles compared to U. maydis, whose unique feature is to cause tumours locally to sites of infection in vegetative tissue. Comparative analysis of these genomes as well as transcriptome profiling approaches allowed the identification of regulatory elements controlling pathogenic development as well as of secreted effector genes with important functions in plant interaction. In this chapter, we summarize the most important findings gained by genome analyses of smut fungi and define important questions that need to be addressed in the future.

The lucky few?

In contrast to most biotrophic plant pathogens, smut fungi belonging to the family of Ustilaginaceae are facultative parasites that have a yeastlike, saprophytic phase contrasting the filamentous, parasitic growth during plant infection. This dimorphic lifestyle allows easy culturing, reverse genetic modifications and cell biological analyses in the saprophytic stage; impact on virulence or pathogenicity can then been monitored easily in infection assays. Such excellent preconditions made the maize smut fungus Ustilago maydis the most advanced model organism for fungal biotrophs. A recent key step in Ustilago research was the high-quality sequencing and manual annotation of its genome, which identified a wealth of novel virulence genes that are arranged in physical gene clusters. In next steps, the genomes of the maize head smut Sporisorium reilianum f. sp. zeae and the barley covered smut Ustilago hordei have been sequenced. These closely related pathogens exhibit significant differences regarding genome organization and gene equipment, likely reflecting different infection styles compared to U. maydis, whose unique feature is to cause tumours locally to sites of infection in vegetative tissue. Comparative analysis of these genomes as well as transcriptome profiling approaches allowed the identification of regulatory elements controlling pathogenic development as well as of secreted effector genes with important functions in plant interaction. In this chapter, we summarize the most important findings gained by genome analyses of smut fungi and define important questions that need to be addressed in the future.

Analysis of differentiation and development of the specialized infection structures formed by biotrophic fungal plant pathogens using monoclonal antibodies

Monoclonal antibodies have been used to study the differentiation and development of the specialized infection structures formed in the Colletotrichum–bean and powdery mildew – pea interactions. In the Colletotrichum lindemuthianum – bean interaction, monoclonal antibodies have been used to show that the extracellular matrices associated with conidia, germ tubes, and appressoria differ in composition and that the extracellular glycoproteins are organized into specific regions of the fungal cell surface. Monoclonal antibody UB27 has been used to show that the plasma membrane of appressoria is differentiated into distinct domains, with the integral membrane glycoprotein identified by UB27 being excluded from the pore region. UB25 recognizes a glycoprotein located specifically in the cell wall/matrix of intracellular hyphae and is expressed only during the biotrophic phase of development. In the Erysiphe pisi – pea interaction, UB8 and UB10 identify glycoproteins specific to the haustorial plasma membrane within the haustorial complex. Monoclonal antibodies that recognize the extrahaustorial membrane have shown that this membrane contains specific components, as well as glycoproteins in common with the host plasma membrane. UB8 has been successfully used to isolate a gene sequence coding for the protein antigen, by immunoscreening a cDNA expression library prepared from infected epidermis. An antibody that recognizes the plant endoplasmic reticulum has been used to show that this structure reorganizes around the developing haustorial complex in pea epidermal cells. Key words: appressorium, biotrophy, Colletotrichum lindemuthianum, Erysiphe pisi, haustorium, monoclonal antibody, powdery mildew.

Purification and characterization of mannitol dehydrogenase from the fungal tomato pathogen Cladosporium fulvum (syn. Fulvia fulva)

As part of our studies to elucidate the mechanisms by which biotrophic plant pathogens obtain and retain carbon from their plant hosts, we have investigated the properties of the mannitol dehydrogenase (E.C. 1.1.1.67) of Cladosporium fulvum (syn. Fulvia fulva). The enzyme has been purified to near-homogeneity and has a subunit mol. wt of 29 kDa and is probably tetrameric. The Michaelis-Menten constants for mannitol and fructose are high and the equilibrium constant lies towards mannitol accumulation. A β-fructosidase (invertase) activity was detected and found to be largely wall-bound. The properties of these enzymes are consistent with a role in the hydrolysis of photosynthetically produced sucrose and the accumulation of mannitol as a storage compound during fungal growth in the plant.

Peronospora hyoscyami f. sp. tabacina . [Descriptions of Fungi and Bacteria

Abstract A description is provided for Peronospora hyoscyami f. sp. tabacina . Information is included on the disease caused by the organism, its transmission, geographical distribution, and hosts. HOSTS: Naturally infected: Capsicum annuum, Nicotiana alpina, N. acuminata, N. attenuata, N. benthamiana, N. bigelowii, N. caesia, N. glauca, N. glutinosa, N. langsdorffi, N. longiflora, N. mesophila, N. nodiflora, N. nudicaulis, N. paniculata, N. quadrivalvis, N. repanda, N. rustica, N .x sanderae, N. stocktoni, N. suavolens, N. sylvestris, N. tabacum, N. tomentosa, N. trigonophylla, N. wigandioides, Solanum lycopersicum, S. melongena, S. nigrum .Infected experimentally: Capsicum frutescens, Dunalia ramiflora, Hyoscyamus muticus, H. niger, Nicotiana alata, N. affnis, N. cavanillesii, N. fragrans, N. gossei, N. goodspeedii, N. ipomopsifolia, N. maritima , N. megalosiphon, N. raimondii, N. tomentosifolius, Lehmania otophora, Physalis alkekangi, P. lanceifolia, P. peruviana, Petunia × hybrida, Schizanthus pinnatus . DISEASE: Blue mould of tobacco plants (48, 3141; 61, 2664) (commercial and ornamental varieties are affected); the fungus is an obligately biotrophic plant pathogen. Chlorotic leaf spots develop on leaves of susceptible seedlings, which become deformed. Undersurfaces of leaves become covered with a layer of sporophores, producing a diagnostic blue-grey felt. Rapid generalized light-brown tissue necrosis follows and the apical meristem ceases growth. Disease symptoms may not appear immediately after infection, however, or during sporophore production. If the fungus encounters any barrier to its growth through the plant tissues (because of a physiological response, high temperature or fungicides), then the infection becomes wholly systemic, being confined to an area close to the veins, or to a necrotic area if the plant is genetically resistant. Similar symptoms develop on mature plants in the field and the entire plant may be destroyed within 3-4 weeks, leaving only a blackened stem. The fungus attacks leaves and stems of tobacco seedlings and mature plants in Europe and Australia, but only leaf tissues in the USA, where weather conditions confine it to being a seedling disease in most years. GEOGRAPHICAL DISTRIBUTION: First reported in Australia in 1891, now worldwide; see CMI Distribution Maps of Plant Diseases 23. TRANSMISSION: By airborne spores, which can remain viable for 2 months at 20-40% RH, and have been reported to be transported over distances of 1, 600 km. Most favourable conditions for sporulation are an average temperature of 20°C and a period of relatively high humidity (95% RH) for at least 3 h. Spore liberation is dependent on a rise in temperature, a decrease in relative humidity and an increase in insolation. There is some evidence for soil-borne transmission by oospores in infected plant debris in the USA, since blue mould appears more often in seedbeds which have previously contained infected tobacco. Although mycelium can survive and overwinter in plant tissues, there are no reports of transmission by a systemic route.