| dc.description.abstracteng | Previous research into Verticillium spp. infection on the model plant Arabidopsis thaliana revealed the existence of three distinct disease classes. Each of these could be characterised by the induction of contrasting phenotypes (Thole, 2016). Infections with strains in the asymptomatic class produce no obvious macroscopic phenotypes whilst wilting strains result in stunting and in the wilting of leaves (Reusche, et al., 2014; Thole, 2016). Interestingly, chlorosis class strain infection results in host developmental reprogramming and is characterised primarily by chlorosis and the trans-differentiation of bundle sheath cells (BSCs) into functional tracheary elements (Reusche, et al., 2012). Comparative genomics and transcriptomics conducted on wilting and chlorosis class strains revealed LSCE2 (herein referred to as XYLEM FORMATION CANDIDATE EFFECTOR 1 (XFORCE1)) as the causal effector responsible for the development of chlorosis class symptoms. An XFORCE1 homolog, XFORCE1-Like, was also found present in Verticillium spp. from all disease classes. Despite a high similarity on both the genomic and amino acid level, XFORCE1-Like was found to be inactive. Additionally, the two proteins exhibited different migration patterns in SDS-PAGE gels despite near identical predicted weights (Weber, 2019).
An aim of this thesis was to investigate the reasons for these differences. To this end, comparative structure function analysis was carried out with heterologously expressed protein from Pichia pastoris. Investigations into post-translational modifications revealed that neither phosphorylation nor glycosylation were responsible for the differences in migration patterns. However, experiments with chimeric proteins could identify an N-terminal non-homologous region in the two proteins responsible for this phenomenon. Although it was found that this region did not result in altered activity, exchanges of a C-terminal region were documented to abolish XFORCE1 activity.
Next, the XFORCE1 protein was used in conjunction with proteomics in order to identify VARICOSE (VCS), a key scaffolding protein of the mRNA decapping complex, as an in planta interactor. Further experiments revealed that by interacting with VCS, XFORCE1 induced VCS phosphorylation in an ABA-independent SNF1-related protein kinase 2 (SnRK2) manner. Additionally, it could be demonstrated that XFORCE1 activated the SnRK2 kinases and that XFORCE1 activity and the induction of VCS phosphorylation was lost in the srk2abgh (Soma, et al., 2017) mutant. Furthermore, putative VCS phosphorylation sites induced by XFORCE1 could be identified with the use of phosphoproteomic approaches.
Lastly, RNAseq was utilised to analyse the transcriptomic changes in XFORCE1 treated A. thaliana plants. It could be shown that the application of XFORCE1 resulted in broad scale transcriptomic changes, with the regulation of many xylogenesis related genes being affected. Additionally, similarities in gene expression could be observed when comparing to VCS mutants. Taken together, results presented here indicate that the Verticillium effector XFORCE1 targets the VCS protein and likely manipulates mRNA turnover which results in host developmental reprogramming. | de |