Incidence of Genome Structure, DNA Asymmetry, and Cell Physiology on T-DNA Integration in Chromosomes of the Phytopathogenic Fungus Leptosphaeria maculans
Bourras, Ahmed Salim, A. S. | Meyer, Michel, M. | Grandaubert, Jonathan, J. | Lapalu, Nicolas | Fudal, Isabelle, Fudal-Grolier | Linglin, Juliette, J. | Ollivier, Bénédicte, B. | Blaise, Francoise, F. | Balesdent, Marie-Helene | Rouxel, Thierry, T. | BIOlogie et GEstion des Risques en agriculture (BIOGER) ; Institut National de la Recherche Agronomique (INRA)-AgroParisTech | French Ministry of Research
The ever-increasing generation of sequence data is accompanied by unsatisfactory functional annotation, and complex genomes, such as those of plants and filamentous fungi, show a large number of genes with no predicted or known function. For functional annotation of unknown or hypothetical genes, the production of collections of mutants using Agrobacterium tumefaciens-mediated transformation (ATMT) associated with genotyping and phenotyping has gained wide acceptance. ATMT is also widely used to identify pathogenicity determinants in pathogenic fungi. A systematic analysis of T-DNA borders was performed in an ATMT-mutagenized collection of the phytopathogenic fungus Leptosphaeria maculans to evaluate the features of T-DNA integration in its particular transposable element-rich compartmentalized genome. A total of 318 T-DNA tags were recovered and analyzed for biases in chromosome and genic compartments, existence of CG/AT skews at the insertion site, and occurrence of microhomologies between the T-DNA left border (LB) and the target sequence. Functional annotation of targeted genes was done using the Gene Ontology annotation. The T-DNA integration mainly targeted gene-rich, transcriptionally active regions, and it favored biological processes consistent with the physiological status of a germinating spore. T-DNA integration was strongly biased toward regulatory regions, and mainly promoters. Consistent with the T-DNA intranuclear-targeting model, the density of T-DNA insertion correlated with CG skew near the transcription initiation site. The existence of microhomologies between promoter sequences and the T-DNA LB flanking sequence was also consistent with T-DNA integration to host DNA mediated by homologous recombination based on the microhomology-mediated end-joining pathway.
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