The identification of lipopolysaccharide (LPS)-binding
proteins in Arabidopsis thaliana plasma membranes.
22/05/2014
Name: Mr. Cornelius Sipho Vilakazi
Supervisor: Dr. Lizelle Piater
Co-supervisor: Prof. Ian Dubery
Lipopolysaccharide (LPS) from the outer membrane of Gramnegative bacteria binds to plasma membrane localized protein
receptor(s) in plants.
∫ Plant Immunity
∫ Pre-formed defenses
∫ Inducible defense responses
Figure 1: Plant innate immunity active defense mechanisms.
(Jones and Dangl, 2006; Muthamilarasan and Prasad, 2013; Zhang et al., 2013; Klemptner et al., 2014)
Lipopolysaccharide (LPS) is a M/PAMP therefore a potent
inducer of innate immunity.
Figure 2: General structure of LPS (taken from Erridge et al., 2002).
(Erridge et al., 2002; Silipo et al., 2010)

Why is the plant plasma membrane an interesting target for
LPS investigations?
Figure 3: Membrane-associated pattern recognition receptors (PRRs) can perceive microbial patterns
(P/MAMP) from different microbes (taken from Mazzotta and Kemmerling, 2011).
Affinity chromatography
(2)
(1)
Endotoxin
removing
columns
MagResyn ™
Strepavidin
(3)
EndoTrap®
HD
Protein identification
(Pierce Biotechnology, RESYN Biosciences; Hyglos GmbH)
Research aims
The objective of the study was to capture, identify and characterize LPSinteracting proteins from Arabidopsis thaliana plasma membranes (PM)
in order to elucidate the LPS receptor/receptor complex leading to the
activation of host plant defense responses.
Materials and methods
PREPARATION OF
CULTURES
LPS EXTRACTION
• Endophytic strain of Burkholderia cepacia (ASP B 2D) was cultivated in Nutrient
broth (Merck, RSA) liquid medium and incubated at 28oC on a continuous rotary
shaker for 10-14 days.
• LPS was extracted from freeze dried bacterial cell walls using an adaptation of the
phenol-water method where the LPS fractionates into the water phase at 65 oC.
• For further purification, the extract was digested with RNase, DNase, Proteinase K
LPS PURIFICATION
(Sigma, USA), dialyzed and lyophilized.
• The 2-keto-3-deoxyoctonate (KDO), carbohydrate as well as the protein content of
the LPS was determined.
LPS
CHARACTERIZATION
• Purified LPS was further analyzed on 10% SDS-PAGE gels containing 4M urea.
Arabidopsis thaliana (Columbia ecotype) were grown in soil
under a 16 h/ 8 h light-dark cycle in a green house.
Plasma membrane isolation
PM ISOLATION
• The plasma membrane was isolated according to a small scale procedure as
described by Giannini et al. (1988) as well as by Abas and Luschin (2010).
PM ISOLATION
• Approximately 20 g of leaf tissue was homogenized and centrifugation was
employed to isolate the microsomal fraction from the homogenate.
PM ISOLATION
• The micosomal fraction was then layered onto a 25/38% sucrose density
gradient and centrifuged at 13 000 xg for 1 h.
H+-ATPase assay
• The plasma membrane H+-ATPase activity was measured following a
method by Ligaba et al. (2004) and Giannini et al. (1988).
Affinity chromatography
• 1 mg/ml LPS in 10mM Tris-Cl pH 7.5 was bound to Endotoxin removing affinity columns.
LPS
Immobilization
PM addition
Elimination of
non-specificity
Elution of target
proteins
Analysis
• 600 pmol biotinylated LPS was bound to 10 µl of streptavidin microspheres.
• 1 mg/ml of plasma membrane was added to each affinity-capture method and incubated for
2 h at RT.
• Non-specifically bound proteins were then washed off using 10mM Tris-Cl, 0.1-0.2 M
NaCl and 100 µg/ml LPS.
• LPS-interacting proteins were eluted out using 1% SDS.
• SDS-PAGE, band excision and MALDI-TOF-MS analysis.
Results and discussion
Extraction and purification of the
bacterial lipopolysaccharides
Table 1: Summary of the characterization of LPS from Burkholderia
cepacia.
(Coventry and Dubery, 2001)
Extraction and purification of the
bacterial lipopolysaccharides
kDa
A
B
150
100
Mature O-antigen, core
oligosaccharide attached with
lipid A
70
50
40
30
Core oligosaccharide attached
with lipid A
20
15
Free lipid A
.
Figure 4: SDS-PAGE analysis of B. cepacia LPS samples Underivatized LPS sample (A) . Biotinylated LPS
sample (B).
Plasma membrane isolation
kDa
1
2
3
150
(A)
(B)
100
70
50
40
1 - Homogenate (HG)
2 - Microsomal fraction (MCF)
3 - Plasma membrane (PM)
30
20
15
10
Figure 5: Sucrose density gradient for the isolation of the PM fraction (A). Comparison by SDS-PAGE of the
HG, MCF and PM proteins isolated from A. thaliana leaves (B).
Plasma membrane H+-ATPase
activity determination
2.5
nmol Pi/min/mg
2
PM H+-ATPase activity
(- Inhibitor)
PM H+-ATPase activity
(+ Inhibitor)
1.5
1
0.5
0
0
10
20
30
40
50
60
Time (min)
Figure 6: H+-ATPase activity of the plasma membrane fractions and vanadate inhibition of the enzyme.
Affinity chromatography
(A)
(B)
1.2
1.2
1
Membrane fraction
0.1 M NaCl fractions
0.8
1% SDS fractions
0.6
0.4
Absorbance (280nm)
Absorbance (280nm)
1
Membrane fraction
0.2 M NaCl fractions
0.8
1% SDS fractions
0.6
0.4
0.2
0.2
0
0
0
5
10
15
Fraction number
20
25
0
5
10
15
Fraction number
20
25
Figure 7: Elution curves of non-specifically bound and LPS-interacting proteins. Fractions were collected
subsequent to affinity chromatography using endotoxin removing columns (A) and streptavidin magnetic
microspheres (B).
SDS-PAGE analysis of eluted
fractions
kDa
1
2
3
4
5
150
100
70
50
40
1 & 2 - Membrane fractions
3 – NaCl fraction
4 & 5 - 1% SDS fractions (LPS-interacting proteins)
30
20
15
10
Figure 8: SDS-PAGE analysis of eluted fractions following the chromatographic experiment with polymixin B
based endotoxin removing columns.
SDS-PAGE analysis of eluted
fractions
kDa 1
2
3
4
5
6
7
150
100
70
50
40
30
1 – PM fraction
2 – PM supernatant
3 – Membrane fraction
4 – NaCl fraction
5 – 100 µl/ml LPS
6-7 – 1% SDS fractions (LPS-interacting proteins)
20
15
10
Figure 9: SDS-PAGE analysis of eluted fractions from the magnetic polymeric microsphere affinity-capture
procedure.
Table 2: List of LPS-interacting proteins after in situ digestion of bands from sample bound fractions.
Table 2 : (Continued)
The novel affinity-capture strategy for the enrichment of LPS-interacting
proteins proved to be effective in specifically binding proteins involved in
plant defense responses .
The identification of MAMP receptors will lead to a better understanding of
pathogen perception in plants and may lead to the development of new
and innovative ways to control plant diseases.
(Giangrande et al., 2013)
Abas, L. and Luschnig, C. (2010). Maximum yields of microsomal-type membranes from small amounts of plant material without
requiring ultracentrifugation. Analytical Biochemistry, 401: 217-227.
Coventry, H.S. and Dubery, I.A. (2001). Lipopolysaccharides from Burkholderia cepacia contribute to an enhanced defensive
capacity and the induction of pathogenesis-related proteins in Nicotiana tabacum. Physiological and Molecular Plant
Pathology, 58: 149-158.
Erridge, C., Bennett-Guerrero, E. and Poxton, I.R. (2002). Structure and function of lipopolysaccharides. Microbes and Infection,
4: 837-851.
Giannini, L., Ruiz-Christin, J. and Briskin, D. (1988). A small scale procedure for the isolation of transport competent vesicles
from plant tissues. Analytical Biochemistry, 174: 561-567.
Giangrande, C., Colarusso, L., Lanzetta, R., Molinaro, A., Pucci, P. and Amoresano, A. (2013). Innate immunity probed by
lipopolysaccharides affinity strategy and proteomics. Analytical Bioanalytical , 174: 561-567.
Jones, J.D.G. and Dangl, J.L. (2006). The plant immune system. Nature, 444: 323-329.
Klemptner, R.L., Sherwood, J.S., Tugizimana, F., Dubery, I.A. And Piater., L.A. (2014). Ergosterol, an orphan fungal microbeassociated molecular pattern (MAMP). Molecular Plant Pathology, 1: 1-15.
Ligaba, A., Yamaguchi, M., Shen, H., Sasaki, T., Yamamoto, Y., and Matsumoto, H. (2004). Phosphorous deficiency enhances
plasma membrane H+-ATPase activity and citrate exudation in greater purple lupin (Lupinus pilosus). Functional Plant
Biology, 31: 1075-1083.
Mazzotta, S. and Kemmerling, B. (2011). Pattern recognition in plant innate immunity. Journal of Plant Pathology, 93: 7-17.
Muthamilarasan, M. and Prasad, M. (2013). Plant innate immunity: An updated insight into defense mechanism. Journal of
Biosciences, 38: 1-17.
Silipo, A., Erbs, G., Shinya, T., Dow, J.M., Parrilli, M., Lanzetta, R., Shibuya, N., Newman, M-A. and Molinaro, A. (2010).
Glycoconjugates as elicitors or suppressors of plant innate immunity. Glycobiology, 20: 406-419.
Zhang, J. and Zhou, J-M. (2010). Plant immunity triggered by microbial molecular signals. Molecular Plant, 3: 783-793.