Footprints of evolution: the dynamics of effector genes in the Phytophthora genome

Phytophthora is a genus comprised of over 65 destructive plant pathogenic species that cause severe damages in agriculture, forestry and natural habitats. Economically important pathogens are Phytophthora infestans (causing potato late blight) and Phytophthora sojae (causing soybean root and stem rot). A newly discovered species, Phytophthora ramorum is destroying oak trees along the west-coast of the USA by causing the Sudden Oak Death syndrome. Phytophthora belongs to the oomycetes, which together with phyopathogenic fungi constitute the major groups of plant pathogens. Oomycetes and fungi resemble each other morphologically but belong to different kingdoms, Stramenopila and Fungi, respectively. Convergent evolution has shaped a similar set of weaponry for attacking plants in oomycetes and fungi.Phytophthora secretes a variety of molecules into the plant apoplast presumably to promote infection. These molecules with a potential role in virulence or pathogenicity are named virulence factors or effectors. Despite their intrinsic virulence functions, effectors may cause failure of infection if plants recognize them and initiate defense responses. Effectors that trigger plant defense responses are called avirulence factors or elicitors. This thesis describes the drastic genome rearrangements at an avirulence locus in P. infestans, the characterization of effector gene reservoirs in the fully sequenced P. sojae and P. ramorum genomes, and evolutionary patterns of effector genes.Potato and P. infestans interact accordmg to the gene-for-gene model. As a first step towards unraveling the molecular interaction mechanisms, P. infestans avriulence (Avr) genes have to be isolated. Chapter 2 describes a transcriptional profiling slrategy to identify avirulence associated transcripts. cDNA-AFLP was used for comparing transcripts in P. infestans strains with different virulence phenotypes. A large number of avirulence-associated TDFs (Transcript Derived Fragments) were cloned and sequenced, and EST and genome databases were mined to generate more sequence data. To identify promising candidates, bioinformatic predictions such as the presence of signal peptides, number of cysteine residues and putative virulence functions were used as important selection criteria. Chapter 3 describes how a combination of transcriptional profiling, and genetic and physical mapping resulted in the characterisation of a complex avirulence locus. Four avirulence associated TDFs were shown to be derived from one gene, named p't3.4. Genetic mapping and physical mapping placed pi3.4 at the Avr3b-Avr10-Avr11 locus on linkage group VIIl. Comparative genomic hybridization (CGH) revealed that this A^rlocus belongs to one of the six loci in the genome that show copy number variation (CNV)- An amplified pi3.4 gene cluster is present in the avirulent haplotype but absent in the virulent haplotype. OnIy the 3' half of the pi3.4 gene is amplified, and this amplification was found to provide diverse modules for assembly of novel full length genes. Among the proteins secreted by Phytophthora. elicitins are produced most abundantly. Elicitins show elicitor activity by causing a hypersensitive response in tobacco. In Chapter 4 and Chapter 5, the diversity and genome organization of elicitin genes in four Phytophthora species are described. Elicitins were found to be encoded by a large complex gene family, and they belong to one of the most highly conserved groups of proteins in the Phytophthora genus. Many elicitin (ELI) and elicitin-like (ELL) genes are clustered in the genome. Phylogeny construction indicated that the complex elicitin gene family existed before the ancestral Phytophthora species gave rise to the current Phytophthora species. Molecular phylogeny classified elicitin family members into 17 different clades, namely 4 ELI clades and 13 ELL clades. Based on expression patterns and bioinformatic predictions, different clades are proposed to possess distinct functions.The two fully sequenced Phytophthora species, P. sojae and P. ramorum, differ in their genome sizes, sexual behavior and host specificity. Comparative genomics was carried out to gain insight into the evolution of effector genes. In Chapter 6 the genome organization of potential effector genes is described. Overall co-linearity was found between large genomic regions in P. sojae and P. ramorum. However, insertions, deletions and expansions revealed some hotspots for genome rearrangements, and such rearrangement hotspots often harbor genes associated with virulence. Contrasting evolutionary patterns were found for neighboring gene families, for example, families encoding extracellular enzymes showed more rearrangements than those encoding intracellular enzymes. Also genes encoding host specific elicitors showed more rearrangements than those encoding general elicitors.In Chapter 7 the whole reservoir of secreted proteins present in the proteome was revealed by bioinformatic-predictions. A large secretome comprised of overthousand proteins was found in both P. sojae and P. ramorum. The majority of secreted protein encoding genes form fami!ies and many of them are clustered in the genome. Comparison between secretomes of P. sojae and P. ramorum showed that different families are evolving at a different pace. The most rapidly evolving families include the surface anchored proteins, mating associated factors and 'RXLR-DEER' proteins. They may piay important roles in either host-pathogen interactions or in reproduction.In Chapter 8 the base compositions of P. sojae and P. ramorum were calculated and compared. This information is usefui for analyzing basic genome features. The coding regions of Phytophthora clearly show high GC3 (3rd position codon usage) and this preference causes codon bias in Phytophthora genes. Evolutionary forces such as selective pressure and mutation bias were found to drive codon bias in Phytophthora. The higher GC3 value of highly expressed genes in different Phytophthora species is indicative for selection pressure, whereas lineage specific GC increase of non-coding regions is reminiscent of whole genome mutation bias. The most widespread groups of mobile elements were retrieved from the genomes and they show a codon bias that is similar to the genes of the host Phytophthora.Finally, the evolutionary implications of the findings presented in this thesis are discussed in Chapter 9. For pathogenic organisms, genes encoding effectors are instrumental for interaction with their hosts. As a result, the evolutionary pace of effector genes is in general faster than that of average genes. The results presented in this thesis demonstrate that comparative genomics is a powerful toot to discover these genes and to point out promising candidates responsible for the process of pathogenesis. The ongoing P. infestans genome sequencing project will provide new resources for fundamental and applied research, and with the b!ueprint of P. infestans in hand, late bight research will gain momentum.