Transient transformation of the obligate biotrophic rust fungus Uromyces fabae using biolistics

2011 ◽  
Vol 115 (7) ◽  
pp. 633-642 ◽  
Author(s):  
Alma Djulic ◽  
Annette Schmid ◽  
Heike Lenz ◽  
Pia Sharma ◽  
Christin Koch ◽  
...  
Keyword(s):  
Rust Fungus ◽  
Transient Transformation ◽  
Uromyces Fabae

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

2005 ◽  
Vol 18 (11) ◽  
pp. 1130-1139 ◽  
Author(s):  
Eric Kemen ◽  
Ariane C. Kemen ◽  
Maryam Rafiqi ◽  
Uta Hempel ◽  
Kurt Mendgen ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Polyclonal Antibodies ◽  
Rust Fungus ◽  
Infected Plant ◽  
Plant Cells ◽  
Host Cells ◽  
Nuclear Localization Signals ◽  
Uromyces Fabae ◽  
Host Cytoplasm ◽  
Functional Efficiency

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.

scholarly journals Differential Regulation of Gene Expression in the Obligate Biotrophic Interaction of Uromyces fabae with Its Host Vicia faba

2001 ◽  
Vol 14 (11) ◽  
pp. 1319-1326 ◽  
Author(s):  
Stefan G. R. Wirsel ◽  
Ralf T. Voegele ◽  
Kurt W. Mendgen
Keyword(s):  
Amino Acid ◽  
Vicia Faba ◽  
Developmental Stages ◽  
Rust Fungus ◽  
Regulation Of Gene Expression ◽  
Sugar Uptake ◽  
Amino Acid Transporters ◽  
Plasma Membrane Atpase ◽  
Uromyces Fabae ◽  
Expression Of Genes

Classical analysis of obligate biotrophic fungi revealed changes of enzyme activities or the concentration of metabolites in infected areas. However, due to the intricate integration of host and parasite metabolism, it was not possible to delineate the individual contributions of the two organisms. Here, we used reverse-transcription-polymerase chain reaction to monitor expression of genes from the rust fungus Uromyces fabae and its host Vicia faba. We focused on genes relevant for amino acid and sugar uptake and metabolism in both organisms. In the fungus, mRNA for plasma membrane ATPase was detected in spores and all infection structures. Two genes for fungal amino acid transporters showed dissimilar regulation. Transcripts for one were detected during all developmental stages, whereas those of the other appeared to be under developmental control. The latter result was also obtained for the so far only hexose transporter known from U. fabae and for one gene of the thiamine biosynthesis pathway. In the host plant, transcripts for two ATPases analyzed generally declined upon infection. Sucrose synthase expression increased in leaves, but decreased in roots. Transcript levels of glucose and sucrose transporter genes appeared unchanged. Markers for amino acid metabolism did not show a uniform trend: transcripts for asparagine synthetase increased, whereas those for two amino acid transporters either decreased or increased. Our analyses revealed that not only expression of genes in the immediate vicinity of the primary infection site is altered, but infection also influences transcription of certain genes in remote organs, like stems and roots. This demonstrates alterations in the source-sink relationships.

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

2001 ◽  
Vol 98 (14) ◽  
pp. 8133-8138 ◽  
Author(s):  
R. T. Voegele ◽  
C. Struck ◽  
M. Hahn ◽  
K. Mendgen
Keyword(s):  
Broad Bean ◽  
Rust Fungus ◽  
Uromyces Fabae

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

1997 ◽  
Vol 10 (4) ◽  
pp. 438-445 ◽  
Author(s):  
Matthias Hahn ◽  
Ulrike Neef ◽  
Christine Struck ◽  
Michael Göttfert ◽  
Kurt Mendgen
Keyword(s):  
Amino Acid ◽  
Plasma Membranes ◽  
Rust Fungus ◽  
Amino Acid Transporter ◽  
Infected Leaf ◽  
Molecular Evidence ◽  
Special Role ◽  
Coding Region ◽  
Uromyces Fabae ◽  
Acid Transporter

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 membrane fractions of isolated haustoria but not of germinated rust spores. With immunofluo-rescence 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.

scholarly journals Cloning and Characterization of a Novel Invertase from the Obligate Biotroph Uromyces fabae and Analysis of Expression Patterns of Host and Pathogen Invertases in the Course of Infection

2006 ◽  
Vol 19 (6) ◽  
pp. 625-634 ◽  
Author(s):  
Ralf T. Voegele ◽  
Stefan Wirsel ◽  
Ulla Möll ◽  
Melanie Lechner ◽  
Kurt Mendgen
Keyword(s):  
Cell Wall ◽  
Expression Analysis ◽  
Biochemical Characterization ◽  
Higher Plants ◽  
Rust Fungus ◽  
Expression Patterns ◽  
Invertase Activity ◽  
Pathogen Infection ◽  
Uromyces Fabae

Invertases are key enzymes in carbon partitioning in higher plants. They gain additional importance in the distribution of carbohydrates in the event of wounding or pathogen attack. Although many researchers have found an increase in invertase activity upon infection, only a few studies were able to determine whether the source of this activity was host or parasite. This article analyzes the role of invertases involved in the biotrophic interaction of the rust fungus Uromyces fabae and its host plant, Vicia faba. We have identified a fungal gene, Uf-INV1, with homology to invertases and assessed its contribution to pathogenesis. Expression analysis indicated that transcription began upon penetration of the fungus into the leaf, with high expression levels in haustoria. Heterologous expression of Uf-INV1 in Saccharomyces cerevisiae and Pichia pastoris allowed a biochemical characterization of the enzymatic activity associated with the secreted gene product INV1p. Expression analysis of the known vacuolar and cell-wall-bound invertase isoforms of V. faba indicated a decrease in the expression of a vacuolar invertase, whereas one cell-wall-associated invertase exhibited increased expression. These changes were not confined to the infected tissue, and effects also were observed in remote plant organs, such as roots. These findings hint at systemic effects of pathogen infection. Our results support the hypothesis that pathogen infection establishes new sinks which compete with physiological sink organs.

scholarly journals Characterization of a novel NADP+-dependent D-arabitol dehydrogenase from the plant pathogen Uromyces fabae

2005 ◽  
Vol 389 (2) ◽  
pp. 289-295 ◽  
Author(s):  
Tobias Link ◽  
Gertrud Lohaus ◽  
Ingrid Heiser ◽  
Kurt Mendgen ◽  
Matthias Hahn ◽  
...  
Keyword(s):  
Plant Pathogen ◽  
Broad Bean ◽  
Rust Fungus ◽  
Plant Defence ◽  
In Vitro System ◽  
Uromyces Fabae ◽  
Factor Specificity ◽  
Defence Reactions

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.

Transient transformation of the rust fungus Puccinia graminis f. sp. tritici

2000 ◽  
Vol 262 (6) ◽  
pp. 911-915 ◽  
Author(s):  
S. Schillberg ◽  
R. Tiburzy ◽  
R. Fischer
Keyword(s):  
Rust Fungus ◽  
Puccinia Graminis ◽  
Transient Transformation

scholarly journals Microarray analysis of expressed sequence tags from haustoria of the rust fungus Uromyces fabae

2006 ◽  
Vol 43 (1) ◽  
pp. 8-19 ◽  
Author(s):  
Mirza Jakupović ◽  
Manuel Heintz ◽  
Peter Reichmann ◽  
Kurt Mendgen ◽  
Matthias Hahn
Keyword(s):  
Microarray Analysis ◽  
Expressed Sequence Tags ◽  
Rust Fungus ◽  
Uromyces Fabae ◽  
Expressed Sequence

scholarly journals 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

1998 ◽  
Vol 11 (6) ◽  
pp. 458-465 ◽  
Author(s):  
Christine Struck ◽  
Claudia Siebels ◽  
Oliver Rommel ◽  
Marcus Wernitz ◽  
Matthias Hahn
Keyword(s):  
Plasma Membrane ◽  
Rust Fungus ◽  
Single Gene ◽  
Functional Expression ◽  
Wild Type ◽  
Ph Optimum ◽  
Uromyces Fabae ◽  
Mutant Derivative ◽  
Regulatory Properties

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 (Δ76) 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 Δ76 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.

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