3.8 VfmBSer strains were more aggressive than VfmBPro strains in soft-rot assays
The 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 ). Soft-rot assays using 10 inoculated potato tubers per strain revealed that the set of the VfmBSerstrains was more aggressive (Kruskal-Wallis test; k=9.5; DF=1; p= 2 x 10-3) than that of the VfmBPro strains (Figure 7b ).
To test the existence of differential expression of virulence genes in tuber tissues, we compared transcriptomes of D. solani IPO2222 (VfmBPro) and Ds0432.1 (VfmBSer) (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 S8 ). 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. solanivirulence 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).