Differential contribution of Clavibacter michiganensis ssp michiganensis virulence factors to systemic and local infection in tomato

Clavibacter michiganensis ssp. michiganensis (Cmm) causes substantial economic losses in tomato production worldwide. The disease symptoms observed in plants infected systemically by Cmm are wilting and canker on the stem, whereas blister-like spots develop in locally infected leaves. A wide repertoire of serine proteases and cell wall-degrading enzymes has been implicated in the development of wilt and canker symptoms. However, virulence factors involved in the formation of blister-like spots, which play an important role in Cmm secondary spread in tomato nurseries, are largely unknown. Here, we demonstrate that Cmm virulence factors play different roles during blister formation relative to wilting. Inoculation with a green fluorescent protein (GFP)-labelled Cmm382 indicates that penetration occurs mainly through trichomes. When spray inoculated on tomato leaves, the wild-type Cmm382 and Cmm100 (lacking plasmids pCM1 and pCM2) strains form blister-like spots on leaves, whereas Cmm27 (lacking the chp/tomA pathogenicity island) is non-pathogenic, indicating that plasmid-borne genes, which have a crucial role in wilting, are not required for blister formation. Conversely, mutations in chromosomal genes encoding serine proteases (chpC and sbtA), cell wall-degrading enzymes (pgaA and endX/Y), a transcriptional regulator (vatr2), a putative perforin (perF) and a putative sortase (srtA) significantly affect disease incidence and the severity of blister formation. The transcript levels of these genes, as measured by quantitative reverse transcription-polymerase chain reaction, showed that, during blister formation, they are expressed early at 8-16 h after inoculation, whereas, during wilting, they are expressed after 24-72 h or expressed at low levels. Plant gene expression studies suggest that chpC is involved in the suppression of host defence.