A family of serine proteases of Clavibacter michiganensis subsp. michiganensis: chpC plays a role in colonization of the host plant tomato

Abstract

Genes for seven putative serine proteases (ChpA-ChpG) belonging to the trypsin subfamily and homologous to the virulence factor pat-1 were identified on the chromosome of Clavibacter michiganensis subsp. michiganensis (Cmm) NCPPB382. All proteases have signal peptides indicating export of these proteins. Their putative function is suggested by two motifs and an aspartate residue typical for serine proteases. Furthermore, six cysteine residues are located at conserved positions. The genes are clustered in a chromosomal region of about 50 kb with a significantly lower G + C content than common for Cmm. The genes chpA, chpB and chpD are pseudogenes as they contain frame shifts and/or in-frame stop codons. The genes chpC and chpG were inactivated by the insertion of an antibiotic resistance cassette. The chpG mutant was not impaired in virulence. However, in planta the titre of the chpC mutant was drastically reduced and only weak disease symptoms were observed. Complementation of the chpC mutant by the wild-type allele restored full virulence. ChpC is the first chromosomal gene of Cmm identified so far that affects the interaction of the pathogen with the host plant.

Characterization of phenotypic variants of Clavibacter michiganensis subsp. michiganensis isolated from Capsicum annuum

“…In addition, a large (~129 kb) pathogenicity island (PAI) with low G+C content was discovered in the chromosome of Cmm NCPPB382 . Numerous genes in the PAI might be involved in Cmm-tomato interactions such as the colonization of the tomato plant, hypersensitive reactions, and suppression of host defence reactions (Burger et al 2005;Gartemann et al 2008;Stork et al 2008).…”

Tomato Fruit and Seed Colonization by Clavibacter michiganensis subsp. michiganensis through External and Internal Routes

“…Depending on the assay, C. michiganensis subsp. michiganensis strains were incubated for 3 to 6 days at 27°C in Luria-Bertani (LB) (19), SB (20,21), or D2ANX (22) media. When required, LB and SB media were supplemented with the antibiotic chloramphenicol (10 g/liter; Fischer Scientific; Pittsburgh, PA).…”

“…michiganensis field strains. Competent cells were prepared as previously described by Stork et al (21). Electroporation was performed with 50 l of competent cells and 1 g of the purified eGFP vector using the Bio-Rad Gene Pulser Xcell electroporation system (Hercules, CA) with the following settings: 0.2-cm electroporation cuvettes, 7.5 to 12.5 kV/cm (Table 1), 25 F, and 600 ⍀ with a time constant between 12 and 16 ms. An increased transformation rate was observed when two pulses were applied with a 20-s interval between pulses (23).…”

Putative Serine Protease Effectors of Clavibacter michiganensis Induce a Hypersensitive Response in the Apoplast of Nicotiana Species

“…Cms contains a gene homologous to celA, which is also required for disease symptom development on potato (Laine et al 2000). Alignment of the amino acid sequences encoded by pat-1 homologs revealed the conserved trypsin family serine protease motifs that contain the predicted catalytic histidine and serine residues (Stork et al 2008). Additional genes encoding putative serine proteases homologous to pat-1 have been identified in the Cmm genome, named chromosomal homology of pat-1 (chp) and plasmidal homology of pat-1 (php) genes (Burger et al 2005.…”