Kurt Mendgen

Plant infection and the establishment of fungal biotrophy

To exploit plants as living substrates, biotrophic fungi have evolved remarkable variations of their tubular cells, the hyphae. They form infection structures such as appressoria, penetration hyphae and infection hyphae to invade the plant with minimal damage to host cells. To establish compatibility with the host, controlled secretory activity and distinct interface layers appear to be essential. Colletotrichum species switch from initial biotrophic to necrotrophic growth and are amenable to mutant analysis and molecular studies. Obligate biotrophic rust fungi can form the most specialized hypha: the haustorium. Gene expression and immunocytological studies with rust fungi support the idea that the haustorium is a transfer apparatus for the long-term absorption of host nutrients.

The role of haustoria in sugar supply during infection of broad bean by the rust fungus Uromyces fabae

Biotrophic plant pathogenic fungi differentiate specialized infection structures within the living cells of their host plants. These haustoria have been linked to nutrient uptake ever since their discovery. We have for the first time to our knowledge shown that the flow of sugars from the host Vicia faba to the rust fungus Uromyces fabae seems to occur largely through the haustorial complex. One of the most abundantly expressed genes in rust haustoria, the expression of which is negligible in other fungal structures, codes for a hexose transporter. Functional expression of the gene termed HXT1 in Saccharomyces cerevisiae and Xenopus laevis oocytes assigned a substrate specificity for d-glucose and d-fructose and indicated a proton symport mechanism. Abs against HXT1p exclusively labeled haustoria in immunofluorescence microscopy and the haustorial plasma membrane in electron microscopy. These results suggest that the fungus concentrates this transporter in haustoria to take advantage of a specialized compartment of the haustorial complex. The extrahaustorial matrix, delimited by the plasma membranes of both host and parasite, constitutes a newly formed apoplastic compartment with qualities distinct from those of the bulk apoplast. This organization might facilitate the competition of the parasite with natural sink organs of the host.

Identification of a Protein from Rust Fungi Transferred from Haustoria into Infected Plant Cells

The formation of haustoria is one of the hallmarks of the interaction of obligate biotrophic fungi with their host plants. In addition to their role in nutrient uptake, it is hypothesized that haustoria are actively involved in establishing and maintaining the biotrophic relationship. We have identified a 24.3-kDa protein that exhibited a very unusual allocation. Rust transferred protein 1 from Uromyces fabae (Uf-RTP1p) was not only detected in the host parasite interface, the extrahaustorial matrix, but also inside infected plant cells by immunofluorescence and electron microscopy. Uf-RTP1p does not exhibit any similarity to sequences currently listed in the public databases. However, we identified a homolog of Uf-RTP1p in the related rust fungus Uromyces striatus (Us-RTP1p). The localization of Uf-RTP1p and Us-RTP1p inside infected plant cells was confirmed, using four independently raised polyclonal antibodies. Depending on the developmental stage of haustoria, Uf-RTP1p was found in increasing amounts in host cells, including the host nucleus. Putative nuclear localization signals (NLS) were found in the predicted RTP1p sequences. However, functional efficiency could only be verified for the Uf-RTP1p NLS by means of green fluorescent protein fusions in transformed tobacco protoplasts. Western blot analysis indicated that Uf-RTP1p and Us-RTP1p most likely enter the host cell as N-glycosylated proteins. However, the mechanism by which they cross the extrahaustorial membrane and accumulate in the host cytoplasm is unknown. The localization of RTP1p suggests that it might play an important role in the maintenance of the biotrophic interaction.

Adhesion Pad Formation and the Involvement of Cutinase and Esterases in the Attachment of Uredospores to the Host Cuticle.

We have investigated the basis of adhesion of uredospores of the obligately parasitic rust fungus Uromyces viciae-fabae to leaves of its broad bean host. Upon contact with an aqueous environment, spores form a structure that we have termed an adhesion pad. The adhesion pad is formed by both living and autoclaved spores, but only adhesion pads formed by living spores adhered to the cuticle of leaves of the host plant. Treatment of living spores with the serine-esterase inhibitor diisopropyl fluorophosphate prevented the adhesion of the pad to the leaf surface, suggesting a functional role for esterase or cutinase in the process of adhesion. A cutinase and two nonspecific serine-esterases were found to be localized on the surface of spores. These enzymes were released rapidly from the spore surface upon contact with an aqueous environment. The addition of the cutinase and the nonspecific esterases to autoclaved spores restored their ability to adhere to the host cuticle. Thus, whereas pad formation appears to be a passive response to the aqueous environment, the actual adhesion of pads to the host cuticle appears to depend on the cutinase and esterases associated with the spore surface. These results suggest a new role for cutinases and serine-esterases in the fungal infection process.

Characterization of in planta-induced rust genes isolated from a haustorium-specific cDNA library

Rust fungi are plant parasites that depend on living host tissue for growth. For invasion of leaves, dikaryotic urediospores differentiate germ tubes and infection structures that penetrate through stomata. Biotrophic growth occurs by intercellular mycelia that form haustoria within host cells. A cDNA library was constructed from haustoria isolated from broad bean leaves infected by Uromyces fabae. Differential screening revealed that a high proportion (19%) of the haustorial cDNAs are specifically expressed in planta but are not expressed, or are much weaker, in germlings or infection structures produced in vitro. A total of 31 different in planta-induced genes (PIGs) were identified. Some of the PIGs are highly expressed in haustoria. The PIGs are single or low copy number genes in the rust genome. A variety of developmentally regulated expression patterns of PIG mRNAs were observed. Sequence analysis of PIG cDNAs revealed similarities to genes encoding proteins involved in amino acid transport, thiamine biosynthesis, short-chain dehydrogenases, metallothioneins, cytochrome P-450 monooxygenases, and peptidyl-prolyl isomerases.

Control of Postharvest Pathogens and Colonization of the Apple Surface by Antagonistic Microorganisms in the Field

ABSTRACT Selected isolates of Aureobasidium pullulans, Rhodotorula glutinis, and Bacillus subtilis reduced the size and number of lesions on wounded apples caused by the postharvest pathogens Penicillium expansum, Botrytis cinerea, and Pezicula malicorticis. Combinations of the antagonistic microorganisms were applied to apple trees in the field late in the growing season of two consecutive years. The population dynamics of the introduced microorganisms and the incidence of fruit decay were determined. Population sizes of introduced antagonists on apple surfaces increased in the field following application of treatments until harvest. After transfer of the fruit from the field into cold storage, the populations of the introduced antagonists remained higher than in the control treatments. Identification of the applied isolates of A. pullulans and R. glutinis during the experiments was achieved by isolate-specific DNA probes generated from random amplified polymorphic DNA. A combination of two strains of A. pullulans and one strain of R. glutinis suppressed rotting of apple to the same extent as the commonly used fungicide Euparen. Our data demonstrate that the application of antagonistic microorganisms in the field represents a promising alternative to fungicide treatments to control post-harvest diseases of apple.

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.

Signal and nutrient exchange at biotrophic plant–fungus interfaces

Biotrophic interfaces are formed in mutualistic and parasitic plant-fungus interactions. They result from coordinated developmental programs in both partners and represent specialized platforms for the exchange of information and nutritional metabolites. New data on the establishment and the components of functional interfaces have been obtained in a number of ways. First, by isolation of symbiotically defective mutants; second, by characterization of new genes and their products; and, third, by the identification and localization of components of biotrophic interfaces, such as cell-wall proteins, H+-ATPases and nutrient transporters.

A Putative Amino Acid Transporter Is Specifically Expressed in Haustoria of the Rust Fungus Uromyces fabae

A cDNA library constructed from haustoria of the rust fungus Uromyces fabae was screened for clones that are differentially expressed in haustoria. One family of cDNAs (in planta-induced gene 2 [PIG2] was isolated and found to encode a protein with high homologies to fungal amino acid transporters. A cDNA clone containing the complete coding region of PIG2 and the corresponding genomic clone were isolated and sequenced, revealing the presence of 17 introns in the PIG2 gene. Expression of PIG2 mRNA appeared to be restricted to haustoria. With antibodies raised against synthetic peptides, the PIG2-encoded protein was found in membranes fractions of isolated haustoria but not of germinated rust spores. With immunofluorescence microscopy, the putative amino acid transporter was localized to plasma membranes of the haustorial bodies, but not detected in the haustorial neck, haustorial mother cells, or intercellular fungal hyphae growing within infected leaf tissue. These data present for the first time molecular evidence that the rust haustorium plays a special role in the uptake of nutrients from an infected host cell.

Electron microscopy of plant pathogens.

This review of all electron microscopical techniques currently used also provides some recent principal research reports. Their application to the study of fine structures of various groups of plant pathogens (fungi, bacteria, viruses, mycoplasma-like organisms, protozoa and nematodes) is described as well as host-pathogen and host-symbiont interactions. In recent years, new preparation and labelling techniques (cryofixation, immunolabelling) have been applied in various disciplines of research, and these yield further specific information on the nature of parasitic interactions as detectable in the electron microscope. The reader will obtain an overview of the modern technical possibilities of studying plant pathogens using an electron microscopical approach.