Diverse plant pathogens secrete cellulases to degrade plant cell walls. Previously, the plasmid-borne cellulase gene celA was shown to be important for the virulence of the gram-positive bacterium Clavibacter michiganensis in tomato. However, details of the contribution of cellulases to the development of wilting in tomato have not been well-determined. To better understand the contribution of cellulases to the virulence of C. michiganensis in tomato, a mutant lacking cellulase activity was generated and complemented with truncated forms of certain cellulase genes, and virulence of those strain was examined. A celA mutant of the C. michiganensis type strain LMG7333 lost its cellulase activity and almost all its ability to cause wilting in tomato. The cellulase catalytic domain and cellulose-binding domain of CelA together were sufficient for both cellulase activity and the development of wilting in tomato. However, the expansin domain did not affect virulence or cellulase activity. The celA ortholog of Clavibacter sepedonicus restored the full virulence of the celA mutant of C. michiganensis. Another cellulase gene, celB, located in the chromosome, carries a single-base deletion in most C. michiganensis strains but does not carry a functional signal peptide in its N terminus. Nevertheless, an experimentally modified CelB protein with a CelA signal peptide was secreted and able to cause wilting in tomato. These results indicate that cellulases are major virulence factors of C. michiganensis that causes wilting in tomato. Furthermore, there are natural variations among cellulase genes directly affecting their function.
Clavibacter michiganensis is a Gram-stain-positive bacterium with eight subspecies. One of these subspecies is C. michiganensis subsp. michiganensis, which causes bacterial canker disease in tomato. Bacterial strains showing very similar canker disease symptoms to those of a strain originally classified as C. michiganensis have been isolated from pepper. In this paper, we reclassified strains isolated from pepper. On the basis of phylogenetic analysis with 16S rRNA gene sequences, the strains isolated from pepper were grouped in a separate clade from other subspecies of C. michiganensis. Biochemical, physiological and genetic characteristics of strain PF008T, which is the representative strain of the isolates from pepper, were examined in this study. Based on multi-locus sequence typing and other biochemical and physiological features including colony color, utilization of carbon sources and enzyme activities, strain PF008T was categorically differentiated from eight subspecies of C. michiganensis. Moreover, genome analysis showed that the DNA G+C content of strain PF008T is 73.2 %. These results indicate that PF008T is distinct from other known subspecies of C. michiganensis. Therefore, we propose a novel subspecies, C. michiganensis subsp. capsici, causing bacterial canker disease in pepper, with a type strain of PF008T (=KACC 18448T=LMG 29047T).
Clavibacter michiganensis ssp. capsici is a Gram-positive plant-pathogenic bacterium causing bacterial canker disease in pepper. Virulence genes and mechanisms of C. michiganensis ssp. capsici in pepper have not yet been studied. To identify virulence genes of C. michiganensis ssp. capsici, comparative genome analyses with C. michiganensis ssp. capsici and its related C. michiganensis subspecies, and functional analysis of its putative virulence genes during infection were performed. The C. michiganensis ssp. capsici type strain PF008 carries one chromosome (3.056 Mb) and two plasmids (39 kb pCM1 and 145 kb pCM2 ). The genome analyses showed that this bacterium lacks a chromosomal pathogenicity island and celA gene that are important for disease development by C. michiganensis ssp. michiganensis in tomato, but carries most putative virulence genes in both plasmids. Virulence of pCM1 -cured C. michiganensis ssp. capsici was greatly reduced compared with the wild-type strain in pepper. The complementation analysis with pCM1 -located putative virulence genes showed that at least five genes, chpE, chpG, ppaA1, ppaB1 and pelA1, encoding serine proteases or pectate lyase contribute to disease development in pepper. In conclusion, C. michiganensis ssp. capsici has a unique genome structure, and its multiple plasmid-borne genes play critical roles in virulence in pepper, either separately or together.
Clavibacter species are gram-positive plant pathogens that belong to phylum Actinobacteria (Eichenlaub and Gartemann, 2011). The genus Clavibacter includes six species: C. capsici causes necrosis, wilting, and bacterial canker in pepper; C. michiganensis causes wilting and bacterial canker in tomato; C. sepedonicus causes ring rot in potato; C. nebraskensis causes wilting and blight in maize; C. insidiosus causes wilting and stunting in alfalfa; and C. tessellarius causes bacterial mo
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