Analysis of the Binding of Expansin Exl1, from Pectobacterium carotovorum, to Plant Xylem and Comparison to EXLX1 from Bacillus subtilis

Abstract

The plant xylem is a preferred niche for some important bacterial phytopathogens, some of them encoding expansin proteins, which bind plant cell walls. Yet, the identity of the substrate for bacterial expansins within the plant cell wall and the nature of its interaction with it are poorly known. Here, we determined the localization of two bacterial expansins with differing isoelectric points (and with differing binding patterns to cell wall extracts) on plant tissue through in vitro fluorophore labeling and confocal imaging. Differential localization was observed, in which Exl1 from Pectobacterium carotovorum located into the intercellular spaces between xylem vessels and adjacent cells of the plant xylem, whereas EXLX1 from Bacillus subtilis bound cell walls of most cell types. In isolated vascular tissue, however, both PcExl1 and BsEXLX1 preferentially bound to tracheary elements over the xylem fibers, even though both are composed of secondary cell walls. Fluorescence correlation spectroscopy, employed to analyze the interaction of expansins with isolated xylem, indicates that binding is governed by more than one factor, which could include interaction with more than one type of polymer in the fibers, such as cellulose and hemicellulose or pectin. Binding to different polysaccharides could explain the observed reduction of cellulolytic and xylanolytic activities in the presence of expansin, possibly because of competition for the substrate. Our findings are relevant for the comprehensive understanding of the pathogenesis by P. carotovorum during xylem invasion, a process in which Exl1 might be involved.

Expansin-related proteins: biology, microbe–plant interactions and associated plant-defense responses

“…In addition, for basic proteins such as BsEXLX1, electrostatic interactions, driven by its positive residues with the negative components of pectin and hemicellulose, result in non-productive binding, while an acidic expansin seems to be repelled by the same acid fraction of the wall [4,31,48]. For instance, experiments with fluorophore-labelled proteins and confocal microscopy showed that whereas BsEXLX1 bound cell walls of most cell types in celery petiole cross sections due to the non-specific electrostatic interaction, PbExl1 signal was confined to the intercellular spaces between xylem vessels and the surrounding cells [50]. PbExl1 localization in xylem structures might be relevant as expansin genes are commonly found in the genomes of xylem-colonizing pathogenic bacteria [4,50].…”

“…For instance, experiments with fluorophore-labelled proteins and confocal microscopy showed that whereas BsEXLX1 bound cell walls of most cell types in celery petiole cross sections due to the non-specific electrostatic interaction, PbExl1 signal was confined to the intercellular spaces between xylem vessels and the surrounding cells [50]. PbExl1 localization in xylem structures might be relevant as expansin genes are commonly found in the genomes of xylem-colonizing pathogenic bacteria [4,50].…”

“…Summarizing, most null mutant characterization reports find a correlation between expansin activity and host colonization, but the molecular basis for this relationship needs further analysis. We recently showed that expansin Exl1 from P. brasiliense specifically locates on cell-cell junctions between xylem vessels and the surrounding cells, which corresponds to a pectin-rich region [50]. Additionally, after incubation of isolated vasculature of Swiss chard with Exl1, a solubilized polysaccharide (possibly pectin or hemicellulose) is detected.…”

“…We recently showed that expansin Exl1 from P. brasiliense specifically locates on cell-cell junctions between xylem vessels and the surrounding cells, which corresponds to a pectin-rich region [50]. Additionally, after incubation of isolated vasculature of Swiss chard with Exl1, a solubilized polysaccharide (possibly pectin or hemicellulose) is detected.…”

Expansin Engineering Database: A navigation and classification tool for expansins and homologues

“…Key sites of wall loosening may be limited contact points between cellulose microfibrils 5 . How these interactions result in cell wall loosening still requires further investigation 6 . Expansins were first discovered in plants and were described as proteins mediating pH‐dependent extension and stress relaxation of cell walls 7 .…”