Summary The identification of phytopathogen proteins that are differentially expressed during the course of the establishment of an infection is important to better understand the infection process. In vitro approaches, using plant extracts added to culture medium, have been used to identify such proteins, but the biological relevance of these findings for in planta infection are often uncertain until confirmed by in vivo studies. Here, we compared the proteins of Pectobacterium carotovorum ssp. carotovorum strain PccS1 differentially expressed in Luria–Bertani medium supplemented with extracts of the ornamental plant Zantedeschia elliotiana cultivar ‘Black Magic’ ( in vitro ) and in plant tissues ( in vivo ) by two‐dimensional electrophoresis coupled with mass spectrometry. A total of 53 differentially expressed proteins (>1.5‐fold) were identified (up‐regulated or down‐regulated in vitro , in vivo or both). Proteins that exhibited increased expression in vivo but not in vitro , or in both conditions, were identified, and deletions were made in a number of genes encoding these proteins, four of which ( clpP , mreB , flgK and eda ) led to a loss of virulence on Z. elliotiana , although clpP and mreB were later also shown to be reduced in growth in rich and minimal media. Although clpP , flgK and mreB have previously been reported as playing a role in virulence in plants, this is the first report of such a role for eda , which encodes 2‐keto‐3‐deoxy‐6‐phosphogluconate (KDPG) aldolase, a key enzyme in Entner–Doudoroff metabolism. The results highlight the value of undertaking in vivo as well as in vitro approaches for the identification of new bacterial virulence factors.