Measuring Pectin Properties to Track Cell Wall Alterations During Plant-Pathogen Interactions.

Plant cell walls act both as a barrier to pathogen entry and as a source of signaling molecules that can modulate plant immunity. Cell walls consist mainly of three polymeric sugars: cellulose, pectin, and hemicellulose (Mohnen et al., Biomass Recalcitrance: deconstructing the plant cell wall for bioenergy, 2008). In Arabidopsis more than 50% of the primary cell wall is pectin (Zablackis et al., Plant Physiol 107:1129-1138, 1995). There are various types of pectin, but all pectins contain galacturonic acid subunits in their backbone (Harholt et al., Plant Physiol 153:384-395, 2010; Mohnen, Curr Opin Plant Biol 11:266-277, 2008). Many pathogens secrete pectin-degrading enzymes as part of their infection strategy (Espino et al., Proteomics 10:3020-3034, 2010; ten Have et al., Mol Plant-Microbe Interact 11:1009-1016, 1998). Pectin is synthesized in a highly esterified fashion and is de-esterified in the cell wall by pectin methylesterases (Harholt et al., Plant Physiol 153:384-395, 2010; Mohnen, Curr Opin Plant Biol 11:266-277, 2008). During plant-pathogen interactions, both the amount and the patterns of pectin methylesterification in the wall can be altered (Bethke et al., Plant Physiol 164:1093-1107, 2014; Lionetti et al., J Plant Physiol 169:1623-1630, 2012). Pectin methylesterifications influence mechanical properties of pectin, and pectins must be at least partially de-methylesterified to be substrates for pectin-degrading enzymes (Levesque-Tremblay et al., Planta 242:791-811, 2015). Additionally, alterations of pectin methylesterification or pectin content affect pathogen growth (Bethke et al., Plant Physiol 164:1093-1107, 2014; Lionetti et al., J Plant Physiol 169:1623-1630, 2012; Bethke et al., Plant Cell 28:537-556, 2016; Raiola et al., Mol Plant-Microbe Interact 24:432-440, 2011; Vogel et al., Plant Cell 14:2095-2106, 2002; Vogel et al., Plant J 40:968-978, 2004; Wietholter et al., Mol Plant-Microbe Interact 16:945-952, 2003). This chapter explains a simple protocol that can be used in any molecular biology laboratory to estimate total pectin content using a colorimetric assay and pectin composition using antibodies raised against specific pectin components.

Pectin Biosynthesis Is Critical for Cell Wall Integrity and Immunity in Arabidopsis thaliana.

Plant cell walls are important barriers against microbial pathogens. Cell walls of Arabidopsis thaliana leaves contain three major types of polysaccharides: cellulose, various hemicelluloses, and pectins. UDP-D-galacturonic acid, the key building block of pectins, is produced from the precursor UDP-D-glucuronic acid by the action of glucuronate 4-epimerases (GAEs). Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) repressed expression of GAE1 and GAE6 in Arabidopsis, and immunity to Pma ES4326 was compromised in gae6 and gae1 gae6 mutant plants. These plants had brittle leaves and cell walls of leaves had less galacturonic acid. Resistance to specific Botrytis cinerea isolates was also compromised in gae1 gae6 double mutant plants. Although oligogalacturonide (OG)-induced immune signaling was unaltered in gae1 gae6 mutant plants, immune signaling induced by a commercial pectinase, macerozyme, was reduced. Macerozyme treatment or infection with B. cinerea released less soluble uronic acid, likely reflecting fewer OGs, from gae1 gae6 cell walls than from wild-type Col-0. Although both OGs and macerozyme-induced immunity to B. cinerea in Col-0, only OGs also induced immunity in gae1 gae6. Pectin is thus an important contributor to plant immunity, and this is due at least in part to the induction of immune responses by soluble pectin, likely OGs, that are released during plant-pathogen interactions.

Arabidopsis PECTIN METHYLESTERASEs contribute to immunity against Pseudomonas syringae.

Pectins, major components of dicot cell walls, are synthesized in a heavily methylesterified form in the Golgi and are partially deesterified by pectin methylesterases (PMEs) upon export to the cell wall. PME activity is important for the virulence of the necrotrophic fungal pathogen Botrytis cinerea. Here, the roles of Arabidopsis PMEs in pattern-triggered immunity and immune responses to the necrotrophic fungus Alternaria brassicicola and the bacterial hemibiotroph Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) were studied. Plant PME activity increased during pattern-triggered immunity and after inoculation with either pathogen. The increase of PME activity in response to pathogen treatment was concomitant with a decrease in pectin methylesterification. The pathogen-induced PME activity did not require salicylic acid or ethylene signaling, but was dependent on jasmonic acid signaling. In the case of induction by A. brassicicola, the ethylene response factor, but not the MYC2 branch of jasmonic acid signaling, contributed to induction of PME activity, whereas in the case of induction by Pma ES4326, both branches contributed. There are 66 PME genes in Arabidopsis, suggesting extensive genetic redundancy. Nevertheless, selected pme single, double, triple and quadruple mutants allowed significantly more growth of Pma ES4326 than wild-type plants, indicating a role of PMEs in resistance to this pathogen. No decreases in total PME activity were detected in these pme mutants, suggesting that the determinant of immunity is not total PME activity; rather, it is some specific effect of PMEs such as changes in the pattern of pectin methylesterification.