PhD Thesis
Creators: name: Jonge, Ronnie De
Publisher: name: figshare
Year: 2013
Subjects: subject: 60102 Bioinformatics
subject: FOS: Computer and information sciences
subject: FOS: Computer and information sciences
subject: Pathology
subject: Biochemistry
subject: Molecular Biology
subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Evolutionary Biology

description: Research on effectors secreted by pathogens during host attack has dominated the field of molecular plant–microbe interactions over the recent years. Effectors are defined as molecules secreted by plant pathogens to modulate host physiology to enable colonization. In contrast to bacterial type III effectors that are delivered inside the host cytoplasm, fungal and oomycete effectors are delivered extracellularly, and we are gradually learning more about the functions of these effectors. While some function outside the host cell to disarm defence, others exploit host cellular uptake mechanisms to suppress defence or liberate nutrients intracellularly. In Chapter 1 we describe the function and evolution of effectors from filamentous plant pathogens, guided by the consecutive stages occurring during disease establishment. In Chapter 2, the occurrence and characteristics of a family of effectors that we named LysM effectors is described, and we show that this family is conserved throughout the fungal kingdom. LysM effectors are secreted proteins that contain no other recognizable protein domains than Lysin motifs (LysMs) that have been recognized as carbohydrate-binding protein domains. We propose that LysM effectors have a role in sequestration of chitin oligosaccharides, breakdown products of fungal cell walls that are released during invasion and act as triggers of host immunity, to dampen host defence. In Chapter 3 we investigated the function of the LysM effector Ecp6 from the plant pathogenic fungus Cladosporium fulvum. We show that Ecp6 binds to chitin and prevents the induction of chitin-triggered host defence responses, such as the alkalinisation of tomato and tobacco cell suspensions and the production of reactive oxygen species in tomato and tobacco leaf disks upon chitin treatment. Consistent with a role as suppressor of chitin-triggered immunity, Ecp6 was found to successfully compete with the rice receptor for binding of chito-oligosaccharides. In conclusion, we show that Ecp6 mediates virulence through scavenging of chitin fragments. As LysM effectors are widely conserved in the fungal kingdom, this may represent a common strategy of host immune evasion by fungal pathogens. In Chapter 4 we describe the identification of the race 1 elicitor of Ve1-mediated resistance in tomato. By high-throughput population genome sequencing of both race 1 and race 2 isolates, a single 50-Kb sequence stretch was identified that only occurs in race 1 strains. Subsequent transcriptome sequencing of Verticillium-infected Nicotiana benthamiana plants revealed only a single highly expressed ORF in this region, designated Ave1 (for Avirulence on Ve1 tomato). Functional analyses confirmed that Ave1 activates Ve1-mediated resistance and demonstrated that Ave1 markedly contributes to fungal virulence, not only on tomato but also on Arabidopsis. Interestingly, we found that Ave1 is homologous to a widespread family of plant natriuretic peptides that, beside plants, can also be found in the plant pathogenic fungi Colletotrichum higginsianum, Cercospora beticola and Fusarium oxysporum f. sp. lycopersici, as well as in the bacterial plant pathogen Xanthomonas axonopodis. The distribution of Ave1 homologs, coincident with the presence of Ave1 within a flexible genomic region, strongly suggests that Verticillium acquired Ave1 from plants through horizontal gene transfer. Remarkably, by transient expression we show that also the Ave1 homologs from F. oxysporum and C. beticola can activate Ve1-mediated resistance. In line with this observation, Ve1 was found to mediate resistance toward F. oxysporum in tomato, showing that this immune receptor is involved in resistance against multiple fungal pathogens. In Chapter 5, a comparative genomics approach was used to study sequence diversity within a population of V. dahliae isolates that is known to reproduce asexually. We found that sequence diversity is generally low among V. dahliae isolates. However, comparative analyses by pairwise alignment between the two highly similar isolates VdLs17 and JR2 (&gt;99.9% percentage identity) revealed regions of extensive synteny that are repeatedly interrupted by intra- and inter-chromosomal rearrangements. Syntenic breakpoints were associated with the presence of retrotransposons and frequently flanked by lineage-specific sequences. Syntenic breakpoints and lineage-specific sequences were found in each isolate, and pulsed-field gel electrophoresis further confirmed considerable chromosome length variation among all sequenced isolates. Apparently, chromosomal rearrangement establishes highly dynamic ‘plastic’ regions that lead to variation. Interestingly, the highly dynamic plastic genomic regions are enriched for in planta-induced genes, including effector genes that contribute to virulence such as the Ave1 effector in strain JR2 and a LysM effector in strain VdLs17. Although it is generally believed that asexual reproduction limits genetic variation, and consequently limits adaptive capability, we propose that chromosomal plasticity is a mechanism that allows asexual haploid genomes to adapt to changing environments. A perspective on next-generation genomics in relation to plant pathogen research and identification of effector genes is provided in Chapter 6. We illustrate the power of comparative genomics, and discuss recent studies describing comparative population genomics to identify effector genes. Studying natural variation by next-generation sequencing can be useful to analyse the evolutionary potential of a pathogen population and can be exploited to develop durable resistance in crop species.