3.7 Characteristics of VfmBPro and
VfmBSer strains
Alignments of VfmB proteins retrieved from genomic databases revealed
the occurrence of the VfmBPro allele inD. dadantii , D. chrysanthemi and D. dianthicolagenomes, while the VfmBSer allele was found in D.
zeae genomes (Figure S6 ). However, the low number of genomes
available let open the possibility of the existence of additional
alleles in each of these species.
Modeling of the D. solani VfmBPro and
VfmBSer protein structures predicted a conformational
difference in the beta-sheet structure of the virulence regulator VfmB
(Figure 7a ). This change is explained by the exceptional
conformational rigidity of proline that strongly affects secondary
structures such as alpha helices and beta sheets. The modeling data
prompted us to test whether
VfmBPro-VfmBSer variation could be
associated with a change in aggressiveness. To compare the
aggressiveness of D. solani VfmBSer and
VfmBPro isolates, we used genomic data to identify a set
of 8 isolates carrying either VfmBPro (IPO2222, MIE35,
AM3a and 3337) or VfmBSer (Ds0432.1, RNS10-27-2A, Sp1a
and M21a), in which the other variations were at different positions
(Tables S3 and S4 ). Using the assignation of five symptom
classes for 10 inoculated potato tubers per strain, virulence assays
revealed that VfmBSer strains were more aggressive
(Kruskal-Wallis test; k=9.5; DF=1; p= 2 x 10-3) than
VfmBPro strains (Figure 7b ).
We used transcriptomics in D. solani IPO2222
(VfmBPro) and Ds0432.1 (VfmBSer) to test
the existence of differential expression of virulence genes in tuber
tissues (Figure 7c ; Table S6 ). The expression of four
genes found differentially expressed based on transcriptomics
(pelE , impC , cytA and budA ) was further
investigated by RT-qPCR in the same samples used for transcriptomics:
they found to be also upregulated (Figure S7 ). Overall, 170
differentially expressed genes (adjusted p-value < 0.05 and
absolute log2 fold change > 2) were identified by
comparative transcriptomics. Most of them (150 genes) were upregulated
in Ds0432.1 carrying the VfmBSer allele (Table
S7 ). We studied more specifically 35 well-characterized D.
solani virulence genes (Raoul des Essarts et al., 2019). We observed an
enrichment of these virulence genes among the upregulated genes inD. solani Ds0432.1, while none of them was found among the
downregulated genes (Table S8 ). These upregulated virulence
genes in D. solani Ds0432.1 included the pectate lyase genespelB, pelC, pelD, pelE and pelL, the protease genesptrA , prtB and prtC, and the T6SS-related
toxin/antitoxin genes hcp (Table S8 ). These upregulated
genes encompassed virulence determinants that are already known to be
regulated by the Vfm quorum-sensing in D. dadantii (Nasser et
al., 2013).