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Signatures of Adaptation to Obligate Biotrophy in the Hyaloperonospora arabidopsidis Genome

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Science  10 Dec 2010:
Vol. 330, Issue 6010, pp. 1549-1551
DOI: 10.1126/science.1195203

Abstract

Many oomycete and fungal plant pathogens are obligate biotrophs, which extract nutrients only from living plant tissue and cannot grow apart from their hosts. Although these pathogens cause substantial crop losses, little is known about the molecular basis or evolution of obligate biotrophy. Here, we report the genome sequence of the oomycete Hyaloperonospora arabidopsidis (Hpa), an obligate biotroph and natural pathogen of Arabidopsis thaliana. In comparison with genomes of related, hemibiotrophic Phytophthora species, the Hpa genome exhibits dramatic reductions in genes encoding (i) RXLR effectors and other secreted pathogenicity proteins, (ii) enzymes for assimilation of inorganic nitrogen and sulfur, and (iii) proteins associated with zoospore formation and motility. These attributes comprise a genomic signature of evolution toward obligate biotrophy.

The oomycete Hyaloperonospora arabidopsidis (Hpa, formerly Peronospora parasitica or Hyaloperonospora parasitica) is a natural pathogen of Arabidopsis thaliana and a model for dissection of A. thaliana pathogen response networks (1, 2). Hpa belongs to a group of “downy mildew” pathogens, comprising more than 800 species that cause disease on hundreds of plant species (3). Downy mildew pathogens are related to other destructive oomycete plant pathogens (e.g., Phytophthora species) (4, 5). Oomycetes belong to the kingdom Stramenopila, which includes brown algae and diatoms. Although oomycetes and fungi share morphological and ecological similarities, they evolved independently to colonize plants.

Hpa hyphae grow between plant cells and establish feeding structures called haustoria, which have also evolved in fungal pathogens (2, 6). Downy mildews are obligately biotrophic and cannot be cultured apart from their hosts. In contrast, Phytophthora species are hemibiotrophic; an initial phase of biotrophic growth is followed by a necrotrophic phase. Molecular phylogenies show that downy mildews arose from a paraphyletic, Phytophthora-like, hemibiotrophic ancestor (4, 5, 7). Thus, insight into the genomic basis and evolution of obligate biotrophy can be obtained through comparison of the Hpa genome to the recently sequenced genomes of Phytophthora species (8, 9).

Genome analysis was performed on the Hpa Emoy2 isolate (1) using DNA from asexual spores (10). Sanger shotgun sequencing at 9.5-fold coverage, combined with 97 sequenced bacterial artificial chromosome inserts, yielded an assembly of 77.8 Mb. Illumina sequencing at 46-fold coverage yielded 3.8 Mb of additional sequence, which was integrated into the Sanger assembly to form the 81.6-Mb final version (v8.3.2). Forty-two percent of the Hpa genome is composed of repetitive elements (table S1). Analysis of Sanger and Illumina read depth suggests that v8.3 contains ~12.7 Mb composed of tandem repeats compressed into reduced copies in the assembly, indicative of a genome size of around 100 Mb (fig. S1). The CEGMA (Core Eukaryotic Genes Mapping Approach) pipeline, combined with manual examination, identified Hpa orthologs of 95% of the 248 conserved single-copy eukaryotic genes (fig. S2). Moreover, 94% of 31,759 expressed sequence tag (EST) reads aligned to the assembly, indicating that the draft genome assembly encompasses a very high percentage of the Hpa gene space.

A total of 14,543 genes were computationally predicted in v8.3, of which 80% are supported by ESTs and/or Illumina cDNA tags. This predicted gene content is similar to P. ramorum (65 Mb, 15,743 genes) and lower than P. sojae (95 Mb, 19,027 genes) (9) or P. infestans (240 MB, 17,887 genes) (8). A total of 6882 predicted genes in Hpa had no identifiable ortholog in sequenced Phytophthora species or similarity to known proteins, and as such represent potentially lineage-specific genes. Some of these genes may play roles that are specific to biotrophy. For example, a novel family of secreted small, cysteine-rich proteins exists in Hpa (PPAT12/24-like) (Table 1) (11).

Table 1

Copy numbers of annotated Hpa genes for hydrolases, PAMPS, and effectors, compared with Phytophthora genomes.

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Pathogenicity genes were compared among Hpa and Phytophthora species, revealing that families encoding host-targeted, degradative enzymes (secreted proteinases, cell wall–degrading enzymes) are reduced in Hpa (Table 1). Two notable examples are the family 12 endoglucanases (EGL12) and pectin methyl esterases (Pect). Phylogenetic analyses delineated several EGL12 and Pect gene clades containing genes from P. sojae and P. ramorum but not Hpa (figs. S6 and S7). Because Hpa and P. sojae likely share a sister group relationship relative to P. ramorum (4, 5), it is probable that a number of EGL12 and Pect genes were lost from Hpa after divergence of the lineage leading to Hpa and P. sojae. Hydrolytic enzymes that target the host cell wall can release cell wall fragments that elicit host defenses. It is conceivable that in evolving a biotrophic lifestyle, Hpa has lost most of the secreted hydrolytic enzymes that were present in a hemibiotrophic ancestor.

Similarly, gene families encoding necrosis and ethylene-inducing (Nep1)–like proteins (NLPs) are significantly reduced in Hpa, compared with P. sojae and P. ramorum. NLPs in Phytophthora and Pythium can trigger plant cell death and defenses (12), which have been implicated in the transition from biotrophy to necrotrophy. Only three of the 13 oomycete NLP clades contain genes from Hpa. However, one clade contains an expanded family that is specific to Hpa (Fig. 1A). All 10 HaNLP genes are supported by transcriptional data. Of these, HaNLP3 is most closely related to the PsojNIP and PiNPP1.1 proteins, but it did not induce necrosis in Nicotiana tabacum (Fig. 1B). These results suggest that downy mildew NLP genes may have evolved a different function than in Phytophthora. Copy number reduction was also evident for genes encoding known pathogen-associated molecular patterns (PAMPs) such as sterol-binding elicitins (13) and carbohydrate-binding CBEL (cellulose-binding, elicitor, lectin-like) genes (14) (Table 1). These examples further suggest that selection for “stealth” (avoidance of host defenses) was a major force during downy mildew evolution.

Fig. 1

Diversity, evolutionary history, and functional analysis of oomycete necrosis and NLPs. (A) Phylogeny of oomycete NLPs. A consensus tree from the Bayesian inference is shown. Thick lines indicate high support in minimum evolution (>90), maximum likelihood (>90), and Bayesian inference (>0.95). Hollow lines indicate branches highly supported in at least two analyses. Branches with high support in less than two analyses are represented by thin lines. (B) An Hpa NLP ortholog does not induce necrosis in plant leaves. NLP genes were transiently expressed in Nicotiana tabacum by agroinfiltration.

Phytophthora genomes encode hundreds of potential effector proteins (9, 15, 16) with RXLR cell entry motifs (1618) that likely function to suppress host defenses (19, 20). The Hpa genome contained 134 high-confidence effector gene candidates (HaRxL genes), including the known effector genes Atr1 and Atr13 (21, 22), significantly fewer than in the Phytophthora genomes (9, 15). Single-nucleotide polymorphisms arising from heterozygosity in v8.3 occurred at a rate 5 times as high in RXLR effector candidates [1 per ~500 base pairs (bp)] than in other genes (1 per ~2500 bp). Only 36% of the high-confidence Hpa effectors had significant matches in any Phytophthora genome (sequence similarity >30%), consistent with strong divergent selection on RXLR effector genes (15, 23). Moreover, Hpa effectors generally were not located in syntenic locations relative to Phytophthora genomes, except for three families of effectors, which have unusually high levels of sequence conservation (Fig. 2).

Fig. 2

Synteny of conserved RXLR effectors. (A) Region around HaRxL23, spanning scaffold_9:467737- 739923 (v6) and supercontig16:325445-40488 (v8.3.2) (B) Region around HaRxL13 and HaRxL136, spanning scaffold_150:3503-183330 (v6) and supercontig35:456072-268293 (v8.3.2). Colored boxes show order of gene models. Noncoding DNA is not represented. Dark green, orthologs; light green, orthologs found only in Phytophthora; dark brown, syntenic paralogs; light brown, syntenic orthologs found only in Phytophthora; white, syntenic gene families; dark blue, syntenic conserved RXLR effectors; cyan, syntenic RXLR effectors conserved only in Phytophthora; yellow, RXLR effectors not syntenic or conserved; blue-gray, other genes not conserved or syntenic. Black lines join syntenic genes with the same orientation; red lines join genes with reversed orientations. Staggered black lines in (A) show scaffold joins predicted from the synteny analysis. HaRxL23, HaRxL13, and HaRxL136 have 47, 38, and 40% amino acid identity, respectively, with their most similar Phytophthora ortholog within the normally hypervariable C terminus.

As obligate biotrophs, downy mildews may have lost some metabolic pathways. We identified several potential metabolic defects in Hpa compared with P. sojae and P. ramorum (fig. S9). For example, genes for nitrate and nitrite reductases, a nitrate transporter, and sulfite reductase were missing (fig. S10 and table S3), which is also a feature of the genomes of obligately parasitic powdery mildew fungi (24). Hpa also lacks genes required for synthesis of arachidonic acid and polyamine oxidases.

Flagellated zoospores are produced by many oomycetes (25). Contrastingly, several downy mildew lineages germinate by extending infective germ tubes from nonmotile conidiospores, although evidence exists for a rare zoosporic stage in some otherwise conidial downy mildews (26, 27). To conclusively determine whether spore motility has been lost from the Hpa lineage, we searched the Hpa genome for 90 flagella-associated genes using Chlamydomonas sequences and their Phytophthora orthologs (28). No matches were detected in Hpa for any of these. Similarly, many Phytophthora adhesion-related genes are reduced in number or absent from Hpa, consistent with the lack of adherent cysts that normally develop from zoospores during infection.

Analysis of Hpa gene space revealed genomic signatures of major alterations in pathogenic strategy, metabolism, and development that occurred during the evolution of obligate biotrophy from a facultative, hemibiotrophic ancestor. Interestingly, some features of Hpa gene space (large numbers of secreted effectors, reduction in degradative enzymes, and loss of N and S assimilation) are mirrored in genomes of biotrophic fungi (24, 29, 30). These similarities indicate that convergent adaptations occurred during the independent evolution of biotrophy in fungal and oomycete lineages.

Supporting Online Material

www.sciencemag.org/cgi/content/full/330/6010/1549/DC1

Materials and Methods

Figs. S1 to S10

Tables S1 to S3

References

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. We thank E. Holub for providing the Emoy2 isolate, D. Greenshields and N. Bruce for technical assistance, A. Heck and M. Slijper for analysis of secreted Hpa proteins, R. Hubley for creating repeat modeller libraries, and participants in the 2007 Annotation Jamboree and in the 2008 and 2009 Oomycete Bioinformatics Training Workshops for sequence annotations. This research was supported by grants EF-0412213, IOS-0744875, IOS-0924861, and MCB-0639226 from the U.S. NSF and 2004-35600-15055 and 2007-35319-18100 from the U.S. Department of Agriculture National Institute of Food and Agriculture to B.M.T. and J.M.M.; Biotechnology and Biological Sciences Research Council (BBSRC) BB/C509123/1, BB/E024815/1, and Engineering and Physical Sciences Research Council/BBSRC Systems Biology DTC student EP/F500025/1 to J.B.; Gatsby GAT2545 and BBSRC BB/F0161901, BB/E024882/1, and BBSRC CASE studentship T12144 to J.D.G.J. Other support is detailed in the supporting online material. Genome browsers are maintained at the Virginia Bioinformatics Institute (vmd.vbi.vt.edu) and the Sainsbury Laboratories (gbrowse2.tsl.ac.uk/cgi-bin/gb2/gbrowse/hpa_emoy2_publication).
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