Christine Struck

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.

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.

The Plasma Membrane H+-ATPase from the Biotrophic Rust Fungus Uromyces fabae : molecular Characterization of the Gene (PMA1) and Functional Expression of the Enzyme in Yeast

To study the molecular basis of biotrophic nutrient uptake by plant parasitic rust fungi, the gene (Uf-PMA1) encoding the plasma membrane H(+)-ATPase from Uromyces fabae was isolated. Uf-PMA1 exists probably as a single gene. However, two nearly identical sequences were identified; the similarity apparently is due to two Uf-PMA1 alleles in the dikaryotic hyphae. Multiple Uf-PMA1 transcripts were observed during early rust development, and reduced amounts of a single Uf-PMA1 mRNA were observed in haustoria and rust-infected leaves. This is in contrast to elevated enzyme activity in haustoria compared to germinated spores (C. Struck, M. Hahn, and K. Mendgen. Fungal Genet. Biol. 20:30-35, 1996). Unexpectedly, the PMA1-encoded rust protein is more similar to H(+)-ATPases from plants (55% identity) than from ascomycetous fungi (36% identity). When the rust PMA1 cDNA was expressed in Saccharomyces cerevisiae, both the wild-type enzyme and a mutant derivative (delta 76) deleted for the 76 C-terminal amino acids were able to support growth of a yeast strain lacking its own H(+)-ATPases. Compared to the wild-type, the delta 76 mutant enzyme displayed increased affinity to ATP, a higher vanadate sensitivity, and a more alkaline pH optimum. These results indicate that the C-terminal region of the rust enzyme exhibits auto-regulatory properties.

The Uromyces fabae UfAAT3 gene encodes a general amino acid permease that prefers uptake of in planta scarce amino acids.

SUMMARY The obligately biotrophic rust fungi are dependent on nutrient supply from their host plants. A cDNA library of infection structures of the rust fungus Uromyces fabae was used to identify a gene (UfAAT3) that encodes a protein with a high degree of sequence similarity to fungal amino acid permeases. The expression profile revealed by RT-PCR shows an up-regulation very early during rust development, with the highest level in haustoria and infected leaves. Heterologous expression of UfAAT3p in Xenopus oocytes revealed an amino acid permease energized by co-transport with protons and exhibiting a broad substrate specificity. Compared to the previously described U. fabae amino acid transporter (AAT1), which represented the highest transport activities for lysine and histidine, electrophysiological measurements with cRNA of UfAAT3-injected oocytes showed substrate preferences for leucine and the sulphur containing amino acids methionine and cysteine. The unique contribution of the amino acid permeases and their substrate affinities might be connected with the availability of amino acids in the leaf tissue. Thus, in order to compare the substrate profiles of AAT1p and UfAAT3p with the natural environment of U. fabae we analysed the amino acid concentration in the apoplastic space, in addition to that in extracts of Vicia faba leaves. The predominant free amino acids were asparagine, alanine, glutamine and glutamate. However, most amino acids were present at low concentrations (between 0.02 and 0.16 mm), including the preferred substrates of the U. fabae amino acid permeases AAT1p and UfAAT3p.

Different Resistance Mechanisms of Medicago truncatula Ecotypes Against the Rust Fungus Uromyces striatus

ABSTRACT A pathosystem consisting of the model plant Medicago truncatula and the rust fungus Uromyces striatus was characterized. From a collection of 113 mostly European accessions of M. truncatula, the vast majority were found to be susceptible to U. striatus, whereas 5 accessions showed strong resistance reactions. Stomatal surface characteristics, even if partly occluded, did not interfere with the ability of U. striatus germ tubes to infect. After penetration, the resistant ecotypes reacted with various degrees of cell death during different stages of haustorial establishment. Whereas four ecotypes showed a typical hypersensitive reaction by developing necrotic lesions, one ecotype (F11.008) exhibited a prehaustorial type of defense without hypersensitive response. This ecotype may be used as a source of nonhost-type of resistance against U. striatus.

History of Oilseed Rape Cropping and Geographic Origin Affect the Genetic Structure of Plasmodiophora brassicae Populations

The soilborne pathogen Plasmodiophora brassicae causes clubroot on Brassica crops, a common disease in many oilseed rape growing regions. Here, we investigate genetic diversity and geographic differentiation of P. brassicae populations from different regions in Germany. We compared three regions that differ in oilseed rape cropping history, oilseed rape acreage, and incidence of clubroot. These regions were either spatially separated or separated by the former inner German border. Plasmodiophora isolates were collected from 59 fields (29, 17, and 13 fields per region, respectively) and 174 amplified fragment length polymorphism (AFLP) markers were analyzed. Every field isolate showed a unique genotype pattern; that is, no genotype was shared among the regions and different fields. The mean gene diversity was 0.27, suggesting that P. brassicae is a genetically diverse species. The comparison of indexes (gene diversity, genotypic diversity, and linkage disequilibrium) between the regions does not support our hypotheses that cropping history, oilseed rape acreage, and incidence of clubroot affect these estimates. Principal component analysis (PCA), fixation index (FST), and generalized linear model (GLM) were suitable to specify regional differences. PCA revealed two clusters of isolates based on the geographic origin of the isolates and FST showed that these clusters were highly differentiated. Hypotheses about association of genotypes with different spatial scales were tested with GLM: the region, reflecting the cropping history, and the individual field had a significant effect on the AFLP pattern. We propose that individual field isolates represent a discrete population and that geographic differentiation results from low levels of gene flow due to the limited dispersal of this soilborne pathogen and from localized selection pressure as unifying force on the genotypes.

Amino acid uptake in rust fungi.

The plant pathogenic rust fungi colonize leaf tissue and feed off their host plants without killing them. Certain economically important species of different genera such as Melampsora, Phakopsora, Puccinia, or Uromyces are extensively studied for resolving the mechanisms of the obligate biotrophy. As obligate parasites rust fungi only can complete their life cycle on living hosts where they grow through the leaf tissue by developing an extended network of intercellular hyphae from which intracellular haustoria are differentiated. Haustoria are involved in key functions of the obligate biotrophic lifestyle: suppressing host defense responses and acquiring nutrients. This review provides a survey of rust fungi nitrogen nutrition with special emphasis on amino acid uptake. A variety of sequences of amino acid transporter genes of rust fungi have been published; however, transport activity of only three in planta highly up-regulated amino acid permeases have been characterized. Functional and immunohistochemical investigations have shown the specificity and localization of these transporters. Sequence data of various genome projects allowed identification of numerous rust amino acid transporter genes. An in silico analysis reveals that these genes can be classified into different transporter families. In addition, genetic and molecular data of amino acid transporters have provided new insights in the corresponding metabolic pathways.

Antipredator Behavioral Traits of some Agriotes Wireworms (Coleoptera: Elateridae) and their Potential Implications for Species Identification

Many insects show a typical antipredator behavior. The two simplest tactics are immobilization and flight. The antipredator behavior of wireworms, the soil-dwelling polyphagous larvae of click beetles, has not yet been investigated. Birds are known to attack wireworms. Our experimental method (picking up and dropping wireworms) mimics the first attack of a predator, such as a bird. In Europe, some Agriotes species are of particular importance as crop pests. As these species differ considerably, in ecology, damage threshold, and pathogen resistance, knowing more about the species specific biology and behavior will support the establishment of successful control measures. In an extensive microcosm study, we observed the antipredator behavior of wireworms belonging to four different Agriotes species (A. lineatus, A. obscurus, A. sordidus, and A. ustulatus) before and during digging into the soil after a startle-stimulus. We recorded wireworm immobility frequency and duration, and locomotor activity at three temperatures. We also analyzed genetic and morphologic attributes of A. lineatus larvae from two different origins. Following behavior types were found reflecting different escape tactics: (a) distinct tonic immobility (A. lineatus), (b) brief/inconsistent tonic immobility (A. obscurus, A. sordidus), and (c) immediate fleeing/burying (A. ustulatus). Additionally, we found small but significant differences in morphology, behavior, and genetics (PCR results) between A. lineatus larvae originating from the Netherlands and those originating from Germany. The biological information we gathered about each species will further increase the understanding of these insects and improve interpretation of future experimental data. In addition, the described behavioral differences between Agriotes obscurus and A. lineatus wireworms may represent a useful additional criterion in morphological species identification.

Risk Potential of Clubroot Disease on Winter Oilseed Rape

Clubroot disease caused by Plasmodiophora brassicae is an important disease of cruciferous plants. Although the pathogen is widespread and has been reported to cause high yield losses, the impact on winter oilseed rape (OSR) has not been experimentally verified. To quantify the risk potential of P. brassicae, we conducted two closely linked experiments. A semicontrolled experiment used artificial soil infestation at inoculum densities between 106 and 108 spores liter-1 of soil to detect the impact on seed yield and yield components of a susceptible and resistant OSR cultivar. A greenhouse experiment was implemented using the soil of the semicontrolled experiment after cropping the two cultivars to quantify the influence of cultivar resistance on soil inoculum. According to cumulative link mixed models, disease rating was positively correlated with the amount of inoculum. Linear regression analyses revealed a negative correlation between seed yield and inoculum density. Yield losses of 60% already appeared at the lowest inoculum density. Plant losses and reduced seed per pod were accountable for yield losses. Although the resistant cultivar showed clubroot symptoms, seed yield was not affected by the pathogen. The greenhouse experiment revealed that clubroot severity in subsequent OSR was reduced after cropping the resistant cultivar. This study showed significant yield damage of P. brassicae already at low infestation levels.

Feeding preferences of the weevils Sitona gressorius and Sitona griseus on different lupin genotypes and the role of alkaloids

The weevils Sitona gressorius and Sitona griseus are specialist herbivores on lupins in Europe. The adult weevils feed on the leaves, and the larvae on the root nodules of the plants. This causes severe damage to lupin crops. In the present study, the feeding preferences of lupin weevil adults on different lupin genotypes were examined with respect to a possible effect of lupin alkaloids on host selection. A total of 12 genotypes from the species Lupinus albus, L. angustifolius, L. luteus, and L. nanus were grown in a field experiment and the feeding damage on the leaves caused by naturally occurring lupin weevil adults was estimated. Additionally, a feeding choice test with S. gressorius adults was performed to examine feeding preferences under laboratory conditions. A gas chromatographic analysis provided information on the alkaloid content and profiles in the leaves of the tested lupin genotypes. In the field experiment, significant differences in the extent of the feeding damage within the 12 lupin genotypes were observed. The dual-choice feeding bioassay did not show discrimination of lupin species, but two L. angustifolius genotypes were significantly less affected than the standard L. luteus “Bornal”. The alkaloid analysis revealed large contrasts in alkaloid concentrations and profiles in the leaves of the tested genotypes. Correlation analysis with the results from the field and laboratory did not indicate a significant influence of the total foliar alkaloid content on the extent of weevil feeding.