The identification of lipopolysaccharide (LPS)-binding proteins in Arabidopsis thaliana plasma membranes.

22/05/2014

Name: Mr. Cornelius Sipho VilakaziSupervisor: Dr. Lizelle PiaterCo-supervisor: Prof. Ian Dubery

Hypothesis

Lipopolysaccharide (LPS) from the outer membrane of Gram-

negative bacteria binds to plasma membrane localized protein

receptor(s) in plants.

Background

∫ 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

Protein identification

EndoTrap® HD

Endotoxin removing columns

MagResyn ™ Strepavidin

(1)(2)

(3)

(Pierce Biotechnology, RESYN Biosciences; Hyglos GmbH)

Research aims The objective of the study was to capture, identify and

characterize LPS-interacting 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

Extraction and purification of bacterial LPS

PREPARATION OF

CULTURES

•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 EXTRACTION

•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 65oC.

LPS PURIFICATIO

N

•For further purification, the extract was digested with RNase, DNase, Proteinase K (Sigma, USA), dialyzed and lyophi

lized.

LPS CHARACTERIZATIO

N

•The 2-keto-3-deoxyoctonate (KDO), carbohydrate as well as the protein content of the LPS was determined.

•Purified LPS was further analyzed on 10% SDS-PAGE gels containing 4M urea.

Preparation of plant material

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 ISOLAT

ION

•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 ISOLAT

ION

•Approximately 20 g of leaf tissue was homogenized and centrifugation was employed to isolate the microsomal fraction from the homogenate.

PM ISOLAT

ION

•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

LPS Immobilization

•1 mg/ml LPS in 10mM Tris-Cl pH 7.5 was bound to Endotoxin removing affinity columns.

•600 pmol biotinylated LPS was bound to 10 µl of streptavidin microspheres.

PM addition

•1 mg/ml of plasma membrane was added to each affinity-capture method and incubated for 2 h at RT.

Elimination of non-specificity

•Non-specifically bound proteins were then washed off using 10mM Tris-Cl, 0.1-0.2 M NaCl and 100 µg/ml LPS.

Elution of targe

t proteins

•LPS-interacting proteins were eluted out using 1% SDS.

Analysis

•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

Figure 4: SDS-PAGE analysis of B. cepacia LPS samples. Underivatized LPS sample (A) . Biotinylated LPS sample (B).

kDa A B

150 100

70

50

40

30

20

15

Mature O-antigen, core oligosaccharide attached with lipid A

Core oligosaccharide attached with lipid A

Free lipid A

Plasma membrane isolation

(A) (B)

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).

150 100

70

50

40

30

20

15

10

kDa 1 2 3

1 - Homogenate (HG)2 - Microsomal fraction (MCF)3 - Plasma membrane (PM)

0 10 20 30 40 50 600

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

PM H+-ATPase activity (- Inhibitor) PM H+-ATPase activity (+ Inhibitor)

Time (min)

nmol

Pi/

min

/mg

Figure 6: H+-ATPase activity of the plasma membrane fractions and vanadate inhibition of the enzyme.

Plasma membrane H+-ATPase activity determination

Affinity chromatography

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).

(A) (B)

0 5 10 15 20 250

0.2

0.4

0.6

0.8

1

1.2

Membrane fraction

0.2 M NaCl fractions

1% SDS fractions

Fraction number

Abso

rban

ce (2

80nm

)14 15 16 17 18 19 20 21 22 23 24

0

0.2

0.4

0.6

0.8

1

1.2

Membrane fraction

0.1 M NaCl fractions

1% SDS fractions

Fraction number

Abso

rban

ce (2

80nm

)

SDS-PAGE analysis of eluted fractions

Figure 8: SDS-PAGE analysis of eluted fractions following the chromatographic experiment with polymixin B based endotoxin removing columns.

150 100

70

50

40

30

20

1510

kDa 1 2 3 4 5

1 & 2 - Membrane fractions 3 – NaCl fraction 4 & 5 - 1% SDS fractions (LPS-interacting proteins)

SDS-PAGE analysis of eluted fractions

Figure 9: SDS-PAGE analysis of eluted fractions from the magnetic polymeric microsphere affinity-capture procedure.

150 100

70

50

40

30

20

15

10

1 – PM fraction 2 – PM supernatant 3 – Membrane fraction 4 – NaCl fraction 5 – 100 µl/ml LPS6-7 – 1% SDS fractions (LPS-interacting proteins)

kDa 1 2 3 4 5 6 7

Table 2: List of LPS-interacting proteins after in situ digestion of bands from sample bound fractions.

Table 2 : (Continued)

ConclusionThe 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)

References 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 microbe-

associated 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.

Muchas gracias