2.5 Characterization of the fitness differences betweenDickeya solani strains carrying the VfmBSer and VfmBPro alleles
Structure of the VfmB proteins was modeled and represented using the Phyr2 and EzMol web portals (Kelley, Mezulis, Yates, Wass, & Sternberg, 2015; Reynolds, Islam, Sternberg, 2018). Given the known role in virulence of the vfm gene cluster (Nasser et al., 2013), we assessed virulence differences between D. solani strains exhibiting either a serine (allele VfmBSer) or a proline (allele VfmBPro) at the position 55 of the VfmB protein. Using genomic data, we chose four isolates carrying VfmBPro (IPO2222, MIE35, AM3a and 3337) and four isolates carrying VfmBSer (Ds0432.1, RNS10-27-2A, Sp1a and M21a), the four isolates in each group differing at other positions in genomes (their characteristics in Tables S3 andS4 ). Plant inoculation assays (on potato tubers and stems) were performed following the same protocols as described above using pure strains and mixtures as inocula. In the figures, aggressiveness data were presented as percentage of symptomatic plants (in potato stem infections assay) and DSI values (in potato tuber infection assays).
To measure their relative fitness, co-inoculation assays were also performed with VfmBSer and VfmBProexperimental populations, and their relative abundance was quantified by shot gun sequencing of DNA extracted from inoculum and from plant tissues. From 12 to 28 million reads were obtained for each sample, corresponding to an average coverage of D. solani genomes ranging from 180× to 420×. Sequencing (75 × 2 cycles) was performed using an Illumina NextSeq500 at the I2BC platform (CNRS, Gif-sur-Yvette, France) and Illumina MiSeq platform (University of Malaya, Kuala Lumpur, Malaysia). The trimmed reads were mapped on D. solani vfmBgene for quantifying the relative abundance of the two VfmBSer and VfmBPro alleles in each sample using the CLC genomic workbench version 10.1.3. The relative abundances of the alleles permitted calculation of CI values of the VfmBSer and VfmBPro populations (seeSM1 ). A CI value equal to one indicated an equal fitness between D. solani VfmBPro andVfmBSer populations, CI value greater than one indicated a fitness advantage to VfmBPro, and CI value less than one indicated a fitness advantage to VfmBSer. Finally, we used transcriptomics to identity the genes that were differentially expressed between IPO2222 (VfmBPro) and Ds0432.1 (VfmBSer) D. solani strains when they grew inside potato tubers (SM2) . Transcriptomes were compared as described by Raoul des Essarts et al. (2019).