Impala-based transposon mutagenesis is influenced by chromatin modifications in the fungal plant pathogen Zymoseptoria tritici

Abstract

Transposition of TC1-mariner impala from F. oxysporum was tested in Zymoseptoria tritici. We used an existing excision assay in which impala is inserted in A. nidulans nitrate reductase gene promoter. This vector was introduced in a Z. tritici nitrate reductase mutant unable to grow on nitrate minimal medium (MM). Inoculating these transgenic strains onto MM allowed recovering revertants (10-30/plate). Almost all revertants corresponded to excisions of impala from niaD (95%). Inoculum culture conditions had a significant effect on impala excision rate, the optimum being obtained with yeast-like cells grown on YPD for 5 days at 18°C. Varying culture conditions during reversion assay (carbon starvation, light, darkness, heat or cold shocks, and copper stress) had no effect on impala excision rate. However, adding histone deacetylase inhibitor trichostatin at a sub-inhibitory concentration (0.1 microM) to MM during reversion assays, significantly increased impala excision rate. This unexpected result suggests that modifying histone acetylation level has an effect on impala excision either as a consequence of modifying chromatin status at vector integration site, or through a direct effect on impala machinery. In most revertants (90%), impala was inserted in Z. tritici genome. 60 impala re-insertion loci were characterized by Ligation-mediated PCR amplification and sequencing. Impala was inserted at random locations in core chromosomes (no hot spots), but not in accessory chromosomes (0/8 expected insertions). Since accessory chromosomes mostly carry repressed chromatin, this result suggests that impala preferentially inserts in regions with open chromatin. Impala also inserts at lower rate in native transposons (7%) compared to random (30%), suggesting that it avoids these regions. Overall, impala was preferentially inserted either at the 5’ end of transcriptionally active genes (5’UTR, promoters; 68%) or in genes (14%). These results show that impala is active in Z. tritici. Its integration patterns make it particularly suitable for insertional mutagenesis.