In vitro differentiation of haustorial mother cells of the wheat stem rust fungus, Puccinia graminis f. sp. tritici, triggered by the synergistic acti

Copyright © 2003 Elsevier Science (USA). All rights reserved.

In vitro differentiation of haustorial mother cells of the wheat stem rust fungus, Puccinia graminis f. sp. tritici, triggered by the synergistic action of chemical and physical signals

Nicola Wiethölter, Susanne Horn, Katrin Reisige, Ursula Beike and Bruno M. Moerschbacher,

Department of Plant Biochemistry and Biotechnology, Westfälische Wilhelms-Universität Münster, Hindenburgplatz 55, 48143, Münster, Germany


Received 17 July 2002; accepted 19 September 2002. ; Available online 11 March 2003.

Abstract
Biotrophic plant pathogenic fungi often develop a sophisticated series of infection structures for non-destructive host tissue penetration. In vitro, early infection structures of rust fungi—germ tube, appressorium, substomatal vesicle, infection hyphae—can easily be induced, but in vitro differentiation rates of late infection structures—haustorial mother cells (hmc), haustoria—are low at best. Under appropriate conditions (humid atmosphere), a combination of physical (mild heat shock) and chemical signals (trans-2-hexen-1-ol) induced the in vitro differentiation of hmc in the wheat stem rust fungus, Puccinia graminis f. sp. tritici. Around two thirds of the in vitro differentiated germlings developed up to three hmc which were cytologically identical to hmc formed in planta. Efficient in vitro differentiation of hmc will allow us to analyse in molecular detail the processes involved in the induction and differentiation of this critically important developmental stage of the economically important plant pathogenic rust fungi.

Author Keywords: Puccinia graminis f. sp. tritici; Wheat stem rust fungus; Haustorial mother cells; Biotrophic fungi; Leaf alcohol; Trans-2-hexen-1-ol; In vitro differentiation; Infection structures

Article Outline
1. Introduction
2. Materials and methods
2.1. Origin of fungal material
2.2. Induction of infection structures
2.3. Staining and microscopy
3. Results
4. Discussion
Acknowledgements
References
1. Introduction
Rust fungi are obligately biotrophic plant pathogens that are highly specialised for growth and development on and in their respective host plant tissues. Their biotrophic nature requires careful host tissue penetration and colonisation in order to prevent premature recognition by the host cells and the ensuing triggering of induced resistance mechanisms such as hypersensitive host cell death. Uredospore germlings of most rust fungi enter their host tissues via the natural openings of the plant stomates, thus preventing host tissue wounding (Mendgen et al., 1996).

Uredospores germinating on a plant surface produce a germ tube which tightly adheres to the cuticle, apparently a prerequisite for oriented germ tube growth towards the stomates (Dickinson, 1969; Maheshwari and Hildebrandt, 1967; Wynn, 1976). Upon reaching a stoma, tip growth of the germ tube is arrested and an appressorium is formed on top of the stoma ( Allen, 1923 and Allen, 1926). The appressorium containing the two fungal nuclei which emerged from the spore is separated from the germ tube by a septum, and a first round of synchronised mitoses occurs in the appressorium. From the appressorium, a narrow penetration peg grows through the stomatal opening and develops into a vesicle in the substomatal cavity.

The cell wall of penetration peg and vesicle differs from the cell wall of germ tube and appressorium (Harder et al., 1986; Littlefield and Heath, 1979). The cytoplasmic content of the appressorium is transferred into the substomatal vesicle where a second round of synchronised mitoses occurs, leading to a total of eight nuclei. Infection hyphae emanating from the vesicle start growing in the intercellular spaces of the host tissue, and pairs of fungal nuclei migrate into these hyphae ( Allen, 1923; Heath and Heath, 1976; Staples et al., 1975). When the tip of an infection hypha reaches the cell wall of an epidermal host cell, tip growth is arrested and a haustorial mother cell is formed. The haustorial mother cell usually containing 2–4 fungal nuclei is separated from the infection hypha by a septum ( Heath and Heath, 1975; Heath et al., 1996). From the haustorial mother cell, a narrow haustorial neck grows through the host plant’s cell wall and develops into a haustorium in the periplasmic space of the host cell. A second change in cell wall characteristics occurs upon differentiation of haustorial mother cells ( Chong and Harder, 1982; Chong et al., 1985 and Chong et al., 1986). Infection hyphae branch just proximal to the haustorial mother cell septum, and the branches develop new haustorial mother cells at their tips upon host mesophyll cell encounters. The infection hyphae of the rust mycelium remain extracellular throughout host tissue colonisation and eventual fungal sporulation. Only the terminally differentiated haustoria reach into the pericellular space of the host cells.

Quite apparently, the sophisticated series of infection structures—germ tube, appressorium, substomatal vesicle, infection hyphae, haustorial mother cells, haustoria—are a prerequisite for the obligately biotrophic rust fungi to penetrate, colonise, and feed from the host plant tissue with a minimum of host cell perturbation. The unique process of infection structure differentiation can be expected to be an absolute conditio sine qua non for the successful rust development and as such, to provide promising targets for the development of novel fungicides.

Rust uredospores readily germinate in the presence of liquid water, producing a germ tube tightly adhering to any hydrophobic surface. Also, the differentiation of infection structures can be induced in vitro when appropriate chemical or physical signals, such as an appropriately structured hydrophobic surface, are provided (Dickinson, 1969; Heath and Perumalla, 1988; Hoch and Staples, 1987; Mendgen et al., 1996; Read et al., 1992). Unlike the in vivo situation, where a series of independent signals can be assumed to regulate the development of infection structures on and in the host leaf ( Heath, 1997; Mendgen, 1982), a single trigger may induce the consecutive differentiation of appressorium, substomatal vesicle, infection hyphae and, albeit often at low frequency only, haustorial mother cells in vitro ( Deising et al., 1991; Heath and Perumalla, 1988). The cowpea rust fungus, Uromyces vignae, has even been reported to build some rare haustoria in vitro upon appropriate chemical triggers (Heath, 1990).

Topographical signals are less effective in triggering the differentiation of infection structures of rust fungi specialised on monocotyledonous host plants, such as the wheat stem rust fungus (Allen et al., 1991; Read et al., 1997). Instead, a number of other physical and chemical signals such as a mild heat shock ( Dunkle and Allen, 1971; Emge, 1958; Maheshwari et al., 1967), organic compounds ( Macko et al., 1978), host epicuticular waxes ( Daniels, 1996; Grambow, 1977; Grambow and Grambow, 1978; Grambow and Riedel, 1977), or leaf volatiles ( Daniels, 1996; Grambow, 1977; Grambow and Riedel, 1977) have been reported to trigger the sequential in vitro development of appressoria, substomatal vesicles, and infection hyphae of the wheat stem rust fungus. Recently, a combination of surface ridges of appropriate sizes and spacings with trans-2-hexen-1-ol was shown to act synergistically in inducing these infection structures (Collins et al., 2001). However, the wheat stem rust fungus does not easily differentiate haustorial mother cells in vitro. We here report on the reproducible, high frequency differentiation of haustorial mother cells of the wheat stem rust fungus in vitro, by the synergistic action of a physical and a chemical signal.

2. Materials and methods
2.1. Origin of fungal material
Uredospores of the wheat stem rust fungus Puccinia graminis f. sp. tritici Eriks. & Henn., race 32, were collected from fully susceptible wheat plants Triticum compactum L. cv. Little Club. Uredospores were frozen in liquid nitrogen and stored at −70 °C. Spores were reactivated for 3 min at 43 °C.

2.2. Induction of infection structures
For the induction of infection structures of the wheat stem rust fungus (appressoria, substomatal vesicles, infection hyphae and haustorial mother cells), 0.5 mg of uredospores were distributed with a brush in the lid of a Petri dish (Ø 6 cm, Greiner, Frickenhausen). Five milliliters of trans-2-hexen-1-ol (Aldrich, Taufkirchen) (0.5 mM in doubly distilled water) was filtered (pore Ø 0.22 μm, cellulose ester membrane, Fisherbrand, Schwerte; 30 ml Luer Lock syringe, Plastipak, Becton–Dickinson, Heidelberg) into the bottom of the Petri dishes. The spore-containing lids were replaced on the Petri dish bottoms and sealed with parafilm (American National Can, Baltimore, Maryland) to prevent evaporation. The Petri dishes were placed in a temperature controlled chamber (Heraeus, Hanau) at 23 °C where the uredospores started to germinate. When the average length of the germ tubes had reached the size of the spore diameter (approx. 50–60 min) the germlings were given a mild heat shock at 30 °C for 2 h (Emge, 1958; Maheshwari et al., 1967), followed by further incubation at 23 °C. All incubation steps were carried out in darkness.

2.3. Staining and microscopy
Fungal infection structures were analyzed using an Olympus BX 40 and a Leica DM RBE microscope both equipped with epifluorescence, and an Olympus IX 50 inverted microscope with phase contrast optics. The filter module U-MNV (excitation filter BP 400–410, dichroic mirror DM 455, barrier filter BA 455 nm) was used for fluorescence microscopy using the Olympus BX 40. Fluorescence microscopy using the Leica DM RBE was carried out with the excitation filter BP 450–490 nm, the dichroic mirror RKP 510 nm and the barrier filter LP 515 nm. Microphotographs were taken on Agfa CT Precisa slide film. Cell walls of the fungus were stained using Calcofluor White (Sigma, Taufkirchen) (Maeda and Ishida, 1967). For this procedure, a stock solution (2.5 mg ml−1 in water) was diluted 1:20 before use. The fungus was incubated with this staining solution for 30 s and washed 10 times with water. Fungal nuclei could be stained afterwards using DAPI (4′,6-Diamin-2′-phenylindol-dihydrochloride; Sigma, Taufkirchen) (0.01 μgml−1 DAPI in water, 10 s) then washed 10 times with water (Butt et al., 1989).

3. Results
Germlings of the wheat stem rust fungus, Puccinia graminis f. sp. tritici, were treated with trans-2-hexen-1-ol, either as a volatile when the germlings were grown in a humid atmosphere, or in dissolved form when the germlings were grown immersed in a liquid medium. In both cases, trans-2-hexen-1-ol induced the differentiation of an appressorium, a substomatal vesicle, and one or two infection hyphae within 24 h. Fig. 1 shows that in a humid atmosphere, induction by trans-2-hexen-1-ol also led to the formation of haustorial mother cells in about 10% of the germlings within about three days. In contrast, no haustorial mother cells were formed when the germlings were grown submerged in a trans-2-hexen-1-ol solution (data not shown).



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Fig. 1. Time course of in vitro differentiation of infection structures by Puccinia graminis f. sp. tritici uredospores induced by the application of the volatile leaf alcohol trans-2-hexen-1-ol (0.5 mM). Around 20% of the sporelings produced infection structures within 24 h, and about half of these formed haustorial mother cells. Symbols represent appressoria (•), substomatal vesicles (▪), infection hyphae (), and haustorial mother cells (). Data given are means ± SD of three independent experiments, with a minimum of 80 sporelings counted per time point in each experiment.


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In vitro differentiated haustorial mother cells of the wheat stem rust fungus appeared as small (15 μm in length), granular structures at the end of the infection hypha, from which they were clearly separated by a septum (Fig. 2E). Their cell walls appeared thicker than those of the infection hypha, and the fluorescent brightener Calcofluor White bound more strongly, leading to bright fluorescence under UV-light. Invariably, two nuclei were observed in each haustorial mother cell (Fig. 2E). These typical characteristics easily allowed their unequivocal identification, e.g. as compared to stress-induced terminal swellings of infection hyphae ( Fig. 2D).


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Fig. 2. In vitro differentiation of infection structures including haustorial mother cells by the wheat stem rust fungus Puccinia graminis f. sp. tritici after application of a mild heat shock (2 h, 30 °C) and trans-2-hexen-1-ol (0.5 mM) in a humid atmosphere. (A) and (B) represent fully differentiated germlings using bright field and phase contrast optics, respectively (gt, germ tube; ap, appressorium; sv, substomatal vesicle; ih, infection hypha; hmc, haustorial mother cell) (bar: 15 μm). When fungal structures were stained with DAPI (E, F) and Calcofluor (C–F), nuclei, septa, and cell wall alterations were visible under fluorescent light, and haustorial mother cells were clearly distinguishable from terminal swellings of infection hyphae (D). When differentiation was induced by trans-2-hexen-1-ol alone, appressoria and subsequent infection structures formed at the end of a long and often branched germ tube (C). When trans-2-hexen-1-ol was combined with a heat shock, infection structures were formed much faster at the end of a short germ tube only (A, B, D–F). When germlings were immersed in liquid culture medium after infection structures had formed (A, F), an occasional outgrowth of haustorial mother cells was observed (F).


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One of the most effective ways of inducing infection structure differentiation in the wheat stem rust fungus is a mild heat shock given shortly after germination. This treatment is active with germlings growing submerged in liquid or in a humid atmosphere, but no haustorial mother cells formed even after prolonged times of incubation (data not shown). However, we found that a combination of the chemical signal trans-2-hexen-1-ol (0.5 mM) and the physical signal of a mild heat shock (30 °C, 2 h) effectively triggered the induction of haustorial mother cells at high frequency, when the germlings were grown in a humid atmosphere. Fig. 3 gives the time course of appearance of the different infection structures induced.


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Fig. 3. Time course of in vitro differentiation of infection structures by Puccinia graminis f. sp. tritici uredospores induced by a mild heat shock (2 h, 30 °C, grey bar) and the application of the volatile leaf alcohol trans-2-hexen-1-ol (0.5 mM). Appressoria (•) started to appear during the heat shock. Substomatal vesicles (▪) and infection hyphae () were first observed 2 and 8 h after the end of the heat shock, respectively. First haustorial mother cells () were formed between 21 and 45 h after the heat shock. Data given are means ± SD of three independent experiments, with a minimum of 80 germlings counted per time point in each experiment.


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Tip growth of the germ tube was stopped by the heat shock so that the germlings had a compact shape (Figs. 2A, B, D–F). Under these conditions, about 60% of the germinated uredospores had differentiated appressoria, substomatal vesicles, and infection hyphae 24 h after germination. Haustorial mother cells emerged 24–48 h after germination, and a maximum hmc-rate of 40% was reached 72 h after spore germination (Fig. 3), after which time no further increase was observed.

Often, haustorial mother cells developed at the tip of both infection hyphae of a rust germling. Some infection hyphae branched just proximal to the haustorial mother cell septum, and a third haustorial mother cell might then form at the tip of the branch (Figs. 2A and B). Fungal growth and development then stopped; we never observed more than three haustorial mother cells on a single germling. In order to test whether failure of further growth could be attributed to exhaustion of the nutrient pool of the uredospores, we dissolved the trans-2-hexen-1-ol in axenic culture medium (Fasters et al., 1993) instead of water, and inverted the Petri dishes at different times after germination, bringing the germlings into contact with the nutrient solution. As a control, Petri dishes with trans-2-hexen-1-ol in water were inverted at the same times. No substantial further growth was observed even in the axenic culture medium. Immersion of the germlings at earlier times after germination (7, 12, or 48 h) prevented or stopped further development of haustorial mother cells.

In axenic medium, we occasionally observed outgrowth of a fungal hypha from the differentiated haustorial mother cell (Fig. 2F). We never observed the differentiation of haustoria from the haustorial mother cells induced in vitro. Failure to resume sustained growth may be due to the trans-2-hexen-1-ol present in the medium, as growth did resume in pure culture medium. However, without the addition of trans-2-hexen-1-ol, no haustorial mother cells were formed.

4. Discussion
In vitro differentiation of haustorial mother cells has been reported previously for several rust species, including the bean rust fungus, Uromyces appendiculatus (Maheshwari et al., 1967), the cowpea rust fungus, U. vignae (Heath and Perumalla, 1988; Stark-Urnau and Mendgen, 1993), and the broad bean rust fungus, Uromyces viciae-fabae (Deising et al., 1991). In these cases, a single inductive signal (oil-collodion membranes or scratched polyethylene membranes) led to the formation of haustorial mother cells in 10–25% of the germlings.

In vitro induction of haustorial mother cell differentiation of the wheat stem rust fungus appears to be strictly dependent on (i) the development in a humid atmosphere—no haustorial mother cells were observed when the fungus grew submerged in water or liquid axenic medium—and (ii) on the presence of a suitable topographical signal (Read et al., 1997) or of a volatile chemical inductor, such as the leaf alcohol trans-2-hexen-1-ol. Under such conditions of a single inductive signal, haustorial mother cell differentiation was sporadic and occurred at low frequency only. A reproducible high frequency induction of haustorial mother cells was reached by the combination of a mild heat shock—which given alone induced appressoria, substomatal vesicles, and infection hyphae, but never haustorial mother cells—with the volatile inductor trans-2-hexen-1-ol. Under these conditions, about two thirds of the germlings developed infection structures, and about two thirds of these differentiated up to three haustorial mother cells. The high frequency of induction combined with the formation of multiple haustorial mother cells per germling allows an unprecedented high number of haustorial mother cells to be differentiated in vitro.

In planta, haustorial mother cells of the wheat stem rust fungus are oval, long and slender (Allen, 1923; Niks, 1986). They are separated from the infection hypha by a septum, they are characterised by an optically dense appearance, and they contain two nuclei ( Allen, 1923). The cell wall of in planta grown haustorial mother cells of the wheat stem rust fungus is more complex than that of the infection hyphae, containing additional layers which are also present in the septum, leading to increased stainability with Calcofluor ( Chong et al., 1985). Haustorial mother cells of the wheat stem rust fungus formed in vitro exhibited all of these typical characteristics.

Although the morphogenetically active signals inducing infection structure differentiation in different rust fungi differ in detail, the genetic program induced appears to share similarities. In vitro, a single trigger usually induces the sequential differentiation of appressoria, substomatal vesicles, and infection hyphae at high frequency, while the differentiation rarely extends to the building of haustorial mother cells. In planta, however, those rust fungi that do not penetrate closed stomata in the dark, e.g. Puccinia graminis f. sp. tritici, differentiate an appressorium upon encounter of a closed stoma, but the development of further infection structures is arrested until the stoma opens (Wynn and Staples, 1981). Moreover, the subsequent development of the infection hyphae appears to respond to additional host factors, as e.g. the intercellular infection hyphae of the oat crown rust fungus have been shown to grow directly into the mesophyll of an infected oat leaf, while they grow parallel to the leaf surface in an infected wheat leaf ( Moerschbacher et al., 1990). Clearly, the morphogenetic program of sequential infection structure differentiation is naturally triggered and regulated by a number of host derived signals.

The concept of multiple recognition extends to the differentiation of haustorial mother cells and, most likely at least, also of haustoria (Heath, 1997). Surface signals from the plant cell walls have been implicated in these differentiation steps ( Fasters et al., 1993; Heath, 1990; Mendgen, 1978 and Mendgen, 1982). While the exact nature of these signals acting in planta is not yet known, the wheat stem rust fungus appears to be a suitable object to study in vitro the combined action of different signals in the triggering of infection structure differentiation. It has been shown that the volatile leaf alcohol trans-2-hexen-1-ol acts synergistically with an inductive factor from epicuticular waxes of the host leaf (Grambow, 1977; Grambow and Grambow, 1978; Grambow and Riedel, 1977), and with topographical signals mimicking a gramineaceous stoma ( Collins et al., 2001). We are currently studying the combined action of all three of these factors on rust differentiation in vitro. We have shown in this study that a mild heat shock combined with the volatile inductor leads to the differentiation of haustorial mother cells. It will be difficult to identify the presumably chemical in planta equivalent of the thermal signal as haustorial mother cells did not develop in submerged culture so that application of putative signal molecules is difficult. We are currently developing methods to reproducibly deposit known amounts of putative non-volatile chemical inductors on appropriate surfaces so that we can study their morphogenetic effects on rust development in a humid atmosphere.

Acknowledgements
We gratefully acknowledge many fruitful discussions with Dr. Nick Read, University of Edinburgh, where some preliminary experiments to this study were carried out.

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