Towards the identification of Verticillium effector molecules involved in host plant developmental reprogramming

Infections of Arabidopsis thaliana Col-0 plants with the Verticillium longisporum isolate VL43 and the Verticillium dahliae isolate VdJR2 results in clearly distinguishable disease symptoms, which are characterized by early senescence or plant wilting phenotypes, respectively. To elucidate whether VL43- and VdJR2-like infection symptoms are pathogen species-specific features, a Verticillium strain collection, composed of 21 near-diploid V. longisporum and 46 haploid V. dahliae isolates was systematically analysed in this study and categorized with regard to disease symptom induction on A. thaliana wildtype accession Col-0. These analyses allowed the classification of three different interaction classes: “early senescence”, “wilting” and “asymptomatic”. For in-depth characterization, Verticillium isolates which robustly induced disease symptoms in the “early senescence” and the “wilting” interaction class were selected. The strains were compared with respect to in planta proliferation and pathogen-induced developmental changes. These analyses showed that strains of the “wilting” interaction class cause enhanced lignification of leaf xylem cell walls. Contrarily, strains of the “early senescence” interaction class trigger developmental reprogramming in the leaf vasculature and induce early senescence. Interestingly, developmental reprogramming in the leaf vasculature represents a common host plant response as this symptom was also induced on Nicotiana benthamiana, a Solanaceae species. Thus, it was conceivable to postulate that interaction class-specific fungal effector molecule repertoires underlie the early senescence plant response. In order to identify potential “early senescence” interaction class-specific fungal effector molecules, comparative genome sequence analyses were conducted with haploid V. dahliae isolates of the three interaction classes. These data indicated that “early senescence” interaction class-specific sequences exist which are absent in genomes of isolates of the “wilting” and “asymptomatic” interaction class and thus, potentially harbor fungal effector genes involved in “early senescence” interaction class-specific disease symptom induction. Moreover, comparative in planta transcriptome studies were performed with V. dahliae isolates inducing wilting or early senescence symptoms and identified genes with a higher transcript abundance in the V. dahliae isolates of the “early senescence” interaction class compared with the “wilting” interaction class. From this group candidate effector genes were selected. Remarkably, the potential effector molecule repertoire included a ligninase, CEL, which possibly hydrolyzes lignin and influences the differences of xylem cell wall lignification observed between the “wilting” and “early senescence” interaction class. Two candidate effector genes, CEL and CE1, were selected for comparison of the promoter sequences between V. dahliae isolates of the two interaction classes. Noteworthy, promoter sequence investigations did not indicate interaction class-specific nucleotide differences which may explain the differential expression of CEL and CE1 in the “wilting” and the “early senescence” interaction classes. Hence, the results lead to the hypothesis that expression of these genes is epigenetically controlled.