Functional Characterization of Two Cellulase Genes in the Gram-Positive Pathogenic Bacterium Clavibacter michiganensis for Wilting in Tomato

Abstract

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.

Global cellulose biomass, horizontal gene transfers and domain fusions drive microbial expansin evolution

“…Like plant expansins, no microbial expansins have been documented to have enzymatic activity (Jahr et al, 2000;Laine et al, 2000;Olarte-Lozano et al, 2014;Georgelis et al, 2015;Cosgrove, 2017;Tancos et al, 2018). The evolutionary history, taxonomic distribution, mechanism(s) of action and ecological function(s) of microbial expansins remain enigmatic in non-Plantae genetic backgrounds, and there is currently no framework for predicting their functional roles (Jahr et al, 2000;Saloheimo et al, 2002;Brotman et al, 2008;Georgelis et al, 2015;Junior et al, 2015;Hwang et al, 2019).…”

A horizontally acquired expansin gene increases virulence of the emerging plant pathogenErwinia tracheiphila

“…The gh5 domain in Enterobacteriaceae is non-homologous to the GH5 domain in Xanthomonadaceae, and the exlx and gh5 domain structure in some Enterobacteriaceae is in reverse orientation compared to the gh5-exlx domain order in Xanthomonadaceae. A distinct gh5 domain that is truncated to 289 amino acids is found in Clavibacter michiganensis ( CelA ), and this is the only known microbial expansin that is fused to both a GH5 and CBM2 domain in a single coding sequence (27). This suggests there have been at least three independent origins of an expansin adjacent or fused to a gh5 family functional domain in bacterial plant pathogens.…”

Virulence and pCM1 plasmid carriage are related to BOX‐PCR fingerprint type in strains of Clavibacter michiganensis subsp. michiganensis that cause bacterial wilt and canker of tomato in Argentina

“…CelA is considered to have an essential role in virulence. Several studies (Meletzus et al , 1993; Thapa et al , 2017; Hwang et al , 2019) showed that nonpathogenic mutant strains lacking celA restored their pathogenicity when the gene was reincorporated. However, in New York State and Uruguay 6% and 2.5% of the strains, respectively, did not amplify celA even though they were pathogenic (Tancos et al , 2015; Croce et al , 2016).…”

“…The PI contains genes coding for serine proteases (e.g., chpC , chpG , and ppaA ), important for host plant colonization, and a tomatinase ( tomA ), which putatively deactivates the plant immune system (Eichenlaub and Gartemann, 2011). Plasmids pCM1 and pCM2 carry genes coding for the endo‐β‐1,4‐glucanase CelA and the serine protease Pat‐1, respectively, which are critical factors for bacterial pathogenicity (Dreier et al , 1997; Hwang et al , 2019). Strains lacking either of these two genes are less virulent or nonpathogenic (Meletzus et al , 1993; Kleitman et al , 2008; Milijašević‐Marčić et al , 2012; Tancos et al , 2015).…”