Mendgen, Kurt
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The haustorial transcriptomes of Uromyces appendiculatus and Phakopsora pachyrhizi and their candidate effector families
Haustoria of biotrophic rust fungi are responsible for the uptake of nutrients from their hosts and for the production of secreted proteins, known as effectors, which modulate the host immune system. The identification of the transcriptome of haustoria and an understanding of the functions of expressed genes therefore hold essential keys for the elucidation of fungus–plant interactions and the development of novel fungal control strategies. Here, we purified haustoria from infected leaves and used 454 sequencing to examine the haustorial transcriptomes of Phakopsora pachyrhizi and Uromyces appendiculatus, the causal agents of soybean rust and common bean rust, respectively. These pathogens cause extensive yield losses in their respective legume crop hosts. A series of analyses were used to annotate expressed sequences, including transposable elements and viruses, to predict secreted proteins from the assembled sequences and to identify families of candidate effectors. This work provides a foundation for the comparative analysis of haustorial gene expression with further insights into physiology and effector evolution.
Nutrient uptake in rust fungi : how sweet is parasitic life?
A better understanding of the fundamental principles of host-pathogen interactions should enable us to develop new strategies to control disease and to eliminate or at least manage their causative agents. This is especially true for obligate biotrophic parasites like the rust fungi. One vital aspect in the field of obligate biotrophic host-pathogen interactions is the mobilization, acquisition and metabolism of nutrients by the pathogen. This includes transporters necessary for the uptake of nutrients as well as enzymes necessary for their mobilization and metabolism. In a broader sense effector molecules reprogramming the host or triggering the infected cell into metabolic shifts favorable for the pathogen also play an important role in pathogen alimentation.
The Uredinales : Cytology, Biochemistry, and Molecular Biology
Fungi belonging to the order Uredinales are commonly referred to as rust fungi. All members of the Uredinales are parasitic on plants, often causing dramatic losses in various important crop plants (Alexopoulos et al. 1996). Together with the powdery mildew fungi and the downy mildew-causing oomycetes, rust fungi form an extremely successful group of parasites, the obligate biotrophs. The term obligate biotrophic characterizes a specific lifestyle in which the pathogen is absolutely dependent on a living host to complete its life cycle. In turn, the host plant as a whole usually suffers only limited damage over an extended period of time (Staples 2000). By contrast, necrotrophic parasites kill their hosts quickly after infection and subsequently thrive on the dead plant material (Staples 2001). Hemibiotrophic fungi, such as Colletotrichum spp., are characterized by a more or less extended biotrophic phase before switching to necrotrophic growth and killing their host (Perfect and Green 2001). In order to separate the true obligate biotrophic pathogens from hemibiotrophs and necrotrophs we suggest the following six criteria:
1. Obligate biotrophs are not culturable in vitro (at least not to a point representing the parasitic phase)
2. They form highly differentiated infection structures (variations of the normally tubular cell shape, which are necessary for pathogenesis)
3. They have limited secretory activity
4. They establish a narrow contact zone separating fungal and plant plasma membranes
5. They engage in a long-term suppression of host defense responses
6. They form haustoria (specialized hyphae that penetrate host cells).
The peculiarities of the lifestyle of obligate biotrophs, paired with their huge economic impact, make rust fungi a versatile field of study at both the fundamental and the applied level. This chapter on Uredinales can by no means cover the complete literature on rust fungi. It is intended to summarize key references, review articles, and books to provide the interested reader with
a gateway to more specialized literature on most aspects of research involving rust fungi. Readers new to the field are encouraged to consult the excellent textbooks by Alexopoulos et al. (1996) and Webster and Weber (2007) to gain easier access into the exciting field of mycology in general and obligate biotrophic plant parasites like the rust fungi in particular.
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 (Darabitol 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.
A novel structural effector from rust fungi is capable of fibril formation
It has been reported that filament‐forming surface proteins such as hydrophobins are important virulence determinants in fungi and are secreted during pathogenesis. Such proteins have not yet been identified in obligate biotrophic pathogens such as rust fungi. Rust transferred protein 1 (RTP1p), a rust protein that is transferred into the host cytoplasm, accumulates around the haustorial complex. To investigate RTP1p structure and function, we used immunocytological, biochemical and computational approaches. We found that RTP1p accumulates in protuberances of the extra‐haustorial matrix, a compartment that surrounds the haustorium and is separated from the plant cytoplasm by a modified host plasma membrane. Our analyses show that RTP1p is capable of forming filamentous structures in vitro and in vivo. We present evidence that filament formation is due to β–aggregation similar to what has been observed for amyloid‐like proteins. Our findings reveal that RTP1p is a member of a new class of structural effectors. We hypothesize that RTP1p is transferred into the host to stabilize the host cell and protect the haustorium from degradation in later stages of the interaction. Thus, we provide evidence for transfer of an amyloid‐like protein into the host cell, which has potential for the development of new resistance mechanisms against rust fungi.
Immunolocalization of pathogen effectors
The use of polyclonal antibodies enables the detection of proteins on a cellular and even subcellular level. Immunolocalization can be used on all pathosystems even if one or both partners of the interaction are unamenable to molecular tools like transformation. This chapter provides detailed information about how to obtain high quality antibodies, how to prepare samples, and finally how to detect the proteins. Methods for light and electron microscopy are presented.
Impact of genomics on fungal biology
Fungi represent an extremely diverse and complex class of organisms, and their categorization as lower eukaryotes should by no means be mistaken as meaning low-end. At present, fungi serve as model systems for various aspects of molecular and cellular biology, for example cell cycle regulation, intracellular signaling, metabolic pathway analysis and transcriptional regulation (Feldbrügge et al., 2004; Jiang, 2006; Oliver, 2006). They are also increasingly used on an industrial scale in the production of chemical compounds or in bioremediation (Grimm et al., 2005; Tortella et al., 2005). Some of the most recent and exciting advances within the field of fungal biology have been linked with genomic studies. To explore these, the IXth International Fungal Biology Conference & 16th New Phytologist Symposium entitled Impact of Genomics in Fungal Biology was held in Nancy, France (http://www.newphytologist. org/fungal-genomics/default.htm). The meeting brought together nearly 100 scientists, from all areas of fungal research, and highlighted a wide range of impacts that genome sequencing has and will have on our understanding of fungal biology.
The rust transferred proteins : a new family of effector proteins exhibiting protease inhibitor function
Only few fungal effectors have been described to be delivered into the host cell during obligate biotrophic interactions. RTP1p, from the rust fungi Uromyces fabae and U. striatus, was the first fungal protein for which localization within the host cytoplasm could be demonstrated directly. We investigated the occurrence of RTP1 homologues in rust fungi and examined the structural and biochemical characteristics of the corresponding gene products. The analysis of 28 homologues showed that members of the RTP family are most likely to occur ubiquitously in rust fungi and to be specific to the order Pucciniales. Sequence analyses indicated that the structure of the RTPp effectors is bipartite, consisting of a variable N‐terminus and a conserved and structured C‐terminus. The characterization of Uf‐RTP1p mutants showed that four conserved cysteine residues sustain structural stability. Furthermore, the C‐terminal domain exhibits similarities to that of cysteine protease inhibitors, and it was shown that Uf‐RTP1p and Us‐RTP1p are able to inhibit proteolytic activity in Pichia pastoris culture supernatants. We conclude that the RTP1p homologues constitute a rust fungi‐specific family of modular effector proteins comprising an unstructured N‐terminal domain and a structured C‐terminal domain, which exhibit protease inhibitory activity possibly associated with effector function during biotrophic interactions.
Monitoring E. amylovora using Real Time PCR
Fire blight, caused by the Gram-negative bacterium Erwinia amylovora is the most damaging bacterial disease of pome fruit world wide. Disease outbreak is only sporadic, disease outcome, however, can be devastating. Aside from physical factors like temperature and humidity, presence of the pathogen in sufficient amounts to cause infection is a crucial aspect in abatement of disease. We have refined a real time PCR based quantification of E. amylovora to be used in the field on a variety of environmental samples. This method allowed us to monitor pathogen abundance during blossom during the last four years. Integration of the amount of pathogen detected into refined prediction algorithms will most certainly improve applied forecasting models. The method also allows the quantification of pathogen outside the blooming period within or on the surface of different tissue, or plant organs. This may allow a monitoring of the pathogen population throughout the year and in the long run may facilitate timely countermeasures. Results also indicate a potential role of fruit mummies in overwintering and dissemination of the pathogen early in the growing season, as well as a role of non-host flowers in the built up of pathogen potential. The method is also capable of monitoring disease progression in infected, but symptomless tissue. Results from this set of experiments might improve recommendations for good cultural practice.
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
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-wallbound invertase isoforms of V. faba indicated a decrease in the expression of a vacuolar invertase, whereas one cellwall-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.