Currently P. ramorum is known only in Europe and North America. Populations on both continents are clonal and belong to three distinct lineages (Table 2). Population genetic analyses have shown them to be genetically distinct and asexually reproducing yet clearly conspecific (Ivors et al., 2004, 2006; Prospero et al., 2007). In 2006 there was an informal agreement within the P. ramorum research community on designated names for these clonal lineages: EU1, NA1 and NA2. The EU1 clonal lineage was first identified on Rhododendron and Viburnum in European nurseries (Werres et al., 2001). It is the only lineage found in Europe to date, but it is now regularly found in nurseries on the west coast of the USA (Grünwald et al., 2008a; Hansen et al., 2003; Ivors et al., 2006). Clonal lineage NA1 is responsible for the natural infestations in California and Oregon and many of the nursery infections in North America (Ivors et al., 2006; Prospero et al., 2007). The third clonal lineage, NA2, has a limited distribution. It has only been isolated in a few instances from nurseries in North America (Ivors et al., 2006). All NA1 and NA2 isolates that have been tested are of mating type A2 (Table 2). Interestingly, EU1 is predominately mating type A1 (Brasier and Kirk, 2004; Werres and Kaminsky, 2005), yet rare A2 EU1 isolates have been identified in Belgium (Werres and De Merlier, 2003). It is unclear how the mating type switch occurred in the EU1 lineage as the genetics of mating type are complex and poorly understood. In the case of P. infestans, the A1 mating type behaves as a heterozygote and the A2 as a homozygote (Judelson and Blanco, 2005). It is thus possible that the A2 genotypes observed in the EU1 lineage are the product of a mutation to the homozygous state. As already discussed, there is currently no evidence for sexual reproduction in the nurseries in which both mating types are present, yet the levels of heterozygosity observed in P. ramorum are consistent with an outcrossing species (Tyler et al., 2006). In addition, sequencing both alleles of several nuclear genes in each clonal lineage reveals that the alleles for each lineage do not cluster together and, in fact, the genealogical relationships among the lineages change with each gene (E. M. Goss and N. J. Grünwald, unpublished data). This suggests that the clonal lineages are descendants of sexually recombining populations.
Table 2. Currently recognized nomenclature and behavioural characteristics of Phytophthora ramorum clonal lineages (adapted from Ivors et al., 2006).
|Clonal lineage||Current distribution||Habitat||Mating type||Colony growth||Colony stability||Aggressiveness|
|EU1||EU, North America||Nurseries||A1 (A2)†||fast||Stable||Higher|
|NA1||North America||Forests, nurseries||A2||slow||Unstable||Lower|
The three clonal lineages also show phenotypic differences (Table 2). The most notable difference is relative homogeneity among EU1 isolates as compared with phenotypic variation and intrinsic instability in NA1. Instability in NA1 has been suggested by variation in colony morphology and vegetative growth rates among subcultures (Brasier et al., 2006) as well as the observation of abnormally shaped sporangia in single colony segments and between subcultures (Werres and Kaminsky, 2005). On average, EU1 isolates have shown faster growth rates in culture and larger chlamydospore size, although the growth rate of NA1 isolates can equal that of EU1 isolates (Brasier, 2003; Brasier et al., 2006; Werres and Kaminsky, 2005). NA2 exhibited a relatively rapid growth rate in culture, similar to EU1 isolates (Ivors et al., 2006). EU1 isolates have been found to produce larger lesions than NA1 isolates on cut stems of Quercus rubra (Brasier et al., 2006) and detached Rhododendron leaves (Fig. 5), but differences in other measures of fitness were not consistent across experiments. No differences in aggressiveness between the two major lineages were observed on a variety of other host species (Denman et al., 2005; Tooley et al., 2004). Variation in aggressiveness among isolates within both NA1 and EU1 lineages has been documented on coast live oak seedlings, detached bay laurel leaves and Q. rubra cut stems (Brasier et al., 2006; Huberli et al., 2006). None of these studies has observed genotype-specific (gene-for-gene-like) variation in virulence.
Figure 5. Difference in aggressiveness of P. ramorum clonal lineages EU1 and NA1. (A) Leaf lesion area 10 days post inoculation on two Rhododendron cultivars as a function of clonal lineage. The difference between EU1 and NA1 was significant in trials 2 and 3 (P < 0.005 and P < 0.05, respectively) (V. T. McDonald and N. J. Grünwald, unpublished data). (B) Mean lesion area in wound inoculated lower stems of Quercus rubra (data from Brasier et al., 2006). The differences between EU1 and NA1 were significant in trials 1 and 3 (P < 0.001 and P < 0.0012, respectively).
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Amplified fragment length polymorphism (AFLP) and fast-evolving microsatellites (simple sequence repeats) have revealed genetic variation within the lineages, which has allowed for studies of population structure and change. Given the asexual reproduction of these populations, this variation is hypothesized to be the result of mutation or mitotic recombination (Goodwin, 1997). The first AFLP and microsatellite studies found the European population to be more variable than the NA1 population (Ivors et al., 2004, 2006). Conversely, Prospero et al. (2007) found microsatellites that are variable within NA1 but not EU1. Yet these loci may be evolving so quickly in NA1 as to be considered unstable, providing an interesting parallel to the phenotypic data. These markers significantly differentiated the nursery and forest populations in Oregon and showed that the genotypic composition of nursery populations had changed from 2003 to 2004 (Prospero et al., 2007). Microsatellite variation has also proven valuable for rapid and accurate diagnosis of clonal lineages and is being used to monitor nursery finds in the USA. There are numerous simple sequence repeats identified in the P. ramorum genome sequence that have not been screened for variation and may yet provide useful markers (Garnica et al., 2006; Tyler et al., 2006) (http://web.science.oregonstate.edu/bpp/labs/grunwald/resources.htm).
A 7X draft whole-genome sequence for P. ramorum (an NA1 isolate from California) was published in 2006 along with that of the soybean pathogen P. sojae (Tyler et al., 2006), and the genome sequences of P. infestans and P. capsici are forthcoming (Broad Institute and Joint Genome Institute, respectively). The complete mitochondrial genome of P. ramorum has also been sequenced (Martin et al., 2007) and there are several other oomycete mitochondrial genomes to which it can be compared (Avila-Adame et al., 2006; Grayburn et al., 2004; Martin et al., 2007; Paquin et al., 1997). The mitochondrial genomes are largely conserved between Phytophthora species, although there are a number of more rapidly evolving genes, some of which may be specific to Phytophthora, as well as apparent hotspots for gene rearrangement. P. ramorum, in particular, contains a short inverted repeat, which among Phytophthora has previously been observed in only one other species, as inversions in other Stramenopile mitochondrial genomes typically represent much larger proportions of the genome (Martin et al., 2007).
Since the first release of the P. ramorum and P. sojae whole genome sequences, there has been rapid progress in the area of Phytophthora genomics, especially as pertains to genes involved in pathogenicity (Kamoun, 2006; Lamour et al., 2007; Morgan and Kamoun, 2007). The follow-up functional studies are mostly being conducted in the established model systems P. infestans and P. sojae, which are both fairly host-specific pathogens and may rely on different strategies for infection than the generalist P. ramorum. As such, it is interesting to note how the P. ramorum genome differs from these other Phytophthora genomes.
The P. ramorum genome size is 65 Mb, smaller than P. sojae (95 Mb) and P. infestans (240 Mb), but approximately the same size as P. capsici (Lamour et al., 2007). Proteins that are secreted from the pathogen are thought to have functions specific to the host–pathogen interaction, and thus the predicted secretomes are of particular interest to the plant pathology community. Comparison of the P. ramorum and P. sojae genomes showed these genes to be evolving faster than the rest of the proteome (Tyler et al., 2006). In addition, there has been family-specific expansion of pathogenicity-related genes. Figure 6 shows the families that are expanded in P. ramorum as compared with P. sojae and the autotrophic diatom Thalassiosira pseudonana. P. ramorum has more protease genes than P. sojae, but fewer of the other groups of hydrolytic enzymes, proteins that may be important to the necrotrophic stage of infection. Among effectors, the necrosis and ethylene-inducing protein (NPP) family shows an expansion in P. ramorum, while P. sojae contains relatively more of the necrosis-inducing PcF and Crn families, which are also large and diverse families in P. infestans (Kamoun, 2006).
Figure 6. Relative number of genes potentially involved in infection in P. ramorum compared with P. sojae (grey bars) and the diatom Thalassiosira pseudonana (white bars). For three gene families, the number of genes in T. pseudonana was not determined (nd). Data are from Tyler et al. (2006: table 1).
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The avirulence genes cloned from oomycetes to date have all contained the conserved amino acid motif RXLR (Arg, any amino acid, Leu, Arg), which has recently been shown to be required for translocation of the protein into the host cell (Dou et al., 2008b; Whisson et al., 2007). Following the RXLR is an additional motif, dEER, which takes on more variable functional forms (Allen et al., 2004; Jiang et al., 2008; Shan et al., 2004) but is also required for translocation (Dou et al., 2008b; Whisson et al., 2007). Fascinatingly, the RXLR motif shares similarity with the motif in the malaria parasite Plasmodium falciparum (RXLX followed by E, D or Q) that serves the equivalent function of exporting pathogen proteins to the host erythrocyte (Bhattacharjee et al., 2006).
When homologues of these genes were examined in the P. ramorum and P. sojae draft genome sequences, a large and diverse family of potential effectors was found (Jiang et al., 2008; Tyler et al., 2006). The number of ‘true’ RXLR-class effectors (i.e. avirulence gene homologues) in the Phytophthora genomes is under debate, as the vast majority of them have not been functionally validated; certainly none has been validated in P. ramorum. As a result, different numbers exist based on the bioinformatic method used to mine them (Table 3). These genes are so variable between Phytophthora genomes that orthology is difficult to determine. Of the nearly 400 genes identified in the P. ramorum and P. sojae genomes by Jiang et al. (2008), only 34 syntenic orthologues could be definitively assigned as such. Most of these genes have only distant homologues based on weak similarity between inferred amino acid sequences.
RXLR-type effectors have a modular structure: the RXLR and dEER motifs are localized to the N-terminus, while the C-terminal domain is thought to be responsible for activity within the host plant cell (Dou et al., 2008a; Jiang et al., 2008; Kamoun, 2007; Whisson et al., 2007). The majority of likely RXLR-class effectors in both P. ramorum and P. sojae contain a conserved C-terminal motif, called the W motif, which has recently been shown in P. sojae to suppress programmed cell death and thus increase virulence in the initial biotrophic phase of infection (Dou et al., 2008a; Jiang et al., 2008). The W motif is often followed by two other conserved motifs (Y and L) forming a W–Y–L module that may be repeated up to eight times (Jiang et al., 2008). As the function of these and other motifs are characterized in model organisms, they may elucidate the function of effectors with these motifs in P. ramorum.
Within genomes, recently duplicated genes (paralogues) can be identified based on sequence similarity. Analysis of paralogous RXLR-class effectors in P. ramorum showed many to be under detectable positive selection, often localized to the C-terminal region and within the W and Y motifs (Jiang et al., 2008; Win et al., 2007). Of 59 groups of closely related paralogues examined by Win et al. (2007), 41 showed evidence of positive selection. Of those 41 groups, 21 had two to four members clustered in the genome (within 100 kb). The P. sojae genome contained a comparable number of putative RXLR-type effectors, but only 28 groups of closely related paralogues were identified, of which 18 had experienced positive selection and only five of these were clustered. These data indicate that P. ramorum may have experienced a more recent expansion of RXLR-class effectors than P. sojae. We examined a group of four paralogues in P. ramorum more closely and found only a single homologue in P. lateralis and P. hibernalis (Fig. 7; E. M. Goss and N. J. Grünwald, unpublished), suggesting that this group is a result of gene duplication since the divergence of P. ramorum and P. lateralis. Win et al. (2007) found that for those P. ramorum RXLR-type effectors that have paralogues, an estimated 67–75% had between 70 and 99% amino acid identity to their closest paralogue. The most divergent of the four genes in Fig. 7 have 72% amino acid identity (21% divergence at synonymous sites in the whole genome sequence isolate Pr102) and thus fall in the lower end of the above range. Therefore, many of the expansions may indeed have occurred since divergence from P. lateralis, although one would expect rates of evolution to vary widely among effector genes.
Figure 7. Maximum likelihood genealogy of a subfamily of RXLR-class effector genes (avr gene homologues, Avh) in Phytophthora clade 8c. PrAvh gene sequences are from P. ramorum isolate Pr102. For heterozygous genes, the two alleles are distinguished by the suffixes ‘a’ and ‘b’. Homologous sequences are in bold typeface. The P. lateralis and P. hibernalis homologues to PrAvh60, PrAvh68, PrAvh108 and PrAvh205 (genes in box) appear to be syntenic to PrAvh205 based on flanking sequence similarity. Bootstrap support is shown for all branches (E. M. Goss and N. J. Grünwald, unpublished data).
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