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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