Fig. 7

Functional complementation assays on a five-gene deletion non-pathogenic Z. tritici mutant support the combined use of pangenome-derived mutation rate and expression level analysis as predictors for important core lifestyle genes. A Growth and infection characteristics of a Z. tritici random T-DNA insertion mutant “23-21”. The strain grows normally on rich nutrient agar but is defective in filamentous growth on poor nutrient agar and severely compromised in wheat leaf disease causing activity. Scale bars represent 1 cm. B Whole genome resequencing of strain 23-21 reveals a T-DNA mediated deletion of a 13 kb genomic region disrupting or deleting five predicted genes from the core pangenome. C Displays the average High and Moderate mutation events for each gene (labelled 1-5) from the pangenome relative to encoded protein length (aa). D Displays the mean relative expression levels of genes 1-5 across 12 strain-specific RNAseq datasets. E Functional complementation assays. Strain 23-21 had each of the five genes re-introduced separately by genetic complementation. Upper panels show effects on filamentous growth on poor nutrient agar. Middle panels show representative plant infection images at 21 dpi for one of multiple tested complemented isolates for each gene. Lower panel shows the quantification of disease levels using Lemna Grid image analysis system. Complementation by gene 4 encoding a predicted nucleoside diphosphate kinase (NDK) alone resulted in full restoration of pathogenicity in the 23-21 mutant. “a” and “b” indicate statistically significant differences (p < 0.001) between green leaf areas for the tested treatments. F RT-PCR analysis of each complemented strain, demonstrating that all constructs were correctly expressed by their native promoters in all tested isolates