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A novel Phytophthora infestans haustorium-specific membrane protein is required for infection of potato

Phytophthora infestans causes late-blight, a devastating and re-emerging disease of potato crops. During the early stages of infection, P. infestans differentiates infection-specific structures such as appressoria for host epidermal cell penetration,
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  A novel  Phytophthora infestans   haustorium-specificmembrane protein is required for infection of potato Anna O. Avrova, 1 ** Petra C. Boevink, 1 Vanessa Young, 1 Laura J. Grenville-Briggs, 2 Pieter van West, 2 Paul R. J. Birch 3 andStephen C. Whisson 1 * 1 Plant Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK. 2 Aberdeen Oomycete Group, College of Life Sciences and Medicine, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB24 2ZD, UK. 3 Division of Plant Sciences, College of Life Sciences,University of Dundee at SCRI, Invergowrie, Dundee DD2 5DA, UK. Summary Phytophthorainfestans  causeslate-blight,adevastat-ing and re-emerging disease of potato crops. Duringtheearlystagesofinfection, P. infestans  differentiatesinfection-specific structures such as appressoria forhost epidermal cell penetration, followed by infectionvesicles,andhaustoriatoestablishabiotrophicphaseof interaction. Here we report the cloning, from asuppression subtractive hybridization library, of a P. infestans   gene called  Pihmp1  encoding a putativeglycosylated protein with four closely spaced  trans  -membrane helices.  Pihmp1  expression is upregulatedin germinating cysts and in germinating cysts withappressoria,andsignificantlyupregulatedthroughoutinfectionofpotato.Transientgenesilencingof Pihmp1 ledtolossofpathogenicityandindicatedinvolvementof this gene in the penetration and early infectionprocesses of  P. infestans  .  P. infestans   transformantsexpressing a  Pihmp1 ::monomeric red fluorescentprotein (mRFP) fusion demonstrated that  Pihmp1  wastranslated in germinating sporangia, germinatingcysts and appressoria, accumulated in the appresso-rium, and was located at the haustorial membraneduring infection. Furthermore, we discovered thathaustorial structures are formed over a 3 h period,maturing for up to 12 h, and that their formation isinitiated only at sites on the surface of intercellularhyphaewhere Pihmp1 ::mRFPislocalized.Weproposethat  Pihmp1  is an integral membrane protein that pro-vides physical stability to the plasma membrane of P. infestans  infectionstructures.Wehaveprovidedthefirst evidence that the surface of oomycete haustoriapossess proteins specific to these biotrophic struc-tures, and that formation of biotrophic structures(infection vesicles and haustoria) is essential to suc-cessful host colonization by  P. infestans  .Introduction Filamentous eukaryotic plant pathogenic microorganismssuch as fungi and oomycetes frequently form highly spe-cialized structures during invasion and colonization oftheir hosts. For many plant pathogens, host invasion com-mences with the differentiation of a domed or swollenstructure, called an appressorium, at the terminus of agermination tube or hypha from a spore (Talbot, 2003;Grenville-Briggs and van West, 2005). The appressoriumsecretes adhesive compounds to maintain contactbetween host and pathogen during infection (Tucker andTalbot, 2001; Grenville-Briggs and van West, 2005), andcan generate mechanical force (Howard  et al  ., 1991;Bechinger et al  .,1999)and/orhydrolyticenzymestopene-trate host cell walls (Francis  et al  ., 1996; Pryce-Jones et al  ., 1999).After invading a compatible host, fungal and oomycetehyphae grow either intercellularly or intracellularly,with or without biotrophic structures called haustoria(Mendgen and Hahn, 2002). For example,  Cladosporium fulvum   produces entirely intercellular hyphae withouthaustoria,  Magnaporthe grisea   grows  in planta   as intra-cellular hyphae without haustoria (Kankanala  et al  .,2007), while obligate biotrophs, such as the rust fungi,produce intercellular hyphae with intracellular haustoria.Haustoria are formed by invagination of the host cellmembrane and are thus in intimate contact with the hostcell. The haustorial plasma membrane of fungal plantpathogens is differentiated from the intercellular hyphalplasma membrane and is distinct in its possession ofproteins for the transport of nutrients such as sugarsand amino acids (Hahn  et al  ., 1997; Struck  et al  ., 1998;Voegele  et al  ., 2001). Haustoria are also the site ofsecretion for fungal and oomycete effector proteins,some of which may act inside the host cell to modulate Received 29 April, 2008; revised 30 June, 2008; accepted 7 July,2008. For correspondence. *E-mail; Tel.( + 44) 1382 562731; Fax ( + 44) 1382 562426; **; Tel. ( + 44) 1382 562731; Fax ( + 44) 1382 562426. Cellular Microbiology (2008) doi:10.1111/j.1462-5822.2008.01206.x  © 2008 Scottish Crop Research InstituteJournal compilation © 2008 Blackwell Publishing Ltd  the host cell environment (Catanzariti  et al  ., 2006;Kamoun, 2006; Whisson  et al  ., 2007). Phytophthora infestans   is an oomycete plant pathogen,best known for its role in precipitating the Irish potatofamines in the mid-19th century. It remains a major con-straint to potato production worldwide. Although sharingmorphological similarities with fungi such as hyphae andspores, oomycetes are more closely allied to the stra-menopiles among the heterokonts, and deeply rooted withthe alveolates (Baldauf  et al  ., 2000; Burki  et al  ., 2007).Asa model oomycete, the life cycle of  P. infestans   is wellcharacterized (van West and Vleeshouwers, 2004;Avrova  et al  ., 2007), as are its interactions with its hostplants. Almost all molecular genetic tools are available todetermine gene function in this pathogen (Birch andWhisson, 2001; Lamour  et al  ., 2007), including reliablemethods for transient gene silencing and occasionalstable gene silencing, reporter gene assays, and a wealthof genetic information including greater than 80 000 ESTsand a genome sequence (van West  et al  ., 1999; Randall et al  ., 2005; Whisson  et al  ., 2005; 2007). This establishes P. infestans   as the model oomycete organism for studyingthe pathogen molecular and biochemical processesunderlying disease development. Asexual sporangiaare the infectious dissemination propagules and maygerminate directly, or cleave to yield motile, biflagellatezoospores. Zoospores encyst at an infection site inresponse to chemical, electrical and physical signals, dis-carding flagella and forming a cell wall (van West  et al  .,2002, 2003). A germ tube emerges from the cyst or spo-rangium, the tip of which swells to differentiate into anappressorium and subsequently a penetration hypha.After the plant cuticle and cell wall have been breached,an intracellular, biotrophic infection vesicle is produced inthe epidermal cell. Intercellular hyphae grow into themesophyll cell layers, producing intracellular, biotrophicfinger-like haustoria as new host cells are encountered.After 36–48 h post inoculation (hpi), the mode of interac-tion between  P. infestans   and initially infected host cellsbecomes necrotrophic and new haustoria are not formed.From 72 to 96 hpi, initially infected areas of the leaf arefully colonized and necrotic. Sporulating hyphae emergefrom stomata to release sporangia, allowing aerial dis-semination of the pathogen. Spatially, this is visualized asa leaf lesion with a biotrophic margin as new cells areinfected. The centre of the lesion is necrotic where initiallycolonized cells have died.Oomycete haustoria differ from those formed by fungalplant pathogens in that cell wall and membrane differen-tiation is less pronounced in oomycetes. For example,fungal haustoria have a distinct neckband that seals theextrahaustorial matrix (EHM) from the apoplast, whereasthe oomycete cell wall is continuous from the intercellularhyphae to the haustoria (Hohl and Suter, 1976; Soylu,2004). Compared with fungal plant pathogens, molecularprocesses underlying oomycete development and patho-genicity are poorly understood. With the exception ofthe AVR3a avirulence protein (Whisson  et al  ., 2007), nooomycete proteins have been demonstrated to localizeto haustoria, either for secretion or for other haustorialfunctions.Several studies have reported differentially expressedcandidate pathogenicity factors from  P. infestans   pre-infection stages, as they are likely to be enriched formolecules involved in successful penetration of the host,and establishment of a compatible interaction (Görnhardt et al  ., 2000; Beyer  et al  ., 2002; Avrova  et al  ., 2003; Birch et al  ., 2003; Bittner-Eddy  et al  ., 2003; Randall  et al  ., 2005;Judelson  et al  ., 2008). A high proportion of oomycetegenes identified through transcript profiling techniquesare entirely novel, and identifying those with a role inpathogenicity remains a significant challenge. Throughgene silencing in stable transformants, a small number of P. infestans   genes have been analysed at the functionallevel, revealing an involvement for some in pathogenicity.However, these have typically encoded proteins involvedin signal transduction or transcriptional regulation (Latijn-houwers and Govers, 2003; Latijnhouwers  et al  ., 2004;Blanco and Judelson, 2005). These genes and theirencoded proteins may form part of the core biology of P. infestans   and their silencing may have resulted inmultiple developmental defects, including reducedpathogenicity. Transient silencing of genes is also pos-sible in  P. infestans  , and may facilitate higher-throughputfunctional analysis of, and more silencing-recalcitrant,genes than previously (Whisson  et al  ., 2005; Grenville-Briggs  et al  ., 2008; Walker  et al  ., 2008). In identifyinggenes encoding proteins contributing directly to patho-genicity, it may be expected that candidate genes encodeproteins that act at the interface between pathogen andhost. Although numerous oomycete genes, such as pro-tease inhibitors, a glucanase inhibitor, avirulence proteinsand necrosis-inducing proteins, have been identified thatinteract with host plant proteins (Rose  et al  ., 2002; Torto et al  ., 2003; Tian  et al  ., 2004; 2005; 2007; Birch  et al  .,2006), their contribution towards oomycete pathogenicityremains to be determined.In a broader transcript profiling study in  P. infestans  targeting candidate pathogenicity genes expressed inpre-infection stages, we selected one transcript for furtherstudy in order to test the hypotheses that it encoded aprotein that may act at the pathogen–host interface, andplay a role in pathogenicity. Here we describe the cloning,homologues in other oomycetes, role in pathogenicity,and localization of a  P. infestans   gene encoding a pre-dicted integral membrane protein. Translational fusion ofthis gene to the monomeric red fluorescent protein(mRFP) in stable  P. infestans   transformants demon-2  A. O. Avrova   et al.  © 2008 Scottish Crop Research InstituteJournal compilation © 2008 Blackwell Publishing Ltd,  Cellular Microbiology   strated localization to the haustorial plasma membraneduring infection of potato. Accordingly, it has been named Pihmp1 , for  P. infestans   haustorial membrane protein 1.The involvement of  Pihmp1  in the penetration process of P. infestans   and establishment of a compatible interactionwas shown using transient gene silencing. Results Cloning of   Pihmp1From an existing suppression subtractive hybridization(SSH) cDNA library (Grenville-Briggs  et al  ., 2005) a198 bp transcript-derived fragment (clone 2c5) wasselected for further analysis, as  BLASTN  and  TBLASTX similarity searches of sequence databases (see  Experi- mental procedures  ) revealed sequence similarity only toexpressed sequence tags (ESTs) from  P. infestans   (4e - 9 to 5e - 24 for  TBLASTX ; 2e - 115 to 1e - 124 for  BLASTN ), and thegenome sequences of  Phytophthora sojae  ,  Phytophthora ramorum   and  Hyaloperonospora parasitica   (3e - 33 to 1e - 40 for  TBLASTX ; 3e - 16 to 6e - 20 for  BLASTN ). As this section ofwork was carried out before the availability of theassembled draft  P. infestans   genome sequence, genomiccopy number, and the sequence of the complete openreading frame (ORF) and promoter of  Pihmp1  were deter-mined from three positively hybridizing clones (Fig. S1)from an existing  P. infestans   bacterial artificial chromo-some (BAC) library (Whisson  et al  ., 2001) representing10 ¥  genome coverage. The  Pihmp1  gene sequencespresent on BAC clones 5J1 and 15J23 were identical, andthe sequence on BAC clone 1B18 was a truncated copy ofthe gene. Since the release of the  P. infestans   genomesequence (see  Experimental Procedures   for URL), boththe intact and truncated copies of the gene have beenfound within the same genomic region (sequence super-contig 1.1), with no additional copies located elsewhere inthe genome.A  TBLASTN  search of the  P. infestans   genome databaseusing the predicted amino acid sequence of Pihmp1as a query revealed 11 supercontigs containing severalhomologous regions with  E  -values of 8  ¥  10 - 43 - 10 - 172 . A TBLASTN  search of the NCBI EST databases, includinghuman and mouse sequences ( with the predicted Pihmp1 protein sequence,identified two additional homologous ESTs (gi58106741and gi58105907) from  P. infestans   germinating sporangiawith  E  -values of 7  ¥  10 - 73 and 2  ¥  10 - 37 , respectively, onehomologous EST (gi58087999) from  P. infestans   sporu-lating mycelium with  E  -value of 2  ¥  10 - 43 , and two homolo-gous ESTs (gi38117833 and gi38062167) from  P. sojae  with  E  -values of 2  ¥  10 - 48 and 2  ¥  10 - 51 respectively.The  Pihmp1  sequence is deposited in GenBank underAccession No. EU680858.Pihmp1  expression is upregulated prior to and during infection  Real-time reverse transcription polymerase chain reaction(RT-PCR) analysis was used to characterize expressionof  Pihmp1  in  P. infestans in vitro   stages of cultured myce-lium, sporangia, zoospores, germinating cysts, germinat-ing cysts with appressoria and  in planta   during theinfection of susceptible potato cv. Bintje with a compatiblerace (isolate 88069) of  P. infestans   at 12, 24, 33, 48, 56and 72 hpi. Microscopic analysis indicated that the timingof  P. infestans   development during these infectionsclosely followed the events described by Vleeshouwers et al  . (2000) and Avrova  et al  . (2007). Thus, at 12 hpi,germinating cysts, appressoria and infection hyphae wereclearly visible. At 24–33 hpi, numerous haustoria werevisible in infected tissue, indicative of the biotrophicphase, whereas few haustoria were visible in infectedtissue by 48 hpi, indicating the transition from biotrophy tonecrotrophy. By 72 hpi, the necrotrophic phase was wellestablished with sporulation and necrosis clearly visible.Primer pairs were designed to anneal to  Pihmp1 ,  PiactA and  PiipiO1  (Table S1). The  PiactA  gene from  P. infestans  was used as a constitutively expressed endogenouscontrol (Grenville-Briggs  et al  ., 2008; Judelson  et al  .,2008). Expression of  PiipiO1  and  Pihmp1  in asexual lifecycle- and infection-stage samples was compared withthe level of their expression in a calibrator sample, whichwas cDNA synthesized from RNA isolated from non-sporulating mycelium grown in rye broth. The expressionof  PiipiO1  and  Pihmp1  in the mycelium cDNA sampleswas assigned the value of 1.0.The  P. infestans PiipiO1  gene, known to be upregulatedininvadinghyphaeduringinfection(vanWest et al  .,1998),wasusedtoconfirmdiseaseprogressionininoculatedleafsamples and yielded the expected expression profile(Fig. 1).The Pihmp1 expressionprofileinpre-infectioncelltypes is very similar to that of  PiipiO1 : upregulated over100-fold in zoospores, peaking at over 1600-fold in germi-natingcystsandthendecreasingto600-foldingerminatingcysts with appressoria (Fig. 1). Unlike  PiipiO1 , the relativeupregulationof Pihmp1 duringinfectionislowerthanthatingerminating cysts with appressoria, suggesting that itsmRNA is transcribed during cyst germination and appres-sorialdevelopment,tobetranslatedfortheearlybiotrophicstage of interaction. Repeated amplifications, on indepen-dent occasions with biologically replicated cDNAsamples,resulted in similar expression profiles.To test whether the truncated paralogue of  Pihmp1  wasexpressed, primers 1B18F and 1B18R were used in real-time RT-PCR across the life cycle stages of  P. infestans  .Aproduct of the expected size was amplified from genomicDNAbut not from cDNAof any  P. infestans   life cycle stage(not shown).Phytophthora infestans  haustorial membrane protein   3  © 2008 Scottish Crop Research InstituteJournal compilation © 2008 Blackwell Publishing Ltd,  Cellular Microbiology   Pihmp1 is a putative glycosylated membrane protein  The 3072 bp ORF of  Pihmp1 , which lacks introns,encodes a predicted 1023-amino-acid, 108.3 kDa protein.A signal peptide for secretion was predicted at theN-terminus by SignalP 3.0 (neural network mean 0.929,hidden Markov model 0.998), with the molecular weight ofthe mature protein estimated at 105.5 kDa. The CBSPrediction Servers ( TMHMM  v. 2.0) and  SOSUI  program(v. 1.11) predicted  Pihmp1  to encode a protein with four trans  -membrane (TM) helices between residues 605 and722, potentially anchoring the protein to the plasma mem-brane (Fig. 2). Both N- and C-termini of Pihmp1 werepredicted to be located outside the plasma membrane,with only 10 amino acids in two loops between TM helicespredicted to extend into the cytoplasm.  TMHMM  posteriorprobabilities 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 TMhelix 4. BLASTP  and  TBLASTN  searches, using default settings, ofNCBI GenBank (non-redundant database) yielded onlyweak similarity (5e - 7 to 1e - 13 ) to a bacterial and fungaldomain in the N-terminal half of the protein, betweenresidues293and505.In BLASTP -returnedsequencesfrom Xanthomonas axonopodis   pv.  citri   (NP_644432.1) and Bradyrhizobium   sp. (YP_001208179.1), this domainyielded modest support for a jacalin-like (mannose/ galactosebinding)lectindomain.Weaksupport,belowthethresholds used to construct the Pfam model, for a jacalin-likelectindomainwasidentifiedinPihmp1usingPfam22.0( E  -value0.0014)betweenresidues374and505.Adomaincontaining three lysine/serine (K/S)-rich repeats of 32–39residues was identified manually between residues 130and 260 (Fig. 2B). The final two K/S repeats contain fourcopies of a DxSxS metal ion-dependent adhesion site(MIDAS) motif (Tozer  et al  ., 1996). An additional threeserine/glycine (S/G)-rich repeats of 15–17 residues followthe K/S-rich repeats. CBS Prediction Servers (NetNGlyc1.0) predicted N-glycosylation at positions 390, 818 and966, and potential O-glycosylation (mucin type; NetOGlyc3.1) at positions 93–95, 134, 135, 139, 142, 145 and 226(Fig. 2B). A mucin domain was also predicted by Pfam inthisregionofPihmp1butwithpoorsupport( E  -value0.55),below the thresholds used to construct the Pfam model.Seven cysteine residues are present in the 99-amino-acidregion between residue 505 and the first TM helix of thepredicted mature Pihmp1 protein, and a further three cys-teines are present in the C-terminal portion of the proteinafter the TM helices (Fig. 2B). These protein sequencefeatures suggest that Pihmp1 is a membrane-anchored,predominantly extracellular, glycosylated protein that mayact at the interface between  P. infestans   and host cellsduring infection. 050100150200250300350M S Z C A B12 B24 B33 B48 B56 B72    R  e   l  a   t   i  v  e  e  x  p  r  e  s  s   i  o  n PiipiO B 0400800120016002000 M S Z C A B12 B24 B33 B48 B56 B72    R  e   l  a   t   i  v  e  e  x  p  r  e  s  s   i  o  n Pihmp1 A Fig. 1.  Pihmp1  is expressed in pre-infection stages and throughoutinfection of potato. Real-time RT-PCR expression profile of (A) Pihmp1  and (B)  PiipiO1  in pre-infection stages (sporangia – S,zoospores – Z, germinating cysts – C, germinating cysts withappressoria – A) and 12 (B12), 24 (B24), 33 (B33), 48 (B48), 56(B56) and 72 (B72) h post inoculation of potato cv. Bintje with acompatible race (88069) of  P. infestans  , relative to their expressionin vegetative non-sporulating mycelium (M). Error bars representconfidence intervals calculated using three technical replicates foreach sample within the RT-PCR assay. 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 shadedgrey. 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 cysteineresidues (vertical blue bars) and  trans-  membrane helices (TM; shaded green). 4  A. O. Avrova   et al.  © 2008 Scottish Crop Research InstituteJournal compilation © 2008 Blackwell Publishing Ltd,  Cellular Microbiology   Phytophthora infestans  haustorial membrane protein   5  © 2008 Scottish Crop Research InstituteJournal compilation © 2008 Blackwell Publishing Ltd,  Cellular Microbiology 
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