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Dynamics and innovations within oomycete genomes: 3
insights into biology, pathology, and evolution 4
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Howard S. Judelson 7
Department of Plant Pathology and Microbiology 8
University of California 9
Riverside, CA 92521 USA 10
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Address correspondence to: [email protected], Tel: 951-827-4199, Fax: 951-12
827-4294 13
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Running title: Dynamics and innovations within oomycete genomes 15
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Copyright © 2012, American Society for Microbiology. All Rights Reserved.Eukaryotic Cell doi:10.1128/EC.00155-12 EC Accepts, published online ahead of print on 24 August 2012
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ABSTRACT 19
The eukaryotic microbes known as oomycetes are common inhabitants of terrestrial 20
and aquatic environments, and include saprophytes and pathogens. Lifestyles of the 21
pathogens extend from biotrophy to necrotrophy, obligate to facultative pathogenesis, 22
and narrow to broad host ranges on plants or animals. Sequencing of several 23
pathogens has revealed striking variation in genome size and content, a plastic set of 24
genes related to pathogenesis, and adaptations associated with obligate biotrophy. 25
Features of genome evolution include repeat-driven expansions, deletions, gene 26
fusions, and horizontal gene transfer, in a landscape organized into gene-dense and 27
gene-sparse sectors and influenced by transposable elements. Gene expression 28
profiles are also highly dynamic throughout oomycete life cycles, with transcriptional 29
polymorphisms as well as differences in protein sequence contributing to variation. The 30
genome projects have set the foundation for functional studies and should spur the 31
sequencing of additional species, including more diverse pathogens and nonpathogens. 32
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INTRODUCTION 34
Oomycetes are known best for their plant pathogens, but also include saprophytes 35
and colonizers of insects, vertebrates, and microbes. Oomycetes outwardly resemble 36
fungi since both exhibit hyphal growth and heterotrophic absorptive nutrition, and reside 37
in similar ecological niches. Oomycetes were misclassified as Mycota until the last part 38
of the 20th century, but are now accepted to have a distinct evolutionary history and 39
belong to the Kingdom Stramenipila, which also includes brown algae and diatoms (7). 40
One theory joins stramenopiles with other protists into the Chromalveolata 41
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superkingdom, linked by an ancestral endosymbiosis of a photosynthetic alga (44). 42
Of the hundreds of known oomycetes, those affecting agriculture and natural 43
ecosystems are the most studied. Most fall into two large orders, the Peronosporales 44
and Saprolegniales (Fig. 1). Most notorious is the potato and tomato late blight agent 45
Phytophthora infestans, a peronosporalean that triggered the Irish Famine in the 1840s 46
and still has major impact worldwide (22). Phytophthora contains over 100 species, all 47
plant pathogens (48). Many are relatively recent discoveries, such as the Sudden Oak 48
Death agent P. ramorum that has severely damaged woodlands in North America and 49
Europe (29). No Phytophthora is known to persist as a saprophyte, in contrast to its 50
sister genus Pythium where most members are saprophytic and opportunistic 51
phytopathogens. Mycoparasites and an animal pathogen also populate Pythium. 52
Saprolegnian genera, like those in the Peronosporales, also include saprophytes and 53
plant pathogens, but there is a greater tendency to an aquatic lifestyle. Most species 54
within Achlya and Saprolegnia, for example, are usually innocuous inhabitants of 55
freshwater ponds and streams, where they grow on dead vegetable matter. They 56
however can also parasitize a range of aquatic animals, particularly when immune 57
systems are stressed. Saprolegnia parasitica in particular has become a growing 58
problem in commercial aquaculture, where high fish density and imperfect water quality 59
increase stress and favor disease (88). 60
Much diversity in the nature of pathogenesis is displayed by different oomycetes. 61
Species such as P. infestans and the soybean pest Phytophthora sojae colonize only a 62
limited number of hosts. Other Phytophthora spp. are more cosmopolitan, such as P. 63
ramorum which infects over 60 trees and shrubs, and P. cinnamomi which has over 64
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1000 hosts spanning both herbaceous and woody plants (19). Broad host range is also 65
typical of most species of Pythium, and Saprolegnia which infects both fish and 66
crustaceans. In contrast, with a few exceptions each downy mildew and white rust 67
colonizes only a single type of host. The white rust Albugo laibachii and the downy 68
mildew Hyaloperonospora arabidopsidis only infect Arabidopsis thaliana, for example. 69
Besides diversity in host range, pathogenic interactions involving oomycetes are also 70
varied, ranging from biotrophy to necrotrophy. Pythium is largely necrotrophic, 71
extracting resources from dying cells. At the other extreme, white rusts and downy 72
mildews feed on living cells and do not directly kill their hosts; one Albugo is even 73
reported as an asymptomatic endophyte (64). Phytophthora are hemibiotrophs, which 74
are biotrophic initially but shift to necrotrophy at the end of the disease cycle. 75
Genome sequences are now available for several oomycetes, and valuable for 76
resolving long-standing issues about their biology and evolution. Questions that can be 77
addressed include: What accounts for the diversity of oomycete lifestyles? How do the 78
genes that oomycetes use to infect hosts compare to those of pathogens in other taxa? 79
To what extent has horizontal gene transfer affected the pathogenic, metabolic, or 80
regulatory characteristics of oomycetes? Do oomycete genomes have features that 81
help oomycetes evolve to survive environmental changes, or defeat efforts to control 82
them? 83
84
Genome size and topography. Oomycete genomes are estimated to range in size 85
from a low of about 37 Mb, as in the cases of Pythium sylvaticum and A. laibachii, to a 86
high of 280 Mb, as in some Phytophthora spp. Repetitive DNA, mostly in the form of 87
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transposable elements (TEs), is responsible for the bulk of variation (45, 55, 65). 88
Assemblies annotated with gene models are now available publicly for eight plant 89
pathogens, including five Phytophthora spp. (P. capsici, P. cinnamomi, P. infestans, P. 90
parasitica, P. ramorum, P. sojae), Pythium ultimum, H. arabidopsidis, and A. laibachii 91
(6, 31, 45, 50, 51, 86). Data are also released for one animal pathogen, S. parasitica, 92
but not for an exclusively saprophytic species. Predicted gene contents range from 93
about 13 to 26 thousand (Fig. 1). A striking feature is the wide variation in repetitive 94
DNA content, which ranges from 7% for Py. ultimum to 74% for P. infestans. Some 95
correlation exists between gene number and genome size in P. infestans, P. ramorum, 96
and P. sojae but the trend loosens when considering other species. The recently 97
released sequence of P. cinnamomi, for example, includes 26,131 predicted genes 98
within a 78 Mb genome, compared to 16,988 for the 95 Mb genome of P. sojae. Initial 99
gene counts are often overestimates, however it is intriguing to consider that the large 100
number of genes in P. cinnamomi relates to its ability to infect more than a thousand 101
plant species. 102
Genome-wide ortholog mapping suggests that a core set of about 8,000 to 9,500 103
genes is conserved between species (31, 75). The variable component thus represents 104
nearly half of some proteomes. Interspecific differences are largely attributable to family 105
expansion and/or gene loss, which to some extent relates to the novel partitioning of 106
oomycete genomes into gene-dense and gene-sparse regions. In P. infestans, for 107
example, about 80% of genes reside in clusters of tightly-spaced genes, with the rest 108
distant to each other (~2 or more kb). This is illustrated in Fig. 2B for a portion of the P. 109
infestans genome, which contains a gene-rich cluster of 24 genes with small intergenic 110
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distances near a gene-sparse region with larger intergenic distances. Insight towards 111
the diversity of pathogenic lifestyles has come from studying the gene-sparse regions, 112
as these are enriched for loci encoding host-modulating "effectors" (31, 66). Many 113
genes in such regions have evolved faster than average in sequence and copy number, 114
possibly aided by the nearby repeated DNA that fosters illegitimate recombination and a 115
low gene density that minimizes lethal events. 116
Some clusters of repeated genes can nevertheless be found in gene-dense regions, 117
including some protein kinases and genes involved in sexual development (14, 34). P. 118
infestans encodes 354 proteins with classic eukaryotic protein kinase domains, with 119
other members of the genus having similar numbers. As with some other gene families, 120
unequal crossing-over appears to have contributed to its expansion as about 20% of 121
kinase loci reside in clusters of 2 to 13 genes. The same clusters tend to occur in other 122
members of the genus, indicating their presence in the last common ancestor of 123
Phytophthora, and a few are shared with H. arabidopsidis. The number of kinases in 124
different oomycetes varies dramatically, ranging from less than 100 in Py. ultimum to 125
about 500 in S. parasitica. 126
127
Transcriptional landscape. A typical oomycete life cycle involves vegetative 128
filamentous growth followed by sporulation, spore germination, and for pathogens the 129
formation of structures used to enter and feed from host tissues (37). Most species also 130
form both sexual and asexual spores. mRNA profiles exhibit dynamic changes 131
throughout these developmental transitions, with many genes expressed specifically in 132
spore or infection stages. In P. infestans, mRNA levels for nearly one-half of genes 133
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change >2-fold during life-stage changes, with about 14% being expressed exclusively 134
in one stage or another (35). Studies in P. infestans, the lima bean pathogen 135
Phytophthora phaseoli, and the cucumber downy mildew agent Pseudoperonospora 136
cubensis also documented changes between growth on artificial media and a plant host 137
(31, 49). Although P. infestans and Ps. cubensis have different lifestyles 138
(hemibiotrophic and obligately biotrophic, respectively), most orthologs had similar 139
expression patterns suggesting that the non-orthologous genes may play roles in 140
determining the mode of pathogenesis (71). 141
Many differentially-expressed genes have functions that are logically required at only 142
some stages of the life cycle. Examples include structural components of asexual or 143
sexual spores, cell cycle proteins that may act to establish spore dormancy, and 144
pathogenicity factors. Others however have roles that are more challenging to discern, 145
such as new isoforms of metabolic enzymes. Many transcription factors also show 146
strong differential expression, especially those in the bZIP and Myb families which have 147
patterns suggestive of a transcription factor cascade (35, 71, 92). 148
Most oomycetes genes are tightly spaced, with median intergenic distances being 149
only 435 nt within the gene-dense regions of P. infestans. By comparison, median 150
intergenic regions in S. cerevisiae, Arabidopsis thaliana, and Homo sapiens are 0.45, 151
1.5 and 35 kb, respectively (13, 47, 94). It is remarkable that despite the close spacing 152
of genes in Phytophthora, neighboring genes typically lack similar patterns of 153
developmental regulation. This can be seen in Fig. 2A, where each color indicates up-154
regulation in a particular developmental stage with black representing constitutive 155
expression. Tandemly duplicated genes are frequently co-expressed, as illustrated in 156
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Fig. 2B where the right border of the gene-rich interval contains a tandemly arrayed co-157
expressed cluster. In contrast, other adjacent genes are not usually up-regulated in the 158
same stage. The transcriptional independence of most neighboring genes in 159
Phytophthora contrasts with that of many eukaryotes, where genes are often organized 160
into co-expressed domains (40, 85, 94). The main mechanism for regulating 161
transcription during oomycete development may therefore not involve chromatin-level 162
effects, which in yeasts and metazoans can influence genes within a 5-50 kb region 163
(23). 164
One effect of high gene density in oomycetes appears to be a bias in gene 165
orientation. Within the gene-dense regions of P. infestans, only about 40% of loci are 166
transcribed divergently, i.e. head-to-head; this is much less than expected by random 167
chance, and less than in yeasts and plants. An example of genes in this orientation is 168
shown in Fig. 2C, where the intergenic region is only 301 nt. About 10% of such 169
closely-spaced genes show some degree of correlated or anti-correlated expression 170
(69). Oomycete promoters are therefore not only compact, but may overlap and 171
possibly antagonize their neighbors. 172
Intergenic distances are also small between the 3' ends of genes transcribed 173
towards each other, averaging 255 nt within the gene-dense regions of P. infestans 174
(69). Such regions must include nearby transcriptional terminators, a configuration 175
which may mechanistically efficient. This also may generate overlapping transcripts for 176
epigenetic regulation. A role for the latter is demonstrated in fungi and metazoans (16), 177
and it would be interesting to see if this also holds true for oomycetes. 178
The tight spacing of genes may also help stabilize genomes. Insertion of a 179
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transposable element or any illegitimate recombination event within such a region would 180
likely be lethal and eliminated quickly from the population. In contrast, if intergenic 181
distances were large then insertions would be tolerable in the short-term, but might 182
foster rearrangements that would be catastrophic during sexual reproduction. The 183
selection for genome stability may counterbalance the normal propensity of 184
transposable elements to insert within regions of high transcriptional activity, where a 185
higher fraction of DNA is uncoiled and exposed (21). The same principle likely 186
underlies the expansion of the gene-poor regions of oomycete genomes, as these but 187
not the gene-dense regions could tolerate transposon-mediated expansion. 188
The small size of most intergenic regions can also be helpful for identifying 189
regulatory motifs through mutagenesis or bioinformatics (1, 69, 81, 93). For example, 190
by searching just upstream of open reading frames for over-represented motifs, over 191
100 putative transcription factor binding sites could be identified from Phytophthora, of 192
which many were verified by functional assays (69). Most were within 200 nt of the 193
transcription start, consistent with the compactness and potential simplicity of oomycete 194
promoters. Nevertheless, sequenced Phytophthora spp. are predicted to encode 195
between 543 and 669 transcription factors, similar to that of fungal phytopathogens 196
such as Fusarium graminearum and Magnaporthe oryzae (659 and 481, respectively; 197
(63)). H. arabidopsidis, however, has fewer predicted transcription factors at 369. This 198
could reflect its simplified life cycle compared to Phytophthora; while the latter form 199
sporangia that release zoospores that encyst and then form germ tubes, H. 200
arabidopsidis conidia germinate directly and lack the zoospore stage. 201
Most oomycete genes have few introns, although intron-rich genes are common. 202
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Means of about 1.6 per gene are predicted for Phytophthora and Pythium, although a 203
lower number was reported for the subset of P. sojae genes with EST support (51, 77, 204
86). Many cases exist of "mis-splicing" in which unexcised introns or alternate junctions 205
would lead to premature translational termination (77). There is, however, only one 206
credible report of alternate splicing generating a new biological function. This comes 207
from Ps. cubensis, where the novel product had a new transporter activity (72). 208
209
Novel gene fusions. Oomycetes are unusually rich in proteins with novel combinations 210
of domains (59, 74). These have several predicted functions, which often involve 211
signaling. Two notable examples are separate families of phosphatidylinositol-4-212
phosphate kinases and aspartic proteases with transmembrane domains characteristic 213
of G protein-coupled receptors (43, 58). While their structures are mostly unconfirmed 214
by cDNA or proteomic analysis, most appear conserved throughout Phytophthora. 215
Protein kinases also have evolved domain combinations that are oomycete-specific or 216
seen only in closely related groups. One example found in Phytophthora, A. laibachii, 217
H. arabidopsidis, and S. parasitica is a MAP kinase with a N-terminal PAS signal 218
sensing fold (34). This combination does not appear in other eukaryotes including 219
diatoms or chromalveolates. Another unusual protein joins an N-terminal cyclin-220
dependent kinase with a C-terminal cyclin, which in other taxa are on separate proteins. 221
This unusual configuration is detected in most oomycetes but otherwise appears only in 222
apicomplexans and ciliates, which are chromalveolates. 223
Due to the tight spacing between oomycete genes, viable fusions can easily result 224
from the loss of a stop codon in an upstream gene. The diploidy of oomycetes provides 225
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a setting in which innovative combinations of domains could persist for long periods 226
during which their benefits could be tested, or be eliminated by gene conversion during 227
asexual growth or replacement with the wild-type allele during sexual reproduction. 228
229
Genes for pathogenesis and host colonization. After an oomycete attaches to a 230
plant or animal host, several events are required for successful infection. The invader 231
must degrade host barriers such as cell walls, usually by secreting hydrolytic enzymes. 232
Nutrients must be acquired, which for necrotrophs such as Py. ultimum involves taking 233
molecules leaked from damaged host cells. In contrast, most biotrophs and 234
hemibiotrophs are assumed to acquire much of their nutrients through haustoria, which 235
are specialized hyphae that insert into a host cell while remaining external to its plasma 236
membrane. Movement through haustoria is presumably bidirectional; not only are 237
nutrients probably moved from the plant, but oomycete proteins move across the 238
interface to alter the physiology of the host including its immune system. It should be 239
noted that data on the role of oomycete haustoria in nutrient acquisition are limited (3), 240
although infection-specific transporters can be identified from microarray data. 241
Analyses of the Phytophthora and Py. ultimum genomes indicate they are largely 242
autotrophic and can use diverse molecules as substrates for metabolism. As described 243
later, obligate biotrophs are deficient in several pathways. Haustorial oomycetes (A. 244
laibachii, H. arabidopsidis, Phytophthora) have lost the gene for thiamine biosynthesis. 245
The gene is present in Py. ultimum and S. parasitica, which as a necrotrophs are not 246
thought to employ haustoria (84). While it seems that haustorial oomycetes have 247
chosen to take this vitamin from their host, this is not the case for rust fungi such as 248
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Uromyces which makes their own thiamine in haustoria (78). 249
Most plant pathogenic oomycetes can not make sterols, due to the absence of the 250
relevant biosynthetic genes. An exception is the legume root pathogen Aphanomyces 251
euteiches, where a biosynthetic pathway was predicted from an expressed sequence 252
tag (EST) project and confirmed biochemically (53). The status of the sterol pathway 253
has been a topic of long-standing interest since such compounds are needed for sexual 254
reproduction. Also, while the agrochemical industry has found sterol biosynthetic 255
enzymes to be frequent fungicide targets, this has not been the case for oomycetes. It 256
follows that a practical application of genome sequencing is the identification of targets 257
for chemicals to defend against oomycete pathogens. 258
Plant defenses against oomycetes include producing enzymes that degrade the 259
pathogen's cell wall. Oomycete proteins inhibiting these were first identified from EST 260
projects, and later catalogued from whole genomes. P. infestans, for example, encodes 261
38 secreted Kazal-like and cystatin protease inhibitors, some of which have been shown 262
to suppress host apoplastic serine and cysteine proteases, respectively. These 263
inhibitor-like genes are present in moderately reduced numbers in the other 264
Phytophthora spp. and Py. ultimum (51). The phytopathogenic oomycetes also make 265
inhibitors of plant β-1,3-glucanases (15). 266
Oomycetes themselves use proteases and other host-degrading enzymes for 267
offense, similar to other phytopathogens. Many have similar numbers in Phytophthora 268
and Py. ultimum, such as cysteine proteases which average 37 in Phytophthora versus 269
42 in Py. ultimum. Others vary widely, such as cutinases and pectin esterases which 270
have about 7-12 copies in each Phytophthora but none in Py. ultimum. This may be 271
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because the latter primarily infects young non-suberized tissue while Phytophthora can 272
colonize plant parts with thick cuticles. In contrast, lipases and subtilisin-related serine 273
and aspartyl proteases are more common in Py. ultimum. As would be expected the 274
opportunistic fish pathogen S. parasitica is largely deficient in enzymes for degrading 275
plant cell walls, although its genome is annotated with two polygalacturonases. The 276
latter might be maintained for saprophytic growth on plant debris. 277
Whether an oomycete consumes plants or animals, host attachment is required for 278
infection; binding to a growth substrate must also be useful for saprophytes. 279
Oomycetes express several factors that may act in this role, such as glue-like proteins 280
released by zoospores, mucins, thrombospondin repeat proteins, jacalin domain 281
proteins, and cellulose-binding proteins including CBEL which genome data indicate are 282
made throughout oomycetes (25, 31, 68, 84). Interestingly, CBELs contain a 283
PAN/Apple domain also used by parasitic apicomplexans (also chromalveolates) to bind 284
their hosts. Recent discoveries in the Py. ultimum and Phytophthora genomes also 285
include cadherins, which play adhesion and secretory roles in animals (51). Except for 286
CBELs, most of these lack proved roles in pathogenesis, and it should be noted that 287
many oomycetes form adherent gametangia during sexual development which might 288
also use such proteins (37). 289
290
Fast-evolving families of effectors. Fungal, bacterial, and oomycete pathogens 291
secrete proteins collectively known as effectors that modify their hosts. Some proteins 292
described above are often also categorized as effectors, but much recent work has 293
focused on families unique to oomycetes especially the RXLRs and Crinklers/CRNs 294
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(41). The CRNs are named after a "crinkling" or necrotic phenotype that many cause 295
when overexpressed in plants, and RXLR after an N-terminal motif (X is any amino 296
acid) involved in uptake by host cells. Many RXLRs suppress plant immunity, such as 297
Avr3a and AvrBlb2 which respectively stabilize an E3 ubiquitin-ligase used in plant 298
defense, and block transport of a plant protease (10). The names of these two effectors 299
indicate that they sometimes act as avirulence factors, which means that some plants 300
encode resistance or R proteins that recognize them and respond with a cell death 301
response (33). These proteins, but not necessarily all RXLRs, also contribute to 302
pathogenesis against plant genotypes lacking the cognate R genes. The reader is 303
directed to recent reviews for more details of their activities (10, 79). 304
RXLRs in particular represent a success story for bioinformatics. The family was 305
recognized because of a short N-terminal motif shared between cloned avirulence 306
proteins, as the rest of the proteins are very dissimilar. A conventional N-terminal signal 307
peptide is required, and a nearby dEER motif often associates with RXLR. Early 308
studies used that information to identify 563, 374, and 396 such proteins in P. infestans, 309
P. ramorum, P. sojae, and then 134 in H. arabidopsidis. It was later observed that the 310
original RXLR consensus may be too restrictive. For example, Ps. cubensis employs 311
RXLQ and A. laibachii both RXLR and RXLQ (45, 82). 312
Identification of the RXLR motif stimulated searches of secretomes for conserved 313
motifs and other hallmarks of host-translocated effectors. The CRNs were shown to 314
have a LXLFLAK motif, Albugo spp. contained a family defined by a CHXC, and Py. 315
ultimum was predicted to encode a group with YXSL[RK] (45, 51, 73). Some of these 316
are not widely distributed, such as the CHXCs which have 29 members in A. laibachii 317
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but much fewer in the other oomycetes. It should be stressed that outside of A. laibachii 318
and P. infestans where functional testing of the CHXCs and CRNs was done, these are 319
just candidate effectors. 320
When examined at a genome-wide level it is clear that effectors are quite variable 321
within and between species. As noted before, their genes tend to reside in gene-poor 322
regions where DNA repeats may contribute to changes in family size, copy number, and 323
sequence. Some RXLR genes for example exist in tandem arrays of near-identical 324
copies, which vary in number in different strains (18). Comparisons of P. infestans with 325
four close relatives that infect different plants found that RXLR genes and CRN genes 326
were highly diversified, often through recombination within C-terminal domains which 327
may confer new activities for adaptation to their hosts (65). Examinations of genome or 328
EST data from other species have also identified many non-synonymous changes that 329
suggest an "arms race" between the pathogens and host resistance proteins that 330
interact with the effectors (4, 45, 65, 73). 331
Not all oomycete pathogens employ RXLRs. While found in most sequenced 332
peronosporalean genomes, RXLRs appear absent from Py. ultimum. This probably 333
relates to its necrotrophic lifestyle (in contrast biotrophs and hemibiotrophs need 334
effectors as part of stealthy infection strategies) and its broad host range (distinct 335
RXLRs are probably needed for different hosts). Only a few candidates were found in 336
the S. parasitica genome and in ESTs from the mycoparasite Pythium oligandrum, and 337
none from ESTs of the basal oomycete and algal pathogen Eurychasma dicksonii and 338
the legume root pathogen A. eutiches (25, 28, 32). However, inferences from EST 339
studies are not definitive. For example, sterol-carrier proteins known as elicitins are 340
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predicted from the S. parasitica genome sequence but were not detected in ESTs (84). 341
Another effector group of note is the NLP family, which encode a phytotoxin active 342
against dicots (26). Like those above it is diversified in copy number and sequence 343
throughout the Oomycota. Unlike the others, NLPs represent an ancient family with 344
members in bacteria and fungi. Counterintuitively, the necrotroph Py. ultimum has 345
about one-sixth the number of NLP genes than the hemibiotrophic Phytophthora spp., 7 346
vs. about 45 excluding pseudogenes. A recent study in P. sojae, however, showed that 347
only half of its NLP genes were expressed and only eight had necrosis-inducing activity 348
(17). This means that a reduction in gene number does not necessarily translate to 349
reduced protein levels, which is an important warning to those making inferences from 350
genome analyses without functional data. 351
352
Adaptations for obligate parasitism. Downy mildews and white rusts are not 353
culturable on artificial media and seem to have evolved to be wholly dependent on their 354
hosts. This occurred multiple times in oomycete history, and genome studies suggest 355
an association with defects in metabolism (6, 45). Both A. laibachii and H. arabidopsidis 356
lack nitrite and nitrate reductase and thus are limited in their use of inorganic nitrogen. 357
A. laibachii but not H. arabidopsidis has also lost genes for making the molybdenum 358
cofactor used by nitrate reductase, and another cofactor-dependent enzyme, sulfite 359
oxidase, which generates ATP from sulfite. Both also lack sulfite reductase. These 360
enzymes are found in the other sequenced oomycetes such as Phytophthora, but 361
interestingly are also missing in Plasmodium, an obligately pathogenic chromaveolate 362
(24). Knowing these defects might lead to the artificial culture of downy mildews and 363
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white rusts, however this may be an oversimplification since the species may have also 364
evolved to use plant signals to regulate their metabolism and development. 365
A. laibachii and H. arabidopsidis also encode reduced numbers of many proteins 366
involved in pathogenesis, including enzymes for degrading plant cell walls. Pectate 367
lyase, for example, is encoded by 30 genes in P. infestans but only 8 and 1 in A. 368
laibachii and H. arabidopsidis, respectively. Reductions are less dramatic for other wall 369
hydrolases, which may be used to form haustoria or remodel the pathogen's own walls. 370
The two species also have a reduced complement of CRNs and RXLRs. A. laibachii 371
and H. arabidopsidis encode only 3 and 20 CRNs compared to 196 for P. infestans. 372
CRNs induce host necrosis, so this is consistent with a greater reliance on biotrophy. 373
Genes for RXLR/Q proteins, which are encoded by >300 genes in each Phytophthora, 374
only number 49 in A. laibachii and 115 in H. arabidopsidis (note that A. laibachii also 375
has 29 of the CHXC proteins). The reduction may be explained by the fact that these 376
species need to interact with just one type of host. It is interesting to speculate whether 377
the loss of effectors was an adaptation to a narrowed host range, or its cause. It is also 378
possible that since these species inflict less damage to host cells than do 379
hemibiotrophs, fewer defenses against the plant immune response are required. 380
Gene content is also reduced in H. arabidopsidis due to loss of the zoospore 381
pathway. This includes at least 200 genes encoding structural components of flagella 382
along with >300 others with roles in zoospore assembly, chemotaxis, and encystment. 383
Fossilized remnants of many of these genes can be detected within the genome, but 384
most appear to have disappeared through slow mutation and genome contraction 385
through recombination. Albugo has however maintained the pathway, as have many 386
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other downy mildews. Curiously, genes for flagella are retained in Py. ultimum despite 387
the apparent rarity of that stage in nature (51). 388
389
Transposable elements (TEs) and their impacts. Oomycetes contain diverse 390
populations of retrotransposon and DNA transposon-like sequences. Their abilities to 391
self-replicate and form recombination-stimulating repeats shape the structure of 392
genomes and induce variation. As noted above, TEs appear to have particularly 393
influenced the gene-sparse regions of oomycetes. Within gene-dense regions where 394
gene order is fairly conserved between species, sequences resembling TEs often reside 395
where microsynteny is disrupted. 396
As in many eukaryotes the most abundant oomycete retrotransposons belong to the 397
long terminal repeat (LTR)-containing gypsy and copia families, with non-LTR LINE 398
elements also present. Most are degenerate and incapable of movement. Genome 399
size in Phytophthora correlates with the number of retroelements. P. infestans contains 400
about 10,000 gypsy and 4,000 copia-like elements which comprise 37% of the genome, 401
while P. sojae and P. ramorum contain only 3,000 and 2,400 in total (31). In contrast, in 402
A. laibachii these only represent 9% of the genome and copia outnumbers gypsy by 3:1, 403
and the two make up only 2% of Py. ultimum chromosomes (45, 51). 404
The ratio of DNA transposons to retroelements is strikingly different between 405
species. For example, these comprise 10% and 37% of the P. infestans genome, 406
respectively, and 12% and 9% of A. laibachii. A massive expansion of retrotransposons 407
and not DNA elements thus primarily accounts for the larger Phytophthora genomes. 408
Nevertheless a diverse population of DNA transposons exists in Phytophthora, including 409
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those that replicate by cut and paste, rolling circle, and self-synthesizing mechanisms. 410
Listed in declining order of copy number in P. infestans, ranging from about 2000 to 8, 411
these represent piggyBac, helitron, hAT, crypton, Tc1/mariner/pogo, MuDR/foldback, 412
Sola, Maverick, and PIF/harbinger families (20, 31, 87). Most are more abundant in P. 413
infestans, particularly piggyBac and helitrons. P. infestans has 273 helitrons of which 414
13 appear intact, which is more than other eukaryotes. P. ramorum and P. sojae 415
contain about one-tenth the number, none being functional. 416
Most of the DNA transposons have representatives in A. laibachii, H. arabidopsidis, 417
and Py. ultimum which suggests they are ancient members of the oomycete lineage. 418
Some such as hAT and Tc1/mariner may have been acquired after Stramenopiles 419
diverged from other eukaryotes as they are reportedly absent from the diatom 420
Thalassiosira pseudonana. The history of the cryptons is also interesting. Two of the 421
six major subfamilies described in eukaryotes, F and S, are throughout oomycetes while 422
diatoms only contain cryptonS. CryptonF in contrast is in Phytophthora but not A. 423
laibachii, H. arabidopsidis, or Py. ultimum. Outside of Phytophthora cryptonF has only 424
been found in ascomycetes, which suggests horizontal transfer (46). 425
There is evidence for some movement of TEs. One P. infestans gene was found to 426
contain an inserted gypsy element, including host target site duplications (31). hAT, 427
helitron, and PIF elements may also have "captured" Phytophthora genes and copied 428
them through the genome, based on the detection of their inverted terminal repeats with 429
target site duplications flanking adjoining transposase/replicase and host genes (87). 430
The latter encode SET domain proteins and AdoMet-dependent methyltransferases, 431
which interestingly are both involved in epigenetic regulation, an ABC transporter, 432
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cysteine protease, and transglutaminase. 433
434
TEs, epigenetics, and transcriptional polymorphisms. Although few TEs are active 435
in oomycetes, many are transcribed (35). Others are silenced based on the detection in 436
P. infestans of small interfering RNAs or siRNA (90). That oomycetes have an active 437
epigenetic system was also shown by RNA interference studies, where silencing was 438
associated with siRNA and heterochromatinization that spread from the target locus (2, 439
39, 89). In non-oomycetes, TEs influence nearby genes by recruiting heterochromatin 440
modifications that repress transcription, or insulating genes from such effects (52). 441
Since a TE is within 2 kb over half of effector genes in P. infestans, their influence on 442
expression should be considered. 443
In fact, certain P. infestans and P. sojae RXLR genes are known to be 444
transcriptionally inactive, with virulent and avirulent alleles distinguished by differences 445
in expression rather than protein sequence (18, 27). Some alleles also vary 446
quantitatively in mRNA level (76). Promoter mutations were associated with one case 447
of RXLR silencing, but others could involve epigenetic events. Both TEs and 448
epigenetics may relate to the phenotypic variability observed within Phytophthora spp., 449
including reports of strains with unstable avirulence phenotypes or losing pathogenicity. 450
In P. ramorum, isolates with unusual colony morphologies and an early senescence 451
phenotype were observed to have higher levels of expression of several copia elements 452
(42), Although causality was not established, this may be a sign of epigenetic flux 453
within their genomes. The contribution of expression level polymorphisms to oomycete 454
biology, due to promoter differences or epialleles, is just starting to be appreciated. 455
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It should be remembered that mobile elements and epigenetic phenomena are not 456
the only mechanisms driving change within oomycete genomes. Changes in ploidy, 457
gene conversion, and loss of heterozygosity are reported in several species (11, 30, 458
50). Unstable supernumerary chromosomes have also been described in Pythium (56). 459
460
Horizontal gene transfer (HGT). Prior to genome sequencing it was known that some 461
fungal and oomycete genes, such as the wall-degrading polygalacturonases, were 462
similar suggesting convergent evolution or HGT (83). As noted above the NLP 463
phytotoxins were also seen as HGT candidates (26). Careful phylogenetic analyses 464
using whole-genome data now provide strong support for HGT. Transfers from fungi to 465
oomycetes appear to have involved about 20 types of genes (67). About two-thirds 466
were predicted to be secreted and/or have potential roles in pathogenesis including 467
lipases, carbohydrate depolymerizing enzymes, sugar and nitrogenous base 468
transporters, and enzymes to degrade plant defense compounds. Since transfers from 469
fungi to oomycetes were much more common than the reverse, it was suggested that 470
plant pathogenesis developed in oomycetes more recently than in fungi. The same 471
study also reported very few cases of HGT for any gene in A. laibachii or S. parasitica 472
(67), and it is interesting to speculate that the larger TE-rich Phytophthora genomes are 473
more tolerant of recombination with foreign DNA. 474
Oomycetes also have obtained genes not related directly to pathogenesis by HGT, 475
including several involved in cofactor metabolism and amino acid or lipopolysaccharide 476
biosynthesis (91). Glucokinases are another example, with different genes coming from 477
plants and bacteroidales (38, 91). The retention of similar genes from two origins is not 478
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very common in eukaryotes, but these may function in distinct cellular locations. 479
480
Uncertain support for chromalveolate hypothesis. In one evolutionary model, the 481
Chromalveolata are a major eukaryotic group derived from a biflagellated cell with a red 482
algal endosymbiont (44). Chromalveolates are proposed to include about half of known 483
protists and algae, mainly cryptophytes, alveolates, haptophytes, and stramenopiles 484
(the latter including various classes of golden-brown or brownish-green algae, diatoms, 485
and oomycetes). The diatom and algal lineages are thought to have retained the red 486
alga as a photosynthetic plastid, with oomycetes losing the endosymbiont but retaining 487
some genes (54). Studies of the first sequenced oomycetes, P. ramorum and P. sojae, 488
reported 855 genes of likely red algal origin based on sequence similarity (86). Combined 489
with the finding that chromalveolate and oomycete proteins share some novel domain 490
combinations, this was taken by some to support the model (44, 59). However, 491
endosymbiotic acquisition is hard to distinguish from other events including HGT. 492
More recent analyses may be leading to a consensus for a polyphyletic origin or 493
falsification of the Chromalveolata. Diatom and oomycete genes having affinity with red 494
alga have limited overlap, so some suggested that separate endosymbiotic events 495
occurred before and after they diverged from their common ancestor (5, 57, 62, 80). 496
Support for endosymbiosis in the diatom lineage appears solid, but its occurrence prior 497
to their divergence from oomycetes was challenged by a study that argued that other 498
evolutionary events provide better explanations for the presence of red algal-like genes 499
in oomycetes (80). 500
Regardless of the validity of the chromalveolate hypothesis, several data indicate 501
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that stramenopiles have a monophyletic origin, including a recent analysis of 108 502
concatenated nuclear proteins (5). Oomycetes and diatoms also clearly contain 503
common sets of genes of plant ("green genes") and bacterial origin (9, 61). These 504
include many membrane transporters associated with nutrient acquisition or metabolism 505
with roles at the environmental interface or in organelles. Examples include 506
transporters for taking up nitrate, phosphate, or sulfate, and moving ADP/ATP across 507
mitochondrial membranes (12). 508
509
CONCLUSIONS AND OUTLOOK 510
Genomics has provided insight into the biology of oomycetes and paved the way for 511
future experimentation, but also raises new questions. The species sequenced so far 512
include agents of globally important diseases such as Phytophthora and Pythium, and 513
pathogens of a model plant. Phytophthora is also a good subject since it is amenable to 514
transformation, enabling functional studies of genes (36). However, data from more 515
species need to be made available publicly to help researchers understand the 516
foundations of pathogenesis and relationships between orders. These should include 517
both nonpathogens and pathogens from additional clades. These need not be limited to 518
species that are culturable in the lab, based on the success with the obligate pathogens 519
A. laibachii and H. arabidopsidis for which spores were taken from infected leaves for 520
analysis. This should also be feasible for the graminicolous (grass and grain-infecting) 521
downy mildews which are of major economic importance. Basal genera such as 522
Eurychasma or Haptoglossa, which include algal and nematode pathogens, are also 523
obligate pathogens but grow intracellularly with few external structures (8). Perhaps 524
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sufficient DNA could be obtained from zoospores, or pathogen sequences separated 525
from host DNA in silico. 526
The extant data indicate that oomycete genomes have had dynamic histories 527
colored by expansions/contractions, birth/death events, gene fusions, and HGT. The 528
acquisition of bacterial, fungal, and organellar genes has given oomycetes unique 529
capabilities, enabling them to occupy multiple environmental niches. Variation within 530
species is also just starting to be understood; one must wonder what pressures are 531
imposed by environmental changes and agronomic practices such as agrochemical 532
treatment, which may select for alterations in coding and regulatory sequences and may 533
also have epigenetic effects. Several Phytophthora spp. appear to be recent 534
interspecific hybrids (48), perhaps fostered by man's movement of plant material, and it 535
would be of interest to see how their genomes evolve. 536
Evidence of adaptations are evident in the genomes of the obligately biotrophic 537
oomycetes, which are partially condensed. They have not undergone the dramatic 538
reductions of obligate pathogens like Giardia and Plasmodium (24, 60), perhaps since 539
plants provide a less stable environment than an animal host. The benefits of genome 540
reduction in H. arabidopsidis due to loss of the zoospore might seem obvious but 541
neither Albugo or Py. ultimum (which rarely if ever makes zoospores) has chosen this 542
path. While much remains to be learned about the ecological and environmental 543
pressures that influence the evolution of oomycetes, it is clear that nature provides 544
niches for both streamlined and expanded genomes. 545
546
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ACKNOWLEDGMENTS 547
This work was supported by grants from the United States Department of Agriculture-548
National Institute of Food and Agriculture, and National Science Foundation. 549
550
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FIGURE LEGENDS 899
Fig. 1. Evolutionary history of oomycetes, lifestyles, and genome sequencing overview. 900
The main part of the figure portrays phylogenetic relationships between the major 901
orders, based on 28S rRNA sequences. Orders comprising the peronosporalean group 902
(~1000 species), saprolegnians (~500 species), and basal clades are noted (8). While 903
Phytophthora was traditionally considered as part of the Pythiales, it is now recognized 904
to belong to the Peronosporales along with the downy mildews (70). Species with 905
publicly released genome data are listed along with their predicted gene number, 906
genome size, and repetitive DNA content if known; values are based on the most recent 907
publication or data on web sites. The phylogram in the lower right shows the position of 908
oomycetes compared to other eukaryotes. 909
910
Fig. 2. Structural and transcriptional landscape of the P. infestans genome. (A) 911
Expression patterns of genes in a portion of a chromosome. Genes with >2-fold higher 912
mRNAs in one life-stage versus average are colored based when expression is highest, 913
according the key in the upper right (HY, hyphae; SP, sporangia; CLSP, sporangia 914
cleaving into zoospores; ZO, swimming zoospores; GC, zoospore cysts germinating 915
and making infection structures, e.g. appressoria). Genes not changing are in dark 916
grey, at half-height. Note that the scaffold is split into two portions, and the horizontal 917
axis represents gene order and not distance. (B) Representative gene-dense and 918
gene-sparse regions. Shown are gene orientations, expression patterns as in panel A, 919
and flanking DNA transposon (T) or retroelement-like sequences (R). The two regions 920
are separated by 450 kb, which is also gene-sparse. (C) Example of closely spaced 921
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opposing promoters from genes with strong EST support from panel B. The 301 nt 922
intergenic region includes a combined 142 nt of 5' UTRs, and has five predicted 923
transcription factor binding sites. The genes have a similar configuration in P. sojae. 924
925
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