Comparative genomic analyses of Clavibacter michiganensis subspecies and characterization of their interactions with nonhost plants

Abstract

Clavibacter is a group of gram-positive coryneform bacteria, containing several subspecies of C. michiganensis which cause diseases on economically important crops worldwide, and non-pathogenic plant-associated isolates. In contrast to the well-studied gram-negative plant pathogenic bacteria, little is known regarding the mechanisms of pathogenesis in gram-positive bacteria. Recent advances in methods and technologies have spawned several exciting discoveries and provided new directions for the research of Clavibacter. This thesis reports the use of a combination of Illumina sequencing and the PacBio Single-Molecule Real-Time sequencing technologies and obtained high quality de novo genome assemblies of three C. michiganensis subsp. insidiosus (Cmi). Comparisons between the genomes of the Cmi isolates revealed chromosomal rearrangements and heterogeneity of plasmids. By comparing the genome of Cmi to other published genomes of Clavibacter, a gene cluster potentially involved in biosynthesis of the blue pigment indigoidine was discovered in the genome of Cmi and a list of candidate genes that may be involved in pathogen virulence was generated. Cmm and Cms secrete serine proteases that are required for triggering hypersensitive responses (HR) on nonhost plants. Using a transient expression assay targeting the recombinant protein to either apoplast or cytosol of tobacco, the serine proteases ChpG from Cmm and Chp-7 from Cms were found to be the HR elicitors and were demonstrated to function in the apoplast of tobacco plants. The predicted catalytic serine residue in Chp-7 was found to be required for triggering an HR. C. michiganensis is non-adapted on Arabidopsis thaliana. This model organism was used to characterize the nonhost resistance against C. michiganensis. Screening of 34 A. thaliana accessions identified 11 accessions that showed a chlorotic response to Cmm inoculation. The ability to induce a chlorotic response in Kas-1 was conserved among all the five tested subspecies of C. michiganensis. The chlorosis elicitor appears to be heat stable and constitutively present in the culture. One single locus controlling the variation was mapped to a 53 kb region on chromosome 4. Variations was also observed in activation of hormone signaling marker genes, and rates of reducing the in planta titer of Cmm.