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Cellular Microbiology (2008) 10(11), 2271–2284 doi:10.1111/j.1462-5822.2008.01206.

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First published online 5 August 2008

A novel Phytophthora infestans haustorium-specific


membrane protein is required for infection of potato

Anna O. Avrova,1** Petra C. Boevink,1 hyphae where Pihmp1::mRFP is localized. We propose


Vanessa Young,1 Laura J. Grenville-Briggs,2 that Pihmp1 is an integral membrane protein that pro-
Pieter van West,2 Paul R. J. Birch3 and vides physical stability to the plasma membrane of
Stephen C. Whisson1* P. infestans infection structures. We have provided the
1
Plant Pathology Programme, Scottish Crop Research first evidence that the surface of oomycete haustoria
Institute, Invergowrie, Dundee DD2 5DA, UK. possess proteins specific to these biotrophic struc-
2
Aberdeen Oomycete Group, College of Life Sciences tures, and that formation of biotrophic structures
and Medicine, Institute of Medical Sciences, University (infection vesicles and haustoria) is essential to suc-
of Aberdeen, Foresterhill, Aberdeen AB24 2ZD, UK. cessful host colonization by P. infestans.
3
Division of Plant Sciences, College of Life Sciences,
University of Dundee at SCRI, Invergowrie, Dundee
Introduction
DD2 5DA, UK.
Filamentous eukaryotic plant pathogenic microorganisms
such as fungi and oomycetes frequently form highly spe-
Summary
cialized structures during invasion and colonization of
Phytophthora infestans causes late-blight, a devastat- their hosts. For many plant pathogens, host invasion com-
ing and re-emerging disease of potato crops. During mences with the differentiation of a domed or swollen
the early stages of infection, P. infestans differentiates structure, called an appressorium, at the terminus of a
infection-specific structures such as appressoria for germination tube or hypha from a spore (Talbot, 2003;
host epidermal cell penetration, followed by infection Grenville-Briggs and van West, 2005). The appressorium
vesicles, and haustoria to establish a biotrophic phase secretes adhesive compounds to maintain contact
of interaction. Here we report the cloning, from a between host and pathogen during infection (Tucker and
suppression subtractive hybridization library, of a Talbot, 2001; Grenville-Briggs and van West, 2005), and
P. infestans gene called Pihmp1 encoding a putative can generate mechanical force (Howard et al., 1991;
glycosylated protein with four closely spaced trans- Bechinger et al., 1999) and/or hydrolytic enzymes to pene-
membrane helices. Pihmp1 expression is upregulated trate host cell walls (Francis et al., 1996; Pryce-Jones
in germinating cysts and in germinating cysts with et al., 1999).
appressoria, and significantly upregulated throughout After invading a compatible host, fungal and oomycete
infection of potato. Transient gene silencing of Pihmp1 hyphae grow either intercellularly or intracellularly,
led to loss of pathogenicity and indicated involvement with or without biotrophic structures called haustoria
of this gene in the penetration and early infection (Mendgen and Hahn, 2002). For example, Cladosporium
processes of P. infestans. P. infestans transformants fulvum produces entirely intercellular hyphae without
expressing a Pihmp1::monomeric red fluorescent haustoria, Magnaporthe grisea grows in planta as intra-
protein (mRFP) fusion demonstrated that Pihmp1 was cellular hyphae without haustoria (Kankanala et al.,
translated in germinating sporangia, germinating 2007), while obligate biotrophs, such as the rust fungi,
cysts and appressoria, accumulated in the appresso- produce intercellular hyphae with intracellular haustoria.
rium, and was located at the haustorial membrane Haustoria are formed by invagination of the host cell
during infection. Furthermore, we discovered that membrane and are thus in intimate contact with the host
haustorial structures are formed over a 3 h period, cell. The haustorial plasma membrane of fungal plant
maturing for up to 12 h, and that their formation is pathogens is differentiated from the intercellular hyphal
initiated only at sites on the surface of intercellular plasma membrane and is distinct in its possession of
proteins for the transport of nutrients such as sugars
and amino acids (Hahn et al., 1997; Struck et al., 1998;
Received 29 April, 2008; revised 30 June, 2008; accepted 7 July, Voegele et al., 2001). Haustoria are also the site of
2008. For correspondence. *E-mail Steve.Whisson@scri.ac.uk; Tel.
(+44) 1382 562731; Fax (+44) 1382 562426; **E-mail Anna.Avrova@ secretion for fungal and oomycete effector proteins,
scri.ac.uk; Tel. (+44) 1382 562731; Fax (+44) 1382 562426. some of which may act inside the host cell to modulate
© 2008 Scottish Crop Research Institute
Journal compilation © 2008 Blackwell Publishing Ltd
2272 A. O. Avrova et al.

the host cell environment (Catanzariti et al., 2006; 2004). Compared with fungal plant pathogens, molecular
Kamoun, 2006; Whisson et al., 2007). processes underlying oomycete development and patho-
Phytophthora infestans is an oomycete plant pathogen, genicity are poorly understood. With the exception of
best known for its role in precipitating the Irish potato the AVR3a avirulence protein (Whisson et al., 2007), no
famines in the mid-19th century. It remains a major con- oomycete proteins have been demonstrated to localize
straint to potato production worldwide. Although sharing to haustoria, either for secretion or for other haustorial
morphological similarities with fungi such as hyphae and functions.
spores, oomycetes are more closely allied to the stra- Several studies have reported differentially expressed
menopiles among the heterokonts, and deeply rooted with candidate pathogenicity factors from P. infestans pre-
the alveolates (Baldauf et al., 2000; Burki et al., 2007). As infection stages, as they are likely to be enriched for
a model oomycete, the life cycle of P. infestans is well molecules involved in successful penetration of the host,
characterized (van West and Vleeshouwers, 2004; and establishment of a compatible interaction (Görnhardt
Avrova et al., 2007), as are its interactions with its host et al., 2000; Beyer et al., 2002; Avrova et al., 2003; Birch
plants. Almost all molecular genetic tools are available to et al., 2003; Bittner-Eddy et al., 2003; Randall et al., 2005;
determine gene function in this pathogen (Birch and Judelson et al., 2008). A high proportion of oomycete
Whisson, 2001; Lamour et al., 2007), including reliable genes identified through transcript profiling techniques
methods for transient gene silencing and occasional are entirely novel, and identifying those with a role in
stable gene silencing, reporter gene assays, and a wealth pathogenicity remains a significant challenge. Through
of genetic information including greater than 80 000 ESTs gene silencing in stable transformants, a small number of
and a genome sequence (van West et al., 1999; Randall P. infestans genes have been analysed at the functional
et al., 2005; Whisson et al., 2005; 2007). This establishes level, revealing an involvement for some in pathogenicity.
P. infestans as the model oomycete organism for studying However, these have typically encoded proteins involved
the pathogen molecular and biochemical processes in signal transduction or transcriptional regulation (Latijn-
underlying disease development. Asexual sporangia houwers and Govers, 2003; Latijnhouwers et al., 2004;
are the infectious dissemination propagules and may Blanco and Judelson, 2005). These genes and their
germinate directly, or cleave to yield motile, biflagellate encoded proteins may form part of the core biology of
zoospores. Zoospores encyst at an infection site in P. infestans and their silencing may have resulted in
response to chemical, electrical and physical signals, dis- multiple developmental defects, including reduced
carding flagella and forming a cell wall (van West et al., pathogenicity. Transient silencing of genes is also pos-
2002, 2003). A germ tube emerges from the cyst or spo- sible in P. infestans, and may facilitate higher-throughput
rangium, the tip of which swells to differentiate into an functional analysis of, and more silencing-recalcitrant,
appressorium and subsequently a penetration hypha. genes than previously (Whisson et al., 2005; Grenville-
After the plant cuticle and cell wall have been breached, Briggs et al., 2008; Walker et al., 2008). In identifying
an intracellular, biotrophic infection vesicle is produced in genes encoding proteins contributing directly to patho-
the epidermal cell. Intercellular hyphae grow into the genicity, it may be expected that candidate genes encode
mesophyll cell layers, producing intracellular, biotrophic proteins that act at the interface between pathogen and
finger-like haustoria as new host cells are encountered. host. Although numerous oomycete genes, such as pro-
After 36–48 h post inoculation (hpi), the mode of interac- tease inhibitors, a glucanase inhibitor, avirulence proteins
tion between P. infestans and initially infected host cells and necrosis-inducing proteins, have been identified that
becomes necrotrophic and new haustoria are not formed. interact with host plant proteins (Rose et al., 2002; Torto
From 72 to 96 hpi, initially infected areas of the leaf are et al., 2003; Tian et al., 2004; 2005; 2007; Birch et al.,
fully colonized and necrotic. Sporulating hyphae emerge 2006), their contribution towards oomycete pathogenicity
from stomata to release sporangia, allowing aerial dis- remains to be determined.
semination of the pathogen. Spatially, this is visualized as In a broader transcript profiling study in P. infestans
a leaf lesion with a biotrophic margin as new cells are targeting candidate pathogenicity genes expressed in
infected. The centre of the lesion is necrotic where initially pre-infection stages, we selected one transcript for further
colonized cells have died. study in order to test the hypotheses that it encoded a
Oomycete haustoria differ from those formed by fungal protein that may act at the pathogen–host interface, and
plant pathogens in that cell wall and membrane differen- play a role in pathogenicity. Here we describe the cloning,
tiation is less pronounced in oomycetes. For example, homologues in other oomycetes, role in pathogenicity,
fungal haustoria have a distinct neckband that seals the and localization of a P. infestans gene encoding a pre-
extrahaustorial matrix (EHM) from the apoplast, whereas dicted integral membrane protein. Translational fusion of
the oomycete cell wall is continuous from the intercellular this gene to the monomeric red fluorescent protein
hyphae to the haustoria (Hohl and Suter, 1976; Soylu, (mRFP) in stable P. infestans transformants demon-

© 2008 Scottish Crop Research Institute


Journal compilation © 2008 Blackwell Publishing Ltd, Cellular Microbiology, 10, 2271–2284
Phytophthora infestans haustorial membrane protein 2273

strated localization to the haustorial plasma membrane Pihmp1 expression is upregulated prior to and
during infection of potato. Accordingly, it has been named during infection
Pihmp1, for P. infestans haustorial membrane protein 1.
The involvement of Pihmp1 in the penetration process of Real-time reverse transcription polymerase chain reaction
P. infestans and establishment of a compatible interaction (RT-PCR) analysis was used to characterize expression
was shown using transient gene silencing. of Pihmp1 in P. infestans in vitro stages of cultured myce-
lium, sporangia, zoospores, germinating cysts, germinat-
ing cysts with appressoria and in planta during the
Results infection of susceptible potato cv. Bintje with a compatible
race (isolate 88069) of P. infestans at 12, 24, 33, 48, 56
Cloning of Pihmp1
and 72 hpi. Microscopic analysis indicated that the timing
From an existing suppression subtractive hybridization of P. infestans development during these infections
(SSH) cDNA library (Grenville-Briggs et al., 2005) a closely followed the events described by Vleeshouwers
198 bp transcript-derived fragment (clone 2c5) was et al. (2000) and Avrova et al. (2007). Thus, at 12 hpi,
selected for further analysis, as BLASTN and TBLASTX germinating cysts, appressoria and infection hyphae were
similarity searches of sequence databases (see Experi- clearly visible. At 24–33 hpi, numerous haustoria were
mental procedures) revealed sequence similarity only to visible in infected tissue, indicative of the biotrophic
expressed sequence tags (ESTs) from P. infestans (4e-9 phase, whereas few haustoria were visible in infected
to 5e-24 for TBLASTX; 2e-115 to 1e-124 for BLASTN), and the tissue by 48 hpi, indicating the transition from biotrophy to
genome sequences of Phytophthora sojae, Phytophthora necrotrophy. By 72 hpi, the necrotrophic phase was well
ramorum and Hyaloperonospora parasitica (3e-33 to 1e-40 established with sporulation and necrosis clearly visible.
for TBLASTX; 3e-16 to 6e-20 for BLASTN). As this section of Primer pairs were designed to anneal to Pihmp1, PiactA
work was carried out before the availability of the and PiipiO1 (Table S1). The PiactA gene from P. infestans
assembled draft P. infestans genome sequence, genomic was used as a constitutively expressed endogenous
copy number, and the sequence of the complete open control (Grenville-Briggs et al., 2008; Judelson et al.,
reading frame (ORF) and promoter of Pihmp1 were deter- 2008). Expression of PiipiO1 and Pihmp1 in asexual life
mined from three positively hybridizing clones (Fig. S1) cycle- and infection-stage samples was compared with
from an existing P. infestans bacterial artificial chromo- the level of their expression in a calibrator sample, which
some (BAC) library (Whisson et al., 2001) representing was cDNA synthesized from RNA isolated from non-
10¥ genome coverage. The Pihmp1 gene sequences sporulating mycelium grown in rye broth. The expression
present on BAC clones 5J1 and 15J23 were identical, and of PiipiO1 and Pihmp1 in the mycelium cDNA samples
the sequence on BAC clone 1B18 was a truncated copy of was assigned the value of 1.0.
the gene. Since the release of the P. infestans genome The P. infestans PiipiO1 gene, known to be upregulated
sequence (see Experimental Procedures for URL), both in invading hyphae during infection (van West et al., 1998),
the intact and truncated copies of the gene have been was used to confirm disease progression in inoculated leaf
found within the same genomic region (sequence super- samples and yielded the expected expression profile
contig 1.1), with no additional copies located elsewhere in (Fig. 1). The Pihmp1 expression profile in pre-infection cell
the genome. types is very similar to that of PiipiO1: upregulated over
A TBLASTN search of the P. infestans genome database 100-fold in zoospores, peaking at over 1600-fold in germi-
using the predicted amino acid sequence of Pihmp1 nating cysts and then decreasing to 600-fold in germinating
as a query revealed 11 supercontigs containing several cysts with appressoria (Fig. 1). Unlike PiipiO1, the relative
homologous regions with E-values of 8 ¥ 10-43-10-172. A upregulation of Pihmp1 during infection is lower than that in
TBLASTN search of the NCBI EST databases, including germinating cysts with appressoria, suggesting that its
human and mouse sequences (http://www.ncbi.nlm.nih. mRNA is transcribed during cyst germination and appres-
gov/blast/) with the predicted Pihmp1 protein sequence, sorial development, to be translated for the early biotrophic
identified two additional homologous ESTs (gi58106741 stage of interaction. Repeated amplifications, on indepen-
and gi58105907) from P. infestans germinating sporangia dent occasions with biologically replicated cDNA samples,
with E-values of 7 ¥ 10-73 and 2 ¥ 10-37, respectively, one resulted in similar expression profiles.
homologous EST (gi58087999) from P. infestans sporu- To test whether the truncated paralogue of Pihmp1 was
lating mycelium with E-value of 2 ¥ 10-43, and two homolo- expressed, primers 1B18F and 1B18R were used in real-
gous ESTs (gi38117833 and gi38062167) from P. sojae time RT-PCR across the life cycle stages of P. infestans. A
with E-values of 2 ¥ 10-48 and 2 ¥ 10-51 respectively. product of the expected size was amplified from genomic
The Pihmp1 sequence is deposited in GenBank under DNA but not from cDNA of any P. infestans life cycle stage
Accession No. EU680858. (not shown).

© 2008 Scottish Crop Research Institute


Journal compilation © 2008 Blackwell Publishing Ltd, Cellular Microbiology, 10, 2271–2284
2274 A. O. Avrova et al.

trans-membrane (TM) helices between residues 605 and


A Pihmp1
2000 722, potentially anchoring the protein to the plasma mem-
brane (Fig. 2). Both N- and C-termini of Pihmp1 were
Relative expression

1600 predicted to be located outside the plasma membrane,


with only 10 amino acids in two loops between TM helices
1200
predicted to extend into the cytoplasm. TMHMM posterior
800 probabilities for the sequence were: 0.98 for TM helix 1,
0.90 for TM helix 2, 0.90 for TM helix 3 and 1.00 for TM
400 helix 4.
BLASTP and TBLASTN searches, using default settings, of
0
M S Z C A B12 B24 B33 B48 B56 B72 NCBI GenBank (non-redundant database) yielded only
weak similarity (5e-7 to 1e-13) to a bacterial and fungal
B domain in the N-terminal half of the protein, between
PiipiO residues 293 and 505. In BLASTP-returned sequences from
350
Relative expression

300 Xanthomonas axonopodis pv. citri (NP_644432.1) and


Bradyrhizobium sp. (YP_001208179.1), this domain
250
yielded modest support for a jacalin-like (mannose/
200
galactose binding) lectin domain. Weak support, below the
150 thresholds used to construct the Pfam model, for a jacalin-
100 like lectin domain was identified in Pihmp1 using Pfam 22.0
50 (E-value 0.0014) between residues 374 and 505. A domain
0 containing three lysine/serine (K/S)-rich repeats of 32–39
M S Z C A B12 B24 B33 B48 B56 B72 residues was identified manually between residues 130
and 260 (Fig. 2B). The final two K/S repeats contain four
Fig. 1. Pihmp1 is expressed in pre-infection stages and throughout
infection of potato. Real-time RT-PCR expression profile of (A) copies of a DxSxS metal ion-dependent adhesion site
Pihmp1 and (B) PiipiO1 in pre-infection stages (sporangia – S, (MIDAS) motif (Tozer et al., 1996). An additional three
zoospores – Z, germinating cysts – C, germinating cysts with
serine/glycine (S/G)-rich repeats of 15–17 residues follow
appressoria – A) and 12 (B12), 24 (B24), 33 (B33), 48 (B48), 56
(B56) and 72 (B72) h post inoculation of potato cv. Bintje with a the K/S-rich repeats. CBS Prediction Servers (NetNGlyc
compatible race (88069) of P. infestans, relative to their expression 1.0) predicted N-glycosylation at positions 390, 818 and
in vegetative non-sporulating mycelium (M). Error bars represent
966, and potential O-glycosylation (mucin type; NetOGlyc
confidence intervals calculated using three technical replicates for
each sample within the RT-PCR assay. 3.1) at positions 93–95, 134, 135, 139, 142, 145 and 226
(Fig. 2B). A mucin domain was also predicted by Pfam in
this region of Pihmp1 but with poor support (E-value 0.55),
below the thresholds used to construct the Pfam model.
Pihmp1 is a putative glycosylated membrane protein
Seven cysteine residues are present in the 99-amino-acid
The 3072 bp ORF of Pihmp1, which lacks introns, region between residue 505 and the first TM helix of the
encodes a predicted 1023-amino-acid, 108.3 kDa protein. predicted mature Pihmp1 protein, and a further three cys-
A signal peptide for secretion was predicted at the teines are present in the C-terminal portion of the protein
N-terminus by SignalP 3.0 (neural network mean 0.929, after the TM helices (Fig. 2B). These protein sequence
hidden Markov model 0.998), with the molecular weight of features suggest that Pihmp1 is a membrane-anchored,
the mature protein estimated at 105.5 kDa. The CBS predominantly extracellular, glycosylated protein that may
Prediction Servers (TMHMM v. 2.0) and SOSUI program act at the interface between P. infestans and host cells
(v. 1.11) predicted Pihmp1 to encode a protein with four during infection.

Fig. 2. Organization, and multiple alignment of homologues, of the predicted Pihmp1 protein.
A. Sequences in multiple alignment are Pihmp1 from P. infestans, and closest homologues from P. ramorum (Prhmp1), P. sojae (Pshmp1) and
H. parasitica (Hphmp1). Protein sequences were aligned using the CLUSTALW accessory program in the BioEdit Sequence Alignment Editor.
Identical residues across three or more homologues are shaded black, and similar residues across three or more homologues are shaded
grey. The signal peptide is marked by solid overhead bracket, conserved cysteine residues are marked with an asterisk (*) and the
trans-membrane helices are marked by solid black lines.
B. Schematic representation of Pihmp1 showing location of features (not to scale): signal peptide (SP; shaded blue), conserved EER motif
(shaded dark blue), predicted O-glycosylation (vertical red bars), 3¥ K/S-rich repeats (3¥ grey shaded squares), 4¥ DxSxS MIDAS motif
(white squares within K/S repeats), 3¥ S/G-rich repeats (shaded pink), predicted N-glycosylation (vertical black bars), conserved cysteine
residues (vertical blue bars) and trans-membrane helices (TM; shaded green).

© 2008 Scottish Crop Research Institute


Journal compilation © 2008 Blackwell Publishing Ltd, Cellular Microbiology, 10, 2271–2284
Phytophthora infestans haustorial membrane protein 2275

© 2008 Scottish Crop Research Institute


Journal compilation © 2008 Blackwell Publishing Ltd, Cellular Microbiology, 10, 2271–2284
2276 A. O. Avrova et al.

Pihmp1 has homologues in other oomycetes dsRNA did not exhibit sequence identity over 20 bp to any
other predicted gene or genomic region to initiate off
A TBLASTN search, individually, of the oomycete genome
target gene silencing. The only other sequence exhibiting
databases held within the Virginia Microbial Database
sequence similarity was the truncated and non-expressed
(VMD; Tripathy et al., 2006) with Pihmp1 identified
copy of Pihmp1. In transient gene silencing experiments
homologues in P. sojae (Assembly V3.0, Scaffold
for Pihmp1, lines exhibiting a phenotype after treatment
Super_7, 1918598–1921645 bp), P. ramorum (Assembly
with Pihmp1 dsRNA also showed reduced Pihmp1 mRNA
V1.0, Scaffold 1, 433071–436238 bp) and H. parasitica
abundance, ranging from 8% to 47% compared with
(Assembly V6.0, Contig 6, 566653–569616 bp) with
the non-homologous dsRNA-treated controls (Fig. 3D).
E-values ranging from 9 ¥ 10-88 to 10-140 (Fig. 2A). Homo-
Therefore, successful transient silencing of Pihmp1 was
logues identified by TBLASTN were verified by BLASTN
achieved using dsRNA treatment of protoplasts, allowing
search of the same oomycete genomes with the Pihmp1
the resultant phenotypes to be studied.
DNA sequence to demonstrate that the genomic locations
Both the Pihmp1 dsRNA-treated (silenced) and the
of closest TBLASTN and BLASTN homologues of Pihmp1
non-homologous dsRNA-treated (control) lines grew nor-
were the same. These analyses yielded a single-copy
mally on rye agar medium and produced similar numbers
Pihmp1 homologue from each oomycete genome
of sporangia and zoospores that encysted normally (data
examined. The Pihmp1 homologue in P. ramorum is the
not shown). In control lines, by 24 hpi, cysts and sporan-
most closely related to Pihmp1, as determined by protein
gia had germinated on the leaf surface, formed appres-
distance (Table S2). A homologous sequence to Pihmp1
soria and begun host penetration, forming infection
also exists in the genome of Phytophthora capsici, with
vesicles and haustoria inside host cells (Fig. 3A). Germi-
E-values of 2 ¥ 10-37-10-105, but this genome sequence
nated cysts and sporangia from Pihmp1 dsRNA-treated
has not been assembled to date and so no further
lines developed appressoria-like structures, but infection
conclusions can be made about Pihmp1 homologues in
vesicles and haustoria for in planta infection were not
this species. Although homologues of Pihmp1 in other
observed (Fig. 3B), suggesting a role for Pihmp1 in cell
oomycetes exhibited similarity across the entire length of
membrane, and perhaps cell wall, differentiation and func-
the protein, the greatest divergence in the amino acid
tion during infection. Hyphae from germinated cysts and
sequence was observed in the N-terminal 504 amino
sporangia of Pihmp1-silenced lines occasionally entered
acids of Pihmp1 (Fig. 2A, Table S2). In comparison, these
host leaf tissue via stomata, but infection did not develop
levels of localized protein divergence were not observed
to the levels of control non-homologous dsRNA-treated
for cellular proteins actinA (actA), translation elongation
lines (Fig. 3C); sparse intercellular hyphae and no visible
factor 1a (tef1a) or the trans-membrane protein cellulose
lesion were observed in these instances. Germ tubes of
synthase 3 (cesA3) (Table S2). All 10 cysteine residues in
cysts from Pihmp1-silenced lines also formed atypical
the C-terminal half of Pihmp1 (Fig. 2; seven preceding
distorted hyphae with numerous bulges and swellings that
and three following the TM helices) are conserved in all
grew across the leaf surface by 48 hpi. This latter pheno-
four homologues, suggesting localized conservation of
type was also occasionally observed in wild-type non-
structure in these proteins across different oomycetes.
silenced lines where penetration was unsuccessful or
Additional protein sequences from P. sojae, P. ramorum
delayed and, although this phenotype was significantly
and H. parasitica showed similarity to the P. infestans
more pronounced in Pihmp1-silenced lines, it may be a
Pihmp1 protein. However, these related sequences did
nutrient starvation response by P. infestans, rather than a
not align across the full length of the protein, with little
direct phenotype of Pihmp1 silencing.
or no similarity observed to the (approximately) 300
The observed microscopic phenotype associated with
N-terminal residues, and with some possessing up to
reduced Pihmp1 expression was accompanied by ap-
two predicted mannose/galactose-binding jacalin lectin
parent loss of pathogenicity on potato leaves (Fig. 3C)
domains in the N-terminal half of the protein sequence.
due to inability of Pihmp1-silenced lines to spread within
leaf tissue and form necrotic, sporulating lesions. In our
experiments, Pihmp1 dsRNA-treated lines expressing
Silencing of Pihmp1 leads to loss of pathogenicity
Pihmp1 mRNA up to 47 ⫾ 6% of wild-type Pihmp1 mRNA
Transient gene silencing, triggered by double-stranded levels were non-pathogenic. Pihmp1 dsRNA-treated lines
RNA (dsRNA; Whisson et al., 2005), was used to deter- expressing Pihmp1 mRNA greater than 63 ⫾ 8% pro-
mine the role of Pihmp1 during P. infestans infection of duced appressoria and infection vesicles, colonized leaf
potato. DsRNA for the green fluorescent protein gene tissue by forming typical intercellular hyphae with hausto-
(gfp) or another non-homologous dsRNA not present in ria, leading to lesions indistinguishable in size to the
the wild-type P. infestans isolates 88069 or CY29 were control dsRNA-treated lines. These results clearly show
used as controls. The sequence of the 200 bp Pihmp1 that silencing of Pihmp1 affected P. infestans pathogeni-

© 2008 Scottish Crop Research Institute


Journal compilation © 2008 Blackwell Publishing Ltd, Cellular Microbiology, 10, 2271–2284
Phytophthora infestans haustorial membrane protein 2277

A B a

iv h

C D 1.8
1.6

Relative expression
1.4

1.2
1

0.8
0.6
0.4
0.2
0
C1 C2 C3 C4 C5 1 2 3 4 5

Independent lines

Fig. 3. Transient silencing of Pihmp1 leads to loss of pathogenicity.


A. Control line treated with non-homologous dsRNA. Host penetration has occurred by 24 hpi with infection vesicles (iv) and haustoria (h)
seen within invaded host cells.
B. Pihmp1 transient silenced line showing germinated cyst (c) with two appressoria-like structures (a), but failed host penetration. Scale bar in
(A) and (B) represents 10 mm.
C. Macroscopic disease symptoms at 7 days post inoculation on potato leaves of Pihmp1 transient silenced lines (bottom) compared with
control lines treated with non-homologous dsRNA (top).
D. Levels of transient Pihmp1 silencing observed in experiment shown in (C). Error bars represent confidence intervals calculated using three
technical replicates for each sample within the RT-PCR assay.

city, with silenced lines unable to form intracellular primary tested by this gene silencing technique (Whisson et al.,
infection vesicles or haustoria and establish infection. 2005; Grenville-Briggs et al., 2008; Walker et al., 2008).
Five independent transient silencing experiments with
P. infestans strains CY29 (three experiments) or 88069
Pihmp1 is localized to the P. infestans haustorial
(two experiments), on different dates and with indepen-
membrane during infection of potato
dent preparations of in vitro synthesized dsRNA (controls
and Pihmp1), yielded the same loss of pathogenicity phe- The Pihmp1 ORF, including 694 bp of promoter
notype for Pihmp1. From these experiments, a total of 100 sequence, was cloned for translational fusion with GFP
Pihmp1 dsRNA-treated lines were generated, of which 44 in P. infestans stable transformants. Three out of six
exhibited the loss of pathogenicity phenotype. Seventy- transformants transcribed gfp in germinated cysts, in-
two control lines, derived from protoplasts treated with dicating the 694 bp region to be sufficient to direct normal
non-homologous dsRNA, were also used, all of which Pihmp1 expression. However, GFP fluorescence was not
exhibited wild-type levels of pathogenicity. The frequency observed in germinated cysts either in vitro or in planta
of transient silencing observed among Pihmp1 dsRNA- following the inoculation of detached potato leaves. In
treated lines is similar to that observed for other genes addition, no GFP fluorescence was observed in inter-

© 2008 Scottish Crop Research Institute


Journal compilation © 2008 Blackwell Publishing Ltd, Cellular Microbiology, 10, 2271–2284
2278 A. O. Avrova et al.
Fig. 4. Localization of Pihmp1 to haustoria by
translational fusion to the monomeric red
fluorescent protein (mRFP) and under control
of the Pihmp1 promoter. Enhanced green
fluorescent protein (eGFP) was expressed
constitutively to define P. infestans cytoplasm.
A. Localization of Pihmp1 in germinating
sporangia and cysts with appressoria on the
surface of a potato leaf. Aggregations of
tagged Pihmp1 can be observed in the cyst or
sporangia germ tube, concentrating in the
swollen appressorium (arrowhead) at the
germ tube tip. Arrows and letters indicate
example cyst (c), sporangia (s), germ tube
(g) and appressorium (a) expressing
Pihmp1::mRFP.
B. Red channel of the image shown in (A).
C. Pihmp1 localizes to biotrophic digit-like
intracellular haustoria (arrow) during
P. infestans infection of potato. Additional
small ‘plaques’ of tagged Pihmp1 can also be
observed in the intercellular hyphae
(arrowhead) and may represent sites of either
early or aborted haustorium formation.
D. Red channel of the image shown in (C).
Scale bars represent 50 mm.

cellular hyphae or haustoria in planta. This might be DNA of transformants, indicating that the mRNA encoding
explained by previously reported low fluorescence yield of the fusion protein was being transcribed under control of
GFP in the apoplast (Zheng et al., 2004) and may confirm the Pihmp1 promoter.
bioinformatic predictions suggesting the C-terminus of Confocal microscopy of these transformants showed
Pihmp1 to be located outside the plasma membrane. that the Pihmp1::mRFP fusion protein was present, in
Pihmp1 with its native promoter was transferred what appeared to be vesicles, inside sporangia and cysts
from p2c5-GFP to pPLmRFP to yield p2c5-mRFP, a germinated both in vitro and on the leaf surface. The
Pihmp1::mRFP translational fusion. Co-transformation of Pihmp1::mRFP-containing ‘vesicles’ moved along the
P. infestans strain 88069 with p2c5::mRFP and pVW2 germ tube, concentrating in the appressorium in contact
(constitutive eGFP expression; van West et al., 1999) with the leaf surface (Fig. 4A and B). After host penetra-
yielded 17 transformants that exhibited normal in vitro and tion and development of infection, mRFP fluorescence
in planta development. Expression of the Pihmp1::mRFP was observed surrounding haustoria, outlining the haus-
fusion in germinated cysts was shown by RT-PCR of torial membrane and concentrated around the haustorial
the entire mRFP transcript for nine transformants base (Fig. 4C and D). Plaques of Pihmp1::mRFP were
(2c5mRFP1, 2, 5, 10, 11, 12, 14, 17 and 19) using mRF- also observed on the surface of intercellular hyphae, and
PclaF and mRFPsacR primers. Of these transformants these may indicate sites for initiation of haustorial growth.
expressing the Pihmp1::mRFP fusion, six exhibited These observations are consistent with Pihmp1 transla-
detectable mRFP fluorescence at varying intensities tion in the pre-infection stages of germinating cysts and
(2c5mRFP1, 2, 10, 11, 12 and 17), and four transformants appressoria for its destined site of localization in the
(2c5mRFP10, 11, 12 and 17) also exhibited cytoplasmic biotrophic infection structures of P. infestans.
eGFP fluorescence. To confirm that the Pihmp1::mRFP To investigate sites of haustorial growth, development
fusion was expressed from the native promoter in trans- of intercellular hyphal tips of transformant 2c5mRFP11
formants exhibiting mRFP fluorescence, PCR amplifying were followed by confocal microscopy over 12 h. This
the region from the Pihmp1 promoter to mRFP stop codon determined that the previously identified plaques of
was performed with the primers SpeI2C5a and mRFP- Pihmp1::mRFP were indeed the sites for initiation of haus-
sacR, using transformant genomic DNA and cDNA from torial growth (Fig. 5). However, not all Pihmp1 plaques
germinated cysts, separately, as templates. The product subsequently developed haustoria. Where haustoria did
of the expected size was amplified only from genomic develop, initial penetration of host cells was rapid, occur-

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Journal compilation © 2008 Blackwell Publishing Ltd, Cellular Microbiology, 10, 2271–2284
Phytophthora infestans haustorial membrane protein 2279

Fig. 5. Development of P. infestans intracellular haustoria in potato leaf tissue, showing that not all potential sites of haustorial growth develop
into mature haustoria. T = 0. Two adjacent intercellular hyphal tips showing at least six Pihmp1 plaques as sites of potential haustorium
formation. T = 3. Plant cells are invaded by haustoria (arrowheads) within 3 h from Pihmp1 accumulation sites on the intercellular hyphae.
T = 7. After 7 h, haustoria continue to grow into the invaded cells and begin to exhibit a characteristic hooked form. After 9 h (T = 9) and up to
12 h (T = 12) the haustoria continue to grow and hook, accumulating more Pihmp1 at the surface. Note that several potential sites of
haustorium formation failed to develop further (long arrows indicate one of these). Scale bar represents 10 mm.

ring within 3 h (Fig. 5), with subsequent elongation and C-terminal half without discernible domains of known
development of the characteristic hooked shape of the function. MIDAS motifs complex metal ions and are found
haustorium occurring for up to 12 h (Fig. 5). The distribu- in animal, and animal pathogen, membrane-localized pro-
tion of Pihmp1 also changed across this time-course, teins (such as integrins), where they are involved in medi-
appearing to become increasingly concentrated in ating cell attachment through interaction with proteins
patches on the haustorial surface during elongation and such as collagen and fibronectin (Tozer et al., 1996;
maturation. Akhouri et al., 2004; Kottom et al., 2008).
Pihmp1 is represented by a single full-length functional
copy in all four oomycete genomes examined. Additional
Discussion
similar proteins are also present in all genomes and, as
We have provided the first evidence that the surface of such, Pihmp1 appears to form part of a group of similar
oomycete haustoria possesses proteins specific to these proteins encoded by dissimilar genes. All of these similar
biotrophic structures, and that formation of biotrophic proteins exhibit the greatest similarity at the C-terminal
structures (infection vesicles and haustoria) is essential to end, typically showing little or no similarity within the
successful host colonization by P. infestans. We used N-terminal 300 amino acids.
SSH to enrich for cDNAs of transcripts upregulated in The precise role of Pihmp1 in the cell biology of
P. infestans germinating cysts with appressoria, struc- P. infestans remains to be determined. One possible func-
tures formed just prior to infection of potato, with the tion for Pihmp1 may be as a receptor/sensor, but this
hypothesis that proteins required for infection would be appears unlikely as only 10 amino acids are located within
translated just prior to host penetration. This strategy has the cell cytoplasm, and no putative phosphorylation sites
proven highly successful for identifying genes encoding were predicted within these short regions to initiate down-
effectors probably required for successful establishment stream signalling. This line of evidence suggests that
of a compatible interaction (Liu et al., 2005; Randall et al., Pihmp1 is more likely to be a structural protein. One model
2005; Judelson et al., 2008). While Pihmp1 was most for the structure of Pihmp1 may involve the anchoring of
highly expressed in germinated cysts and appressoria, the protein to the plasma membrane, with the cysteine-rich
the encoded protein was translated in these pre-infection domain potentially spanning the cell wall. The K/S-rich,
structures and transported towards the penetration site to DxSxS MIDAS-like motifs, S/G-rich, and O-glycosylated
be later incorporated into the plasma membrane of either amino acids would thus be exposed to the EHM, and also
the infection vesicle or haustoria. extracellular host surveillance and defences. This latter
Pihmp1 is predicted to encode a putative outer mem- consideration may in part explain the N-terminal diver-
brane protein with four TM helices in the middle of the gence in the homologues of Pihmp1 between P. infestans,
peptide sequence, anchoring the protein to the plasma P. sojae, P. ramorum and H. parasitica; host species selec-
membrane (Fig. 2). Both N- and C-termini of Pihmp1 are tion may have acted to diversify this region. Although a
predicted to be located outside the membrane. Several Pfam search failed to recognize any other well-supported
domains and features were identified or predicted in known functional domains in Pihmp1, this protein might still
the sequence of Pihmp1, being (from N- to C-terminus): contain domains specific to oomycetes, the function(s) of
signal peptide, mucin-like O-glycosylation, K/S-rich which remains to be elucidated.
repeats, DxSxS MIDAS-like motifs, S/G-rich repeats, Consistent with a suggested role as a structural protein,
N-glycosylation, cysteine-rich domain, TM helices and a transient gene silencing of Pihmp1 in P. infestans led to an

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2280 A. O. Avrova et al.

inability to form biotrophic structures after appressorium tion, becoming increasingly aggregated by 12 h, showed
formation. Pihmp1 may stabilize the P. infestans cell that the structures of the haustorial membrane and wall
membrane, and perhaps cell wall, during penetration. The are likely to be in constant flux over the course of its
observed microscopic phenotype was accompanied by an development and function, possibly in response to host
apparent loss of pathogenicity, even though Pihmp1- cues.
silenced lines were able to enter the leaf via stomata but The hypersensitive response, triggered by recogni-
were unable to spread further, indicating that Pihmp1 is tion of specific pathogen avirulence proteins by host
required for successful establishment of infection in addi- resistance proteins, targets the biotrophic stages of
tion to penetration. P. infestans infection, with host programmed cell death
Haustoria of fungal and oomycete plant pathogens leading to control of infection. The inability of Pihmp1-
differ in at least one major respect: differentiation of a silenced lines to form haustoria, and thus infect potato,
neckband close to the intercellular hyphae. The neckband also highlights the importance of haustorium formation to
functions to seal the EHM from the apoplast such that the the success of P. infestans infection. These observations
matrix becomes a unique and isolated compartment indicate that haustoria, and the essential functions they
(Szabo and Bushnell, 2001). Electron microscopy studies perform, provide ideal targets for late-blight control.
of the haustorium-forming oomycetes Albugo candida
and H. parasitica (syn. Peronospora parasitica) revealed Experimental procedures
some differences in electron-dense material between
Growth of P. infestans, potato plants and
intercellular hyphae and the intracellular haustorial cell
plant inoculation
wall and membrane (Soylu, 2004; Soylu et al., 2004),
although these differences were not as pronounced as for Growth of potato plants, P. infestans isolates CY29 and 88069,
fungal haustoria. The localization of Pihmp1 has revealed and plant inoculation were carried out as described in Grenville-
the first evidence that although the P. infestans hausto- Briggs et al. (2005). Life cycle stages of P. infestans (axenic
rium membrane and wall are apparently undifferentiated cultured mycelium, sporangia, zoospores, germinated cysts with
or without appressoria) were also prepared as described by
(Hohl and Suter, 1976), it does have a different protein
Grenville-Briggs et al. (2005).
complement to intercellular hyphae. This finding is con-
sistent with haustoria of rust fungi where it has been
RNA isolation, cDNA synthesis, SSH, real-time RT-PCR
shown that the uptake of sugars and amino acids occurs
expression analysis and mRFP RT-PCR
only in the haustoria through membrane-localized
transporters (Hahn et al., 1997; Voegele et al., 2001). As Total RNA was extracted using the QIAGEN RNeasy Plant Mini
stated, fungal haustoria are considered to be sealed from Kit using the protocol supplied by the manufacturer, with the
the host apoplast by a neckband. However, recent results addition of on-column DNase digestion. Synthesis of first-strand
with mRFP-tagged, translocation-deficient, Avr3a aviru- cDNA was carried out using the First Strand cDNA Synthesis kit
(GE Healthcare, UK) using oligo dT priming, following the proto-
lence protein showed that some leakage of proteins from
col supplied by the manufacturer.
the P. infestans EHM to the apoplast could occur
Suppression subtractive hybridization using the PCR-Select
(Whisson et al., 2007), indicating that it does not have a cDNA subtraction kit (Clontech) was performed to generate a
sealed EHM. Furthermore, those results highlighted that cDNA library enriched for sequences specifically expressed
the haustorium is likely to be a major site of effector during appressorium formation. SSH conditions were as
protein secretion during infection. described by Grenville-Briggs et al. (2005).
Also highlighted in this study was the rapidity with which For gene expression analysis, SYBR green real-time RT-PCR
haustoria are formed in plant cells and the time taken for assays were carried out as described in Avrova et al. (2003). The
a haustorium to fully mature. From the initial formation of P. infestans actinA (PiactA) gene was used as a constitutively
expressed endogenous control and relative expression of
Pihmp1::mRFP plaques on intercellular hyphae, haustoria
Pihmp1 was normalized against expression levels in non-
are nearly fully formed within 3 h, but continue maturing sporulating mycelium (assigned a relative expression value of
for up to 12 h. The time-courses also suggested that not 1.0) as described in Grenville-Briggs et al. (2008) and Judelson
all available sites for haustoria are utilized, or that the et al. (2008).
earliest events in initiation of haustorium formation may Pihmp1::mRFP fusion gene expression was detected by
be halted if a site is sensed as unsuitable. This observa- RT-PCR using the following conditions: each 20 ml of PCR con-
tion is in contrast with those of Hohl and Suter (1976) tained 0.5 U Taq DNA polymerase (Promega, UK), 2 ml of 10¥
reaction buffer, 1.5 mM MgCl2, 15 mM deoxynucleotide triphos-
where haustorial development was thought to only be
phates (Promega), 30 mM mRFPClaF and mRFPSacR primers
halted by host wall appositions after host cell penetration.
(Table S1), and 10 ng of cDNA. Thermocycling conditions were
Our results imply that sensing of suitable sites for haus- as follows: 94°C for 5 min, followed by 40 cycles of 94°C for 30 s,
torium development may occur earlier. The variation in 60°C for 30 s and 72°C for 1 min. A final extension step of 72°C
Pihmp1 localization during haustorial growth and matura- for 10 min was included.

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Journal compilation © 2008 Blackwell Publishing Ltd, Cellular Microbiology, 10, 2271–2284
Phytophthora infestans haustorial membrane protein 2281

P. infestans DNA manipulations Additional internet sites used for the structural analysis of the
predicted proteins are (all using default parameters): CBS Pre-
Transfer of the P. infestans BAC library to nylon membranes, diction Servers (TMHMM v. 2.0; NetOGlyc v. 3.1; NetNGlyc v. 1.0;
colony and Southern hybridization was as described in Whisson SignalP v. 3.0; http://www.cbs.dtu.dk/services/), SOSUI program v.
et al. (2001). BAC clone plasmid preparations were as described 1.11 (http://bpnuap.nagoya-u.ac.jp/sosui/) and Pfam v. 22.0
in Whisson et al. (2001; 2005). For fingerprinting, BAC clones (http://pfam.sanger.ac.uk/). Protein alignments were performed
were restriction digested with HindIII restriction endonuclease, using the CLUSTALW v. 1.4 program, using default parameters,
and fragments were separated by 1% agarose gel electrophore- within the BioEdit Sequence Alignment Editor v. 7.0.5.3. Protein
sis and stained with SYBR Gold nucleic acid stain (Invitrogen, distance calculations between homologues of Pihmp1 were per-
UK). formed on aligned protein sequences using default parameters
Phytophthora infestans genomic DNA was extracted from (Jones Taylor Thornton matrix; Jones et al., 1992) for the
freeze dried mycelium using the QIAGEN DNeasy Plant Mini Kit, PROTDIST v. 3.5c accessory program in the BioEdit Sequence
using the protocol supplied by the manufacturer. Southern Alignment Editor.
transfer of either restriction endonuclease (HindIII)-digested
P. infestans genomic DNA or BAC DNA from agarose gels to
Hybond N+ filters (GE Healthcare) was by standard methods Transient RNAi and trypan blue staining of
(Sambrook et al., 1989). Pihmp1-silenced P. infestans in planta
Preparation of single-sequence hybridization probes used
25 ng of probe DNA, 50 mCi of [a-32P]-dCTP and a single reaction Transient RNAi was performed as described in Whisson
of the High Prime labelling kit (Roche). et al. (2005). Primer T7SSHac2C5Fs in combination with
DNA from BAC clones 1B18, 5J1 and 15J23 was partially SSHac2C5Rs, and SSHac2C5Fs in combination with
T7SSHac2C5Rs (Table S1) were used to amplify an approxi-
digested with Sau3AI (Promega) and subcloned into vector
mately 200 bp section of Pihmp1 for in vitro synthesis of dsRNA
pGEM-3Z (digested with BamHI restriction endonuclease and
using the Megascript RNAi kit (Ambion, UK) following the proto-
alkaline phosphatase; Promega, UK) as described in Bell et al.
col supplied by the manufacturer.
(2002). For each BAC clone, 768 recombinant Escherichia coli
strain DH10B (Invitrogen) subclones were selected, grown at After treatment with dsRNA (Pihmp1 or controls), single colo-
37°C in 384-well microtitre plates in freezing medium (Whisson nies from regenerated (typically 5–6 days after treatment) proto-
et al., 2001) with ampicillin for 24 h, and then stored at -70°C. plasts were transferred to rye agar medium. Thirteen days after
BAC subclone plasmids were prepared using the QIAGEN the exposure of protoplasts to dsRNA, these colonies were
Plasmid Miniprep kit and sequenced in both directions with SP6 flooded with cold sterile distilled water to induce zoospore
and T7 primers (Table S1) using the Perkin Elmer ABI PRISM formation. After 3 h at 4°C, the suspension was collected and
BigDye Terminator 3.1 cycle sequencing kit, manufacturer- used for leaf inoculations. Leaf samples were taken at 48 hpi
recommended thermal cycling conditions, and ABI Model 377 (15 days after exposure to dsRNA) for RNA extraction. Subse-
DNA sequencer. The 5′ end of the Pihmp1 sequence was quently real-time RT-PCR was used to determine whether the
obtained by direct sequencing from BAC clones 1B18, 5J1 and Pihmp1 dsRNA-treated lines were silenced. Given the small
15J23 using primers 1B18R, 5J1R, 5J1R1, 5J1R2, 15J23R3, amounts of biological material available from each dsRNA-
2C5prR2 and 2C5prF2 (Table S1). treated line, the individual lines formed the biological replicates
for the experiment as a whole, while three technical replicates
were performed for each real-time RT-PCR assay.
DNA and protein sequence analyses Leaf samples were taken at 16, 24 and 48 hpi for trypan blue
staining (Koch and Slusarenko, 1990) and light microscopy. Both
DNA and protein sequence similarity searches were made with the infection site and developing lesion margin (where infection
BLASTN, TBLASTN, TBLASTX and BLASTP algorithms, using default progressed) were examined. Digital photomicrographs were cap-
search parameters, at NCBI GenBank (http://www.ncbi.nlm.nih. tured on an Olympus BH2 light microscope using Olympus DP70
gov/blast/Blast.cgi; non-redundant nucleotide and protein or C5050 digital cameras with automatic exposure settings.
databases, ESTs including human and mouse sequences),
Phytophthora Functional Genomics Database (PFGD; ESTs from
P. infestans and P. sojae; http://www.pfgd.org/), Virginia Microbial GFP and mRFP tagging of Pihmp1, and P. infestans
Database (individual genome databases for P. sojae, P. ramorum transformation
and P. capsici and H. parasitica; http://phytophthora.vbi.vt.edu/)
and P. infestans genome database (http://www.broad.mit.edu/ The Pihmp1 ORF preceded by 694 bp was amplified from
annotation/genome/phytophthora_infestans). BLASTN algorithm P. infestans BAC clone 5J1 using primers SpeI2C5 and
parameters were: expect threshold = 10, word size = 28, linear NotI2C5R. Each 50 ml of PCR contained 1 U Taq DNA poly-
gap cost, match and mismatch scores of 1 and -2 respectively. merase (Promega, UK), 5 ml of 10¥ reaction buffer, 1.5 mM
BLASTP and TBLASTN algorithm parameters were: expect MgCl2, 15 mM deoxynucleotide triphosphates (Promega), 30 mM
threshold = 10, word size = 3, BLOSUM62 matrix, gap costs of forward and reverse primers (Table S1), and 10 ng of BAC DNA.
existence = 11 and extension = 1, and conditional compositional Thermocycling conditions were as follows: 94°C for 5 min, fol-
score matrix adjustment. Algorithm parameters for TBLASTX were lowed by 40 cycles of 94°C for 30 s, 62°C for 30 s and 72°C for
as for BLASTP, but without conditional compositional score matrix 4 min. A final extension step of 72°C for 10 min was included. The
adjustment. ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf. Pihmp1 PCR product was digested with SpeI and NotI restriction
html) and DNA Tools (http://biology.semo.edu/cgi-bin/dnatools.pl) endonucleases, and introduced into the pSam SpeI and NotI
were used for predicting the ORFs in the sequences obtained. cloning sites in the sense orientation to yield construct p2C5-

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2282 A. O. Avrova et al.
GFP. pSam is a promoterless GFP construct also containing the Biological Sciences Research Council (BBSRC) (L.J.G.-B.) and
neomycin phosphotransferase gene (nptII) under control of the The Royal Society (P.v.W.). The authors thank L. Pritchard at SCRI
Bremia lactucae hsp70 promoter for expression in oomycetes. for bioinformatics advice. Genetic modification of P. infestans was
Pihmp1 with its native promoter was transferred from p2C5-GFP carried out under plant health licence PH/11/2008.
into pPL-mRFP to yield p2c5-mRFP. pPL-mRFP was constructed
by excision of the B. lactucae Ham34 promoter from pTor (Blanco
and Judelson, 2005) and ligation of the mRFP gene into the KpnI References
and SacII sites, with a linker for KpnI, NotI and ClaI cloning sites
at the 5′ end of the gene to yield a promoterless mRFP vector for Akhouri, R.R., Bhattacharyya, A., Pattnaik, P., Malhotra, P.,
oomycete expression. Pihmp1 was excised from p2c5-GFP with and Sharma, A. (2004) Structural and functional dissection
SpeI and SacII restriction endonucleases, blunt-ended with of the adhesive domains of Plasmodium falciparum
Klenow polymerase (New England Biolabs), cut with NotI restric- thrombospondin-related anonymous protein (TRAP).
tion endonuclease, purified and ligated into the pPL-mRFP Biochem J 379: 815–822.
vector. All fusions were sequenced in both directions to verify that Avrova, A.O., Venter, E., Birch, P.R., and Whisson, S.C.
the fusions were in frame and without introduced cloning errors. (2003) Profiling and quantifying differential gene transcrip-
Oomycete expression vector pVW2 for constitutive eGFP tion in Phytophthora infestans prior to and during the early
expression has been described elsewhere (van West et al., stages of potato infection. Fungal Genet Biol 40: 4–14.
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and Birch, P.R.J. (2007) A novel, non-protein coding
Stable transformation of P. infestans was achieved using a
infection-specific gene family is clustered throughout the
modified PEG-CaCl2-Lipofectin protocol described by Judelson
genome of Phytophthora infestans. Microbiology 153:
et al. (1991). An updated version of this protocol can be found at
747–759.
http://138.23.152.128/protocols/protocols.html. For use here,
Baldauf, S.L., Roger, A.J., Wenk-Siefert, I., and Doolittle,
the protocol was modified by substituting Novozym 234 with
W.F. (2000) A kingdom-level phylogeny of eukaryotes
a mixture of 5 mg ml-1 lysing enzymes (from Trichoderma
based on combined protein data. Science 290: 972–977.
harzianum; Sigma L1412) and 2 mg ml-1 cellulase (from Tricho-
Bechinger, C., Giebel, K.-F., Schnell, M., Leidere, P.,
derma reesei; Sigma C8546) to release protoplasts from germi-
Deising, H.B., and Bastmeyer, M. (1999) Optical measure-
nating sporangia. For each transformation, 50 mg of plasmid DNA
ments of invasive forces exerted by appressoria of a plant
was mixed with Lipofectin reagent (Invitrogen, UK) in the propor-
pathogenic fungus. Science 285: 1.
tions described in the protocol. Protoplasts were regenerated for
Bell, K.S., Avrova, A.O., Holeva, M.C., Cardle, L., Morris, W.,
48–72 h in pea broth (Whisson et al., 2005) containing 20 g l-1
De Jong, W., et al. (2002) Sample sequencing of a
sucrose and 1 M mannitol, prior to selection of transformants on
selected region of the genome of Erwinia carotovora
rye agar amended with 5 mg ml-1 geneticin. Transformants were
subsp. atroseptica reveals candidate phytopathogenicity
then transferred to, and maintained on, rye agar amended with
genes and allows comparison with Escherichia coli.
20 mg ml-1 geneticin.
Microbiology 148: 1367–1378.
Samples of mycelium, and cysts germinated in vitro for 16 h at
Beyer, K., Jimenez, S.J., Randall, T.A., Lam, S., Binder, A.,
10°C, were taken for DNA or RNA extraction and PCR or RT-PCR
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in 10 ml droplets onto detached potato leaflets. Inoculated leaflets Birch, P.R.J., Avrova, A.O., Armstrong, M., Venter, E., Taleb,
were incubated at 20°C for 24–72 h to allow cysts to germinate N., Gilroy, E., et al. (2003) The potato–Phytophthora
on the leaf surface, form appressoria, penetrate host cells and infestans interaction transcriptome. Can J Plant Pathol 25:
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six leaflets were inoculated on two independent occasions. Birch, P.R., Rehmany, A.P., Pritchard, L., Kamoun, S., and
All GFP (488 nm excitation; emission 500–530 nm) and mRFP Beynon, J.L. (2006) Trafficking arms: oomycete effectors
(561 nm excitation; emission 600–630 nm) imaging was con- enter host plant cells. Trends Microbiol 14: 8–11.
ducted on a Leica TCS-SP2 AOBS confocal microscope using Bittner-Eddy, P.D., Allen, R.L., Rehmany, A.P., Birch, P., and
HC PL FLUOTAR 63X0.9, HCX APO L U-V-I 40X0.8 or HCX APO Beynon, J.L. (2003) Use of suppression subtractive hybrid-
L U-V-I 20X0.5 water dipping lenses. For all images shown in this ization to identify downy mildew genes expressed during
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Burki, F., Shalchian-Tabrizi, K., Minge, M., Skjaeveland, A.,
This work was supported by the Scottish Government Rural and Nikolaev, S.I., Jakobsen, K.S., and Pawlowski, J. (2007)
Environment Research and Analysis Directorate (RERAD) Phylogenomics reshuffles the eukaryotic supergroups.
(A.O.A., P.C.B., V.Y., P.R.J.B. and S.C.W.), the Biotechnology and PLoS ONE 2: e790. doi:10.1371/journal.pone.0000790.

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Supporting Information
Tucker, S.L., and Talbot, N.J. (2001) Surface attachment and
pre-penetration stage development by plant pathogenic Additional Supporting Information may be found in the online
fungi. Ann Rev Phytopathol 39: 385–417. version of this article:
Vleeshouwers, V.G., van Dooijeweert, W., Govers, F.,
Fig. S1. Copy number and alleles of Pihmp1 in P. infestans
Kamoun, S., and Colon, L.T. (2000) The hypersensitive
isolate T30-4.
response is associated with host and nonhost resistance to
A. Pihmp1-hybridizing BAC clones digested with HindIII restric-
Phytophthora infestans. Planta 210: 853–864.
tion endonuclease, separated on 1% agarose gel and stained
Voegele, R.T., Struck, C., Hahn, M., and Mendgen, K. (2001)
with SYBR Gold.
The role of haustoria in sugar supply during infection of
B. Southern blot of gel in (A) hybridized to radiolabelled probe to
broad bean by the rust fungus Uromyces fabae. Proc Natl
Pihmp1, revealing three hybridizing restriction fragments of dif-
Acad Sci USA 98: 8133–8138.
fering sizes. Fragments shown in BACs5J1 and 15J23 represent
Walker, C.A., Köppe, M., Grenville-Briggs, L.J., Avrova, A.O.,
different RFLP alleles and contain the full Pihmp1 ORF, while the
Horner, N.R., McKinnon, A.D., et al. (2008) A DEAD-box
hybridizing fragment shown in BAC 1B18 represents a truncated
RNA helicase is required for normal zoospore development
non-expressed copy of Pihmp1. Molecular sizes are given at left
in the potato late blight pathogen Phytophthora infestans.
as kilo base pairs.
Fungal Genet Biol 45: 954–962.
Table S1. Oligonucleotide primers used in sequencing, real-time
van West, P., and Vleeshouwers, V.G.A.A. (2004) The Phy-
RT-PCR and transient RNAi.
tophthora infestans–host interaction. In Plant Pathogen
Table S2. Protein distance among homologues of hmp1, cellu-
Interactions. Annual Plant Reviews, Vol. 11. Talbot, N.J.
lose synthase 3 (cesA3), actin A (actA) and translation elongation
(ed.). Oxford: Blackwell Publishing UK, pp. 219–242.
factor 1a (tef1a) from Phytophthora infestans (Pi), P. ramorum
van West, P., de Jong, A.J., Judelson, H.S., Emons, A.M.C.,
(Pr), P. sojae (Ps) and Hyaloperonospora parasitica (Hp). Above
and Govers, F. (1998) The ipiO gene of Phytophthora
diagonal for each gene: protein distance measured across full
infestans is highly expressed in invading hyphae during
length of protein. Below diagonal for each gene (top to bottom):
infection. Fungal Genet Biol 23: 126–138.
protein distance measured across N-terminal half of protein,
van West, P., Kamoun, S., Van’t Klooster, J.W., and Govers,
protein distance measured across C-terminal half of the protein,
F. (1999) Internuclear gene silencing in Phytophthora
difference between protein distance from N- and C-terminal
infestans. Mol Cell 3: 339–348.
halves. Protein distance was calculated from CLUSTALW align-
van West, P., Morris, B.M., Reid, B., Appiah, A.A., Osborne,
ment using the PROTDIST accessory program in the BioEdit
M.C., Campbell, T.A., et al. (2002) Oomycete plant patho-
Sequence Alignment Editor.
gens use electric fields to target roots. Mol Plant Microbe
Interact 15: 790–798. Please note: Blackwell Publishing are not responsible for the
van West, P., Appiah, A.A., and Gow, N.A.R. (2003) content or functionality of any supporting materials supplied by
Advances in research on oomycete root pathogens. Phys the authors. Any queries (other than missing material) should be
Mol Plant Pathol 62: 99–113. directed to the corresponding author for the article.

© 2008 Scottish Crop Research Institute


Journal compilation © 2008 Blackwell Publishing Ltd, Cellular Microbiology, 10, 2271–2284

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