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Fungi are organisms that form a distinct kingdom within the eukaryotes. Although most fungi are saprophytes that decay organic matter, the fungal kingdom also includes species that are able to cause considerable yield losses in crop production systems worldwide. In fact, fungi are the most significant type of plant pathogens. Chapter 1 is a general introduction about fungal plant pathogens. Recent advances in genome sequencing and assembly strategies have facilitated the availability of high-quality genome assemblies of many fungal species. Consequently, comparative genomics studies have provided novel insight in the evolution of pathogen genomes.
The fungal genus Verticillium contains ten species, some of which are notorious causal agents of vascular wilt diseases, while others are mainly known as saprophytes that have only been reported to cause disease occasionally as opportunistic pathogens. Whereas the genome of the most notorious plant pathogen in the genus, Verticillium dahliae, has been quite well characterized, genome characteristics of the other species have received little attention. With comparative genomics, we aimed to identify genomic features in pathogenic Verticillium species that confer the ability to cause vascular wilt disease. In Chapter 2, we sequenced and assembled one of the saprophytic Verticillium species, V. tricorpus, based on a hybrid approach that combines second and third generation sequencing. The resulting near-gapless genome assembly was subsequently used for a comparative genomics analysis with V. dahliae. Unexpectedly, both species encode similar effector repertoires and share a genomic structure with genes encoding secreted proteins clustered in genomic islands. In conclusion, we highlight the technical advances of a hybrid sequencing and assembly approach and reveal that the saprophyte V. tricorpus shares many hallmark features with the pathogen V. dahliae. Subsequently, in Chapter 3, we extended our comparative genomics study to the whole genus-level to try and identify genomic features that can be associated with pathogenicity. We sequenced the genomes of all haploid Verticillium spp. and demonstrated that all species display similar genomic features, including the occurrence of extensive genomic rearrangements and the presence of extensive effector catalogs. Overall, this chapter failed to identify particular genomic features that can be associated with pathogenicity in the genus Verticillium.
Previously, comparative genomics of V. dahliae revealed that lineage-specific genomic regions are hotspots for presence/absence polymorphisms, chromosomal rearrangements active transposable elements and in planta-induced effector genes. This finding suggests that V. dahliae evolved according to a so-called two-speed genome model as is similarly observed for other filamentous plant pathogens. In Chapter 4, we demonstrate the occurrence of differential sequence divergence between core and lineage-specific genomic regions of V. dahliae. Surprisingly, we observed that lineage-specific regions display markedly increased sequence conservation, suggesting that host adaptation is merely achieved through presence/absence polymorphisms. Increased sequence conservation of genomic regions that are important for pathogenicity is an unprecedented finding in filamentous pathogens and sheds new light on genomic dynamics in host-pathogen co-evolution. We provide evidence that disqualifies horizontal transfer between Verticillium species to explain the observed sequence conservation and conclude that sequence divergence occurs at a slower pace in lineage-specific regions of the V. dahliae genome. We hypothesize that differences in chromatin organisation may explain reduced SNP frequencies that occur in the plastic LS regions of V. dahliae. Furthermore, we also show that the clustering of absence/presence polymorphisms in rapid-evolving genome sections of the V. dahliae genome was similarly found for V. tricorpus, suggesting that this increased sequence conservation is not confined to pathogenic Verticillium species.
One of the previously characterized V. dahliae lineage-specific effector genes, Ave1, previously shown to be horizontally acquired from a plant donor. In Chapter 5, we systematically searched for evidence of inter-kingdom HGT events in the genome of V. dahliae using an alien index score that provides a score for genes that are phylogenetically more related to a defined outgroup when compared with an in-group. This analysis resulted in 42 likely HGT events, with 41 being of bacterial origin and one (Ave1) being of plant origin. Although most HGT candidates were not found to localize in lineage specific regions, they were significantly more likely to be found within 1 kb distance of repetitive elements than other genes. Overall, we show a high number of inter-kingdom gene acquisitions in V. dahliae.
In Chapter 6, we reveal the secondary metabolite (SM) gene cluster catalog of V. dahliae. We first performed in silico predictions using distinctive traits of gene clusters and the conserved signatures of core genes, resulting in 25 potential SM gene clusters. Subsequently, we used phylogenetic- and comparative genomics analyses, revealing that two putative siderophores, ferricrocin and TAFC, DHN-melanin and fujikurin compounds may belong to the SM repertoire of V. dahliae.
In Chapter 7, the major results in this thesis are discussed and placed in a broader perspective. I show that genomics studies enable us to detect features underlying various mechanisms involved in the adaptive evolution of fungal plant pathogens. Intriguingly, however, most of the features that were identified in diverse plant pathogenic fungi can also be found in non-pathogenic species. This finding suggests that the basis of pathogenicity is rather subtle and that pathogens and non-pathogens should not be seen as two distinct classes of microorganisms but rather form a continuum.
|Qualification||Doctor of Philosophy|
|Award date||3 May 2019|
|Place of Publication||Wageningen|
|Publication status||Published - 2019|