Gene Gain and Loss during Evolution of Obligate Parasitism in the White Rust Pathogen of Arabidopsis thaliana
[['For more than 150 years , attempts to culture downy mildews , powdery mildews , and rusts on artificial nutrient media have been unsuccessful .', 'The terms obligate parasitism and obligate biotrophy are used to denote organisms that live in such an obligatory association with living hosts [1] , [2] .', 'Recent research on the obligate biotroph powdery mildew fungus Blumeria graminis or downy mildew oomycete Hyaloperonospora arabidopsidis reveals a close correlation between the biotrophic life style and massive gene losses in primary and secondary metabolism [3] , [4] .', 'Obligate biotrophs form an intimate haustorial interface with parasitized cells .', 'Haustoria are differentiated intercellular hyphae , but little is known about their functionality and evolution beyond their involvement in nutrient uptake [5] , [6] .', 'The obligate biotroph oomycete Albugo laibachii is a member of the Chromalveolata , which incorporates Dinophyta , Ciliophora , Heterokonts ( containing the oomycetes ) , Haptophyta , Cryptophyta , and Apicomplexa ( which includes human parasites like Plasmodium falciparum and Toxoplasma gondii [7] , [8] ) .', 'Within the oomycetes , A . laibachii belongs to a lineage known as peronosporalean , which includes the hemibiotrophic pathogen of potato Phytophthora infestans [9] and the necrotroph pathogen Pythium ultimum [10] .', 'Within this lineage , obligate biotrophy evolved twice independently in white blister rusts ( Albuginales ) and downy mildews ( part of the Peronosporaceae ) [11] .', 'The downy mildew pathogen H . arabidopsidis and A . laibachii are both pathogens of the model plant Arabidopsis thaliana [12] .', 'While both show similar infection structures within the host [13] , [14] , A . laibachii releases motile zoospores from asexual spores and sexual oospores , while H . arabidopsidis lacks all motile stages [4] , [15] .', 'Both pathogens are regularly found to co-infect plants and sporulate on the same leaf [16] .', 'A remarkable consequence of infection by Albugo sp .', 'is enhanced host plant susceptibility to other parasites to which the host is resistant in the absence of Albugo infection , and also impairment of cell death mechanisms [16] .', 'Albugo sp .', 'infect 63 genera and 241 species [17] , including economically important Brassica rapa ( canola ) , B . juncea ( oilseed mustard ) , and B . oleracea ( cabbage family vegetables ) [18] , [19] .', 'Recent analysis of oomycete evolutionary history [11] suggest that Albugo is more closely related to necrotrophs such as Pythium than to downy mildews , and thus provides a unique system to study the evolution and consequences of biotrophy , and to identify new defence-suppressing effectors and their host targets .'], ['Since prolonged culture of pathogen strains can result in genetic changes [20] , we sequenced a fresh highly virulent field isolate of A . laibachii .', 'The strain was selected from a heavily infected Ar .', 'thaliana field plot ( Norwich , United Kingdom ) [21] , and strains were single zoospore purified .', 'Isolate Norwich 14 ( Nc14 ) was determined as A . laibachii [19] and used for further analyses .', 'In contrast to Nc14 , A . laibachii isolate Em1 ( formerly Acem1 , A . candida East Malling 1 [19] ) is an established Albugo strain that was collected 15 y ago [16] , [22] , [23] , and we resequenced this strain .', 'Both strains show identical ITS ( internal transcribed spacer of ribosomal RNAs ) and COX2 ( cytochrome C oxidase subunit II ) sequences .', 'To ensure that sequence differences observed between these strains are of biological relevance not just the result of background mutations , we tested the host range for both isolates on 126 Ar .', 'thaliana accessions and identified 12 that show resistance to only one of the A . laibachii isolates ( Table S1 ) .', 'Nc14 is virulent on more accessions than the Em1 isolate is ( Table 1 ) .', 'The A . laibachii Nc14 genome was sequenced using Illumina 76-bp paired reads with ∼240-fold coverage ( Figure 1 ) .', 'In order to assemble the diploid heterozygous genome , an assembly pipeline was developed using Velvet [24] as primary assembler and Minimus [25] as meta-assembler ( Figure S1 ) .', 'Short read assembly programs are sensitive to heterozygous positions depending on read depth and kmer-length .', 'Reads not aligning to bacterial or plant sequence in public databases were used to estimate the genome size as ∼37 Mbp .', 'Using the estimated genome size , 50% of the resulting assembly is contained in 164 contigs with an N50 of 56 . 5 kbp .', 'A comparative analysis of contig size classes versus frequency indicates that 90% of the assembled genome shows a high degree of continuity in only 585 contigs , while 10% of the genome is fragmented in 3 , 231 contigs ( Figure 2A ) .', 'Read depth indicates that this 10% of the genome shows elevated levels of nucleotide coverage that are likely to comprise unresolved repeats ( Figure 2B ) .', 'Aligning Illumina cDNA reads from different stages of infection to reveal transcriptionally active regions in the assembly shows that few transcripts arise from the unresolved repetitive regions of the genome ( Figure 2D ) , suggesting that the gene space of a genome can be reliably defined using Illumina-only approaches .', 'A CEGMA [26] analysis revealed a high degree of completeness of assembly of core eukaryotic genes , as well as a continuity within the core genes comparable to high-quality Sanger read assemblies ( Figure S2; Table S2 ) .', 'We designed 32 primer pairs for regions between 0 . 6 and 5 kb based on our assembly ( Table S3 ) .', 'Thirty-one genomic regions could be amplified and were Sanger sequenced from both ends .', 'All PCR products had the predicted size , and sequences showed 100% identity to the genome assembly .', 'The mitochondrial draft genome was assembled in a separate attempt because of its high repeat content and therefore higher coverage compared to the core genome .', 'The assembled genome comprises 26 . 7 kb in 11 contigs and shows a high degree of synteny to the P . infestans mitochondrion Ia [27] and the Py .', 'ultimum mitochondrion [10] ( Figure S3 ) .', 'Considering the node coverage of the Velvet primary assembly ( ∼150× ) , 15 . 6 kb of the mitochondrial genome have >300× node coverage and seem to be duplicated .', 'This might indicate , comparable to the Py .', 'ultimum mitochondrion genome [10] , that ∼50% of the genome is duplicated , leading to an estimated genome size of ∼43 kb .', 'While the highly repetitive tRNAs are not resolved within the A . laibachii mitochondrial genome , regions of high synteny between the Py .', 'ultimum and the P . infestans mitochondrial genome are found in ribosomal proteins and subunits of the NADH dehydrogenase as well as cytochrome C oxidase .', 'Approximately 22% of the A . laibachii Nc14 genome assembly consists of repetitive regions ( Figure 3; Tables S4 and S5 ) .', 'The majority of repeats are represented by transposable elements ( 96% ) , while 4% of all repeats are A . laibachii-specific ( Table S5 ) .', 'Compared to other obligate biotrophs , the number of repeats is low .', 'H . arabidopsidis , for example , with an estimated genome size of 100 Mb , contains ∼43 . 3% repeats [4] , while transposable elements account for 64% of the ∼120-Mb Bl .', 'graminis ( powdery mildew ) genome [3] .', 'We identified 45 contigs carrying telomeric repeats; amongst these , 25 contigs have telomeric repeats located at one end of a contig .', 'We therefore postulate that the A . laibachii Nc14 genome is distributed over 12 or 13 chromosomes ( Table S6 ) .', 'tRNA genes are difficult to resolve because of their high copy number [28] .', 'Within our Illumina assembly , 153 tRNA genes were detected with 48 distinct anticodons ( Figure S4; Table S7 ) .', 'Our ability to resolve all these repeats within the Illumina short read assembly illustrates its quality .', 'Based on read depth , both Nc14 and Em1 isolates possess ∼6 Mbp of hemizygous or highly heterozygous regions ( 6 . 2 and 5 . 6 Mbp for Nc14 and Em1 , respectively ) ( Figure 1B and 1D ) as well as ∼13 , 000 heterozygous loci ( 13 , 116 and 13 , 523 for Nc14 and Em1 , respectively ) ( Figure 2C ) .', 'Remarkably , most of the hemizygous/highly heterozygous regions are shared between Nc14 and Em1 .', 'Compared to other sequenced oomycetes like P . infestans ( 240 Mbp ) , H . arabidopsidis ( 100 Mbp ) , or even Py .', 'ultimum ( 42 . 8 Mbp ) , A . laibachii has a highly compact genome structure ( Figure 4A ) .', 'Approximately 50% of the A . laibachii genome assembly matched cDNA reads , and transcriptionally active regions are further clustered , resulting in transcriptional hot spots and silent genomic regions ( Figure 4B ) .', 'A reference set of 13 , 032 gene models was generated incorporating cDNA reads from different stages of infection ( Figure S5A ) .', 'From extensive cDNA sequencing of infected Arabidopsis leaves , approximately 20 M ( ∼1 . 5 Gbp ) unique Illumina reads match the Nc14 genome assembly but not Ar .', 'thaliana TAIR 9 . 0 , and these were used to generate training sets for ab initio gene predictions and as evidence sets for consensus gene prediction .', 'In all , 88 . 3% of all gene models are supported by at least three cDNA hits .', 'For validation of these gene models , a set of 860 annotated core eukaryotic orthologous groups ( KOGs ) [29] was compiled and tested .', 'In all , 75% of these groups are present in the current annotation .', 'For comparison , 78% of KOGs were present in P . infestans , 73% in H . arabidopsidis , 42% in Pl . falciparum , and 85% in Ar .', 'thaliana ( Figure S5B ) .', 'In addition , 49 . 9% of all gene models show Pfam support , resulting in 2 , 505 Pfam domains , and 803 genes were functionally assigned to pathways using ASGARD [30] and manual annotation .', 'Transcriptional units show an even more compact , clustered occurrence than P . sojae or P . ramorum and an occurrence pattern clearly different from that of P . infestans [9] ( Figure 4C ) .', 'From our annotations using ASGARD we identified major enzymes of the lipopolysaccharide biosynthesis pathway , as have been described for P . infestans [31] .', 'These analyses revealed , in addition , the possibility that A . laibachii is able to synthesize brassinosteroids .', 'We identified potential homologues to the Ar .', 'thaliana brassinosteroid biosynthesis genes Dwf4 and DET2 ( Table S8 ) .', 'Although ASGARD identified homologues of Br6ox , D2 , and CPD , manual annotation revealed that assigning function to members of the superfamily of cytochrome P450 enzymes in A . laibachii is difficult based on homology alone ( Table S8 ) .', 'It has been hypothesized that the frequency of functionally redundant genes is reduced in obligate biotrophs , as reported for Bl .', 'graminis [3] .', 'Combining ASGARD and manual annotation we identified the absence of the whole steroid biosynthesis pathway , and , like other oomycetes , A . laibachii probably relies on the host as a source of sterols .', 'We hypothesize that A . laibachii would need to take up campesterol from the plant as a precursor for brassinosteroid synthesis .', 'During evolution , plastids of both red algae and green algae were transferred to other lineages by secondary endosymbiosis .', 'How often and when secondary endosymbiosis occurred is difficult to address but of importance to clarify the origin of chromalveolates and their gain and loss of endosymbionts .', 'There are two distinct hypotheses for what took place .', 'The monophyletic hypothesis posits that a red alga was taken up only once , followed by repeated losses of this algal genome , giving rise to the highly divergent group of chromalveolates [32] .', 'An alternative and more common view hypothesizes polyphyletic origins of the Chromalveolata , with in some cases multiple events of secondary endosymbiosis [33]–[35] .', 'Molecular divergence of A . laibachii from other species within the Chromalveolata was assessed by examining the percentage of amino acid identity between orthologous gene pairs ( Figure 5 ) .', 'These analyses demonstrate that the green alga Chlamydomonas reinhardtii , the brown alga Ectocarpus siliculosus , and the diatom Phaeodactylum tricornutum show the same distribution of percentage amino acid identity to A . laibachii Nc14 regarding the cumulative frequency of orthologous pairs .', 'In contrast , previous systematic analyses suggested that brown algae and diatoms are the closest relatives of oomycetes and that secondary endosymbiosis occurred with a red alga [32] , although there are suggestions that oomycetes diverged before this event [36] .', 'Using a set of >1 , 700 genes that are of “green” origin ( from green algae ) or “red” origin ( from red algae ) and that have been integrated into the diatom nuclear genome [37] , we found more oomycete genes that show significant BLAST hits to green algae than to red algae ( 34 “green” compared to five “red” ) ( Figure S6; Table S9 ) .', 'These findings are consistent with the results published by Moustafa et al . [37] for diatoms .', 'In a separate approach we identified genes showing high similarity between oomycetes , green algae , and red algae that are absent from diatoms ( 32 “green”; 11 “red” ) ( Tables S10 and S11 ) .', 'This result might indicate the presence of all these genes in a common ancestor , followed by loss or expansion of the gene family depending on adopted live style .', 'To address this question , we further analysed genes absent from A . laibachii Nc14 and studied their presence/absence in three other oomycetes , Pl . falciparum , and the brown alga E . siliculosus ( Table S12 ) .', 'The majority of genes absent from A . laibachii Nc14 are absent from other oomycetes and from Pl . falciparum but are present in the brown alga .', 'These genes are involved in the photoautotrophic , aquatic life style of diatoms and algae , such as a sodium/bile acid cotransporter , a haloacid dehalogenase-like hydrolase , fatty acid biosynthesis genes , a zeaxanthin epoxidase and a fucoxanthin chlorophyll a/c binding protein .', 'In contrast to the genes lost , we found that certain gene families like aspartic proteases or proteases containing MORN ( membrane occupation and recognition nexus ) repeats [38] show expansion in A . laibachii Nc14 compared to in diatoms .', 'Although our results fit the hypothesis of a common ancestor , we cannot exclude horizontal gene transfer and uptake of an endosymbiont after the divergence between a brown algal ancestor and an oomycete ancestor , given the low number of diagnosed genes that we could analyse .', 'Potentially green-algae-derived proteins carrying MORN repeat domains ( Figure S7 ) are involved in the complex process of internal budding in apicomplexans [39] , which may be similar to the zoospore formation of oomycetes within oospores or zoosporangia or gamete formation in diatoms [40] .', 'While oomycetes with a motile zoospore stage like A . laibachii and P . infestans carry the MORN repeat proteins , these proteins are absent in the non-motile H . arabidopsidis and absent in the non-motile red alga Cyanidioschyzon merolae [41] .', 'We therefore hypothesize that loss of this gene of hypothetical green algal origin could have led to the evolutionary loss of the whole flagellum apparatus in H . arabidopsidis [4] .', 'However , we cannot rule out that depletion of any major flagellar protein could have caused evolutionary loss of the whole flagellum apparatus .', 'Inspection of the flagellar inner arm dynein 1 heavy chain alpha , which is absolutely necessary for flagellum function , reveals that genomic regions carrying flagellar inner arm dynein 1 heavy chain alpha genes show a high degree of synteny between oomycetes like Py .', 'ultimum and A . laibachii .', 'In contrast , a syntenic region in H . arabidopsidis shows replacement of the flagellar dynein by Mariner- or Gypsy-like transposable elements ( Figure S8 ) .', 'Since within the peronosporalean lineage , biotrophy evolved twice independently [11] , we compared A . laibachii with the other obligate biotroph H . arabidopsidis [4] , hemibiotroph P . infestans [9] , and necrotroph Py .', 'ultimum [10] ( Figure 5; Tables S13 and S14 ) .', 'We found that H . arabidopsidis is the most diverged from A . laibachii .', 'H . arabidopsidis shares the fewest ( 4 , 826 ) orthologous genes with A . laibachii , versus the average of 5 , 722 in A . laibachii/P .', 'infestans and A . laibachii/Py .', 'ultimum comparisons .', 'Meanwhile , H . arabidopsidis genes show the highest amino acid identity with the genes of P . infestans , on average 73% of amino acid identity between all single copy orthologous pairs .', 'Py .', 'ultimum shares the highest number of orthologous genes with A . laibachii ( 5 , 910 pairs ) .', 'P . ultimum proteins also have a slightly higher percentage of amino acid identity with A . laibachii proteins than with other oomycetes ( Figure 5 ) .', 'Yet , Py .', 'ultimum itself is closer to H . arabidopsidis and P . infestans than to A . laibachii , sharing with them more orthologous genes with higher mean amino acid identity .', 'These analyses support the hypothesis that A . laibachii and H . arabidopsidis evolved biotrophy independently; genes missing in one or the other genome compared to the necrotroph Py .', 'ultimum or hemibiotroph P . infestans may be correlated with biotrophy ( Table S15 ) .', 'One of these genes is that for molybdenum-cofactor-dependent nitrate reductase .', 'Nitrate reductase catalyzes pyridine-nucleotide-dependent nitrate reduction for nitrogen acquisition [42] .', 'Both biotroph pathogens have a set of transporters showing homology to amino acid transporters , but other uptake mechanisms or sources could also enable nitrogen acquisition from their hosts [43] .', 'While H . arabidopsidis lost only the nitrate reductase , A . laibachii also lost the sulphite oxidase and the whole molybdopterin ( a cofactor required for nitrate reductase and sulphite oxidase function ) biosynthesis pathway .', 'In Pl . falciparum , which shows a high degree of adaptation to parasitism , nitrate reductase , sulphite oxidase , and the whole molybdopterin biosynthesis pathway are also missing .', 'Most likely the loss of the two Mo-containing enzymes and the Mo-cofactor biosynthesis is the outcome of biotrophy and not the reason for biotrophy , though conceivably there may have been selection against this pathway if other nitrogen or sulphate sources are less energy-consuming and therefore enhance fitness during parasitism .', 'Molybdenum has been reported to interfere with function of chaperones like Hsp90 [44] , [45] .', 'Avoiding the uptake of molybdenum might prevent this Hsp90 inhibition and increase fitness on Ar .', 'thaliana accessions with high molybdenum levels like Col-0 [46] .', 'H . arabidopsidis therefore could be in a less advanced stage of host adaptation compared to A . laibachii and Pl . falciparum .', 'Besides biotrophy , the formation of haustoria and haustorium-like structures evolved several times in peronosporalean biotroph and hemibiotroph pathogens .', 'Haustoria in fungi are sites of enhanced nutrient uptake [47] and metabolism , such as thiamine biosynthesis [48] .', 'In the oomycetes , all haustorium-forming species have lost the thiamine biosynthetic pathway .', 'We infer that haustorial oomycetes obtain thiamine from the host .', 'We therefore hypothesize that evolution to biotrophy is initiated not by gene loss , but rather from the ability to build a haustorium and therefore differentiate a sophisticated interface with a host .', 'The critical step to adopting biotrophy is likely to be efficient defence suppression to enable persistence of functioning haustoria; subsequent loss of biosynthetic pathways is likely to be secondary .', 'Well-adapted human pathogens like Pl . falciparum and plant pathogenic fungi like Ustilago maydis have small secretomes ( 320 [49] and 426 [50] proteins , respectively ) compared to necrotrophic fungi like Aspergillus fumigatus ( up to 881 proteins [51] ) .', 'We found that the same is true for oomycetes .', 'Using SignalP [52] to predict potential secretion signal peptides and MEMSAT [53] to predict transmembrane ( TM ) domains , we identified 2 , 473 ( 2 , 136 without TM domains ) potentially secreted proteins in the hemibiotroph P . infestans and 1 , 636 ( 1 , 222 without TM domains ) in the necrotroph Py .', 'ultimum .', 'For H . arabidopsidis only 1 , 350 ( 1 , 054 without TM domains ) and for A . laibachii 949 ( 672 without TM domains ) were identified .', 'Analysing the secretome for pathogenicity-related proteins like proteases , glucosyl hydrolases , and potential elicitins or lectins reveals a significant reduction in the H . arabidopsidis and A . laibachii secretome ( Tables 2 and S16 ) .', 'We postulate that biotrophs reduce their activation of host defence by reducing their inventory of secreted proteins , particularly cell wall hydrolyzing enzymes .', 'The ability to establish a sophisticated zone of interaction like the parasitophorous vacuole in Pl . falciparum or the haustorium in oomycetes and fungi requires sophisticated host defence suppression [54] , which is predominantly achieved via secreted proteins delivered into the host cell [55] , [56] .', 'The A . laibachii secretome comprises 672 secreted proteins without TM domains .', 'Genetically identified oomycete avirulence ( Avr ) proteins are secreted proteins that have signal peptide and RXLR motifs [57] , [58] .', 'In many oomycete genomes the RXLR motif is over-represented and positionally constrained within the secreted protein [59] .', 'We identified 25 RXLR and 24 RXLQ effector candidates in the A . laibachii secretome .', 'To determine the likelihood that RXLR or RXLQ motifs occur merely by chance in the A . laibachii secretome based on amino acid content , we performed in silico permutation of the motifs ( Figure 6A and 6B ) .', 'We concluded that the RXLR and RXLQ motifs were not likely to occur merely by chance , and that the likelihood of occurrence by chance is higher in the proteome as a whole than among secreted proteins .', 'It was shown for P . infestans that effectors are often located in gene-depleted repetitive regions of the genome [9] .', 'We therefore investigated RXLR candidate proteins in highly repetitive regions of the genome .', 'We identified two RXLRs , one in a highly conserved repeat region with ∼10 repeats in Nc14 and one in a more diverged repeat region with >80 repeats within the genome .', 'The first region also exists in A . laibachii isolate Em1; the diverged repeat of the second identified region exists but without the RXLR gene-containing region ( Figure S9 ) .', 'There are 563 RXLR effector candidates identified in P . infestans [9] , so RXLR effectors are less likely to be relevant for A . laibachii virulence .', 'Similar conclusions can be drawn for the CRN protein family , which shows expansion in P . infestans [9] , [60] but not A . laibachii , where only three members of the CRN family could be identified with signal peptides .', 'Eight additional CRN-like proteins were identified where no signal peptide has been predicted .', 'To identify new classes of effectors in the Albuginales clade , the secretome of A . laibachii was computationally screened for genes either showing heterozygosity or showing nucleotide polymorphisms between Nc14 and Em1 .', 'We identified a new class carrying a “CHXC” motif by inspection of the first 80 amino acids after the signal peptide cleavage site .', 'CHXC candidates are significantly enriched within the secretome ( Figure 6C ) .', 'Comparisons of the N-terminal part of the CHXC proteins revealed additional conserved amino acids , particularly a glycin at +6 to the CHXC motif ( Figure 6D ) .', 'In host–pathogen interactions , intraspecies comparisons enable the search for virulence alleles that undergo positive selection and fixation within the population [61] , [62] .', 'Secreted proteins with close contact to the host cell , such as effector proteins , often show enhanced levels of positive selection [63] , [64] .', 'By comparing the two A . laibachii isolates Nc14 and Em1 , we identified a significantly higher frequency of non-synonymous to synonymous mutations within the predicted secretome compared to the rest of the proteome .', 'Our analyses showed that this was particularly true for heterozygous positions and less convincing for homozygous SNPs ( Table S17 ) .', 'Genes that are highly conserved between species , like KOGs , showed comparable non-synonymous and synonymous substitution rates , with a slight excess of synonymous mutations .', 'There are significantly more genes within the KOGs showing a non-synonymous/synonymous ratio less than 1 than genes with values greater than 1 .', 'Comparing this to candidate effector classes like RXLRs , RXLQs , and CHXCs reveals that in particular the CHXCs show significantly higher frequencies of non-synonymous to synonymous mutations .', 'This supports the idea that the CHXC sub-class of secreted proteins is under positive selection , similar to other described oomycete effectors like ATR1 or ATR13 from H . arabidopsidis [57] , [65] .', 'Further to this we identified Nc14 genes absent or highly diverged from the Em1 complement .', 'We defined a gene as absent or highly diverged if >10 bp showed 0 coverage in the Em1 alignment .', 'Out of the 672 secreted proteins without TM domains , we identified seven as absent from Em1 ( 1 . 04% ) .', 'We also detected two with a predicted TM domain ( 0 . 73% ) that are absent from Em1 .', 'Regarding all gene models , 96 were absent ( 0 . 74% ) .', 'This finding is a further indication for a greater selection pressure on secreted than on non-secreted proteins , as has been found in species or interspecies comparisons in Phytophthora sp .', '[66] and Ustilago/Sporisorium [67] .', 'We tested A . laibachii effector candidates ( one CHXC , one RXLR , and one CRN effector candidate ) for their host delivery efficiency using a P . capsici–Nicotiana benthamiana translocation assay [68] .', 'Briefly , N-terminal domains of candidate effectors were fused to the P . infestans Avr3a effector domain , transformed into P . capsici , and tested for whether they confer translocation of Avr3a into N . benthamiana carrying R3a , resulting in avirulence .', 'Statistical analyses of the delivery efficiency ( Figure 7 ) clearly indicate that the A . laibachii CRN3 N-terminus and CHXC9 N-terminus are as efficient as the Avr3a N-terminus in Avr3a translocation , while the RXLR1 N-terminal domain is less efficient .', 'An alanine replacement construct of the CHXC motif supports the importance of this motif for delivery efficiency .', 'The Avr3a C-terminus alone confers a low basal delivery level without the need for the N-terminal enhancer .', 'These findings reveal the potential of the CHXC proteins to be delivered into the host cell , similar to RXLRs and CRNs , though the delivery mechanism for all these effector classes requires further investigation .', 'To assay the effectors for virulence function , we used Pseudomonas syringae pv .', 'tomato ( Pst ) DC3000 luciferase [69] carrying “effector detector vector” ( EDV ) constructs to deliver effectors into the plant cytoplasm via type III secretion [70] ( Figure 8 ) .', 'Tests on Ar .', 'thaliana Nd-0 plants revealed that several selected A . laibachii RXLRs , CRNs , and CHXCs enhance virulence compared to a non-functional AvrRps4 ( AvrRps4[AAAA] ) .', 'On Ar .', 'thaliana Col-0 , in contrast , the CRN and one RXLR ( RXLR1 ) do not enhance virulence while RXLR2 and CHXCs still do .', 'These tests indicate that CHXCs carry the capacity to enhance virulence in phytopathogenic bacteria , perhaps by suppression of host resistance mechanisms [54] , [70] .', 'These virulence assays together suggest that A . laibachii uses at least three different major effector classes .', 'To try to identify the evolutionary source of CHXCs , we investigated enrichment of CHXC-motif-containing proteins in the secretomes of P . infestans , Py .', 'ultimum , H . arabidopsidis , Saprolegnia parasitica , Thalassiosira pseudonana ( diatom ) , Pl . falciparum ( Apicomplexa ) , E . siliculosus ( brown alga ) , C . merolae ( red alga ) , Ch .', 'reinhardtii ( green alga ) , Volvox carteri ( green alga ) , and Ar .', 'thaliana .', 'Only A . laibachii contained a significant enrichment of CHXCs in its secretome .', 'Although not significantly enriched , both the fish pathogen S . parasitica and the land plant Ar .', 'thaliana contained more than ten CHXC proteins carrying potential secretion signals ( 14 and 11 , respectively ) ( Figure S10 ) .', 'In contrast to CHXC-containing proteins , almost all inspected organisms show a high number of CXHC-containing potentially secreted proteins; a common CXHC protein is protein disulphide isomerase ( Table S18 ) .', 'Given that A . laibachii CHXCs show the closest clustering with S . parasitica , V . carteri , Ch .', 'reinhardtii , and Ar .', 'thaliana CHXCs ( Figure 9 ) , conceivably this candidate effector class evolved from an ancestral green-alga-derived gene .', 'Whatever their origin , we conclude that CHXC proteins are present in all organisms analysed but evolved effector function only in Albuginales and possibly Saprolegniales .', 'In Albuginales , one N-terminal sub-class of CHXCs ( CHxCLx ( 4 ) Gx ( 5–6 ) L ) shows significant expansion , with 23 members , while other CHXCs are distinct from this clade .', 'S . parasitica CHXCs are distinct from this major A . laibachii clade and therefore remain to be tested in future experiments .', 'The A . laibachii genome assembly sheds light on the evolution of biotrophy since it allows the first comparison , to our knowledge , of two oomycete obligate biotroph pathogens ( A . laibachii and H . arabidopsidis ) that evolved biotrophy independently .', 'In addition , A . laibachii shows the highest overall amino acid identity to the necrotroph pathogen Py .', 'ultimum and the hemibiotroph P . infestans .', 'One of the striking results of this comparison is that all organisms able to build haustoria have lost their thiamine biosynthesis pathway , presumably because thiamine is easily obtained from hosts via the haustorial interface .', 'A closer interface requires effective host defence suppression .', 'We therefore hypothesize that the evolution of biotrophy involves a series of steps: step 1 , involving progressively more effective effectors to suppress defence , step 2 , attenuated activation of defence by reduction in the inventory of cell wall hydrolyzing enzymes , resulting in , step 3 , weak selection to maintain certain biosynthetic pathways if the products of the pathways can be directly obtained from the host .', 'This results in progressively more comprehensive auxotrophy and culminates in irreversible biotrophy ( Figure 10 ) .'], ['An infected leaf was harvested from an Ar .', 'thaliana plant grown in a heavy infected field plot in Norwich ( UK; 52 . 6236 , 1 . 2182 ) [21] in December 2007 .', 'Zoosporangia were washed off the leaf surface and used to infect Ar .', 'thaliana Ws-0-eds1 plants .', 'After 1 wk one pustule was punched out , and spores were placed on ice for 30 min to release zoospores .', 'Unhatched zoosporangia were removed by filtration , and zoospores were diluted to ∼10 zoospores/ml and sprayed on Ar .', 'thaliana Ws-0 plants ( ∼100 µl/plant ) .', 'This procedure was repeated 4× until spores were bulked up on Ar .', 'thaliana Ws-0 plants .', 'Zoosporangia were harvested using a home-made cyclone spore collector [71] .', 'Zoospores were suspended in water ( 105 spores/ml ) and incubated on ice for 30 min .', 'The spore suspension was then sprayed on plants using a spray gun ( ∼700 µl/plant ) , and plants were incubated in a cold room in the dark over night .', 'Infected plants were kept under 10-h light and 14-h dark cycles with a 20°C day and 16°C night temperature .', 'High molecular weight DNA was extracted from zoosporangia using a phenol/chloroform-based purification method after grinding in liquid nitrogen , adapted from [72] .', 'Library preparation for Illumina sequencing was performed as described [28] .', 'All data were generated using paired-end reads .', '800 bp and 400 bp paired-end sequencing libraries were constructed , and 8 . 8 Gbp of usable data were generated ( for read and insert length , see Figure 1A ) .', 'Figure 1A lists all reads after purification from plant and bact
['Introduction', 'Results/Discussion', 'Materials and Methods']
['Biotrophic eukaryotic plant pathogens require a living host for their growth and form an intimate haustorial interface with parasitized cells .', 'Evolution to biotrophy occurred independently in fungal rusts and powdery mildews , and in oomycete white rusts and downy mildews .', 'Biotroph evolution and molecular mechanisms of biotrophy are poorly understood .', 'It has been proposed , but not shown , that obligate biotrophy results from', '( i ) reduced selection for maintenance of biosynthetic pathways and', '( ii ) gain of mechanisms to evade host recognition or suppress host defence .', 'Here we use Illumina sequencing to define the genome , transcriptome , and gene models for the obligate biotroph oomycete and Arabidopsis parasite , Albugo laibachii .', 'A . laibachii is a member of the Chromalveolata , which incorporates Heterokonts ( containing the oomycetes ) , Apicomplexa ( which includes human parasites like Plasmodium falciparum and Toxoplasma gondii ) , and four other taxa .', 'From comparisons with other oomycete plant pathogens and other chromalveolates , we reveal independent loss of molybdenum-cofactor-requiring enzymes in downy mildews , white rusts , and the malaria parasite P . falciparum .', 'Biotrophy also requires “effectors” to suppress host defence; we reveal RXLR and Crinkler effectors shared with other oomycetes , and also discover and verify a novel class of effectors , the “CHXCs” , by showing effector delivery and effector functionality .', 'Our findings suggest that evolution to progressively more intimate association between host and parasite results in reduced selection for retention of certain biosynthetic pathways , and particularly reduced selection for retention of molybdopterin-requiring biosynthetic pathways .', 'These mechanisms are not only relevant to plant pathogenic oomycetes but also to human pathogens within the Chromalveolata .']
['Plant pathogens that cannot grow except on their hosts are called obligate biotrophs .', 'How such biotrophy evolves is poorly understood .', 'In this study , we sequenced the genome of the obligate biotroph white rust pathogen ( Albugo laibachii , Oomycota ) of Arabidopsis .', 'From comparisons with other oomycete plant pathogens , diatoms , and the human pathogen Plasmodium falciparum , we reveal a loss of important metabolic enzymes .', 'We also reveal the appearance of defence-suppressing “effectors” , some carrying motifs known from other oomycete effectors , and discover and experimentally verify a novel class of effectors that share a CHXC motif within 50 amino acids of the signal peptide cleavage site .', "Obligate biotrophy involves an intimate association within host cells at the haustorial interface ( where the parasite penetrates the host cell's cell wall ) , where nutrients are acquired from the host and effectors are delivered to the host .", 'We found that A . laibachii , like Hyaloperonospora arabidopsidis and Plasmodium falciparum , lacks molybdopterin-requiring biosynthetic pathways , suggesting relaxed selection for retention of , or even selection against , this pathway .', 'We propose that when defence suppression becomes sufficiently effective , hosts become such a reliable source of nutrients that a free-living phase can be lost .', 'These mechanisms leading to obligate biotrophy and host specificity are relevant not only to plant pathogenic oomycetes but also to human pathogens .']
['genome expression analysis', 'functional genomics', 'genome evolution', 'plant biology', 'microbiology', 'host-pathogen interaction', 'genome sequencing', 'plant science', 'model organisms', 'microbial evolution', 'plant pathology', 'arabidopsis thaliana', 'plant microbiology', 'proteins', 'comparative genomics', 'biology', 'pathogenesis', 'biochemistry', 'plant and algal models', 'plant pathogens', 'genomics', 'computational biology', 'genetics and genomics', 'defense proteins']