A. thaliana root cortex
4.1 The pathogenic V. longisporum isolate Vl43 possesses a genomic insertion
4.1.3 Host specificity of virulence and putative attenuating factors in Verticillia
interaction of V. longisporum with rapeseed plants was identified. The pathogenesis-reducing mechanism of this region depends either on the fungal species or the host.
The genome of the haploid V. dahliae isolate JR2 contains a Vl43LS20kb homologous region and Vl43LS20kb homologous subregions could be identified rearranged in the genome of the V. dahliae strain VdLs.17. In contrast, the Vl43LS20kb region is absent from the genome of V. alfalfae VaMs.102, which is more closely related to the V. longisporum parental lineage A1, and from the V. longisporum isolate Vl32 (A1/D3).
These findings corroborate the origin of the Vl43LS20kb region in the genome of the V. longisporum lineage A1/D1 parental species D1.
The Vl43LS20kb homologous region encoded in the genome of V. dahliae JR2 displays an indifferent role in pathogenicity towards tomato plants (Figure 10, Figure 30). The V. dahliae JR2 wild type does not induce disease symptoms in rapeseed plants. Two V. dahliae isolates were able to colonize the roots of rapeseed plants, but the isolates remained in the lower parts of the plants without induction of disease symptoms (Zhou et al., 2006). Whether the homologous Vl43LS20kb region in V. dahliae affects the colonization behavior and pathogenicity of this species against rapeseed or other host plants is currently unknown.
The genome of V. dahliae JR2 harbors an additional copy of the gene LSG1 encoded in the Vl43LS20kb homologous region, which was not deleted in V. dahliae JR2 ∆LS. Since all constructed deletion strains in V. longisporum Vl43, ∆LSI, ∆LSII, and ∆LS, resulted in induction of more severe disease symptoms in rapeseed plants and the phenotype was even stronger in ∆LSII than in ∆LSI (Figure 9), the presence of an additional copy of LSG1 located in the LSI subregion does not represent a sufficient explanation for the unaltered pathogenicity of V. dahliae JR2 in absence of the homologous Vl43LS20kb region.
Even if V. dahliae species have a broad host range, the single isolates of the species differ in their host ranges and in virulence towards different host plants (Resende et al., 1994; Zeise & von Tiedemann, 2002; Pegg & Brady, 2002; Gibriel et al., 2019). For isolates of the same V. longisporum lineage, differences in their pathogenicity on different host plants was observed as well (Novakazi et al., 2015). Therefore, one can suggest that strains of both species generally display a high degree of specialization and adaptation to a certain host. This specialization might not always be the result of the acquirement of novel genes, but also the differential regulation of existing mechanisms in response to certain environmental factors.
In other plant colonizing fungi, gene losses due to chromosomal rearrangements with significant roles in niche adaptation were described (Sharma et al., 2014; Hartmann et al., 2017; Plissonneau et al., 2018), which might be a possible mechanism in Verticillia as well. Further, asymptomatic or less virulent isolates might contain several pathogenicity-related genes in their genomes, which are inactive due to certain regulatory mechanisms.
Several studies investigating LS regions from subgroups of V. dahliae isolates revealed effector genes required for colonization of particular hosts as, for example, the effector NLP-2 (necrosis and ethylene-inducing peptide 1-like protein 2) which is involved in virulence of the strain JR2 on tomato and A. thaliana, but dispensable for tobacco infection (Santhanam et al, 2013). The V. dahliae effector Tom1 is essential for tomato
infection only and the effector Sun1 contributes to virulence specifically in sunflower colonization (Li, 2019, Dissertation, University Wageningen).
Figure 30: The Vl43LS20kb region attenuates pathogenicity of V. longisporum Vl43 in rapeseed and the homologous region is indifferent for virulence of V. dahliae JR2 in tomato plants. The genomes of V. longisporum lineages evolved by hybridization from two haploid parental species, which are V. dahliae strains (D2, D3) or close relatives of V. alfalfae (A1) and V. dahliae (D1). The hybrid genomes differ in their karyotypes due to chromosomal rearrangements and display different chromosome numbers (15 for Vl43; 16 for Vl32). The species V. dahliae JR2 and V. longisporum Vl43 (A1/D1) harbor the Vl43LS20kb region in their genomes, which is absent in V. longisporum Vl32 (A1/D3) and V. alfalfae VaMs.102.
V. dahliae JR2 exhibits an additional copy of the gene LSG1. Deletion of the Vl43LS20kb homologous region in V. dahliae JR2 region results in an unaltered virulent phenotype on tomato plants (indicated by the light red arrow for both, the wild type and the LS deletion strain), whereas deletion of the Vl43LS20kb region in V. longisporum Vl43 results in hypervirulence (indicated in dark red), suggesting a species- or host-specific role in attenuation of disease symptoms. V. longisporum isolates are unable to colonize tomato as a host (indicated by dashed lines), whereas V. dahliae remains in the roots without induction of disease symptoms and is not able to colonize the entire plant (indicated by dashed lines;
Zhou et al., 2006). V. alfalfae colonizes alfalfa and hops and is not known as a colonizer of tomato or rapeseed plants (indicated by dashed lines; Bhat & Subbarao, 1999).
Further, seven LS genes acquired by horizontal gene transfer from F. oxysporum, contributed to the virulence of V. dahliae Vd991 in cotton, but were dispensable for virulence on tomato and lettuce (Chen et al., 2018). Integration of these genes into V. dahliae strains JR2 or VdLs.17 resulted in increased virulence on cotton, whereas there was no effect on tomato or lettuce hosts (Chen et al., 2018). The underlying mechanisms regulating the observed host-specific induction of disease symptoms is still unknown for all of these examples and remains to be elucidated. One could assume that fungal virulence factors, which are inducing disease symptoms in a certain host, target host-specific mechanisms for regulation of defense responses. The absence of the corresponding target in a different host would make the fungal tool dispensable. Further, recognition of host-specific external signals sent by the plant might be essential for induction of a fungal mechanism that is regulating its virulence.
In summary, the Vl43LS20kb region makes V. longisporum Vl43 more tolerable for rapeseed plants. Presence of the homologous region in V. dahliae JR2 is indifferent for induction of disease symptoms in tomato plants. Different impacts of the genomic region on the host-fungus interaction might be caused either by differential gene regulation in the different organisms, by host dependent gene regulation, or by host-specific targets of virulence factors regulated by the Vl43LS20kb region.