Oomycetes comprise a diverse group of beings that morphologically resemble Fungis but belong to the stramenopile line of descent within the supergroup of chromalveolates. Recent surveies have shown that works pathogenic Oomycetess have expanded cistron households that are perchance linked to their infective life style. We analyzed the protein sphere organisation of 67 eucaryotic species including four oomycete and five fungal works pathogens.
We detected 246 expanded spheres in fungous and oomycete works pathogens. The analysis of cistrons differentially expressed during infection revealed a important enrichment of cistrons encoding expanded spheres every bit good as signal peptides associating a significant portion of these cistrons to pathogenicity. Overrepresentation and bunch of sphere copiousness profiles revealed spheres that might hold of import functions in host-pathogen interactions but, as yet, have non been linked to pathogenicity. The figure of distinguishable sphere combinations ( bigrams ) in Oomycetes was significantly higher than in fungi. We identified 773 oomycete-specific bigrams, with the bulk composed of spheres common to eucaryotes.
The analyses enabled us to associate sphere content to biological procedures such as host-pathogen interaction, alimentary consumption, or suppression and evocation of works immune responses. Take together, this survey represents a comprehensive overview of the sphere repertory of fungous and oomycete works pathogens and points to fresh characteristics like sphere enlargement and species-specific bigram types that could, at least partly, explicate why Oomycetess are such singular works pathogens.
Oomycetes are a diverse group of beings that live as saprophytic organisms or as pathogens of workss, insects, fish, craniates, and bugs ( Govers & A ; Gijzen 2006 ) . The legion works infective oomycete species cause lay waste toing diseases on many different host workss and have a immense impact on agribusiness. A outstanding illustration is Phytophthora infestans, the causal agent of late blight of murphy ( Solanum tuberosum ) and tomato ( Solanum Lycopersicon ) and responsible for the Irish murphy dearth in the nineteenth century.
Plant infective Oomycetess include a big figure of different species that vary in their life style, from obligate biotrophic and hemibiotrophic to necrotrophic. In add-on, they show great differences in host selectivity, runing from wide to really narrow ( Erwin & A ; Ribeiro 1996 ; Agrios 2005 ) . Oomycetes have morphological characteristics similar to filiform Fungis, and the two groups exploit common infection constructions and mechanisms ( Latijnhouwers & A ; Govers 2003 ) . Together with diatoms, brown algae, and golden-brown algae, Oomycetess are classified as stramenopiles, a line of descent that is united with alveolates in the supergroup of chromalveolates ( Baldauf et al. 2000 ; Yoon et Al. 2002 ) .
The monophyly of this supergroup, nevertheless, is under argument ( Baurain et al. 2010 ) . The genomes of Oomycetess sequenced so far are variable in size and content, runing from 65 Mb in Phytophthora ramorum to 240 Mb in P. infestans ( Haas et al. 2009 ) , and merely include works infective species. Analysis of these genomes revealed that several cistron households easing the infection procedure are expanded ( Martens et al. 2008 ) . Extreme illustrations are cistron households encoding cytoplasmatic effecter proteins such as RXLR effecters, which portion the host cell-targeting motive RXLR and suppress defence responses in the host, and the necrosis-inducing proteins classified as Crinklers ( Crn ; Haas et Al. 2009 ) .
To day of the month, a few oomycete genomes have been sequenced, and this enables a comprehensive comparing of genomic characteristics present in Oomycetess, Fungis, and other eucaryotic species such as cistron households and protein spheres. Experimentally derived functional cognition of the bulk of cistron merchandises in Oomycetess in a comparable deepness as for theoretical account species like Saccharomyces cerevisiae and Arabidopsis ( Arabidopsis thaliana ) will probably non be accessible in the close hereafter. Hence, comparative genomics provides an of import model to functionally qualify oomycete cistron merchandises and generate hypotheses on the basic cellular maps every bit good as the complex interactions of these works pathogens with their hosts and environment.
In this survey, we focus on protein spheres because these are the basic functional, evolutionary, and structural units that shape proteins ( Rossmann et al. 1974 ; Orengo et Al. 1997 ; Vogel et Al. 2004 ) . Domains map independently in single-domain proteins or synergistically in multidomain proteins ( Doolittle 1995 ; Vogel et Al. 2004 ; Bashton & A ; Chothia 2007 ) . Accordingly, some spheres ever occur with a defined set of functional spouses, whereas others are extremely various and form combinations of two consecutively happening spheres ( besides called bigrams ) with different N- or C-terminal spouses ( Marcotte et al. 1999 ; Basu et Al. 2008 ) .
Here, we analyzed the sphere repertory predicted from the genome sequences of 67 eucaryotic species and compared filiform works pathogens with other eucaryotes with a particular accent on Oomycetess. We show how differences in the sphere repertory of Oomycetess, particularly in the ex32 Chapter 2 pansion of certain sphere households and the formation of species-specific bigram types, can be linked to the biological science of this group of beings. This allowed the coevals of candidate sets of proteins and spheres that are likely to play functions in the life style of Oomycetess or their interaction with workss.
The Domain Repertoire of Oomycete Plant Pathogens and its Comparison with Other Eukaryotes
We analyzed the sphere architecture of the predicted proteomes in 67 eucaryotes covering all major groups of the eucaryotic tree of life with the exclusion of the supergroup Rhizaria ( Figure 2-1A ; Supplementary Table S2-1 ) . We included seven strameno Phytophthora infestans Phytophthora ramorum Phytophthora sojae Hyaloperonospora arabidopsidis Aureococcus anophagefferens Thalassiosira pseudonana Phaeodactylum tricornutum Haptophytes 1 species Alveolates 5 species Plants9 speciesExcavates 4 species Fungi 22 species Metazoans 19 species Chromalveolates 13 species Oomycetes 4 species Stramenopiles 7 speciesA B Phytophthora infestans Phytophthora ramorum Phytophthora sojae Hyaloperonospora arabidopsidis Eremothecium ( Ashbya ) gossypii Ustilago maydis Magnaporthe grisea Sclerotinia sclerotiorum Fusarium graminearum Oomycete plant-pathogens Fungal plant-pathogens Plant-pathogens biotroph hemi-biotroph necrotroph.
( A ) The major eucaryotic groups considered in the analysis and the figure of species represented in every group. For the exact species used in the analysis, see Auxiliary Table S2-1. The tree is adapted from Simpson and Roger ( 2004 ) and incorporates the evolution for the stramenopiles based on Blair et Al. ( 2008 ) . ( B ) Fungal and oomycete plant-pathogenic species used in this analysis. The works pathogens include species with different life styles, indicated by the symbol following the species name. The evolution for the Fungi is based on James et Al. ( 2006 ) .33 Domain Analysis in Oomycete Plant Pathogens
hemorrhoids, four of which are works infective Oomycetess, viz. the obligate biotrophic downy mold Hyaloperonospora arabidopsidis and three hemibiotrophic Phytophthora species. The choice besides contained five fungous works pathogens, including rice ( Oryza sativa ) blast fungus ( Magnaporthe grisea ) and maize ( Zea Mayss ) carbon black ( Ustilago maydis ) , both species with a ( hemi ) biotrophic lifestyle comparable to the oomycete works pathogens used in the analysis.
The sphere architecture of all 1,250,996 predicted proteins in the 67 eucaryotic genomes was analyzed utilizing HMMER ( Eddy 1998 ) and a local Pfam-A database ( Finn et al. 2010 ) . Overall, 59 % ( 737,851 ) of all proteins have one or more predicted spheres. We detected a sum of 1,464,807 spheres in all species, 80,180 within the stramenopiles and 51,030 in Oomycetess.
In order to qualify the sphere repertory of eucaryotes, we used two prosodies: Proteome species X Abundance sphere type A sphere type Bdomain type Cdomain type Ddomain type E N C N C N C bigrams: 2x ( E|C ) , 2x ( E|E ) , 2x ( E|D ) , 1x ( C|D ) , 1x ( D|C ) , 1x ( D|E ) , 1x ( D|B ) , 1x ( E|B ) , 1x ( B|D ) N C N C
In the illustration shown, we observe five different sphere types. The copiousness of a sphere type is defined as the figure of happenings of the single entity within the species ( e.g. sphere type B has an copiousness of two ) . The versatility is defined as the figure of different direct next N- or C-terminal neighbours. We distinguish between N- and C-terminal spouses ( e.g. the versatility of sphere type C is three ) . A bigram is a set of two straight next spheres, and we besides consider two entities of the same sphere a bigram ( e.g. we observe nine different bigram types in the proteome, of which three have an copiousness of two ( right panel ) ) .34 Chapter 2
the figure of sphere types and the figure of different combinations of next spheres, besides called bigrams ( Figure 2-2 ) . In entire, 13,994 bigram types were identified in the 67 eucaryotic genomes, dwelling of 6,356 different sphere types. As described by Basu et Al. ( 2008 ) , the figure of bigram types increases superlinearly comparative to the figure of sphere types, with the highest Numberss in multicellular beings ( Figure 2-3 ) . We observed separate bunchs for metazoans, Fungis, and workss ( including land workss and mosses ) . Oomycetes and Fungis have similar Numberss of sphere types, runing from 2,000 to 2,500 ; nevertheless, Oomycetess, in peculiar Phytophthora species, contain significantly more bigram types.
The three analyzed Phytophthora species appeared to hold about 50 % more bigram types compared with other beings that have similar Numberss of sphere types ( Figure 2-3 ; P = 0.0019, by nonreversible Wilcoxon rank-sum trial ) . This even holds when we apply a more conservative attack by flinging all sphere and bigram types that occur one time in each predicted proteome ( Auxiliary Figure S2-1A ) . We observed that the figure of sphere types every bit good as the figure of bigram types increases with proteome size and reaches impregnation for larger proteomes ( Auxiliary Figure S2-1, B and C ; Cosentino Lagomarsino et Al. 2009 ) .
The mean figure of different bigrams of species that have between 2,000 and 2,500 different sphere types is indicated with the bottom horizontal ruddy saloon. The top horizontal ruddy saloon indicates the mean figure of different bigrams for Phytophthora species. The full species names matching to the abbreviations can be found in Supplemental Table S2-1. A magnification of the country embracing the oomycete and fungous works pathogens is shown ; the species of involvement are highlighted. The points are colored harmonizing to the major eucaryotic groups as indicated in the text box.35 Domain Analysis in Oomycete Plant Pathogens
Although Oomycetess and in peculiar Phytophthora species contain a similar figure of sphere types as Fungi, they have a larger predicted proteome ( Auxiliary Figure S2-1B ) . However, they contain more bigram types than Fungis but less than other species with predicted proteomes of similar size ( e.g. Drosophila melanogaster ; Auxiliary Figure S2-1C ) .
Domain Overrepresentation Provides a Snapshot of Pathogen-Host Interaction
Apart from a broad and abundant repertory of spheres related to permutable elements ( Haas et al. 2009 ) , the most abundant sphere types in Oomycetess are similar to those in other eucaryotes.. Hence, absolute sphere copiousness entirely is non declarative plenty to correlate spheres to the life style of both fungous and oomycete works pathogens. Alternatively, we identified spheres that are overrepresented in works pathogens relative to other eucaryotes.
Our analysis inferred 246 overrepresented spheres in works pathogens that are observed in 24,970 proteins. Since we analyzed the enlargement in works pathogens at the degree of a group instead than an single species, spheres that are reported as being expanded in the group are non needfully expanded in all species of the group or may even be absent ( Auxiliary Table S2-3 ) . For illustration, secreted proteins encoding carbohydrate-binding household 25 spheres ( IPR005085 ) are merely found in Phytophthora species and non in fungous works pathogens, whereas secreted proteins incorporating the Cys-rich sphere ( CFEM ;
( A ) The log2-fold overrepresentation of the spheres in works pathogens is shown in the saloon chart. The absolute figure of happenings in works pathogens and the per centum of all predicted spheres in works pathogens are displayed in the bars, and the corrected P values are shown at the tip of the bars. The fold overrepresentation and the P value for the Kazal peptidase inhibitor sphere were based on the overrepresentation in Oomycetess com-pared with works pathogens ( indicated by the white saloon and stars ) .
Many proteins involved in host-pathogen interaction are secreted in the apoplast or, like the RXLR effecter proteins, translocated into host cells following their secernment from the pathogen ( Haas et al. 2009 ) . Hence, we besides predicted the presence of possible N-terminal signal peptide sequences in the whole proteomes of the analyzed species. The combined secretome encompasses 100,521 potentially secreted proteins, of which 11,352 are predicted in works pathogens. Approximately 20 % ( 2,478 ) of these proteins contain overrepresented spheres ; hence, proteins incorporating overrepresented spheres are 1.85-fold enriched in the predicted secretome of the analyzed works pathogens ( P = 2.57 X 10-231, by Fisher ‘s exact trial ) .
Oomycete proteins with significantly expanded spheres are premier campaigners for being pathogenicity associated. To measure this hypothesis, we tested if P. infestans cistrons that are differentially expressed during infection of the murphy host are enriched for the aforesaid expanded spheres. For this, we utilized NimbleGen microarray informations that include genome-wide look degrees of P. infestans cistrons at different yearss station vaccination ( dpi ) of murphy foliages every bit good as from mycelium grown in vitro on different media ( Haas et al. 2009 ) .
We identified in entire 1,584 cistrons that are significantly induced or repressed in P. infestans during infection ( differentially expressed for at least one of the clip points 2-5 dpi ) compared with those grown in vitro ( three different growing media ; P & lt ; 0.05, Q & lt ; 0.05, by t trial ; Supplementary Table S2-4A ) . Of the 1,584 differentially expressed cistrons, 259 encode proteins incorporating significantly expanded spheres ( Auxiliary Table S2-4B ) , which is 1.2-fold more than expected ( P = 8.8 X 10-5, by Fisher ‘s exact trial ) . Furthermore, 44 of these 259 cistrons besides encode proteins with a predicted signal peptide, which is a important enrichment ( 1.8-fold ; P = 4.38 X 10-5, by Fisher ‘s exact trial ) .
The bulk ( 41 ) of these 44 cistrons are differentially expressed early in infection ( 2 dpi ; Figure 2-5A ) . All cistrons differentially expressed at 3 dpi are besides differentially expressed at 2 dpi ( Figure 2-5, A and B ) . Consequently, the 44 differentially expressed cistrons coding for proteins with both predicted signal peptides every bit good as overrepresented spheres are assuring campaigners for pathogenicity-associated proteins, of which several will be discussed in item below.
For several groups of overrepresented spheres, a direct or indirect function in host-pathogen interaction and/or works pathogen life style has already been hypothesized or demonstrated ( Dean et al. 2005 ; Tyler et Al. 2006 ; Haas et Al. 2009 ) . About 18 % of the 246 overrepresented spheres belong to three groups of spheres: hydrolase domains ; spheres involved in substrate conveyance over membranes, such as the general ATP-binding cassette ( ABC ) transporter-like sphere ( IPR003439 ) but besides more specialised transporters of sulphate ( IPR011547 ) and aminic acids ( IPR004841/ IPR013057 ) ; and spheres present in proteases, such as the metalloprotease-type M28 sphere ( IPR007484 ) found in many secreted proteins. Of the hydrolases, which encompass 9 % of the overrepresented spheres, the bulk is present in enzymes that hydrolyze glycosidic bonds. An illustration is the glycoside hydrolase ( GH ) household 12 sphere ( IPR002594 ) . This sphere is observed 34 times in works pathogens, which overall 37 Domain Analysis in Oomycete Plant Pathogens
Genes with important cistron look alterations at different clip points after infection ( 2-5 dpi ) relation to the look strengths of different growing media are displayed ( P & lt ; 0.05, Q & lt ; 0.05, by t trial ) . Heat maps demo the significantly differentially expressed cistrons at different clip points comparative to growing media. Genes were clustered utilizing Spearman rank correlativity and mean linkage bunch. Gene identifiers every bit good as sphere descriptions are displayed. Gene look profiles are displayed for the look strengths relative to the mean strengths of the growing media for each clip point after infection.
Novel Candidate Domains Significantly Expanded in Plant Pathogens
Following to spheres that were already straight or indirectly implied in host-pathogen interaction, we identified fresh campaigners that are besides expanded in works pathogens, several of which are encoded in P. infestans cistrons differentially expressed during infection of the host. Genes encoding the significantly expanded intoxicant dehydrogenase ( zinc binding ; IPR013149 ) every bit good as a GroES-like intoxicant dehydrogenase ( IPR013154 ) spheres are omnipresent in all analyzed eucaryotes, and besides the combination of these two spheres is present in all species with merely a few exclusions.
Nine of these cistrons in P. infestans are induced during infection. Sixty-five cistrons in works pathogens encode proteins with FAD-linked oxidase ( IPR006094 ) and berberine/ berberine-like ( BBE ) domains ( IPR012951 ) , of which three out of six in P. infestans are induced during infection ( PITG_02928, PITG_02930, and PITG_20764 ) . The BBE sphere is involved in the biogenesis of the alkaloid berberine ( Facchini et al. 1996 ) . The cistrons encode a predicted N-terminal signal peptide, although molecular analysis of proteins incorporating these spheres in workss indicated that at least some of these are non secreted but alternatively are targeted to specialised cysts ( Amann et al. 1986 ; Kutchan & A ; Dittrich 1995 ; Facchini et Al. 1996 ) . Furthermore, Moy et Al. ( 2004 ) observed induced look 39 Domain Analysis in Oomycete Plant Pathogens
of a soya bean ( Glycine soap ) cistron ( BE584185 ) shortly after infection with Phytophthora sojae incorporating these two spheres. A recent analysis from Raffaele et Al. ( 2010 ) concentrating entirely on the secretome in P. infestans corroborates our consequences and besides concludes that proteins with BBE and FAD-linked oxidase spheres are candidate virulency factors. Three cistrons encoding secreted metallophosphoesterases ( IPR004843 ; PITG_20454, PITG_07720, and PITG_10322 ) show induced cistron look. These metallophosphoesterase spheres are found in phosphatases and hence are involved in the ordinance of protein activity, since they work as adversaries of kinase activity.
For about 6 % of all overrepresented spheres, no or limited functional information is available in Pfam. These are the alleged DUFs: spheres of unidentified map. Given their enlargement in works pathogens and the fact that other overrepresented spheres are known to map in diverse facets of plant-pathogen interactions, these DUFs are besides likely to play a function in the life style of works pathogens and hence are assuring marks for farther experimental proof ( Auxiliary Table S2-3 ) . Secreted proteins incorporating a combination of two overrepresented DUFs, DUF2403 ( IPR018807 ) and DUF2401 ( IPR018805 ) , are entirely found in Fungi and in Oomycetess, with the bulk ( about 75 % ) in Oomycetess. The N-terminal DUF2403 contains a Gly-rich part without farther functional note, whereas five extremely conserved Cys residues qualify the C-terminal DUF2401. Proteins incorporating both DUFs have been characterized in S. cerevisiae and in Candida albicans as being covalently linked to the cell wall ( Terashima et al. 2002 ; Yin 2005 ; Klis et Al. 2009 ) .
Another overrepresented DUF within works pathogens and chiefly found in Oomycetess is DUF953 ( IPR010357 ) . This sphere is present in several eucaryotic proteins with thioredoxin-like map, and two cistrons in P. infestans incorporating this sphere are differentially expressed during infection ( PITG_07008 and PITG_07010 ) . DUF590 ( IPR007632 ) , which is omnipresent in about all eucaryotes, is observed in proteins incorporating eight putative transmembrane spirals. These proteins exhibit calcium-activated ion channel activity and are involved in diverse biological procedures ( Yang et al. 2008 ) . The P. infestans cistron PITG_06653 that contains the DUF590 sphere is differentially expressed during infection, and this provides farther support for a function in host-pathogen interaction. The exemplified DUFs every bit good as other overrepresented spheres with less or no functional note are interesting campaigners for farther functional surveies to decode their precise function in works pathogens.
Domain Overrepresentation in Oomycete Plant Pathogens
Since the old analysis grouped both fungous and oomycete works pathogens, spheres specifically enriched in Oomycetess were non straight discernable. Hence, we compared the comparative sphere copiousness predicted in works pathogens ( Figure 1B ) with the purpose to place spheres specifically enriched in Oomycetess. Of the 75 spheres that are overrepresented in Oomycetess, 20 are non observed in any fungous works pathogen and therefore can be considered oomycete particular within works pathogens ( Auxiliary Table S2-5 ) . In general, the copiousness of expanded spheres in Phytophthora species is higher than in H. arabidopsidis. A well-described illustration is the NPP1 do40 Chapter 2
chief ( IPR008701 ) that is present in secreted ( SignalP: 122 ) necrosis-inducing proteins. It shows a important overrepresentation in Oomycetess ( 1.68-fold [ 0.75 log2-fold ] enriched ) , in peculiar in Phytophthora species, but is besides ascertained 10 times in fungous works pathogens every bit good as in a few instances in nonpathogenic Fungi as noted before ( Gijzen & A ; Nurnberger 2006 ) . Four P. infestans cistrons encoding this sphere are induced early during infection ( 2-3 dpi ) , whereas a individual cistron ( PITG_18453 ) is induced late ( 5 dpi ) . Several proteases ( e.g. incorporating the protease S1/S6 and C1A spheres ) are overrepresented compared with other works pathogens. S1/S6 ( IPR001254 ; 1.6-fold [ 0.74 log2-fold ] ) is predicted in 91 proteins, of which 67 have a predicted secernment signal, while C1A ( IPR000668 ; 1.79-fold [ 0.85 log2-fold ] ) is predicted in 78 proteins, of which 31 are potentially secreted. C1A is present in several eucaryotic species, but within the works pathogenic group it is entirely found in Oomycetess.
Several secreted protease inhibitors of the Kazal household incorporating the Kazal I1 ( IPR002350 ) and Kazal-type ( IPR011497 ) spheres are significantly expanded in Oomycetess and are within the group of analyzed works pathogens specific to Oomycetess. This suggests that they provide an increased degree of protection of the pathogen against host-encoded defense-related peptidases ( Tyler et al. 2006 ) . Another sphere that is oomycete particular within the works pathogens is the Na/Pi cotransporter ( IPR003841 ) involved in the consumption of phosphate. Several other transporters that have already been described as being overrepresented in works pathogens ( e.g. the ABC-2-type transporters ) are significantly expanded within oomycete works pathogens, since these species are the major subscribers to the overall copiousness of this sphere in works pathogens.
The copiousness of predicted Ser/Thr-like kinase spheres ( IPR017442 ) compared with other works infective species is surprisingly high, and this sphere is specifically expanded in the Phytophthora species. Even if several expanded spheres are observed in both oomycete every bit good as fungous works pathogens, the geographic expedition of spheres chiefly expanded in Oomycetess ( e.g. certain transporter households and defense- and signaling-related spheres ) highlights functional entities that discriminate between these groups of works pathogens.
Bunch of Abundance Profiles Reveals Additional Potential Pathogenicity Factors
We extended the set of campaigner spheres that might be of import for host-pathogen interaction beyond overrepresented spheres by seeking for extra spheres that show presence, absence, and enlargement profiles similar to overrepresented spheres, since these spheres are likely to be functionally linked or involved in similar biological procedures ( Pellegrini et al. 1999 ) . We calculated a normalized profile of sphere copiousness and clustered similar copiousness profiles utilizing hierarchal bunch ( Auxiliary informations S2-1 ) . Several bunchs contained a mix of significantly overrepresented spheres and spheres whose enlargement in works pathogens is non important. We exemplify this with three bunchs that contain 20 % of all overrepresented spheres in works pathogens ( Figure 2-6 ) .41 Domain Analysis in Oomycete Plant Pathogens
In the first bunch ( Figure 2-6 ) , spheres are chiefly expanded in oomycete works pathogens. The copiousness of some spheres in works pathogens is excessively low to be identified as being overrepresented. For illustration, the PcF sphere ( IPR018570 ) , which is present in a little, about 50-amino acerb necrosis-inducing protein found in assorted Phytophthora species ( Orsomando et al. 2001 ; Liu et Al. 2005 ) , was non identified in the initial overrepresentation analysis. Besides in this bunch is the sugar agitation stimulation sphere ( IPR005224 ) , which is chiefly found in bacteriums and involved in the ordinance of maltose metamorphosis ( Kawamukai et al. 1991 ) . In this first bunch, we observed a high figure ( about 40 % ) of spheres without functional characteriza.
tion that are chiefly present in bacterium. An illustration is DUF1949 ( IPR015269 ) , a sphere that is merely found in the three analyzed Phytophthora species. This sphere is observed in functional uncharacterized bacterial proteins like YIGZ in Escherichia coli K12 and adopts a ferredoxin-like crease ( Park et al. 2004 ) . The Phytophthora and bacterial proteins incorporating DUF1949 besides contain a 2nd, N-terminal uncharacterized protein household, UPF ( UPF00029, IPR001498 ) . This sphere is besides found in the human protein Impact and is conserved from bacteriums to eucaryotes ( Okamura et al. 2000 ) . The P. infestans cistron ( PITG_00027 ) incorporating both spheres is induced early in infection ( Supplementalry Table S2-4B ) . Since these DUFs bunch with overrepresented spheres, they are assuring campaigners for farther survey.
The spheres in the 2nd bunch chiefly show an enlargement of the copiousness in both fungous and oomycete works pathogens. This bunch contains, for illustration, cell wall-degrading spheres like cutinases, pectate lyases, and other hydrolases and besides the NPP1 sphere that is found in necrosis-inducing proteins. The glycosyl hydrolase household 88 comprises unsaturated glucuronyl hydrolases thought to be involved in biofilm debasement and is chiefly found in bacteriums and Fungis ( Itoh et al. 2006 ) . Interestingly, homologs are besides observed in works infective bacteriums ( e.g. Pectobacterium atrosepticum ) , in Fungi ( e.g. M. grisea ) , and in all three Phytophthora species.
The 3rd bunch contains spheres that are non entirely found in works pathogens but have a broader copiousness profile. This bunch includes a assortment of overrepresented hydrolases, epimerases, and the ABC-2-type transporter sphere ( IPR013525 ) that is observed about 500 times in works infective species. Another sphere that is found in this bunch is the dienelactone hydrolase sphere ( IPR002925 ) , observed in all works pathogens and besides in other eucaryotic species, with a high copiousness in workss every bit good as in Fungi. This sphere hydrolyzes dienelactone to maleylacetate in bacteriums ( Pathak et al. 1991 ) and is besides detected in a putative 1,3:1,4-I?-glucanase from P. infestans that is proposed to be involved in cell wall metamorphosis ( McLeod et al. 2003 ) .
Quantification of Oomycete-Specific Bigrams
Spheres by and large do non move as individual entities in proteins but instead synergistically with other spheres in the same protein or with spheres in interacting proteins ( Park et al. 2001 ; Vogel et Al. 2004 ) . Domains involved in signaling, feeling, and generic interactions are various and form combinations with several different spouse spheres ( Auxiliary Table S2-6 ) . As described by others ( Vogel et al. 2005 ) , we observed that the versatility of spheres is relative to their copiousness ( Auxiliary Figure S2-3 ) . Hence, we applied a leaden bigram frequence that corrects for copiousness to observe spheres that are promiscuous or prone to organize combinations with different spouses ( Basu et al. 2008 ) . The mean figure of promiscuous spheres in Oomycetess is 424 and in Phytophthora is 464. This is higher than the mean figure of promiscuous spheres ( 357 ) in all other species.
We observed that Oomycetess have a higher figure of bigram types than species 43 Domain Analysis in Oomycete Plant Pathogens with a comparable figure of sphere types ( Figure 2-3 ) . We identified in entire 13,994 different bigram types throughout the 67 analyzed species. The bulk of these bigram types ( i.e. 7,724, or 55.2 % ) are predicted in merely a individual species. In Oomycetess, bigram types formed by spheres that are associated with permutable elements showed a high copiousness ( Auxiliary Tables S2-8 and S2-9 ) .
We identified 1,107 bigram types happening entirely in works pathogens, the bulk of which ( 773 ) are merely observed in the analyzed Oomycetess ( Auxiliary Table S2-10 ) . These oomycete-specific bigram types are identified in entire 1,511 times in 1,375 predicted proteins. Of the 773 oomycete-specific bigram types, 53 are present in all Oomycetess ( Figure 2-7A ) . The biggest convergence in oomycete-specific bigram types is observed between the Phytophthora species, particularly between P. ramorum and P. sojae. A recent analysis of sphere combination in P. ramorum and P. sojae already revealed several proteins involved in metamorphosis and regulative webs incorporating fresh bigrams ( Morris et al. 2009 ) .
We to boot observed in entire 43 bigram types that are shared either between P. infestans and P. sojae or between P. infestans and P. ramorum. However, the bulk of oomycete-specific bigrams ( 467 ) are specific for a individual species. The figure of oomycete-specific bigram types extremely exceeds the figure of oomycete-specific sphere types ( 41 ) . Interestingly, merely six of the oomycete-specific spheres take part in organizing the specific bigrams. Therefore, common sphere types form the bulk of the ascertained species-specific sphere combinations, stressing the importance of fresh sphere combinations instead than fresh sphere types as a beginning for species-specific functionality. Even when we selectively look at the bigrams that occur at least twice in the same proteome or one time in at least two different proteomes, we still observe 320 bigram types that are specific to Oomycetess and occur in 982 predicted proteins.
Approximately 8 % of the proteins incorporating an oomycete-specific bigram have a predicted secernment signal ( 9.2 % of all oomycete proteins contain a predicted secernment signal ) . An illustration that is observed in a secreted putative Cys peptidase nowadays in all analyzed Oomycetess is the combination of the protease C1A sphere ( IPR000668 ) and the ML sphere ( IPR003172 ) . The ML sphere is known to be involved in lipid binding and unconditioned unsusceptibility and has been observed in workss, Fungis, and animate beings ( Inohara & A ; Nunez 2002 ) .
The proteins incorporating this bigram besides have an N terminus cathepsin inhibitory sphere ( IPR013201 ) that is frequently found following to the protease C1A sphere and prevents entree of the substrate to the binding cleft ( Groves et al. 1996 ) . Another bigram that is found in secreted proteins predicted in the analyzed Phytophthora species is the combination of the carbohydrate-binding sphere household 25 ( IPR005085 ; CBM25 ) with a GH-31 sphere ( IPR000322 ) every bit good as the tandem combination of CBM25 domains Nterminal to the glycosyl hydrolase sphere.
The presence of the secreted CBM25 and GH-31 combination has late been noted in Pythium ultimum ( Levesque et al. 2010 ) . We farther tried to clarify the presence of RXLR or Crn motives in proteins incorporating oomycete-specific bigrams. We predicted the presence of one of these motives utilizing single HMMER theoretical accounts for both the RXLR and the Crn motive ( see Materials and Methods ) . We overall predicted 746 proteins incorporating an RXLR and 99 proteins with a Crn motive. None of these proteins is predicted to incorporate an oomycete-specific bigram type.44 Chapter 2
The most abundant oomycete-specific bigram type that occurs in 64 proteins is a combination of the phosphatidylinositol 3-phosphate-binding Zn finger ( FYVE type ) and the GAF sphere. The presence of this oomycete-specific bigram in P. ramorum and P. sojae has been noted before ( Morris et al. 2009 ) . The GAF sphere is described as one of the most abundant spheres in small-molecule-binding regulative proteins ( Zoraghi et al. 2004 ) . It is present in a big figure of different proteins with a broad scope of cellular maps, such as cistron ordinance ( Aravind & A ; Ponting 1997 ) and light sensing and signaling ( Sharrock & A ; Quail 1989 ; Montgomery & A ; Lagarias 2002 ) . A typical eucaryotic sphere composing affecting the GAF sphere is N terminus to the 3 ‘ 5′-cyclic phosphodiesterase sphere found in phosphodiesterases that regulate tracts with cyclic nucleotide-monophosphate as 2nd courier.
( A ) Venn diagram picturing the presence of oomycete-specific bigram types in the analyzed oomycete proteomes and bespeaking the figure of shared bigram types between different proteomes. The entire figure of oomycete-specific bigram types in each proteome is shown in parentheses. The Venn diagram was produced utilizing Venny ( Oliveros, 2007 ) . ( B ) Domain architecture of illustration proteins incorporating a GAF sphere. The top two architectures resemble common protein architectures: the cGMP-dependent 3′,5’-cyclic phosphodiesterase ( observed 111 times in eucaryotes and five times in Oomycetess ) and phytochrome A ( ascertained 21 times in eucaryotes ) .
The bottom two architectures depict oomycete-specific architectures: the FYVE-GAF merger is observed 53 times independent of other spheres, and the myosin motor caput in combination with the FYVE-GAF merger is observed four times, a individual transcript in each of the Oomycetess included in this survey. aa, aminic acids. ( C ) Simplified evolutionary tree based on the phyletic analysis of the GAF sphere in procaryotes and eucaryotes. GAF domains from proteins with a FYVE-GAF merger are entirely found to be near to bacterial GAF spheres. Other oomycete proteins that merely incorporate the GAF sphere without the FYVE sphere besides bunch with other eucaryotic sequences.
Al. 2002 ) . This organisation is observed in entire 111 times, and five times in Oomycetess ( Figure 2-7B ) . The GAF-FYVE bigram is either observed as a individual bigram ( in 53 proteins ) or in combination with other spheres ( in 11 proteins ) , for illustration with myosin ( Richards & A ; Cavalier-Smith 2005 ) . In P. infestans, two cistrons ( PITG_07627 and PITG_09293 ) encoding proteins with this combination are induced early during infection of the works ( Supplementary Table S2-4B ) . A phyletic analysis of the GAF sphere in eucaryotes and procaryotes showed that all GAF spheres in Oomycetess that are involved in the merger with FYVE entirely bunch with procaryotic GAF spheres, whereas other GAFs besides cluster with eucaryotes. Hence, this suggests a horizontal cistron transportation from bacteriums to Oomycetess of those GAF spheres that are involved in the merger with FYVE ( Figure 2-7C ; see Materials and Methods ) .
The FYVE-type Zn finger is non identified in procaryotic species ; therefore, we suggest two independent events, viz. a horizontal cistron transportation of the GAF sphere from bacteriums to Oomycetess and later a merger to the Zn finger sphere. Horizontal cistron transportation seems to play an of import function in the development of eucaryotes ( Keeling & A ; Palmer 2008 ) , and recent grounds indicates that these events besides have a important part to the genome content of protists and Oomycetess, as they received familial stuff from different beginnings ( Richards & A ; Talbot 2007 ; Martens et Al. 2008 ; Morris et Al. 2009 ) . Because GAF spheres are known to be involved in many different cellular procedures, we can merely theorize about the biological map of proteins harbouring the GAF-FYVE bigram. A possible map is the targeting of proteins to lipid beds by the Zn finger sphere in response to 2nd couriers sensed by the GAF sphere.
Several spheres involved in the phospholipid signaling were found to be overrepresented in the filiform works pathogens and in peculiar in Oomycetess. These included the phosphatidylinositol 3-/4-kinase, PIK ( IPR000403 ) , the phosphatidylinositol 4-phosphate 5-kinase sphere, PIPK ( IPR002498 ) , every bit good as the phosphatidylinositol 3-phosphate-binding FYVE. Novel sphere composings in proteins involved in phospholipid signaling and metamorphosis in Phytophthora species have been reported antecedently ( Meijer & A ; Govers 2006 ) .
Signing spheres like the FYVE and the PIK, every bit good as spheres like the IQ-calmodulin-binding sphere ( IPR000048 ) and the phox-like sphere ( IPR001683 ) , form extremely abundant oomycete-specific bigram types ( Supplementary Table S2-10 ) . Furthermore, other spheres, like the Ser/Thr protein kinase-like ( IPR017442 ) , pleckstrin homology ( IPR001849 ) , and DEP ( IPR000591 ) domains, are involved in several oomycete-specific bigram types ( e.g. the DEP-Ser/Thr protein kinase-like sphere merger is predicted in the proteomes of all analyzed Oomycetess ).
We predicted the sphere repertory encoded in the genomes of four oomycete 46 works pathogens and compared it with a wide assortment of eucaryotes crossing all major groups, including several fungous works pathogens that have a similar morphology, life style, and ecological niche as oomycete works pathogens. We quantified and examined sphere belongingss observed in Oomycetess and particularly emphatic differences and common subjects within fungous and oomycete works pathogens and their likely part to a infective life style.
We observed that oomycete works pathogens, in peculiar Phytophthora species, have significantly higher Numberss of alone bigram types compared with species with a similar figure of sphere types. However, Oomycetess besides have on mean 50 % more predicted cistrons than most of the analyzed Fungi, but at the same clip they encode a comparable figure of sphere types and hence exhibit similar sphere diverseness ( Auxiliary Figure S2-1B ) . The high figure of cistrons observed in Oomycetess suggests enlarged complexness compared with Fungis, which is non straight obvious from the sphere diverseness but alternatively from the figure of alone bigram types ( Auxiliary Figure S2-1C ) . This observation has two possible accounts:
- the larger figure of cistrons predicted from oomycete genomes provides the flexibleness to organize new sphere combinations based on a limited set of already bing spheres that are in measures similar to fungi ;
- the sphere theoretical accounts that screen particular spheres are uncomplete and hence do non supply the needed sensitiveness for oomycete genomes.
Hence, we would undervalue the figure of discernible sphere types ( and to a certain extent the figure of predicted bigram types ) . Additionally, Oomycetess, particularly Phytophthora species, are no longer following the ascertained tendency that organisms with a higher figure of cistrons ( proteins ) contain a larger figure of sphere types. Consequently, they are shifted when comparing the figure of predicted sphere and bigram types. Nevertheless, both possible accounts and the ascertained Numberss allow us to reason that oomycete genomes, particularly Phytophthora species, harbor a big repertory of cistrons encoding different bigram types compared with species of comparable complexness and, in the instance of filiform Fungis, even similar morphology.
Oomycetes and fungous works pathogens seem to be really similar to other eucaryotes with regard to absolute sphere copiousness ( Auxiliary Table S2-2 ) , and this metric is therefore non sufficiently declarative to correlate spheres straight or indirectly with the infective life style. Therefore, we predicted overrepresented spheres in works pathogens and identified 246 spheres that are significantly expanded ( Auxiliary Table S2-3 ) . Proteins incorporating overrepresented spheres are significantly enriched in the predicted secretome of the analyzed works pathogens, confirming the thought that expanded sphere households are involved in host-pathogen interaction and that these proteins are chiefly moving in the extracellular infinite. It has to be noted that the presence of a predicted signal peptide does non needfully intend that these proteins are found extracellularly, since some proteins are retained in the endoplasmic reticulum/Golgi and hence are non secreted ( Bendtsen et al. 2004 ) .
Since we anticipate that proteins that are straight involved in host-pathogen interaction are differentially regulated upon infection, we utilized the NimbleGen microarray 47 Domain Analysis in Oomycete Plant Pathogens informations of P. infestans ( Haas et al. 2009 ) and identified 259 induced/repressed cistrons encoding proteins incorporating overrepresented spheres. Genes incorporating overrepresented spheres are significantly enriched within the set of differentially expressed cistrons incorporating a predicted sphere. Furthermore, this subset contains a significantly higher copiousness of cistrons with a predicted N-terminal signal peptide than expected. These observations high spot and confirm the ab initio emerging nexus between sphere enlargement and host-pathogen interaction.
The bulk of the 246 expanded spheres are present in proteins that are involved in general saccharide metamorphosis, alimentary consumption, signaling webs, and suppression of host responses and hence might lend to set uping and keeping pathogenesis ( Figure 2-4 ) . The assortment of overrepresented spheres involved in substrate conveyance over membranes is of particular involvement. Filamentous works pathogens and particularly oomycetes exhibit a complex and expanded repertory of these spheres, enabling them to absorb foods from their environment and host.
The look of P. infestans cistrons encoding ABC-2-like transporters, aminic acid transporters, and Na/Pi cotransporter is induced early in infection of the works, proposing that these proteins act during the biotrophic stage of infection. Several other cistrons encoding proteins with a predicted extracellular localisation are induced during infection and contain overrepresented spheres. For illustration, three P. infestans cistrons encoding the predicted N-terminal signal peptide every bit good as FAD-linked oxidase and BBE spheres are induced during infection.
The BBE sphere is involved in the biogenesis of the alkaloid berberine ( Facchini et al. 1996 ) . Moy et Al. ( 2004 ) showed that a soybean homolog of this cistron is induced after infection with P. sojae. Molecular surveies of proteins incorporating BBE spheres in workss have indicated that several proteins incorporating these spheres are in fact non secreted but alternatively targeted to specific alkaloid biosynthetic cysts where the proteins accumulate ( Amann et al. 1986 ; Kutchan & A ; Dittrich 1995 ; Facchini et Al. 1996 ) . The enlargement of sphere households with possible direct or indirect functions in host-pathogen interaction in filiform works pathogens strongly suggests version to their life style at the genomic degree.
In add-on to cognize spheres, the set of overrepresented spheres besides revealed spheres that, as yet, have non been implicated in pathogenicity nor are functionally characterized. An illustration is the DUF953 sphere, which, within works pathogens, is chiefly found in Oomycetess. This sphere is observed in eucaryotic proteins with a thioredoxin-like map, and P. infestans cistrons encoding these spheres are differentially expressed during infection. The important enlargement of these spheres in works pathogens, and the fact that other well-described spheres with a map in works pathogenicity are besides overrepresented, make proteins encoding ill described but expanded spheres interesting campaigners to decode their function in filiform works pathogens in general and oomycetes in peculiar.
We determined domain overrepresentation on the footing of species groups ( works pathogens and Oomycetess ) instead than on the degree of single species. We are cognizant that, as a effect of this attack, we might hold identified spheres as being 48 Chapter 2 overrepresented in one group even if they do non necessitate to be present or expanded in all the members ( Auxiliary Tables S3 and S5 ) . Hence, we might falsely generalize the functional function of a sphere in a subset of species to the whole group ( e.g. a sphere that is entirely found in works infective Fungis and non in Oomycetess would still be overrepresented in the works infective group ) .
Particularly when comparing oomycete with fungous works pathogens, the dominant enlargement of sphere households within Phytophthora species over households in H. arabidopsidis might bias the inferred overrepresented sphere ( Auxiliary Table S2-5 ) . Since we in general want to place campaigner spheres that might be straight or indirectly involved in host-pathogen interaction, either at the degree of filiform works pathogens or Oomycetess, we think our group-based attack is appropriate to set up a set of campaigner proteins and spheres.
Furthermore, the bunch of presence, absence, and enlargement forms of spheres known or implicated to be involved in a works infective life style with spheres that have no known or direct connexion to host-pathogen interactions AIDSs in spread outing this set of fresh campaigner spheres ( Figure 2-6 ) . For illustration, DUF1949 is within our species selection entirely found in Phytophthora species and adopts a ferredoxin-like crease. The N-terminal part of proteins incorporating this sphere shows similarity to another sphere ( UPF00029 ) that has been found in the human Impact protein.
The P. infestans cistron incorporating both spheres is induced early during infection of the works, supplying extra, independent grounds for the possible function of cistrons encoding this uncharacterized sphere in host-pathogen interaction. However, spheres that are besides abundant in nonpathogenic species ( e.g. other stramenopiles ) might non be related to or merely indirectly involved in pathogenicity. Hence, the exact nature of the part of these spheres to pathogenesis or to general lifestyle requires more in-depth experimental surveies of the campaigner spheres and cistrons predicted to incorporate these functional entities.
Protein domains by and large do non move as individual entities but in synergism with other spheres in the same protein or with other spheres in interacting proteins. We identified 773 oomycete-specific bigrams, of which 53 are observed in all analyzed Oomycetess ( Figure 2-7A ; Supplementary Table S2-10 ) . Based on our species choice, we can non reason that the oomycete-specific bigrams are common to all Oomycetess, since they might merely be specific for works infective Oomycetess or even for the selected Oomycetess analyzed in this survey.
The bulk of the 773 bigrams, nevertheless, are specific for a subset of the tried oomycete species or even a individual species. The 320 bigram types that are observed in more than a individual species or twice in the same proteome are observed in 982 predicted proteins. These bigrams are less likely to be the consequence of a incorrect cistron note and include already well-described illustrations of oomycete-specific sphere combinations, such as the FYVE-PIK bigram observed in Phytophthora phosphatidylinositol kinases ( Meijer & A ; Govers 2006 ) , the AP2-histone deacetylase bigram that is specifically found in P. ramorum and P. infestans ( Iyer et al. 2008 ) , and the myosin caput domain-FYVE bigram every bit good as the FYVE-GAF bigram found in myosin proteins in all analyzed Oomycetess ( Richards & A ; Cavalier-Smith 2005 ) . Still, some of 49 Domain Analysis in Oomycete Plant Pathogens
the bigrams could be unreal due to false negatives or false positives in the sphere anticipations. The staying, species-specific bigrams could be the consequence of unreal merger of cistrons due to incorrect cistron note or an existent biological signal in one of the analyzed oomycete species. The derived consequences are non merely dependant on the quality of the genome sequences of the analyzed Oomycetess but besides on that of the other eucaryotes. Incorrect anticipations of bigrams in these species would take to false negatives in Oomycetess. Hence, the figure of derived oomycete-specific bigrams is merely an estimate, and the true set of oomycete-specific bigrams demands to be farther analyzed.