ENZYMES FOR THE DEGRADATION OF RHAMNOGALACTURONAN I AS VIRULENCE FACTORS OF PHYTOPATHOGENIC BACTERIUM Pectobacterium atrosepticum

Plant pathogenic pectobacteria (Pectobacterium genus) are well-known all over the world as the causal agents of the cultural plant diseases called soft rots. Rot symptoms are related to the extensive plant tissue maceration due to the production by microorganisms of the plant cell wall degrading enzymes. Most of the pectobacteria-secreted enzymes catalyze the cleavage of homogalacturonan. This polysaccharide, that is a linear homopolymer, consists of galacturonic acid residues and is the most abundant (by mass) pectic polysaccharide of plant cell walls. The knockout of genes of homogalacturonan-degrading enzymes is known to lead to reduced virulence of pectobacteria. In addition, the modification of another pectic compound — rhamnogalacturonan I also occurs in the course of infection process caused by pectobacteria. This compound is a ramified heteropolymer, the backbone of which consists of alternate rhamnose and galacturonic acid residues, and side chains are represented by galactose or arabinose residues. However, the role of pectobacterial enzymes for rhamnogalacturonan I degradation in the development of soft rots has not been previously ascertained. The present study is dedicated to the investigation of the necessity of P. atrosepticum SCRI1043 enzymes degrading rhamnogalacturonan I for a full development of soft rots in the plants infected by pectobacteria. By directed mutagenesis, we have obtained mutant forms of P. atrosepticum SCRI1043 deficient in genes encoding rhamnogalacturonyl hydrolase (genome locus eca3749) that cleaves the backbone of rhamnogalacturonan I, and galactanase (genome locus eca0852) that breaks side chains of this polymer. For the target gene knockout, mutant loci were constructed by overlap-extension PCR. Most of the original gene was replaced by kanamicin-resistance cassette. The obtained construction was ligated into a mobilized suicide vector and the resulting plasmid was transferred into donor E. coli СС118 strain cells. The recombinant plasmid with the mutant locus was introduced into P. atrosepticum SCRI1043 cells by three-parental mating. The P. atrosepticum SCRI1043 clones, in which the original locus was replaced by the mutant one, and the donor plasmid was eliminated, were selected on the selective media. The mutant strains P. atrosepticum SCRI1043∆3749 and P. atrosepticum SCRI1043∆0852 caused significantly less damage to the plant tissues of Brassica rapa spp. pekinensis Cha Cha cv. compared to parental wild-type strain. Herewith, the strain mutant in eca0852 locus encoding galactanase, the enzyme that cleaves side chains of rhamnogalacturonan I, was least virulent. The reduction in virulence, in this case, was not related to the suppression of homogalacturonan-degrading enzyme activity or less motility of bacteria. Thus, we have demonstrated that, first, rhamnogalacturonan I-degrading enzymes may be attributed to virulence factors of phytopathogenic pectobacteria, and second, the hydrolysis of the sides chains of rhamnogalacturonan I contributes more to the process of tissue maceration than the decay of the polymer backbone.