Identification of a Mastigoneme Protein from Phytophthora nicotianae

Protist, Vol. 162, 100–114, January 2011 http://www.elsevier.de/protis Published online date 21 July 2010

ORIGINAL PAPER

Identification of a Mastigoneme Protein from Phytophthora nicotianae Leila M. Blackmana,1 , Mikihiko Arikawab,c , Shuhei Yamadab , Toshinobu Suzakib , and Adrienne R. Hardhama aPlant

Sciences Division, Research School of Biology, College of Medicine, Biology and Environment, Australian National University, Canberra ACT 2601 Australia bDepartment of Biology, Graduate School of Sciences, Kobe University, Nada-ku, Kobe 657-8501, Japan cDepartment of Cardiovascular Control, Kochi Medical School, Nankoku, Kochi 783-8505, Japan Submitted November 21, 2009; Accepted January 23, 2010 Monitoring Editor: George B. Witman

Tripartite tubular hairs (mastigonemes) on the anterior flagellum of protists in the stramenopile taxon are responsible for reversing the thrust of flagellar beat and for cell motility. Immunoprecipitation experiments using antibodies directed towards mastigonemes on the flagella of zoospores of Phytophthora nicotianae have facilitated the cloning of a gene encoding a mastigoneme shaft protein in this Oomycete. Expression of the gene, designated PnMas2, is up-regulated during asexual sporulation, a period during which many zoospore components are synthesized. Analysis of the sequence of the PnMas2 protein has revealed that, like other stramenopile mastigoneme proteins, PnMas2 has an Nterminal secretion signal and contains four cysteine-rich epidermal growth factor (EGF)-like domains. Evidence from non-denaturing gels indicates that PnMas2 forms large oligomeric complexes, most likely through disulphide bridging. Bioinformatic analysis has revealed that Phytophthora species typically contain three or four putative mastigoneme proteins containing the four EGF-like domains. These proteins are similar in sequence to mastigoneme proteins in other stramenopile protists including the algae Ochromonas danica, Aureococcus anophagefferens and Scytosiphon lomentaria and the diatoms Thalassiosira pseudonana and T. weissflogii. © 2010 Elsevier GmbH. All rights reserved. Key words: Phytophthora; stramenopiles; mastigoneme; flagella

Introduction Development of cilia and flagella was an important step in the evolution of eukaryotes. In many protists, some fungi and reproductive cells of animals and lower plants, the primary function of cilia and flagella is to move the cell through an aqueous environment (Ginger et al. 2008). In non-motile cells, cilia function in the movement of liquids over

1 Corresponding author; fax +61 2614331. e-mail [email protected] (L.M. Blackman).

© 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.protis.2010.01.005

their surface, act as sensory structures or may be involved in signal transduction (Ginger et al. 2008; Johnson 1995; Marshall and Nonaka 2006). Eukaryotic cilia and flagella have the same fundamental structure. They consist of a shaft or axoneme containing a central pair of microtubules surrounded by nine microtubule doublets linked by a range of proteinacious structures (Silflow and Lefebvre 2001). Recent molecular and proteomic analyses have shown that they typically contain over 400 proteins (Pazour et al. 2005). The term cilia is used when they are present in large numbers and the term flagella is used when only one

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to four occur on a single cell (Marshall and Nonaka 2006). The surface of flagella may be smooth or decorated with fibrils, scales or hairs (Andersen et al. 1991; Melkonian et al. 1991). Flagellar hairs, or mastigonemes, may be non-tubular or tubular, with the latter category including bipartite and tripartite structures (Andersen et al. 1991; Andersen 2004; Bouck 1971; Bouck et al. 1978; Hardham 1987; Leedale et al. 1970). Tripartite mastigonemes consist of a hollow tubular shaft composed of a defined arrangement of globular subunits surrounding a hydrophilic lumen (Bouck 1969, 1972; Hill and Outka 1974), a solid tapering basal region (Bouck 1971) and one or more thin terminal filaments (Bouck 1969; Maier 1995). Biochemical studies of isolated mastigonemes have shown they consist of a number of proteins and glycoproteins (Bouck 1971; Chen and Haines 1976; Honda et al. 2007; Robold and Hardham 1998; Yamagishi et al. 2007). The possession of tripartite tubular mastigonemes is a defining characteristic of protists classified within the stramenopiles, a phylum or kingdom level taxon that includes the oomycetes, coloured algae and diatoms (Adl et al. 2005; Patterson 1989; Van de Peer and De Wachter 1997). The mastigonemes occur in two opposite rows along the forward-projecting flagellum and are essential for normal cell motility. Movement of the mastigonemes caused by wave propagation during flagellar beating, reverses the thrust of the anterior flagellum, resulting in the cell being pulled through the medium (Cahill et al. 1996; Christensen-Dalsgaard and Fenchel 2004; Hardham 1987; Jahn et al. 1964). The class Oomycetes contains a number of genera of destructive plant pathogens. Foremost among these are species of Phytophthora which include the causative agents of late blight of potato, sudden oak death and a wide range of other economically important plant diseases (Erwin and Ribeiro 1996). In most cases, motile Phytophthora zoospores are responsible for disease dissemination and initiation of plant infection. Phytophthora zoospores can swim over considerable distances to reach a suitable infection site (Erwin and Ribeiro 1996). For soil-borne species, the zoospores are attracted by the chemical gradients and electric fields surrounding plant roots, with reception of chemical and electrical signals possibly involving flagellar surface proteins (van West et al. 2002). The crucial role of mastigonemes in Phytophthora zoospore motility has been demonstrated experimentally (Cahill et al. 1996). Treatment of

living P. cinnamomi zoospores with monoclonal antibodies (designated Zg1-4) directed towards the mastigoneme shaft causes the mastigonemes to become detached from the anterior flagellum. Loss of mastigonemes leads to disruption of normal motility (Cahill et al. 1996). In studies of P. nicotianae, another monoclonal antibody designated Pn14B7 was raised against the mastigonemes and shown to be specific for a 40 kDa glycoprotein (Robold and Hardham 1998). In the present paper, we describe the use of the Pn14B7 antibody in immunoprecipitation experiments to obtain amino acid sequence data for the protein antigen. These data were then used to identify putative genes encoding mastigoneme proteins in species of Phytophthora for which complete genome sequence data are available. This information was in turn used to clone, sequence and study the expression of the gene encoding the mastigoneme protein in P. nicotianae. Antibodies raised against a synthetic peptide based on the predicted mastigoneme protein sequence confirmed that the cloned gene, designated PnMas2 for P. nicotianae mastigoneme protein 2, did indeed encode the 40 kDa mastigoneme shaft protein. Bioinformatic characterisation has yielded clues to PnMas2 structure and function.

Results Identification of a Putative Mastigoneme Protein Immunoblots of proteins precipitated by Pn14B7 confirmed the precipitation of the Pn14B7 mastigoneme protein, along with two other co-precipitated proteins with relative molecular weights of 63 kDa and 200 kDa (Supplementary Fig. S1). Bands containing Pn14B7 protein were excised from 19 gel strips and submitted for sequencing. Two regions of peptide sequence were obtained from Pn14B7-immunoprecipitated P. nicotianae protein. The sequence AXPNK/LXS was obtained by N-terminal sequencing and the sequence DMVQAIVINQAGGSGYK/LTYHDPY was obtained from internal sequencing. The latter was used to identify two predicted transcripts from the Joint Genome Initiative (JGI) P. sojae database (estExt_fgenesh1_pg.C_950010 [Physo1_1:141162] and estExt_fgenesh1_pg.C_ 950012 [Physo1_1:141164]), hereafter called PsMas2a (for P. sojae mastigoneme) and PsMas2b, respectively, and one predicted transcript from the JGI P. ramorum database (estExt_

102 L.M. Blackman et al.

fgenesh1_pg.C_950012 [Physo1_1:141164]), hereafter designated as PrMas2a (Fig. 1). All predicted proteins have a secretion signal, and the N-terminal peptide sequence obtained from the Pn14B7-immunoprecipitated protein corresponds to the region immediately after the secretion cleavage site in these three predicted proteins (Fig. 1). A large gene model, which had partial similarity to PsMas2b, with two potential start codons was also found by a tblastn search of P. ramorum scaffolds and was named PrMas2b (fgenesh1_pg.C_scaffold_46000006 [Phyra1_1:80095]). Single homologous genes, designated PiMas2 and PcapMas2, were found in the Broad Institute P. infestans (PITG_09306.1) and JGI P. capsici (estExt_fgenesh1_kg.C_30649 scaffold_3:1382120-1383451) databases. Two ESTs matching PsMas2b were identified in the NCBI EST database and confirmed the predicted coding region of this gene (NCBI accessions CF845434, CF849965). ESTs matching PiMas2 (NCBI accessions CV948191, CV922481, GR287006 and GR289859) and PcapMas2 (NCBI accessions FG040404 and FG040403) were also identified. No ESTs were found for PsMas2a or for the two P. ramorum genes, PrMas2a and PrMas2b. The EST data and Genscan software, which creates gene models based on a number of transcriptional, translational and splicing signals plus characterisations of introns, exons and intergenic regions (Burge and Karlin 1997), suggested that the predicted gene models for PiMas2 (PITG_09306.1) and PrMas2b are incorrect. The revised predicted PiMas2 protein was used in the phylogenetic analysis and this and the predicted protein for PcapMas2 are shown in Figure 1. As no ESTs for PrMas2b were identified and the predicted gene models from JGI and Genscan for this gene did not agree with EST data for the mastigoneme genes from other Phytophthora species, the gene model for PrMas2b could not be refined and this gene was omitted from any further analysis. Several ESTs for a single mastigoneme gene were found for P. brassicae (NCBI accessions ES286559, ES286311, ES284884, ES285445).

Identification of the P. nicotianae Mastigoneme Gene, PnMas2 A 301 bp fragment was amplified from P. nicotianae gDNA using degenerate primers based on the Phytophthora Mas2 gene sequences described above. Sequencing confirmed the PCR fragment was a partial gene homologous to the putative mastigoneme genes from the other Phytophthora species. The gene was designated PnMas2. Screening of a genomic P. nicotianae BAC library with the PCR fragment as a probe identified four positive BACs. The complete sequence of PnMas2 could not be obtained from these BACs using gene specific primers. This situation could arise because there were two copies of the gene on each BAC, thus the copy number of this mastigoneme gene in the P. nicotianae genome and in the BACs was investigated. A P. nicotianae genomic DNA blot probed with a 669 bp PCR fragment showed a single major band after digestion with BamHI and HindIII and two bands after digestion with XhoI (Fig. 2A). Since none of these enzymes are predicted to cut within the probe, the possibility of two closely linked PnMas2 genes remained. A Southern blot of DNA from the four BACs restricted with XhoI yielded two different hybridisation patterns (Fig. 2B). Three BACs had a single 7 kb XhoI hybridising band and one BAC had a single 8 kb XhoI band. To confirm that only one copy of PnMas2 exists in P. nicotianae, 6.28 kb of the 7 kb XhoI fragment from BAC 21J4 and the entire 8 kb XhoI fragment from 27H21 (NCBI accession GQ475286) were sequenced. Both fragments contained only one copy of the PnMas2 gene. The surrounding noncoding sequence was highly similar (results not shown). These data indicate that the four BACs each contain only a single copy of PnMas2. The two different BAC hybridization patterns are indicative of the cloning of the two alleles of the PnMas2 gene. Polymorphism for an Xho1 restriction site in the two alleles in the region of PnMas2 gives rise to the two hybridizing bands after Xho1 digestion in the genomic Southern blot.

➛ Figure 1. Alignment of predicted Phytophthora mastigoneme shaft proteins. PsMas2a (estExt_fgenesh1_pg. C_950010 [Physo1_1:141162]), PsMas2b (estExt_fgenesh1_pg.C_950012 [Physo1_1:141164]), PrMas2a (estExt_fgenesh1_pg.C_950012 [Physo1_1:141164]), PiMas2 (PITG_09306.1), PcapMas2 (estExt_ fgenesh1_kg.C_30649 scaffold_3:1382120-1383451) and PnMas2. Regions corresponding to the Nterminal secretion signal (underlined region), the two regions corresponding to the amino acid sequencing data from P. nicotianae immunoprecipitated protein (shaded region) and the EGF-like signature sequences (bold text) are shown.

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PnMas2 PsMas2a PrMas2a PsMas2b PiMas2 PcapMas2

PnMas2 PsMas2a PrMas2a PsMas2b PiMas2 PcapMas2

PnMas2 PsMas2a PrMas2a PsMas2b PiMas2 PcapMas2

PnMas2 PsMas2a PrMas2a PsMas2b PiMas2 PcapMas2

PnMas2 PsMas2a PrMas2a PsMas2b PiMas2 PcapMas2

PnMas2 PsMas2a PrMas2a PsMas2b PiMas2 PcapMas2

PnMas2 PsMas2a PrMas2a PsMas2b PiMas2 PcapMas2

10 20 30 40 50 60 | | | | | | MTKWGFVLALTVLLATPSLVLGACPNKCSGHGKCGLNDVCQCMQNMIGGDCAGRQCPFTR MTKWWFVAVLTALLATPSLVLGACPNKCSGHGKCDLNDVCDCMQNWIGGDCSGRQCPFTR MTKWWFVAVLTALVVTPSVVLGACPNKCSGHGRCGLNDVCDCMQNWVGGDCSGRQCPFTR MAKWWLVATLSASFAS--VVLGACPNKCSGHGKCGLNDVCDCMQNWIGGDCSGRQCPFTR MTKWGVVVALTLLLATPSLVLGACPNKCSGHGKCGLNDVCQCMQNWVGGDCSGRQCAFTR MAKW--LVTLTVLLAIPSVVMGACPNKCSGHGKCGLNDVCQCMQNWIGGDCSGRQCPFTR *:** : .*: .. :*:***********:*.*****:**** :****:****.*** 70 80 90 100 110 120 | | | | | | AWQDTAQRDDDAHYYAECGNRGTCDRATGECTCDSGFIGSGCRRMQCPNDCSGHGTCEYI AWHDTAQRDDDAHYYAECANRGSCDRSTGECACDAGFVGSGCRRMQCPNDCSGHGTCEFI AWQDTAQRKDDAHYSAECGNRGTCDRTIGECSCDAGFVGSGCRRMQCPNDCSGHGTCEFI AWHDTAQRDDDAHYYAECANRGSCDRSTGECACDAGFVGSGCRRMQCPNDCSGHGTCEFI AWQDTAQRDDDAHYHAECGSRGTCDRATGECTCDAGFIGSGCRRMQCPNDCSGHGTCEFI AWHDTAQRKDDAHYYAECGNRGTCDRTSGECSCDPGFVGSGCRRMQCPNDCSGHGTCEYI **:*****.***** ***..**:***: ***:**.**:********************:* 130 140 150 160 170 180 | | | | | | EELAGDAYHKRIGGVANRKYTLWDQEKIMGCVCDGGYEGHDCSSRTCPKGDDPLTPNQKD EELAGDNFHKRIQGVSGRKYSLWDQEKIMGCVCDANYEGHDCSLRTCPKGDDPLTPNQKD EELASDDYHKRIKGTAGTTYQLWDQEKIMGCVCDANYEGHDCSLRTCPKGDDPLTPNQFD EELAGDNFHKRIQGVSGRKYSLWDQEKIMGCVCDANYEGHDCSLRTCPKGDDPLTPNQFD EELATDTAHKRIGGVAGRKYTLWDQEKIMGCVCDANYEGHDCSMRSCPRGDDPLTPNQYD EELASNDYNKRVGGKSGTTYEVWDQEKIMGCVCDGGYEGHDCSLRSCPKGDDPLTPNQVD **** : :**: * :. .* :************..******* *:**:********* * 190 200 210 220 230 240 | | | | | | MVQAIVINQAGGSGYLTYHDPYGNTYTTEKITFG-----AALGTNDVTTCDNIETALRRL MVQAIKIKQSGGSGYLTYHDPYGNAYTTEKITFG-----AAAGTNDDTTCANIQQALRRL MVQAIVITKPGGTGYLTYYDPYGNAYTTEKIVFGGSG-ATFAASDDDTTCANIETALRRL MVQAIVITAAGGSGFLTYYDPYGNAYTTEKITFG-----AALGTNDDTTCSNIQQALRRL MVQAIILDKAGGEGYLTYYDPYGNAYTTEKITLGGTLSVAFQPTDDDTTCANIQKALRRL MVQAIVIDQAGGSGYLTYHDPYGNSYTTDKITFD-------TTANVKATCDSIQVALRRL ***** : .** *:***:*****:***:**.:. :: :** .*: ***** 250 260 270 280 290 300 | | | | | | PNNVLNNVEVSPASRFYAFTRTDPTD-PNGYGTVSDIHFNDGTSG--------SAVALRV PNNVLNGVTVSAAASFYSFLRTDPTD-PKGYGTVLPIYYSVANTA--------AATQDSV PNNVLNTVNVEVSARFYSFTRTLPLDLVLGVGTTTKLFNDDNVVTSPDLPWSGKGVQNKV PNNVLNTVTVEVAARVYGFTRKDPRG-VSGEGTTTKIFNDDNTGT---LPYDGTGTQDKV PNNVLNTVTVVAVDRFYAFKRSDPTD-SLGYGTLNKIVNDDAAAY------AGTGTQIKA PNNVLNGVMVSLSASFNSFTRTNIAD-PSGAGQLNTVLNNAIAGAPG---LVITATPSKT ****** * * . .* *. . * * : . .. . 310 320 330 340 350 360 | | | | | | ICEVVFNSEPGITGYQNLFECNVATH-TTVGQHPLSGGATGD---TCAVYEVYP--DANV ICEVVFNSEPGGTGYQNMLECNVALH-TAVGQHPVSGVGTG----SCEVFEVYA--DADV ICDVQFTSEPGTTGYQQLLECNALAHIDTTGHHPISAGVAGADATTCKVYEVYP--VAVV ICEVQFTSEAGTTGFQNLLDCNVLAHNDAGGQHPMSAGITGADATTCKVYEVYP--VDVV MCEVIFTSEPGTTGYQNLLDCNVAAHGDTKGQHPLTTGVT---SGTCVVKEVYP--VTLG VCLVEFKSGPGTTGYQNLLECNVALH-TATGQHPVSGRSTD----TCKVYEAAPGIDIPT :* * *.* .* **:*::::**. * : *:**:: : :* * *. . 370 380 390 400 410 420 | | | | | | VVG---------SIIPATTVLQRPLTELTECAGRGACDYDTGTCECFAGHMGLACQKQEALV VAG---------SIISATTVVQRPLTELAECSGRGTCDYSTGACTCFAGHMGLACESQEALV VTDTNANGAVVDEQIPDGTKAYRPLTELAECSGRGTCDYSTGTCTCFAGHMGLACQKQEALV VTDLNTDGSVLDEQIADGTTVYRPLTELAECSGRGACDYSTGTCECFAGHMGLACQKQEALV TSN----------MLAEDTPAYRPLTELTECAGRGTCDYDTGTCECFAGHMGLACQKQEALV VTDNTPSPAVVSYVPMVSTSGYRLLTEQTECSGRGTCDYSTGTCSCFAGHMGLACQSQEALV . . * * *** :**:***:***.**:* **********:.*****

104 L.M. Blackman et al.

Figure 2. Southern blot analysis of PnMas2 in P. nicotianae gDNA cut with BamHI, HindIII and XhoI (A) and in four BACs (21K13, 21J4, 26G20, 27H21) restricted with XhoI (B). Hybridising band patterns indicate that there is only one copy of PnMas2 in the P. nicotianae genome. The two hybridizing bands after XhoI digestion correspond to the two alleles of this gene. Numbers on the left indicate the approximate molecular weight in kB.

PnMas2 Gene Analysis Gene modeling and analysis of a single EST from a P. nicotianae appressorium cDNA library identified in NCBI (accession number FK936062, Kebdani et al. 2009) together with EST data from other Phytophthora species indicate that PnMas2 consists of a single open reading frame. There are three nucleotide differences between the genes encoding the two P. nicotianae alleles, resulting in a single amino acid difference, methionine versus tryptophane at position 47. The predicted protein has a molecular weight of 42 kDa and a pI of 4.86, contains an Nterminal secretion signal and has amino acid

sequences corresponding to the peptide sequence data obtained from the Pn14B7-immunoprecipated protein (Fig. 1). The PnMas2 protein has 79% amino acid identity with PsMas2a, 71% with PsMas2b and 68% with PrMas2a. A search of ESTs in NCBI also identified a partial homologue from another oomycete, the downy mildew Plasmopara halstedii (NCBI accession CB174681). A tblastn search of the NCBI database showed that PnMas2 showed homology with E values less than 1E-21 to four previously identified mastigoneme proteins from the heterokont chrysophyte Ochromona danica (Ocm1: NCBI accession BAF65668, Ocm2: NCBI accession BAH60835, Ocm3: NCBI accession BAI39491, Ocm4: NCBI accession BAH60836, Yamagishi et al. 2007, 2009). PnMas2 also showed homology to three sexually-induced (sig) proteins from the centric diatoms Thalassiosira weissflogii and T. pseudonana (sig1: NCBI accessions AF154499, XM_002286197; sig2: NCBI accessions AF154500, XP_00229659; sig3: NCBI accession AF154501, Armbrust 1999; Honda et al. 2007) (Supplementary Fig. S2) and two proteins in the brown alga Aureococcus anophagefferens (JGI accessions e_gw1.14.241.1 and fgenesh2_pg. C_scaffold_6000087) (Supplementary Fig. S2). No homologues were found in the genome of the pennate diatom Phaeodactylum tricornutum ( http://genome.jgi-psf.org/Phatr2/Phatr2.home.html). PnMas2 was also similar to the predicted protein sequence of a small fragment of the gene encoding the 115 kDa sig1 homologue from the brown alga Scytosiphon lomentaria (Honda et al. 2007). Recent characterization of the Ocm gene family in O. danica has shown that two Ocm proteins, Ocm1 and Ocm4, are located in the shaft of the mastigoneme, while the other two proteins, Ocm2 and Ocm3, are located in the basal region (Yamagishi et al. 2009). In order to determine if there are also other members of the mastigoneme gene family in Phytophthora and other stramenopile protists, tBlastn was used to search NCBI and the complete genomes of stramenopile species where available with Ocm1-4 as the query sequences. These searches identified two additional homologous genes at E values less than 6.8E-34 in each of the following species: P. capsici, P. infestans, P. ramorum, P. sojae, T. pseudonana and A. anophagefferens (Supplementary Table S1). Additional homologous ESTs were also found for P. nicotianae (NCBI accessions FK934823 and FK934881) and P. brassicae (NCBI accession ES287929).

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PnMas2 Protein Characterisation Like the other stramenopile mastigoneme proteins discussed above, the putative Phytophthora mastigoneme proteins are cysteine rich and all cysteine residues are predicted to participate in the formation of disulphide bonds, with the majority of these occurring within epidermal growth factor (EGF)-like domains (Supplementary Fig. S2). Three EGF-like domains were identified in PnMas2 by automated analysis using Pfam23 but manual examination of aligned proteins indicated the presence of a fourth EGF–like domain containing three cysteine residues (C-X-C-X8 -C) in all mastigoneme proteins (Fig. 1, Supplementary Fig. S2). In all Phytophthora proteins for which full-length sequence data are available, three of the EGF-like domains were tandomly arranged at the N-terminus and the fourth occurred at the C-terminus (Fig. 1, Supplementary Fig. S2). Comparison of all stramenopile mastigoneme proteins showed that the region between the third and fourth EGF-like domains was the most variable in sequence and length (Supplementary Fig. S2). The first domain had a structure similar to other well characterised EGF-like modules (Campbell and Bork 1993). The second EGF-like domain had 21 residues between the first and second cysteine residues and the third EGF-like domain had 33 residues between the third and fourth cysteine residues. The fourth EGF-like domain lacks the first cysteine residue. The general structure for all four EGF-like domains in Phytophthora can be given as: -C1 -X(3,21,3, -) -C2 -X3 -G-X-C3 -X(5,6,33,6) -C4 -X-C5 -X8 -C6 where X is any amino acid, C is cysteine, G is glycine and values in parentheses show the number of residues in each EGF-like domain (1-4). Searches for motifs for three types of posttranslational modifications known to occur in EGF-like domains, namely calcium-binding motifs, O-glycosylation sites and O-fucosylation sites, showed that only the latter occurred in stramenopile mastigoneme proteins. Two O-fucosylation motifs between the second and third cysteines were identified in the second and fourth EGF-like domains (C2 -A/G-N-R-G-T-C3 and C2 -S-G-R-GT/A-C3 ). Additional residues, including phenylalanine and tyrosine residues in the second and third EGF-like domains, respectively, and two aspartic acid residues at position 85 and 144 and a glutamic acid residue at position 158, were conserved.

Phylogenetic Analysis of Mastigoneme Proteins The Phytophthora mastigoneme protein consensus sequence had some similarity to tenascin C from a number of eukaryotic species including rat (NM 053861, 5E−12 ) and mouse (NM 011607, 5E−12 ) and a tenascin-like protein from P. infestans (PITG 10517, 1.08E−42 ). No proteins with a sequence similar to that of the Phytophthora mastigoneme proteins were identified in organisms closely related to the stramenopiles (e.g. the alveolates, cryptomonads and haptophytes). Phylogenetic relationships between the stramenopile mastigoneme proteins were investigated using two approaches. In the first, a distance matrix algorithm (ProtDist) compared the conserved residues of all the full length stramenopile mastigoneme protein sequences available, resulting in the tree shown in Supplementary Figure S3. In this tree, the mastigoneme proteins from Phytophthora species form three clusters, designated as PxMas1, PxMas2 and PxMas3 proteins (where x is substituted by a letter or letters indicative of the species). The cluster of PxMas2 proteins from Phytophthora groups with Ocm2 from O. danica, the Sig2 proteins from the two species of Thalassiosira and with a protein from A. anophagefferens. The PxMas3 proteins from Phytophthora cluster with Ocm3 and Ocm4 from O. danica and with one protein from each of the diatoms and brown alga. The relationship of the PxMas1 proteins with any of the other stramenopile mastigoneme proteins is not clear. In the second approach, the CLANS algorithm (Frickey and Lupas 2004) was used in pairwise comparisons of the stramenopile mastigoneme proteins and approximately 2500 other proteins identified by Blast searches that contain EGF-like domains, including the tenacins (data not shown). This latter analysis indicated that the stramenopile mastigoneme proteins form a loosely associated group that is distinct from other proteins with EGF-like domains but no information on relationships between the stramenopile mastigoneme proteins was obtained nor did this approach identify any other functional domains.

Antibodies Confirm PnMas2 is the Mastigoneme Shaft Protein The monoclonal antibody Pn14B7 recognises a single band at approximately 40 kDa in P. nicotianae protein extracts (Fig. 3A) (Robold and Hardham

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1998). Antibodies raised in two rabbits against a synthetic peptide based on DNA sequence data from the fourth EGF-like domain in the C-terminus of the putative mastigoneme protein labelled a single band of the same molecular weight as labelled by Pn14B7 (Fig. 3A). This reaction was seen at all antibody dilutions tested and no other bands were labelled even at high antibody concentrations. The pre-immune sera did not label any polypeptides in the blots (Fig. 3A). These results indicate that the rabbit antibodies are specific and label the Pn14B7 antigen. Western blot analysis of zoospore proteins separated under non-denaturing condi-

Figure 3. A. Western blot analysis of zoospore proteins separated by SDS-PAGE and labelled with the Pn4B7 monoclonal antibody and two rabbit polyclonal antisera (I-1 and I-2) raised against a synthetic peptide the sequence of which corresponded to the fourth EGF-like domain in the C-terminus of the predicted Phytophthora PnMas2 sequence. All three antibodies recognise a protein of the same size. Preimmune sera (PI-1, PI-2) do not react with any protein in the zoospore extracts. B. A protein complex greater than 450 kDa in relative molecular weight is recognised by Pn14B7 antibodies in extracts of zoospore proteins separated by Blue Native PAGE. The addition of 50 or 100 mM ␤-mercaptoethanol (50, 100) does not disrupt the PnMas2 protein complex. Protein size standards are shown in kDa on the left.

tions by Blue native PAGE, allowed the oligomeric state of the mastigoneme protein to be investigated. Labeling with Pn14B7 showed that the PnMas2 protein forms a large oligomeric complex of approximately 450 kDa relative molecular weight. Addition of 50 or 100 mM ␤-mercaptoethanol to the sample buffer failed to disrupt this large complex, indicating the existence of strong intermolecular interactions between the proteins within the complex (Fig. 3B). Immunofluorescence experiments showed that the rabbit anti-PnMas2 antibodies labelled the mastigonemes on the anterior flagellum of P. nicotianae zoospores (Fig. 4 A, C). In double immunolabelling experiments, the polyclonal antibodies co-localised with the Pn14B7 monoclonal antibody (Fig. 4 B, D; Supplementary Fig. S4). The polyclonal antisera also led to fluorescence of the zoospore cytoplasm but the significance of this is unclear as polyclonal antibodies often give higher background labelling of the zoospore cytoplasm than observed with monoclonal antibodies. Labelling of mastigonemes or other components was not observed with the preimmune sera (Fig. 4 E, F). Immunogold labelling of the P. nicotianae zoospores with Pn14B7 confirmed that the PnMas2 protein occurred only in the tubular shaft of the mastigonemes and was not present in the terminal hairs or in the basal segment (Fig. 4 G, H). When purified Pn14B7 antibody is added to living P. nicotianae zoospores, it causes mastigoneme detachment and inhibition of zoospore motility in a concentration-dependent manner (concentration range tested was 0.015 to 2.5 ␮g/ml) as observed with Zg monoclonal antibodies and P. cinnamomi zoospores (Cahill et al. 1996).

Expression of PnMas2 Transcription of PnMas2 was measured at the four main stages in the asexual life-cycle of P. nicotianae using quantitative real time PCR (qPCR) (Fig. 5A). PnMas2 was highly expressed in sporulating hyphae, whereas lower levels of expression were detected in vegetative hyphae and zoospores and no expression was detected in 3 h germinated cysts (Fig. 5A). The onset of PnMas2 expression during sporulation was assessed during the first 3 days after induction of sporulation by transferring the cultures to mineral salts solution (Fig. 5B). In parallel with the marked appearance of sporangia, PnMas2 expression levels increased substantially at 4 and 5 days after induction of sporulation.

Phytophthora Mastigoneme Protein 107

Discussion The tripartite tubular mastigonemes that project from the surface of flagella of many protists are key components on a number of levels. In terms of phylogeny, tripartite tubular mastigonemes are the characteristic that unites a diverse range of protists into the stramenopile clade (Patterson 1989). In terms of biology, mastigonemes alter the mechanics of flagellar function, allowing a forward-projecting flagellum to pull cells through the medium (Jahn et al. 1964). In terms of pathogenicity, mastigoneme-based motility generates strong swimming abilities that allow infective spores to cover considerable distances in their search for suitable infection sites (Cahill et al. 1996). In this paper, we report the first identification and cloning of a gene encoding a mastigoneme protein in the Oomycetes, a major class of stramenopiles that includes a large number of species that cause serious diseases in animals and plants (Lamour and Kamoun 2009). We show that the oomycete mastigoneme shaft protein is homologous to mastigoneme proteins in algal and diatom stramenopile groups, giving further evidence to support the proposal that organisms with tripartite tubular mastigonemes form a monophyletic group (Dick 1997). Analysis of mastigoneme protein structure provides clues to mastigoneme assembly. Analysis of PnMas1 transcript levels shows that enhanced expression of this gene coincides with the appearance of packets of mastigonemes in the sporulating hyphae (Cope and Hardham 1994). The mastigonemes occur in an anti-parallel arrangement within specialized regions of the ER in the hyphal and sporangial cytoplasm before being secreted and becoming distributed along the anterior flagella during sporangial cleavage (zoosporogenesis) (Cope and Hardham 1994) as seen during flagellar formation in other stramenopiles (Bouck 1969, 1971; Heath et al. 1970; Hill and Outka 1974).

➛ Figure 4. Immunofluoresence and immunogold localisation of anti-mastigoneme shaft antibodies in P. nicotianae zoospores. Polyclonal antibodies raised against a synthetic peptide corresponding to the fourth EGF-like domain in the C-terminus of the predicted PnMas2 protein (A, C) co-localise with the

Pn14B7 monoclonal antibody (B, D) and label the hairs along the anterior flagellum. Preimmune serum does not label any zoospore structures (E, F). At the ultrastructural level, Pn14B7 followed by anti-mouse immunoglobulin conjugated to 10 nm gold particles labels the tubular shaft but not the proximal base or the terminal hairs of the mastigonemes (H). Image H is an enlargement from the boxed region in G. Scale bar in A-F = 10 ␮m. Scale bars in G and H = 4 ␮m and 1 ␮m, respectively.

108 L.M. Blackman et al.

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Figure 5. Relative transcript levels of PnMas2 as measured by qPCR during development. (A) Expression levels relative to the normalising gene WS41 in key developmental stages of vegetative hyphae (V), sporulating hyphae (S), zoospores (Z) and 3 h germinated cysts (GC). (B) Expression level of PnMas2 relative to WS41 during sporulation (bars) and average numbers of sporangia per field of view (diamonds). Data show average values from three biological replicates. Error bars show standard deviations for expression data.

With only one exception (Ocm3), all the stramenopile mastigoneme proteins whose genes have been cloned to date contain an N-terminal secretion signal and four EGF-like domains. Bioinformatic analysis shows this to be the case for six species of Phytophthora, for the downy mildew P. halstedii, for two species of centric diatoms (T. weissflogii and T. pseudonana) and for three algal species (O. danica, S. lomentaria and A. anophagefferens). Ocm3 differs in lacking an N-terminal secretion signal

(Yamagishi et al. 2009). Interestingly, homologous proteins were not found in the pennate diatom, P. tricornutum, which forms non-flagellate amoeboid gametes (Bowler et al. 2008). Mastigoneme protein homologues were also absent from the genomes of protists in phylogenetically related taxa outside the stramenopiles (e.g. the alveolates). The functions of proteins containing EGF-like domains are varied (Campbell and Bork 1993; Engel 1991). The six cysteines within the EGF-like domain form disulphide bridges that create a highly stable, two-stranded ␤-sheet that protects the protein from harsh extracellular conditions (Sandal et al. 2006). EGF-like domains can occur as a single module, but more often exist in multiple copies together with other domain types (Campbell and Bork 1993). The three dimensional structure and disulphide bonding pattern have been determined for a number of EGF-like modules. These studies have shown that bridges are typically formed between the first and third, second and fourth, and fifth and sixth cysteines resulting in four loops (Appella et al. 1988; Rao et al. 1995; SelanderSunnerhagen et al. 1992; Werner et al. 2000). While the primary sequence within the EGF-like domain can be variable, apart from the first intercysteine region, the length of the intercysteine regions is conserved (Campbell and Bork 1993). There are a number of features of the stramenopile mastigoneme proteins that differ from typical proteins containing EGF-like domains. Firstly, they are unusual in that the only identifiable domains they contain are the four EGF-like modules. Secondly, two of the EGF-like domains have intercysteine regions that are larger than usual. Thirdly, one of the EGF-like domains consists of only five cysteine residues. EGF-like domains with large intercysteine regions and with fewer or more than six cysteines, although not common, have been found in proteins from other organisms (Campbell and Bork 1993; Cheek et al. 2006). Structural extracellular proteins containing EGFlike domains, such as fibronectin (Patel et al. 2006) and fibrillin (Trask et al. 1999), form oligomers held together by disulphide bonds. The prediction that all cysteine residues in the Phytophthora mastigoneme proteins, including those outside the EGF-like domains, can form disulphide bonds means that the Phytophthora mastigoneme protein may also form disulphide-bonded oligomers. The fact that addition of ß-mercaptoethanol does not disrupt the 450 kDa mastigoneme protein complex seen in native gels indicates that strong intermolecular interactions are linking components of the complex. The immunoprecipitation experiments

Phytophthora Mastigoneme Protein 109

all showed that proteins of 63 kDa and 200 kDa co-precipitated with PnMas2. Although the binding of calcium to EGF-like domains promotes proteinprotein interactions in some proteins (e.g. fibrillin; Rao et al. 1995), only 25% of all EGF-like domains are thought to bind calcium (Stenflo et al. 2000) and so the predicted lack of calcium binding by the Phytophthora mastigoneme EGF-like domains is not unusual. It is possible that the conserved phenylalanine/tyrosine residue found between the fifth and sixth cysteine in the second EGF-like domain of all stramenopile mastigoneme proteins may be involved in hydrophobic packing and domain stabilization as seen in the tandomly arranged EGF-like domains of fibrillin (Handford et al. 2000) and the merozoite surface protein 1 (MSP-1) from P. falciparum (Morgan et al. 1999). Protein-protein interactions and formation of oligomers could also be enhanced by addition of O-fucose at the predicted O-fucosylation sites in two of the EGF-like modules in the mastigoneme proteins (Luther and Haltiwanger 2009; Shao et al. 2003). Immunolabelling with the Pn14B7 monoclonal antibody and the anti-PnMas2 polyclonal antibodies confirms that PnMas2 is located in the shaft of the mastigonemes in P. nicotianae but not in the tapering basal region or the terminal thin filaments. However, of the four mastigoneme proteins in O. danica the one that is the most similar to PnMas2 in amino acid sequence according to the protein distance and neighbour-joining algorithm, Ocm2, does not occur in the mastigoneme shaft. In O. danica, 85 kDa Ocm1 and 22 kDa Ocm4 have been localised to the shaft while 41 kDa Ocm2 and 24 kDa Ocm3 have been localised to the basal segment (Yamagishi et al. 2007, 2009). Antibodies raised against the 115 kDa (SL115) mastigoneme protein from S. lomentaria also label the mastigoneme shaft in this species and in S. biplastida (Honda et al. 2007). The clustering of PxMas3 proteins with Ocm3 raises the possibility that the Mas3 proteins in Phytophthora may occur in the basal segment. Antibodies, designated Zt1-Zt4, raised against mastigoneme proteins in P. cinnamomi have shown that one or more antigenically-distinct proteins are located in the basal region of the Phytophthora mastigonemes (Robold and Hardham 1998) but whether or not these antigens correspond to either PxMas1 or PxMas3 proteins remains to be determined. The inability of the CLANS analysis to show phylogenetic relationships between these proteins indicates that functional homologies between the proteins in the different species will need to be determined empirically from cell biological studies rather than from sequence analyses.

In this regard, it is of interest to note that the 63 kDa protein that co-precipitates with PnMas2 is of the same molecular weight as that predicted for Mas3 proteins from the five Phytophthora species in which the Mas3 gene has been sequenced. In future studies, amino acid sequencing of the two coprecipitated proteins may shed further light on the nature of the PnMas2 protein complex and on the localization and function of its component proteins.

Methods Phytophthora culture: P. nicotianae isolate H1111 (Gabor et al. 1993: ATCC MYA-141), which was originally isolated from Nicotiana tabacum by David I. Guest (isolate M4951), was cultured according to Robold and Hardham (1998) and samples taken for both protein and gene expression. Vegetative hyphae were collected after growth for 24 h in 40 ml of V8 broth in 20 mm deep Petri dishes. Zoospores were released from sporulating hyphae after 3 weeks of culture as described in Blackman et al. (2005) and 3 h germinated cysts were produced according to Gan and coworkers (2009). All samples were frozen in liquid nitrogen immediately after harvesting and stored at -80 ◦ C until protein or RNA extraction. In order to obtain relatively synchronous sporulation, cultures were grown in V8 broth for 9 days, in fresh V8 broth for 4 days and in a final change of V8 broth for 24 h. Sporulation was then induced by transferring the hyphae to mineral salts solution as described in Blackman et al. (2005). Development of sporangia was followed by counting the number of sporangia at low magnification in five fields of view in a light microscope for each culture plate. Average values for sporangial number were calculated for three biological replicates of three plates each. Sporangial counts were made immediately after transfer to mineral salts solution (0 h) and at 9, 12, 18 and 24 h and 2, 3 and 4 days later. Hyphal samples from the three biological replicates of three plates each were collected at these time points and at 5 days after transfer to mineral salts solution. Immunoprecipitation and protein sequencing: Proteins for immunoprecipitation with monoclonal antibody Pn14B7 were prepared from sporulating hyphae grown on miracloth for 19 to 26 days in V8 broth. The harvested sporulating hyphae (from a total of 143 plates) were resuspended in a homogenization buffer (5 mM EDTA, 1 mM phenylmethanesulphonyl fluoride, 0.01 mg/ml leupeptin in PBS) and disrupted by sonication five times for 30 seconds on ice at 30 seconds intervals with an Ultrasonic Disrupter (Tomy UR-20P, Tokyo, Japan) followed by incubation at 4 ◦ C with shaking for 1 hour. Cell debris was removed by centrifugation for 10 min at 10,000 g at 4 ◦ C, followed by filtration through a 0.45 ␮m Millipore (Billerica, MA, USA) membrane. The cleared cell lysate was preincubated with PBS-rehydrated Protein G Sepharose (GE Healthcare, Japan). One part sepharose was added to 10 parts lysate and incubated for 1 hour at 4 ◦ C. After gentle centrifugation with a hand-driven centrifuge, the supernatant was filtered through a 0.45 ␮m Millipore membrane to remove the sepharose. An aliquot of 10 ␮l of the Pn14B7 antibody (1.2 mg/ml purified immunoglobulin, giving a final concentration of 2.4 ␮g/ml) was then added to 5 ml cleared lysate and incubated with shaking at 4 ◦ C for 1 hour. To precipitate immune complexes formed between Pn14B7 antibody and its specific antigen, 30 ␮l Protein G Sepharose was added to the mixture and incubated with

110 L.M. Blackman et al. shaking at 4 ◦ C for 1 hour, followed by manual centrifugation at low speed. The Protein G Sepharose/antibody/antigen pellet was washed three times with PBS and mixed with 40 ␮l 2x Sample Buffer (SB, 0.15 M Tris-HCl pH 6.75, 20% v/v glycerol, 4% w/v sodium dodecyl sulfate, 10% v/v ␤-mercaptoethanol, 0.08% w/v bromophenol blue). The remaining lysate was re-extracted by adding a fresh aliquot of Pn14B7 and Protein G Sepharose and the incubation and centrifugation repeated. The pellet was mixed with the same aliquot of 2x SB obtained above and proteins were released from the sepharose by heating to 100 ◦ C for 5 min. The protein sample (40 ␮l) was separated on an 11% SDS-PAGE gel and visualized by Coomassie staining. The 40 kDa protein band corresponding to the size of the putative mastigoneme protein previously identified as the antigen of the Pn14B7 antibody in immunoblots (Robold and Hardham 1998) was present between two bands of immunoglobulins (light and heavy chains) and excised carefully from the gel. The peptides fragmented by in-gel lysylendopeptidase digestion were separated with a Waters-HPLC system (Waters Corporation, USA) and N-terminal and internal amino acid sequence determined with Procise 494 cLC (Applied Biosystems, USA). Pn14B7 bands were excised from 19 gel strips for the sequencing analysis. DNA and RNA isolation and analysis: Pn14B7 antigen peptide sequences were used to identify homologous genes by tblastn (Altschul et al. 1997) queries of publicly available Phytophthora sequences. Databases searched included the JGI P. sojae (http://genome.jgi-psf.org/Physo1 1/Physo1 1.home.html), P. ramorum (http://genome.jgi-psf. org/Phyra1 1/Phyra1 1.home.html) and P. capsici (http:// genome.jgi-psf.org/PhycaF7/PhycaF7.home.html) sequenced genomes, the Broad Institute P. infestans database (http:// www.broadinstitute.org/annotation/genome/phytophthora infestans/MultiHome.html) and the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/) nucleotide database. Primers were designed against two putative mastigoneme genes, one from P. sojae (estExt fgenesh1 pg.C 950012 [Physo1 1:141164]) and one from P. ramorum fgenesh1 pg.C scaffold 46000008 [Phyra1 1:80097] (CCTGCCCCAACAAGT-CACAGCCCATGATCTTCTC). The primers were used to amplify partial putative mastigoneme genes from P. nicotianae gDNA which had been prepared according to the method described in Dudler (1990). PCR fragments were cloned into pGEM T-Easy (Promega) and sequenced at the Australian Genome Research Facility (St Lucia, Queensland). A P. nicotianae genomic BAC library (Shan and Hardham 2004) was screened and DNA made from four positive BACs. These were partially sequenced using gene specific primers (TGGCAGGATACAGCTC, CGTACCACAAGCGCA, CCGCTGCCGATAAAT, GGCTACGGCACAGTC, ACATCCGGTAGTGCC, GAACACTACCTCACAAATCAC). Gene copy number was estimated from a Southern blot of P. nicotianae gDNA restricted with BamH1, HindIII and XhoI, transferred onto Hybond-N+ and probed with a 669 bp PCR product amplified from P. nicotianae gDNA using mastigoneme gene specific primers (GACGACGCACACTACTACGC, GAACACTACCTCACAAATCAC) as described in Blackman et al. (2005). A Southern blot was also made from DNA isolated from the four BACs using a HiPure Plasmid Kit (Invitrogen, Carlsbad, CA), cut with BamHI and XhoI, transferred to Hybond-N+ (GE Healthcare, Rydalmere, Australia) and probed with the P. nicotianae PCR fragment by the method described in Shan and Hardham (2004). Two representative BACs (21J4 and 27H21) were digested with XhoI, separated on a 1% Tris-acetate-EDTA (TAE; Sambrook & Russell 2001) agarose gel, and bands corresponding to the

size of gDNA hybridising XhoI bands, excised, purified with a PureLink Quick Gel Extraction kit (Invitrogen) and subcloned into pBluescript and sequenced using T7, T3 and gene specific primers. For bioinformatic analysis, putative mastigoneme genes from P. sojae were named PsMas2a and PsMas2b and other Phytophthora mastigoneme genes named according to the degree of amino acid similarity to the P. sojae predicted proteins as found by ClustralW (http://npsapbil.ibcp.fr/cgi-bin/npsa automat.pl?page=npsa clustalw.html, Thompson et al. 1994). The presence of predicted introns in gene models was checked by analysis of ESTs for these genes and by intron-exon modelling using Genscan (http://genes.mit.edu/GENSCAN.html Burge and Karlin 1997). Based on the results gene models were confirmed or refined. ClustralW was also used to determine the Phytophthora mastigoneme protein consensus sequence using the alignment of putative mastigoneme proteins from P. nicotianae, P. sojae, P. infestans, P. ramorum and P. capsici. Phylogenetic analysis of mastigoneme proteins: The consensus sequence for the putative Phytophthora mastigoneme protein was used to search the sequenced genomes from the chromalveolates grouping (Keeling 2009), namely the alveolates (Plasmodium species, Toxoplasma gondii, Neospora caninum, Theileri species and Cryptosporidium at http://eupathdb.org/eupathdb/; and the ciliate Tetrahymena thermophila at http://www.ncbi.nlm. nih.gov/Genomes/), stramenopiles (the diatoms P. tricornutum http://genome.jgi-psf.org/Phatr2/Phatr2.home.html and T. pseudonana http://genome.jgi-psf.org/Thaps3/Thaps3.home. html and the brown alga A. anophagefferens http://genome. jgi-psf.org/Auran1/Auran1.home.html), cryptomonads (Guillardia theta and Hemiselmis andersenii at http://www.ncbi.nlm. nih.gov/Genomes/) and haptophytes (Emiliania huxleyi at http://genome.jgi-psf.org/Emihu1/Emihu1.home.html) for homologous mastigoneme genes. In addition, the predicted protein sequence of four mastigoneme proteins identified in O. danica (Yamagishi et al. 2009) were used to search NCBI, the four Phytophthora species, the P. tricornutum, the T. pseudonana and the A. anophagefferens databases. Proteins were considered to be putative mastigoneme proteins if they had a similar domain structure to those identified from other stramenopiles (Armbrust 1999; Honda et al. 2007; Yamagishi et al. 2007) including the predicted Phytophthora mastigoneme proteins. Putative mastigoneme proteins were labelled according to their homology with the mastigoneme proteins from O. danica (Yamagishi et al. 2009). Full length predicted mastigoneme proteins were aligned using Dialign (http://bibiserv.techfak.uni-bielefeld.de/dialign/, Morgenstern 2004), manually edited and a phylogenetic tree constructed using the ProtDist (protein distance matrix) and neighbourjoining tree function in BioEdit (version 7.0.9.0, Hall 1999) and examined with Treeview (version 1.6.6). Phylogenetic relationships between the mastigoneme proteins from different species of stramenopiles and between these proteins and other proteins containing EGF-like domains was also investigated using the CLANS program that determines pairwise similarities between proteins (Frickey and Lupas 2004). Predicted proteins were analysed for secretion signals using Signal3.0 (http://www.cbs.dtu.dk/services/SignalP/, Emanuelsson et al. 2007) and the molecular weight and pI determined by the Compute PI/MW tool (http://www.expasy. ch/tools/pi tool.html). Pfam 23.0 (http://pfam.sanger.ac.uk/, Finn et al. 2008) and Scanprosite (http://www.expasy. ch/tools/scanprosite/) were used to identify motifs that may be important for protein function. Tertiary structure analysis was

Phytophthora Mastigoneme Protein 111 performed using a number of programs found on the ExPASy Proteomics Tools site (http://www.expasy.ch/tools/). DiANNA software (http://clavius.bc.edu/∼clotelab/DiANNA/, Ferrè and Clote 2006) was used to predict the formation of disulphide bonds between cysteine residues. Residues that were highly conserved among mastigoneme proteins but to which automated annotation did not assign a function were examined manually for cryptic motifs. Epidermal growth factor-like (EGF) domains, which were identified by Scanprosite or by visual scanning for the EGF-like signature domain (C-X-C-X8 -C) (Appella et al. 1988), were examined for consensus sequences for the three post-translational changes that can occur in these domains (Harris and Spellman 1993; Luther and Haltiwanger 2009). Putative sites for the addition of O-fucose to serine or threonine were identified by the motifs C2 -X(4-5) [S/T]-C3 where C2 and C3 are the conserved second and third cysteines. Oglucose glycan addition sites were identified by C1 -X-S-X-P-C2 where C1 and C2 are the first and second cysteines. Calciumbinding relies on the hydroxylation of Asp/Asn residues and this is identified by the consensus sequence [D/N]-X-[D/N]-[E/Q]-C1 and C3 -X-[D/N]-Xn-[Y/F]-C4 (Maurer and Hohenester 1997). Antibody production: To confirm that the P. nicotianae gene, identified through searches using the peptide data from the Pn14B7-immunoprecipitated protein, did encode the mastigoneme protein, polyclonal antibodies were generated in two rabbits against a synthetic peptide based on the predicted protein sequence within the fourth EGF-like domain at the C terminus (CDYDTGTCECFAGHMG) of PnMas2. Immunolabelling: Zoospores were fixed in 4% v/v formaldehyde or 4% v/v formaldehyde/0.2% v/v glutaraldehyde as described in Hardham (2001) and labelled with the rabbit antibodies, diluted 1 in 200 in 1% w/v bovine serum albumin (BSA) plus 0.1% v/v fish skin gelatin (Sigma) in PBS. Zoospores were double-labelled with the rabbit polyclonal antibodies and mouse monoclonal Pn14B7 according to Robold and Hardham (1998). The secondary antibodies were sheep anti-rabbit Ig conjugated to fluorescein isothiocyanate (FITC; Chemicon, Temecula, NJ) diluted at 1 in 50 and sheep anti-mouse Ig conjugated to Cy3 (Jackson ImmunoResearch Lab. Inc., Pennsylvania) diluted 1 in 200. Fluorescence was observed using a Zeiss Axiophot microscope with FITC and Cy3 filter sets and images recorded with a digital camera (Princeton Instruments, Trenton, NJ) with the software Metamorph version 4.1.7. Zoospores were also labelled with pre-immune sera and images recorded and reproduced at the same settings used for immune sera. For immunogold labeling of whole zoospores, 5 ␮l of zoospore suspension were placed on a formvar-coated gold grid and 5 ␮l of 2% v/v glutaraldehyde in 200 mM PIPES buffer (pH 7.0) added, giving a final fixative concentration of 1% v/v glutaraldehyde in 100 mM PIPES. The cells were left for 30 minutes during which time they settled onto the formvar. The grids were rinsed in PBS containing 0.2% v/v Tween 20 (PBST) before being labeled with Pn14B7 followed by goat anti-mouse Ig conjugated to 10 nM gold particles (GE Healthcare; diluted 1:5 in 1% w/v BSA, 0.1% v/v fish skin gelatin in PBS). Both primary and secondary antibody incubations were for 1 h at room temperature. The grids were rinsed in PBST after each antibody incubation, with a final rinse in distilled water before staining in 1% uranyl acetate for 20 minutes. Western Blot analysis: Proteins were extracted from zoospores pelleted in the presence of 0.2 M LiCl2 , in 5 M urea, 2 M thiourea, 4% w/v CHAPS, 40 mM Tris-HCl pH 6.8, 65 mM DTT plus Complete Protease Inhibitor Cocktail (Roche Applied Science, Penzberg, Germany). The homogenate was centrifuged for 10 min at 15,800 g at 4 ◦ C to remove insoluble material. The protein concentration was determined by a

modified Bradford assay (Bradford 1974) using Bio-Rad Protein Reagent (Bio-Rad, Hercules, CA). Proteins were separated on 12% SDS-PAGE gels (Laemmli 1970) with approximately 25 ␮g protein loaded in each lane, with one lane containing 5 ␮l of Precision Plus Protein Kaleidoscope Standard (Bio-Rad). Proteins were transferred overnight onto Hybond-C (GE Healthcare) at 15 V according to the manufacturer’s instructions. Protein loading and transfer was checked by Ponceau S staining. Membranes were blocked with 1% w/v BSA and 2% w/v skim milk powder in PBS for 60 min and rinsed three times with PBS for 5 min. Membranes were labelled with the polyclonal antibodies raised against the C-terminal peptides, diluted 1 in 500, 1 in 1000 and 1 in 2000 in 1% w/v BSA in PBS or undiluted Pn14B7 monoclonal antibody for 60 min. In controls, pre-immune serum from both rabbits was used to label membranes at the same dilution as the immune sera. Membranes were rinsed three times in PBT (PBS plus 0.05% v/v Tween-20) for 5 min and then labelled with goat anti-rabbit or sheep anti-mouse Ig conjugated to alkaline phosphatase (Chemicon) diluted 1 in 1000 in 1% v/v BSA in PBS. Bound antibodies were detected with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate in alkaline phosphate buffer (100 mM Na2 CO3 , 1 mM MgCl2 , pH 9.8). Blue native PAGE was employed to examine the oligomeric state of the putative mastigoneme protein from P. nicotianae (Wittig et al. 2006). For this analysis, zoospore proteins were extracted in 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 10% v/v glycerol and separated on a NativePAGETM Novex 4-16% Bis-Tris gel (Invitrogen Carlsbad, CA) according to the manufacturer’s instructions. NativePAGE sample buffer supplemented with 0, 50 or 100 mM ␤-mercaptoethanol was added to protein samples prior to electrophoresis. Separated proteins were transferred onto Immobilon-PSQ membrane (Millipore Corp. Billerica, MA) and labelled with Pn14B7 using the rapid immunodetection method as described by the manufacturer. A duplicate gel which contained a lane of NativeMarkTM unstained protein standard (Invitrogen) was stained with Coomassie R-250 (Sigma Chemical Co. St Louis, MO) as described in the NativePAGE Novex Bis-Tris Gel System user manual. Expression analysis: Total RNA was isolated from material frozen and ground in liquid nitrogen using Trizol (Invitrogen) according to the manufacturer’s instructions. Contaminating gDNA was removed and cDNA made using oligo dT primers as described in Blackman and Hardham (2008). cDNA was checked by PCR using primers against the normalising gene WS41 (Shan et al. 2004; Blackman and Hardham 2008) and to ensure that all gDNA had been removed, cDNA reactions which omitted the reverse transcriptase were also tested. Expression levels of the putative P. nicotianae mastigoneme gene, PnMas2, were determined in vegetative hyphae and hyphae 0, 9, 12, 18, 24 h and 2, 3, 4 and 5 days after induction of sporulation, zoospores and 3 h germinated cysts by qPCR with primers designed using Oligo Explorer (http://www.genelink.com/tools/gl-oe.asp) and NetPrimer (http://www.premierbiosoft.com/netprimer/). Four sets of PnMas2 primers were tested using a Corbett Rotor-Gene 3000 Real-time Cycler (Qiagen) and the best pair determined by the comparative quantification function and melt curve analysis (Rotor-gene qPCR Analysis Software, version 6.1.81). PnMas2 gene expression levels were determined relative to the expression of WS41 using 150 nM primers (Pnmas1: CGTCGTGGGTAGTATTATTCCGGCGGAGGATTCTTGGTAGTCT, WS41: CGGTCCCATTGTGCTCTATT-TTGTGTCTTTGTGTGATGCG), approximately 30 ng cDNA and FastStart SYBR Green Master Mix (Roche Applied Science, Mannheim, Germany) in a final reaction volume of 20 ␮l using four technical replicates. The qPCR cycling

112 L.M. Blackman et al. conditions were 40 cycles of 95 ◦ C for 15 s (with initial step of 95 ◦ C for 6 min), 20 s at 60 ◦ C and 25 s at 72 ◦ C. A melt curve (65–95 ◦ C) was carried out at the end of each PCR run to check for primer-dimer formation.

Acknowledgements This study was conducted with the support of the Australian Research Council. The authors also wish to thank Corinna Paeper and Andrea Robold for their earlier contributions to this project and Tancred Fricke for advice on the phylogenetic analysis.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.protis.2010.01.005.

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