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).