A novel structural effector from rust fungi is capable of fibril formation

A novel structural effector from rust fungi is capable of fibrilformation

Eric Kemen1,†,*, Ariane Kemen1,†, Andreas Ehlers2, Ralf Voegele3 and Kurt Mendgen4

1Max Planck Institute for Plant Breeding Research, Carl von Linne Weg 10, Cologne 50829, Germany,2Department of Chemistry, Universitat Konstanz, Universitatsstraße 10, Konstanz 78457, Germany,3Fachgebiet Phytopathologie, Fakultat Agrarwissenschaften, Institut fur Phytomedizin, Universitat Hohenheim, Otto Sander

Straße 5, Stuttgart 70599, Germany, and4Phytopathologie, Universitat Konstanz, Universitatsstraße 10, Konstanz 78457, Germany

Received 5 April 2013; revised 29 May 2013; accepted 8 May 2013.

*For correspondence (e-mail [email protected]).†These authors contributed equally to this paper.

SUMMARY

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 b–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.

Keywords: rust transferred protein 1, effector protein, amyloid-like, rust fungi, biotrophy, haustorium.

INTRODUCTION

A characteristic feature of numerous plant pathogens is

their biotrophic lifestyle. Biotrophic pathogens depend on

a living host for successful colonization and completion of

their lifecycle (Mendgen and Hahn, 2002). Sequencing sev-

eral genomes of obligate biotrophic pathogens including

oomycetes and fungi has led to a broader understanding

of biotrophy. While metabolic pathways often show con-

vergent gains and losses between biotrophic pathogens,

the ‘effector’ complement has significantly diverged

(Kemen et al., 2011a; McDowell, 2011). Effectors are

defined as small secreted molecules/proteins that facilitate

infection as virulence factors on some hosts but trigger

defence responses as avirulence factors on others

(Hogenhout et al., 2009). Biotrophic fungi and oomycetes

form haustoria, specialized structures that provide intimate

contact with host cells (Voegele and Mendgen, 2003).

Haustoria are hyphal branches that penetrate the plant cell

wall and invaginate the host plasma membrane so that

pathogen and plant cytoplasm are separated by the haus-

torial membrane, the extra-haustorial matrix and a modi-

fied plant plasma membrane called the extra-haustorial

membrane (Mendgen and Hahn, 2002). Haustoria are con-

nected to intercellular hyphae by the haustorial neck. A

neckband seals the extra-haustorial matrix and membrane

from the apoplast and plant plasma membrane (Chong

et al., 1985). The extra-haustorial matrix represents a

unique structure for exchange of nutrients as well as the

transfer of effector proteins into the host, as has been

shown for rust transferred protein 1 (RTP1p) from Uromy-

ces sp. (Kemen et al., 2005) and AvrM from Melampsora

lini (Rafiqi et al., 2010). It has been postulated that the

extra-haustorial matrix may represent an equivalent to

the parasitophorous vacuole of apicomplexans (Birch

et al., 2006). For Plasmodium falciparum, parasitophorous

© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd

1

The Plant Journal (2013) doi: 10.1111/tpj.12237

membrane-derived structures called Maurer’s clefts (Tilley

et al., 2008) and a pore-like structure (de Koning-Ward

et al., 2009) have been described as being involved in

effector delivery. In comparison with the plant plasma

membrane, the extra-haustorial membrane lacks several

proteins and therefore appears smooth after freeze fractur-

ing in the electron microscope (Knauf et al., 1989). Tubular

invaginations of the extra-haustorial membrane reaching

far into the plant cytoplasm have been observed in several

rust–host interactions (Mims et al., 2002), but their function

remains elusive. Similar tubular structures connect the

parasitophorous vacuolar membrane with Maurer’s clefts

(Henrich et al., 2009). Little is known about the composi-

tion of the extra-haustorial matrix or the parasitophorous

vacuole, as isolating these membrane structures and their

contents is difficult due to limited knowledge of their com-

position. It has been reported that the extra-haustorial

matrix consists of a thin fungal wall and a matrix that con-

sists of plant cell wall-related carbohydrates and enzymes

(Mendgen and Hahn, 2002) but excludes fungal cell-wall

proteins of inter-cellular aerial hyphae and spores such as

hydrophobins or repellents (Teertstra et al., 2006). Hydro-

phobins are typical cell-wall proteins of hyphae that are

capable of forming amyloid-like filaments and higher

aggregates called ‘rodlets’ (Mackay et al., 2001). It has

been reported that these filament-forming surface proteins

are important virulence determinants that are secreted dur-

ing pathogenesis (Whiteford and Spanu, 2002; Kim et al.,

2005). This property of filament-forming amyloid-like sur-

face proteins is not only known for fungi, but has also

been reported for human pathogens such as P. falciparum

(MSP2) (Anders et al., 2009). Amyloid-like fibrils may be

formed by numerous proteins, showing high structural

heterogeneity (Udgaonkar and Kumar, 2010). The mecha-

nism that allows certain proteins to adopt rare conforma-

tional structures that allow amyloids to be formed was

described recently (Radford et al., 2011).

In this study, we show that RTP1p is a representative of

a new class of structural effectors that is able to form

filaments inside the extra-haustorial matrix and the host

cytoplasm. We further show that RTP1p accumulates in

sub-compartments of the extra-haustorial matrix and is

distributed throughout the host cytoplasm in late stages of

infection.

RESULTS

RTP1p specifically localizes to matrix protuberances at the

interface between pathogen and host

Uromyces fabae and Uromyces striatus RTP1p, two hausto-

ria-specific proteins, are localized inside the host cytoplasm

(Kemen et al., 2005). It was further found that transfer into

the host is dependent on the developmental stage of the

haustorium. Immunoelectron microscopy revealed that

RTP1p accumulates in the extra-haustorial matrix before

being transferred into the host cytoplasm (Kemen et al.,

2005). In this study, we performed detailed analyses to

identify sites of RTP1p localization and translocation. We

used a specific antibody that had been tested by Kemen

et al. (2005). Looking at the distribution of RTP1p within the

matrix using the ApoTome technique (Carl Zeiss, Jena, Ger-

many), we were able to reconstruct the 3D distribution of

the immuno signal. This method revealed that RTP1p pre-

dominantly accumulates in mature parts of the extra-haus-

torial matrix and not at the growing haustorial tip

(Figure 1a). No signal was detectable beyond the neck-

band (Figure 1a, arrow, and Figure S1a). Immunoelectron

microscopy revealed that RTP1p is predominantly local-

ized at the most outer layer of the extra-haustorial matrix

(Kemen et al., 2005). We used isolated haustoria that have

been stripped of the extra-haustorial membrane (Hahn

and Mendgen, 1992) to analyse the outermost layer of the

extra-haustorial matrix. Using electron microscopy

combined with anti-RTP1p antibodies and immunogold

labelling on whole-mount samples of isolated haustoria,

we detected a signal on the haustorial surface, including

labelling of protuberances arising from the extra-

haustorial matrix (Figure 1b).

We established a high resolution cryo-scanning electron

microscopy technique following high-pressure freezing to

further investigate the nature of the identified protuber-

ances and to exclude artefacts due to chemical fixation and

embedding. Applying this technique to infected plant

material, we were able to visualize protuberances of the

extra-haustorial matrix reaching far into the plant cyto-

plasm (Figure 1c). These protuberances were in close prox-

imity to the plant endomembrane system such as the

Golgi (Figure 1c, inset), as well as to the nuclear outer

membrane (Figure 1c, left arrow) and resembled those

described for other rust fungi and oomycetes (Harder and

Chong, 1984; Mendgen et al., 1991; Mims et al., 2004).

Consistent with previous findings (Mims et al., 2002), these

protuberances are restricted to mature parts of the hausto-

rium, and were not observed in young haustoria or the

growing tip of older haustoria.

To link structures seen in whole-mount samples with the

protuberances that RTP1p localizes to, we used high

pressure-frozen, freeze-substituted samples for immunolo-

calization. We detected RTP1p signals within protuber-

ances and close to the membrane of these structures

(Figure 1d and Figure S1b). To address whether our

observations are species-specific, we used the pathosys-

tem U. striatus on Medicago sativa to detect RTP1p

localization (UsRTP1p) and obtained comparable results

(Figure S2a,b).

In summary, we conclude that protuberances of the

extra-haustorial matrix are sub-compartments that reach

into the host cytoplasm and contain RTP1p.

© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), doi: 10.1111/tpj.12237

2 Eric Kemen et al.

RTP1p accumulates in the entire cytoplasm of infected

cells

Most interaction studies between haustoria-forming patho-

gens and their host are restricted to early stages of infection

(Heath, 1997; Catanzariti et al., 2007), although reproduction

and therefore completion of the lifecycle of obligate bio-

trophs relies on a much longer phase of interaction. To

close this gap in knowledge, we performed live-cell imaging

at various stages of haustorial differentiation (Figure 2 and

Table S1). We observed the plant nucleus moving towards

the haustorium upon haustorial cell penetration, but mov-

ing away after the initial penetration event. At this stage, we

did not detect any disturbance in cyclosis (Figure 2, tA, and

Video S1). Once the rust haustorium starts to show second-

ary growth, cyclosis slows down and chloroplasts start to

accumulate around the haustorium (Figure 2, tB). These

results are in accordance with previous observations (Heath

et al., 1997; Kemen et al., 2005). To ensure RTP1p is not dif-

fusing into the host cell due to a ruptured extra-haustorial

membrane, we tested the semi-permeability of this mem-

brane. We showed that the extra-haustorial membrane

osmotically expands in the presence of 0.9% NaCl and 2%

sucrose (Figure S3). In later stages of infection, when spo-

rogenous tissue starts to develop and mature haustoria

with extensive secondary growth and branching are visible

within the cells, cyclosis of the nucleus and chloroplasts

ceases (Figure 2, tC). We performed immunolocalization of

cells at stages where cyclosis of chloroplast and nucleus

ceased, and found that the whole cytoplasm shows high con-

centrations of RTP1p (Figure 3a–c).

Based on these findings indicating a correlation between

the localization of RTP1p and cessation of cyclosis, we

hypothesize that RTP1p may be involved in causing the

cessation in nuclear and chloroplast movement. Our

(a)

(c)

(d)

(b)Figure 1. Distribution of RTP1p in the extra-

haustorial matrix.

(a) Top view of an immunostained haustorium

(H) with a haustorial mother cell (HM) and

infection hyphae (IH) using the ApoTome tech-

nique for 3D reconstruction of RTP1p localiza-

tion. RTP1p is restricted to the haustorial matrix

of the mature haustorial body and absent from

the growing tip (asterisk). The neckband (arrow)

delimits RTP1p localization towards the hausto-

rial mother cell.

(b) Immunoelectron microscopy on isolated

haustoria reveals that RTP1p is localized to pro-

tuberances of the extra-haustorial matrix. H,

haustorium.

(c) Protuberances of the extra-haustorial matrix

reach far into the plant cytoplasm (arrows), and

are in close proximity to the plant endomem-

brane system (inset) and organelles as demon-

strated by high-resolution cryo-scanning

electron microscopy. N, nucleus; PZ, plant cell

wall; HV, haustorial vacuole; PV, plant vacuole;

H. haustorium; D, dictyosome; V, vesicles.

(d) Localization of RTP1p (arrows) to protuber-

ances in thin sections of U. fabae-infected

V. faba parenchymatic cells. PC, plant cyto-

plasm; H, haustorium; EHM, extra-haustorial

matrix. Sections were probed using purified

serum S744p in combination with secondary

Cy3 goat anti-rabbit antibody (a) or 10 nm gold-

labelled goat anti-rabbit antibody [(b) and (d)].

Scale bars = 2 lm (a), 20 nm (b), 1 lm (c),

400 nm (c, inset) and 100 nm (d).

© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), doi: 10.1111/tpj.12237

Novel structural effectors in rust fungi 3

live-cell imaging from early to late stages (Videos S1–S3)

reveals a strong cytoplasmic current that is still visible in

the form of fast-moving microbodies despite the fact that

movement of chloroplasts and nucleus ceases in late

stages of infection.

Incompatible interactions, tested using the pathosystem

U. striatus on M. truncatula ecotype GRC.098, revealed an

even stronger secretion of RTP1p once defence reactions are

induced (Figure 3d–f and Figure S4). We therefore hypothe-

size that RTP1p is of importance for the pathogen, especially

once defence cannot be efficiently suppressed anymore.

RTP1p forms filament-like structures in the extra-

haustorial matrix and within the plant cytoplasm

Using cross-linking experiments on infected plant material

and detection by anti-RTP1p antibodies, we revealed that

most RTP1p present in the native system exists either

bound to high-molecular-weight interaction partners or in

the form of multimers (Figure 4a). In addition to the high-

molecular-weight band, we detected monomers (approxi-

mately 25 kDa) and dimers (approximately 50 kDa). To

obtain more information about localization and association

as higher-order structures of RTP1p within the extra-hausto-

rial matrix and within the cytoplasm, we used two methods:

(i) a negative-stain electron microscopy whole-mount

technique on isolated haustoria to investigate RTP1p within

the extra-haustorial matrix, and (ii) a deep-etch

immunolocalization method to obtain insights from the

host cytoplasm. As RTP1p first accumulates within the

extra-haustorial matrix, we used ConA affinity purification

to isolate haustoria that have been stripped of their extra-

haustorial membrane (Hahn and Mendgen, 1992). Using

this approach, the extra-haustorial matrix became accessi-

ble to immunocytochemistry (Figure 4b,c). Immunogold

signal was detected across the matrix (Figure 4b) and asso-

ciated with protuberances extending from isolated hausto-

ria (Figure 1b). Close-ups of the matrix revealed signals

associated with filamentous structures (Figure 4c).

Despite our deep-etch method being destructive to

membranes, it enhances antigenicity of proteins and

therefore enables observation of proteins and protein

aggregates within the cytoplasm, particularly if structures

extend into three dimensions (Figure 4d). In the cyto-

plasm surrounding the haustorium, we identified microfil-

aments in close proximity to the extra-haustorial matrix

as previously described (Heath and Skalamera, 1997;

Takemoto et al., 2003). Immunogold grains labelling RTP1p

showed a pearl necklace-like localization within the host

Figure 2. Live-cell imaging at various stages of

U. fabae haustorial differentiation.

Snapshots from movie sequences taken from

three developmental stages of haustorial differ-

entiation (tA, tB and tC; see Videos S1–S3). tA,

undifferentiated haustorium (H) shortly after

penetration into the cell. The pathogen does

not have any impact on cyclosis (CS, cytoplasm

strand). tB, stage at which the haustorium

shows secondary growth and the haustorial

body starts to elongate. In this stage, chlorop-

lasts start to accumulate close to the hausto-

rium, and the nucleus (N) stays at the

haustorial body while surrounding it. tC, the

host cell is paralysed. Chloroplasts show close

association with the haustorial body, and the

nucleus, which is still closely associated with

the haustorium, is hampered in its movement.

All images were taken using differential inter-

ference contrast microscopy on V. faba leaf

disks infected by U. fabae. Scale bars = 2 lm.

© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), doi: 10.1111/tpj.12237

4 Eric Kemen et al.

cytoplasm, comparable to what was identified within the

extra-haustorial matrix (Figure 4d,e). Stronger contrasted

areas revealed that the immuno signal followed filament-

like structures of approximately 2 nm diameter (Fig-

ure 4f). These results indicate that either RTP1p is

attached to filamentous structures within the extra-haus-

torial matrix and the host cytoplasm, or is able to form fil-

amentous structures itself. As the signal in the cytoplasm

is lower than expected from immuno light microscopy, we

compared conventionally fixed samples used for immuno

light microscopy with high pressure-frozen samples used

for our deep-etch method by probing with anti-RTP1p and

anti-phosphoenolpyruvate carboxylase (PEPC), an antibody

recognizing the cytoplasmic enzyme phosphoenol pyruvate

carboxylase (Figure S5). There was no difference in recog-

nition of phosphoenol pyruvate carboxylase between sam-

ples but a significant difference in recognition of RTP1p,

indicating that cytoplasmic RTP1p is present in a folded

or multimeric configuration, and is only weakly

recognized by our antibody under such conditions.

Heterologously expressed RTP1p forms aggregates and

filaments

As RTP1p has been shown to be glycosylated (Kemen

et al., 2005) and over-expression in Escherichia coli results

in formation of inclusion bodies, we used the expression

system Pichia pastoris for structural studies. Performing

cross-linking experiments as described for the native pro-

tein but using purified heterologously expressed RTP1p,

we obtained similar results: RTP1p was detected as a

monomer and as multimers (Figure 5a). Even if no

cross-linker was added, we observed RTP1p precipitation

after several hours. These findings are independent of

glycosylation, as de-glycosylated protein still showed

aggregates in Western blotting (Figure S6), suggesting that

de-glycosylated homomers are sufficient for the formation

of high-molecular-weight multimers. Expression of full-

length UfRTP1p fused to GFP in the cytoplasm of P. pasto-

ris revealed that, in this heterologous system, RTP1p

shows punctate localization and is not distributed over the

(a)

(d) (e) (f)

(b) (c)

Figure 3. Immunolocalization of RTP1p in late stages of the biotrophic interaction and during post-haustorial incompatibility.

(a–c) Sections through U. fabae-infected V. faba parenchymatic cells probed using purified serum S744 in combination with secondary Cy3-labelled goat anti-

rabbit antibody. NU, nuclear counter-stain; IM, immunolocalization. (a) In late stages (Table S1, stage D), the haustorium branches (two branches of a hausto-

rium are visible close to the nucleus). During this stage, the entire cytoplasm contains RTP1p. (b) Not only do chloroplasts accumulate around the haustorium,

the whole cytoplasm starts to accumulate close to the haustorial body (Table S1, stage E). Only the vacuole does not contain any RTP1p and there is no leakage

to neighbouring non-infected cells. (c) Later in the development of the haustorium (Table S1, stage F), the nucleus starts to disintegrate, but cells infected by

U. fabae do not show signs of collapse of the tonoplast or leakage of RTP1p into neighbouring cells.

(d) Differential interference contrast image of parenchymatic M. truncatula ecotype GRC.098 cells infected with the incompatible rust U. striatus.

(e) UV autofluorescence of the same cells showing accumulation of phenolic compounds once the haustorium starts to differentiate (Table S1, stage C). While

the upper cell harbouring a haustorium that shows secondary growth in terms of haustorial elongation shows massive accumulation of fluorescent, probably

phenolic, compounds, the lower cell with an early haustorium does not.

(f) Immunolocalization of the same interaction showing localization of RTP1p, probed using purified serum S849 in combination with secondary Cy3-labelled

goat anti-rabbit antibody. Cells with strong defence reactions and accumulation of phenolic compounds show accumulation of RTP1p. In this ecotype, RTP1p is

even released into the intercellular space. An early infection in the cell below only shows RTP1p accumulation in the extra-haustorial matrix.

Scale bars = 5 lm. H, haustorium; N, nucleus; V, vacuole; IH, intercellular hyphae; HM, haustorial mother cell; IR, intercellular space.

© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), doi: 10.1111/tpj.12237

Novel structural effectors in rust fungi 5

cytoplasm (Figure S7a). The cytoplasmic spots are not

associated with degradation (Figure S7b), and probably

correspond to the cytoplasmic spots that have been impli-

cated in amyloid formation in Saccharomyces cerevisiae

(Alberti et al., 2009). Applying UfRTP1p to Vicia faba

protoplasts revealed a comparable localization, with RTP1p

being localized in speckles rather than freely diffused

within the cytoplasm (Figure S7c).

We identified amorphous aggregates by analysing the

precipitate of purified heterologously expressed RTP1p by

electron microscopy after negative staining (Figure 5b). We

then used the method described by Lee and Eisenberg

(2003) that allows conversion of amorphous aggregates of

prion proteins into filaments. This method (known as

‘seeded conversion’) resulted in RTP1p forming filament-

like structures (Figure 5c). The difference compared with

the method described by Lee and Eisenberg (2003) was

that RTP1p was able to form filaments de novo without

adding filaments as a starter. Using electron microscopy,

we found that the smaller twisted filaments formed larger

filamentous structures (Figure 5d). To confirm that our

antibodies were capable of detecting both forms, amor-

phous aggregates and filaments were mixed prior to

immunodetection (Figure 5e). High antigenicity was

observed for amorphous aggregates, while filamentous

structures showed varying antigenicity based on their

structural integrity. Folded filaments were barely detected,

but some signal was observed for partially unfolded

filaments. Completely unwound filamentous structures

showed the best antigenicity (Figure 5e,f).

We conclude that RTP1p is able to form filamentous

structures without seeding or the help of other proteins.

(a) (b) (c)

(d) (e)

(f)

Figure 4. RTP1p aggregation in the native sys-

tem.

(a) Dimers (47 kDa) and higher multimers (black

arrow) of RTP1p were detected using immuno-

blotting after cross-linking of U. fabae-infected

V. faba plant material.

(b) Immunolabelling of the extra-haustorial

matrix of whole-mount U. fabae haustoria

stripped of their extra-haustorial membrane

after ConA affinity purification.

(c) Close-up of the immuno signal on filamen-

tous structures in the matrix of purified hausto-

ria.

(d) Overview of the plant cytoplasm between

the extra-haustorial matrix and a chloroplast

after immunolocalization of RTP1p (arrows)

using the deep-etch method whereby samples

are embedded in acrylic resin that is entirely

removed using acetone to increase the accessi-

bility of antigens. Microfilaments were found in

close proximity to the extra-haustorial matrix.

(e) Close-up of (d) (hatched box) showing the

10 nm gold beads (arrows) close to a microfila-

ment.

(f) Filament-like structures and immunogold

beads (arrowheads) after extended uranylace-

tate staining to increase the contrast in the

plant cytoplasm.

All samples (b–f) were probed using S744 and

6 nm gold-labelled goat anti-rabbit antibody.

Scale bars = 50 nm (b), 20 nm (c), 200 nm (d),

50 nm (e) and 20 nm (f). PC, plant cytoplasm;

EHM, extra-haustorial matrix; CH, chloroplast;

MF, microfilament; H, haustorium.

© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), doi: 10.1111/tpj.12237

6 Eric Kemen et al.

A b–aggregation domain computationally identified in

RTP1p is able to form filaments

To unravel the filament-forming mechanism in RTP1p, we

performed computational analyses using Tango and Waltz

algorithms (Fernandez-Escamilla et al., 2004; Maurer-Stroh

et al., 2010). We identified two potential aggregation

domains in positions 139–151 (domain I) and 204–209

(domain II) (Figure S8). Based on Tango analysis and

further secondary structure predictions, domain I resem-

bled a b–aggregation domain consisting of two antiparallel

b–strands and one loop. To analyse properties of this

domain, we used a synthetic peptide covering positions

135–155 (RTP1p135-155). We added four amino acids to both

sides of the predicted aggregation domain to increase

solubility and stabilize the predicted structure. Using CD

spectroscopy, we confirmed our secondary structure pre-

dictions and revealed that the peptide has two antiparallel

b–strands with 40–50% of the amino acids contributing to

this structure, 15–25% being involved in b–turns, and

20–30% being involved in random coils (Figure 6a,b).

The numbers showed variability depending on the

solvent.

We observed macroscopic precipitates of RTP1p135-155

after 24 h. Using negative staining for electron microscopy,

we observed a precipitated peptide showing long filamen-

tous structures consistent with the predicted b–aggregationproperties (Figure 6c,d). As a control to determine whether

our method favours filament formation of peptides

non-specifically, we tested several RTP1p135-155 peptides in

which amino acids have been exchanged (Figure S9).

Replacing the phenylalanine in position 17 of the peptide

by alanine (RTP1p135-155 F151A) did not block filament for-

mation, but replacing the phenylalanine at position 7 by

alanine (RTP1p135-155 F141A) or replacing both phenylala-

nines (RTP1p135-155 F141A F151A) lead to a complete block of

filament formation. These results reveal sequence-

dependent aggregation and highlight a possible role for

phenylalanine at position 7 of RTP1p135-155 in filament for-

mation. We also used thioflavin T to obtain biochemical

evidence for b–aggregation of RTP1p135-155 and mutated

peptides. Thioflavin T shows fluorescence at 510 nm when

intercalated into filaments formed by b–aggregation(Voropai et al., 2003). We observed a significant increase in

fluorescence using RTP1p135-155, thus validating our

hypothesis (Figure S10). While the mutated form RTP1p135-

(a) (b)

(c) (d)

(e) (f)

Figure 5. Conversion of RTP1p aggregates into

filaments.

(a) High-molecular-weight amorphous aggre-

gates (arrow) of RTP1p after purification and

cross-linking detected using immunoblot

analysis.

(b) Electron microscopy analysis of the amor-

phous aggregates that form when RTP1p is

purified and concentrated from P. pastoris

supernatant after over-expression.

(c) Filament-like RTP1p structures after denatur-

ation of heterologous expressed and purified

RTP1p in 2.5 M guanidine/HCl, 3 M NaCl, 1 M

dithiothreitol, and a redox pH shift by two-step

dialysis into 50 mM sodium acetate.

(d) Close-up of filamentous structures.

(e) Immunolabelling of RTP1p after mixing

amorphous aggregates (AA) and filaments

(arrows). Solid black arrows show part of a

highly packed filament as in (c); solid white

arrows show partially unpacked structures.

(f) Pearl necklace-like immunogold signal indi-

cating an RTP1p filament.

The samples in (e) and (f) were probed using

S744 and 6 nm gold-labelled goat anti-rabbit

antibody.Scale bars = 25 nm (b), 100 nm (c),

50 nm (d), 50 nm (e) and 20 nm (f).

© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), doi: 10.1111/tpj.12237

Novel structural effectors in rust fungi 7

155 F151A showed only a minor reduction in endpoint

fluorescence (Figure S11a) and no significant difference in

aggregation rates (Figure S11b), fluorescence levels and

aggregation rates were low for RTP1p135-155 F141A, comple-

menting our electron microscopy studies that revealed fila-

ment formation for RTP1p135-155 and RTP1p135-155 F151A but

not RTP1p135-155 F141A.

To link these findings to in vivo aggregation, we used

a non-denaturating tissue printing method (modified

from Smallwood et al., 1994) on microscope slides, in

combination with a thioflavin T-based fluorescent detec-

tion method for amyloid-like proteins (Westermark et al.,

1999). The RTP1p immunofluorescence signal and the

thioflavin T signal co-localized in the matrix surrounding

the haustorium (Figure S12). Parts of the haustorium

that did not show RTP1p signal also did not show thio-

flavin T staining. This finding suggests that, in addition

to high concentrations of monomeric RTP1p, amyloid-

like RTP1p structures exist within the extra-haustorial

matrix.

We conclude that RTP1p forms aggregates and filamen-

tous structures within the extra-haustorial matrix and the

host cytoplasm based on b–aggregation.

DISCUSSION

RTP1p as a tool to study effector transfer into the host

Unlike bacterial effectors that are directly secreted into the

host cytoplasm via type III secretion systems (Galan and

Collmer, 1999), eukaryotic effectors have to cross at least

one membrane to enter the host cell after being secreted

by the pathogen (Catanzariti et al., 2007; Leborgne-Castel

et al., 2010). In obligate biotrophic interactions, the most

likely place of transfer is the extra-haustorial membrane

that separates the haustorium from the host cytoplasm

(Leborgne-Castel et al., 2010). We showed that, compara-

ble to effector localization within the biotrophic interfacial

complex of Magnaporthe grisea (Khang et al., 2010),

RTP1p accumulates in distinct parts of the extra-haustorial

matrix, particularly the older parts towards the neck of the

haustorium.

Some biochemical evidence of how eukaryotic effectors

may be internalized into host cells has been published

(Kale et al., 2010; Yaeno et al., 2011; Wawra et al., 2012),

but the mechanisms remain controversial (Ellis and Dodds,

2011; Yaeno and Shirasu, 2013). It is therefore crucial to

use markers to understand where the transfer occurs and

(a) (b)

(c) (d)

Figure 6. Secondary and ternary structure of

RTP1p135-155.

(a) Circular dichroism (CD) spectroscopy was

used to predict the possible secondary structure

of the soluble fraction of RTP1p135-155. The

spectrum shown was taken after solving the

peptide in water. The shape of the curve with a

single broad minimum between 205 and

220 nm indicates mainly b–sheet structure.(b) Deconvolution of the CD spectrum of

RTP1p135-155 measurements using various buf-

fers. White, grey and black shaded columns

show deconvoluted measurements, while

hatched columns show calculated results using

secondary structure prediction software CDNN-

2.1 (B€ohm et al., 1992). Predictions and mea-

surements indicate that the peptide consists of

antiparallel b–sheets that are probably linked by

b–turns (b–turn–b-structure), while some amino

acids of the peptide form random coils.

(c) RTP1p135-155 filaments after negative staining

showing the unbranched character of the

aggregated peptide.

(d) Close-up of a long curled filament formed

by self-aggregation of the RTP1p135-155 filament

in 10 mM potassium phosphate buffer at pH 6.0.

Scale bars = 50 nm (c) and 20 nm (d).

© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), doi: 10.1111/tpj.12237

8 Eric Kemen et al.

hence obtain an indication of the conditions under which

effector proteins are transferred into the host cell. Studies

using electron microscopy have been performed that

showed potential effector proteins accumulating within the

extra-haustorial matrix before being transferred into the

host cytoplasm (Kemen et al., 2005; Rafiqi et al., 2010).

Immunocytochemistry is one of the major tools to study

obligate biotroph plant pathogens as functional tests are

limited due to the lack of stable transformation systems.

Unlike previous studies, we improved the resolution of

membrane structures within the host cytoplasm signifi-

cantly, and used RTP1p as a marker for pathogen-to-host

protein transfer. We observed RTP1p in protuberances of

the extra-haustorial matrix. Protuberances of the extra-

haustorial matrix are a common feature of haustoria.

These structures have been observed in rust fungi

(Mendgen et al., 1991; Mims et al., 2002) and oomycetes

(Mims et al., 2004; Baka, 2008). They morphologically

resemble Maurer’s clefts, protein trafficking compartments

that are induced by P. falciparum inside red blood cells

(Tilley et al., 2008). In our study, we showed that occur-

rence of protuberances correlates with sites of RTP1p

immuno signals in the haustorium. In order to cross the

membrane, it is likely that proteins need to bind to lipids

or receptors within the extra-haustorial membrane in order

to be internalized (Grouffaud et al., 2010; Kale et al., 2010).

This is consistent with our findings showing that RTP1p

localizes to the membrane within protuberances. We

hypothesize that sub-compartmentalization within the

extra-haustorial matrix is relevant for transfer of RTP1p

into the host. It is therefore crucial to understand how the-

ses protuberances emerge inside infected cells and how

they are protected from fusion with the host endomem-

brane system or lytic compartments.

Rust infection has an inhibitory effect on plant chloroplast

cyclosis

Using live imaging of infected cells during various stages

of haustorial development, we focused on chloroplast and

nuclear movement. For necrotrophic interactions, move-

ment of the nucleus and chloroplasts to the site of infec-

tion has been reported (Oliver et al., 2009). In biotrophic

interactions, chloroplasts and the nucleus do not stay at

the side of pathogen penetration, but the nucleus moves

towards the haustorium once haustoria show secondary

growth (Heath et al., 1997). This is consistent with our live-

cell imaging results. Further, we observed accumulation of

chloroplasts surrounding the haustorial complex in late

stages of infection while the haustorium is still growing

inside the host cell. The mechano-triggered accumulation

caused during penetration shows directed movement of

chloroplasts towards the signal source (Sato et al., 2003;

Wada et al., 2003). Unlike mechano-triggered accumula-

tion, we observed normal cyclosis of chloroplasts in U. fa-

bae-infected cells. Only during haustorium maturation did

we observe a slow down of chloroplast cyclosis in close

proximity to the haustorial complex, resulting in complete

cessation in late stages of infection. Our immunolocaliza-

tion results revealed that, during this stage, RTP1p

becomes distributed over the complete cytoplasm and

nucleoplasm of the host cell. Using live-cell imaging of

young stages during which RTP1p has been observed to

accumulate to restricted cytoplasmic zones around the

haustorium (Kemen et al., 2005) revealed that cytoplasmic

streaming is strong and therefore accumulation may only

be explained by RTP1p being attached to structures that

are not undergoing cyclosis or by RTP1p forming higher

aggregates that are not affected by cytoplasmic streaming.

Analysing resistant plants that show cell death prior to

secondary growth of haustoria revealed a strong accumu-

lation of RTP1p inside the cytoplasm. From these findings,

we conclude that RTP1p secretion is not triggered by the

haustorial developmental status but by the status of its

host cell. It has been hypothesized that biotrophic patho-

gens can keep their host cell alive as long as they are able

to suppress host-induced cell death (Heath and Skalamera,

1997). As cyclosis and the resulting cytoplasmic streaming

enables distribution of molecules and vesicles (Verchot-

Lubicz and Goldstein, 2010), blocking chloroplast and

nuclear cyclosis probably reduces the signal exchange

between organelles, autophagy and the collapse of lytic

vacuoles, and may therefore be a mechanism to slow

down host cell death.

RTP1p forms amyloid-like fibrillar structures

Our observation that native and heterologously expressed

RTP1p form filamentous polymers, alongside identification

of a b–aggregation domain, are crucial in understanding

RTP1p function. Cross-b–aggregation as predicted by the

TANGO algorithm may either lead to amorphous aggrega-

tion or amyloid-like filamentous structures (Rousseau

et al., 2006). In the native system, we observed filament-

like structures only, but protein heterologously expressed

in P. pastoris forms amorphous aggregates when purified

under non-denaturating conditions. After denaturation and

controlled renaturation, amyloid-like filaments were

formed. For amyloid-like proteins, it has been shown that

seeding with infectious multimers initiates filament growth

in vitro (Taylor et al., 1999; Lee and Eisenberg, 2003;

Nonaka et al., 2010). In cases where the monomer adopts

an amyloid-like conformation, filaments may grow without

seeding, after a lag phase that is required to rearrange the

nucleation complex (Wu and Shea, 2011). Most fungal

amyloid-like proteins are cytoplasmic (Wickner et al.,

2007), and seeding is therefore possible by existing fila-

ments or chaperone assistance (Kryndushkin et al., 2011).

However, RTP1p is targeted to the extracellular space,

where self-assembly and therefore self-seeding are crucial

© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), doi: 10.1111/tpj.12237

Novel structural effectors in rust fungi 9

to induce filament formation. Comparable to RTP1p, yeast

cell adhesion molecules are secreted into the extracellular

space and show b–aggregation in combination with amy-

loid-like filament formation (Ramsook et al., 2010). These

proteins mediate attachment, colony and biofilm forma-

tion, and are therefore important virulence factors deter-

mining host specificity (Nobbs et al., 2010; Martin et al.,

2011). RTP1p may have evolved from an adhesin-like haus-

torial cell-wall protein and gained further functions by

being delivered into the host cell. Heat shock proteins

(HSPs) have been shown to regulate the status of cytoplas-

mic amyloid-like proteins between monomeric and fila-

mentous (Chernoff, 2007). Recent findings revealed HSPs

not only within the haustorial cytoplasm but also within

the extra-haustorial matrix of oomycete pathogens [E.

Kemen, A. Kemen, M. E. Jørgensen, J. D. G. Jones (The

Sainsbury Laboratory, Norwich, UK)]. In yeasts, the status

of cytoplasmic amyloid-like proteins depends on the con-

centration of HSPs: low and normal concentrations favour

filaments, whereas high concentrations favour monomers

(Chernoff, 2007). Under stress conditions, human cells

release HSPs into the extracellular space (Lancaster and

Febbraio, 2005). Based on our results and previous find-

ings, we hypothesize RTP1p is secreted conventionally into

the extra-haustorial matrix in an amyloid-like stage, which

leads to aggregation and accumulation within the extra-

haustorial matrix. Secondary haustorial growth triggers

defence reactions, as seen by the movement of the nucleus

towards the haustorium (Heath et al., 1997). These defence

reactions may cause stress to the haustorium and trigger

the release of HSPs. As for cytoplasmic prion proteins

(Chernoff, 2007), the increase in HSPs may release mono-

mers that are transferred into the host cytoplasm, where,

in the absence of fungal HSPs, formation of higher aggre-

gates occurs upon reaching a critical concentration. This

dualism of RTP1p as monomer or multimer without being

degraded by plant proteases in the apoplast even during

strong plant defence reactions is possible, as the mono-

mer, and likely to an even greater extent the aggregated

form of RTP1p, show protease resistance or even protease

inhibitor function (Pretsch et al., 2013). The amorphous

aggregates that are formed by expressing RTP1p in P. pas-

toris support the existence of a postulated rust-specific

HSP protein that may be substituted by a controlled con-

version process.

A new class of structural effectors

In this study, we have identified RTP1p as a filament-

forming protein that accumulates in the host cell in late

stages during biotrophic interaction. RTP1p may therefore

be involved in the inhibition of cyclosis observed during

late stages of infection (Figure S13). As chloroplasts are

important in plant defence (Padmanabhan and Dinesh-

Kumar, 2010), accumulating chloroplasts and therefore

simulating high-light conditions may lower photosyn-

thetic activity by shading. This in turn may be beneficial

in reducing production of reactive oxygen species. Once

cell death suppression is not effective anymore and

hydrolases are released from collapsed lytic vacuoles

(van Doorn et al., 2011), protease-resistant RTP1p fila-

ments may be relevant to protect the haustorial complex

from degradation.

We hypothesize that RTP1p is an effector protein that is

relevant for extending the biotrophic phase and protecting

the haustorium from defence by delivering host-stabilizing

multimers during the process of infection. RTP1p is there-

fore a representative of a new class of structural effectors

whose function is to stabilize the zone of interaction

between pathogen and host. Our findings suggest a new

class of targets for plant protection that have not been

exploited although extensive resources for amyloid-like

proteins are available.

EXPERIMENTAL PROCEDURES

Cultivation of plants and micro-organisms

Cultivation of Vicia faba cv. con Amore and inoculation with U. fa-bae uredospores was performed as described previously (Deisinget al., 1991; Hahn and Mendgen, 1992). Cultivation of Medicagosativa L. ‘Europe’, M. truncatula Gaertn. ‘Jemalong’ A17, andM. truncatula Gaertn. GRC.098 and U. striatus uredospore infec-tion were performed as described by Kemen et al. (2005).

Isolation of haustoria

Haustoria were isolated using ConA affinity purification asdescribed by Hahn and Mendgen (1992). Pichia pastoris strainKM71 was used for heterologous protein over-expression accord-ing to the manufacturer’s protocol for P. pastoris overexpressionsystems (Life Technologies GmbH, Darmstadt, Germany).

Plasmid construction and heterologous RTP1p expression

For expression of recombinant RTP1p in P. pastoris, UfRTP1p wasamplified using the primers 5′-CGTAGAATTCCATTATGTCAAACCTTCGCTTAC-3′ and 5′-GCCGCCCTAGGTCAGTGGTGGTGGTGGTGG-3′, introducing unique EcoRI and AvrII sites (underlined) aswell as a C–terminal His tag. After digestion with the respectiveenzymes, RTP1 was introduced into EcoRI/AvrII-digested pPIC3.5(Invitrogen). Constructs expressing GFP or N–terminal GFP-taggedRTP1p (-signal peptide) were introduced into pPIC3.5 usingBamHI/NotI digests from GFP–RTP1 fusion constructs used fortransient expression in tobacco protoplasts (Kemen et al., 2005).Pichia pastoris strain KM71 was used for heterologous proteinover-expression according to the manufacturer’s instructions (LifeTechnologies GmbH). For deglycosylation studies, 30 ll proteinsamples were treated with 3000 units endoglycosidase Hf (NewEngland Biolabs, Frankfurt, Germany, in 5 mM sodium citrate, pH5.5, at 37°C for 3 h.

Incubation of V. faba protoplasts with heterologous

UfRTP1p

Vicia faba protoplasts were prepared as described by Okuno andFurusawa (1977), with modifications as described by Obi et al.

© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), doi: 10.1111/tpj.12237

10 Eric Kemen et al.

(1989). His-tag-purified protein was re-dialysed into protoplastbuffer (500 mM D–sorbitol, 1 mM CaCl2, 5 mM MES, pH 5.5), anddiluted 1:16 v/v. A protoplast suspension (200 ll) was obtainedby centrifugation at 80 g in a swing-out rotor, and re-suspendedin 500 ll RTP1p-containing protoplast buffer. Protoplasts wereincubated for 2 h at room temperature after two washing steps,prior to fixation using 3.7% v/v formaldehyde in protoplast buffer.Protoplasts were permeabilized for immunolocalization usingTSW (10 mM Tris/HCl pH 7.5, 154 mM NaCl, 0.25% w/v gelatine,0.02% w/v SDS, 0.1% w/v Triton X-100) buffer as described byFrigerio et al. (2000). Protein was detected using S844p as theprimary antiserum for immunolabelling.

Cross-linking experiments

His-tagged RTP1p was purified from the filtered supernatant ofP. pastoris cultures by immobilized metal ion affinity chromato-graphy using a two-step gradient (113 and 181 mM imidazole). Forcross-linking of heterologous RTP1p, a glutaraldehyde concentra-tion of 0.1% v/v or 1.5 mM Ethylene glycol bis (sulfosuccinimidyl-succinate) were used. Native protein was extracted from U. fabae-infected leaves at 8 days post-infection. Leaves were finely cut inbuffer (2.5 mM Tris/HCl pH 7.2, 0.1% v/v Triton X–100 and 200 lMphenylmethanesulfonyl fluoride) using a razor blade. The superna-tant was cross-linked using a final glutaraldehyde concentration of0.05% v/v. Samples were incubated for 1–60 min. Reactions werequenched using 10 mM glycine for 5 min.

Western blotting

Proteins were separated by 12% SDS–PAGE (Laemmli, 1970), andRTP1p was detected by immunoblotting using purified serumS746 (Kemen et al., 2005) at a 1:10 000 dilution. Anti-GFP antibodywas kindly provided by R. Kissmehl (Department of Biology, Uni-versity of Konstanz, Germany). Visualization was performed usingperoxidise-conjugated anti-guinea pig IgG (Sigma-Aldrich, Ham-burg, Germany) as the secondary antibody and ECL Western blotdetection reagent (GE Healthcare Europe, Freiburg, Germany).

Immunocytochemistry

For immuno light and electron microscopy, plants were fixed andembedded as described previously (Kemen et al., 2005, 2011b).

In brief, for light microscopy, samples were fixed using aceticacid/ethanol (1:3) and embedded in acrylic resin. Prior to immuno-staining, samples were deep etched using acetone, and developedusing purified primary antibody S844p (UfRTP1p) or S849p(UsRTP1p) or anti-PEPC antibody (Rockland Immunochemicals,Gilbertsville, PA, USA) as a control. All primary antibodies weredetected using Cy3-labelled goat anti-rabbit secondary antibody.For electron microscopy, samples were high pressure-frozen andfreeze-substituted prior to embedding in epoxy resin. Sampleswere sectioned and developed using purified S844 as primaryantibody, and 10 nm gold-labelled goat anti-rabbit secondary anti-body.

For deep etching in electron microscopy, samples were highpressure-frozen and freeze-substituted followed by acrylic resinembedding. Sections were mounted on carbon-coated meshgrids. Prior to immunostaining, resin was removed using acetone,and samples were treated as described for conventional immuno-staining.

For immuno whole-mount samples, leaves were cleared withacetic acid/ethanol (1:5) for 1 1/2 h, washed in ethanol and re-hydrated (80, 60, 40, 20 and 10% v/v ethanol/water) and trans-ferred into 2.5 mM Tris/HCl pH 7.2. Samples were developed as

described for light microscopy except that the primary antibodyincubation step was extended to overnight at 4°C and secondaryantibody incubation was extended to to 3 h. For optical slices, anAxioplan2 imaging system equipped with the ApoTome techniquewas used (Carl Zeiss).

Live-cell imaging

For live-cell imaging, infected Vicia faba leaves were syringe-infil-trated with BG11 (Rippka et al., 1979) supplemented with 2 mM

Tris/HCl and 1% sucrose, pH 6.2. Prior to mounting, the epidermiswas removed. For observation, a Zeiss Plan Apochromat100 9 1.4 oil-objective was used, and images were taken at 30 secintervals.

High-resolution cryo-scanning microscopy

For high-resolution cryo-scanning electron microscopy, sampleswere high pressure-frozen and mounted on a Gatan cryo-stagesample holder with lockable clamp (Alto 2500; Gatan, Munich,Germany) under liquid nitrogen. We covered one clamp withindium foil to avoid cracks within the sample when the clamp waslocked. Samples were fractured on the cryo-stage using a coldrotary fractioning device prior to etching and platinum coating.Samples were scanned using a Hitachi S–4700 cold-field emissionscanning electron microscope (Hitachi High-Technologies Europe,Krefeld, Germany) with attached cryo-stage at 1–2 kV.

Seeded conversion

For conversion of amorphic protein aggregates into filaments, themethod described by Lee and Eisenberg (2003) was modified. Het-erologously expressed, purified RTP1p aggregates from P. pasto-ris supernatant were concentrated 3.6-fold using a Vivaspin 15Rultrafiltration unit (Vivascience, Littleton, MA, USA), and dialysedinto monomerization buffer (50 mM Tris/HCl pH 7.6, 2.5 M guani-dine/HCl, 3 M NaCl, 1 M dithiothreitol), followed by incubation for14 h at room temperature. After a 24 h dialysis step into oxidationbuffer (50 mM sodium acetate, 1 M guanidine/HCl, pH 3.8), dialysisinto aggregation buffer (50 mM sodium acetate, pH 3.8) was per-formed. The filaments were incubated at 4°C for 24 h, and trans-ferred onto Pioloform (Agar Scientific, Stansted, UK)-coated grids(200 mesh) for transmission electron microscopy. After 10 minsedimentation, the supernatant was replaced by H2Odistilled, nd0.05% uranyl acetate was used for negative staining.

Negative staining of peptide aggregates

For detection of filaments in electron microscopy, the followingpeptides were dissolved in 50 mM potassium phosphate buffer,pH 6.0, and incubated for 4 h at room temperature: RTP1p135-155

(NH2-SPGDYVFVSYGTCATVFQNPQ-OH), RTP1p135-155 F151A

(NH2-SPGDYVFVSYGTCATVAQNPQ-OH), RTP1p135-155 F141A (NH2-SPGDYVAVSYGTCATVFQNPQ-OH) and RTP1p135-155 F141A F151A

(NH2-SPGDYVAVSYGTCATVAQNPQ-OH) (GenScript, Piscataway,NJ, USA), all with a purity of >95%. Peptide solutions (50 ll) weretransferred to Pioloform-coated grits (200 mesh). After 10 min sed-imentation, the supernatant was replaced by H2Odest, and 0.05%uranyl acetate was used for negative staining.

Spectroscopy

For CD and fluorescence spectroscopy, synthesized RTP1p135-155

peptide (NH2-SPGDYVFVSYGTCATVFQNPQ-OH) (Bio-SynthesisInc., Lewisville, TX, USA) with a purity of >85% was used. Thepeptide was dissolved in four different media (see Figure 6b),

© 2013 The AuthorsThe Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), doi: 10.1111/tpj.12237

Novel structural effectors in rust fungi 11

and measured at 20°C using a JASCO-600 CD spectrophotometer(JASCO, Easton, MD, USA). Each measurement was performedusing a 1 mm cuvette and a 0.1 mm cuvette. Each measurementwas repeated three times. Secondary structures were calculatedfrom peptides in solution using a range for measurementbetween 260 and 185 nm, and CDNN 2.1 analysis software(B€ohm et al., 1992).

For fluorescence spectroscopy, the RTP1p135-155, TP1p135-155 F151A

and RTP1p135-155 F141A peptides were dissolved in 50 mM potassiumphosphate buffer, pH 6.0, and mixed 1:1 v/v with a thioflavin Tstock solution (0.05 lM thioflavin, 50 mM potassium phosphatebuffer, pH 6.0) (see above). Fluorescence was measured afterexcitation at 450 nm, or at 400 nm as a control.

Tissue printing and thioflavin T detection

The tissue printing method was modified from that describedby Smallwood et al. (1994). In brief, infected V. faba leaves werecut on slides coated with Biobond (BBI International, Cardiff,UK), and immediately pressed at a 90° angle against the slide.Slides were air-dried for 1 min and immunostained as previ-ously described (Kemen et al., 2005, 2011b). A 0.05 lM thioflavinT solution in Tris-buffered saline was used, and samples wereincubated for 20 min prior to two 10 min washing steps in Tris-buffered saline. To separate fluorescence signals, we used Cy3-labelled secondary antibodies in combination with a Cy3 filterset (F41–007, AHF Analysentechnik, Tubingen, Germany), and afluorescein isothiocyanate filter set (F41–012, AHF Analysentech-nik) for detection of thioflavin T. Chlorophyll and backgroundfluorescence were detected using filter set 05 (488005-0000, CarlZeiss). Bisbenzimide was detected using a 4,6–diamidino-2–phenylindole filter set (F31–000, AHF Analysentechnik). Pictureswere merged and analysed using ImageJ (Rasband, W.S., Ima-geJ, U. S. National Institutes of Health, Bethesda, Maryland,USA, http://imagej.nih.gov/ij/, 1997–2012).

Acknowledgements

We would like to thank Marie-C�ecile Caillaud (The SainsburyLaboratory, Norwich, UK) and Sebastian Schornack (The Sains-bury Laboratory, University of Cambridge, Cambridge, UK) forcritically reading the manuscript. We thank Ewald Daltrozzo(Department of Chemistry, University of Konstanz, Konstanz, Ger-many) for his guidance in fluorescence spectroscopy, Ulla Neu-mann (Central Micrsocopy, Max Planck Institute for Plant BreedingResearch, Cologne, Germany) for her technical support, andRudolf Heitefuss (Plant Pathology, University of Gottingen, Ger-many) for his impact in rust research.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the online ver-sion of this article.Figure S1. Immunolocalization of UfRTP1p in the extra-haustorialmatrix.Figure S2. Immunolocalization of UsRTP1p in protuberances ofthe extra-haustorial membrane.Figure S3. Test for semi-permeability of the extra-haustorial mem-brane.Figure S4. Comparison of RTP1p antibodies S746p, S844p andS849p in the pathosystems U. fabae on V. faba and U. striatus onM. sativa.Figure S5. Immunolocalization using denaturating andnon-denaturating conditions on U. fabae-infected V. faba plants.Figure S6. Multimer formation of purified de-glycosylated RTP1p.

Figure S7. Heterologous expression of GFP–RTP1p and localiza-tion of RTP1p in protoplasts.Figure S8. In silico prediction of aggregation domains in RTP1p.Figure S9. Capability of filament formation of the RTP1p135-155

peptide and phenylalanine ? alanine mutants.Figure S10. Thioflavin T measurement to validate b–aggregationin RTP1p135-155.Figure S11. Comparative analyses of b–aggregation efficiencyusing the RTP1p135-155 peptide and phenylalanine ? alaninemutants.Figure S12. Thioflavin T stain co-localizes with the RTP1p immunosignal in the extra-haustorial matrix.Figure S13. Model summarizing structural features at the hausto-rial interface and localization of RTP1p.

Table S1. Defining the interaction within the U. fabae/V. fabapathosystem at the cellular level.

Video S1. Early undifferentiated haustorium. Cycloses inside theinfected cell is not effected by the first step of compatible interac-tion.

Video S2. Haustorium inside a parenchymatic cell that show initialstage of elongation. During this stage, the plant nucleus stays inclose contact with the pathogen.

Video S3. Late stage of infection with a mature haustorium. Notonly the nucleus stays in close proximity to the haustorium, butchloroplasts accumulate showing a slow down of cyclosis of theseorganelles.

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