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.