A Gel Diffusion Assay for Quantification of Pectin Methylesterase Activity

Downie, Bruce; Dirk, Lynnette M.A.; Hadfield, Kristen A.; Wilkins, Thea A.; Bennett, A. B.; Bradford, Kent J.

doi:10.1006/abio.1998.2847

Cited by 100 publications

(81 citation statements)

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“…We further monitored total PME activity during pattern-triggered immunity initiated by treatment with the MAMPs flg22 or elf18 or the DAMP PEP1. PME activity was determined by extracting cytoplasmic and cell wall-bound proteins from homogenized plant samples and measuring PME activity in these samples using a gel diffusion assay (Downie et al, 1998;Supplemental Fig. S1).…”

Section: Resultsmentioning

confidence: 99%

“…Protein concentration was measured using Bradford reagent (BioRad), diluted to equal concentration and used to perform a gel diffusion assay (Downie et al, 1998). For the gel diffusion assay, petri dishes (10-cm diameter) were filled with 13 mL of a gel made of highly esterified pectin (0.1% pectin $ 85% esterified [Sigma P9561], 1% agarose, 12.5 mM citric acid, 50 mM Na 2 HPO 4 , pH 7.0; Lionetti et al, 2007) and four holes per plate were cut out using a number 2 cork borer.…”

Section: Pme Activity Assaymentioning

confidence: 99%

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Arabidopsis PECTIN METHYLESTERASEs Contribute to Immunity against Pseudomonas syringae

et al. 2013

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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.The plant cell wall determines cell shape, facilitates cell-cell interaction, and provides mechanical strength to plant cells. De Bary (1886) first observed that a plant pathogen, Sclerotina sclerotiorum, degraded host cell walls during infection. Later, it was concluded that plant cell walls act as preformed structural barriers against pathogen entry, because it was noticed that many plant pathogens produced various types of cell wall-degrading enzymes and that some of those were required for optimal infection of host plants (Albersheim et al., 1969).Arabidopsis mesophyll cells are surrounded by primary cell walls consisting of three major components: cellulose, hemicelluloses, and pectins. Pectins make up approximately 50% of Arabidopsis leaf cell walls (Zablackis et al., 1995;Harholt et al., 2010). They are complex GalA-containing polysaccharides composed of homogalacturonan (HG), rhamnogalacturonan I and II, and xylogalacturonan (Mohnen, 2008). HG is typically the most abundant polysaccharide, constituting approximately 65% of the pectin (Mohnen, 2008;Harholt et al., 2010). HG is a linear homopolymer of 1,4-linked GalA and is synthesized in the Golgi in a highly methylesterified form (Caffall and Mohnen, 2009). Pectin methylesterases (PMEs) demethylesterify HG in the apoplast (Mohnen, 2008;Harholt et al., 2010). Demethylesterification of pectin is considered t...

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Section: Resultsmentioning

confidence: 99%

Section: Pme Activity Assaymentioning

confidence: 99%

Arabidopsis PECTIN METHYLESTERASEs Contribute to Immunity against Pseudomonas syringae

et al. 2013

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“…Enzymatic activity of solutions of commercially available citrus PME and PME plus PP60 was tested using ruthenium red staining of pectin-infused plates of agarose medium (Downie et al, 1998). A serial dilution of PME solution was incubated in agarose plates with and without PP60.…”

Section: Resultsmentioning

confidence: 99%

“…Pectin/agarose medium plates were used to test for enzymatic activity in solutions of PME and PME combined with the hypothesized inhibitor as described by Downie et al (Downie et al, 1998). In preparation for each experiment, two mm diameter wells were created in the agarose medium using a cork-borer.…”

Section: Pectin Infused Agarose Plate Methods For Testing Enzymatic Acmentioning

confidence: 99%

“…For each agarose plate well, these solutions were mixed individually in 96-well plates for transfer into the agarose plates. All plates for a given experiment received identical incubation periods (after Downie et al, 1998). The plates were then rinsed with HPLC-grade water, filled with 0.05% ruthenium red solution and incubated for 45 minutes.…”

Section: Pectin Infused Agarose Plate Methods For Testing Enzymatic Acmentioning

confidence: 99%

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