Plant Science 155 2000 31 – 40
Characterization of a new antifungal non-specific lipid transfer protein nsLTP from sugar beet leaves
Anne K. Kristensen
1
, Janne Brunstedt, Klaus K. Nielsen
2
, Peter Roepstorff
3
, Jorn D. Mikkelsen
Danisco Biotechnology, Langebrogade
1
, P.O. Box
17
, DK-
1001
Copenhagen K, Denmark Received 20 August 1999; received in revised form 27 October 1999; accepted 16 December 1999
Abstract
A novel protein IWF5 comprising 92 amino acids has been purified from the intercellular washing fluid of sugar beet leaves using cation exchange chromatography and reversed phase high performance liquid chromatography. Based on amino acid
sequence homology, including the presence of eight cysteines at conserved positions, the protein can be classified as a member of the plant family of non-specific lipid transfer proteins nsLTPs. The protein is 47 identical to IWF1, an antifungal nsLTP
previously isolated from leaves of sugar beet. A potential site for N-linked glycosylation present in IWF5 Asn-Xxx-SerThr was found not to be glycosylated. The amino acid sequence data were used to generate a polymerase chain reaction PCR clone,
employed for the isolation of a corresponding cDNA clone. According to the cDNA clone, IWF5 is expressed as a preprotein with an N-terminal signal sequence of 26 amino acid residues. The protein shows a strong in vitro antifungal activity against
Cercospora beticola causal agent of leaf spot disease in sugar beet and inhibits fungal growth at concentrations below 10 mg ml
− 1
. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Antifungal protein; nsLTP; Mass spectrometry; Beta 6ulgaris; Cercospora beticola www.elsevier.comlocateplantsci
1. Introduction
In the last decade, many plant proteins with antimicrobial activity in vitro have been identified.
These include chitinases [1], b-1,3-glucanases [2], chitin-binding proteins [3], thionins [4], plant de-
fensins [5] and non-specific lipid transfer proteins nsLTPs [6]. nsLTPs are basic, 9 – 10 kDa proteins
which are able to bind and transfer a variety of very different lipids between membranes in vitro
[6]. The plant nsLTPs are synthesized as precur- sors with N-terminal extensions having the se-
quence characteristics of a signal peptide [7] and recent studies have demonstrated that some plant
nsLTPs are secreted [8] or bound in cell walls [9]. This extracellular localization of plant nsLTPs
makes the originally proposed role in membrane biogenesis and turnover unlikely and it has instead
been suggested that they are actively participating in the biosynthesis of the cutin layer and surface
wax by transporting acyl monomers [10]. In addi- tion, it has been shown that the expression of
nsLTPs can be induced by environmental stress factors, such as heat shock or NaCl treatment [11]
and several nsLTP genes in barley are upregulated
Abbre6iations
:
DTT, dithiotreitol;
EDTA, ethylenediaminete-
traacetic acid; FPLC, fast protein liquid chromatography; HPLC, high performance liquid chromatography; IC
50
, concentration re- quired for 50 growth inhibition; IWF, intercellular washing fluid;
MW, molecular weight; nsLTP, non specific lipid transfer protein; PCR, polymerase chain reaction; SDS PAGE, sodium dodecyl poly-
acrylamide gel electrophoresis; TFA, trifluoroacetic acid. Corresponding author. Tel.: + 45-32-662200; fax: + 45-32-
662167. E-mail addresses
:
akme.dk A.K. Kristensen, g7jdmdanisco. com J.D. Mikkelsen
1
Present address: ME Biotech AS, Kogle Alle 6, DK-2970 Horsholm, Denmark. Tel.: + 45-45-162525; fax: + 45-45-162500.
2
Present address: DLF-Trifolium, Hoejerupvej 31, P.O. Box 19, DK-4660 Store Heddinge, Denmark.
3
Present address: Department of Molecular Biology, Odense Uni- versity, Campusvej 55, DK-5230 Odense M, Denmark.
0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 1 9 0 - 4
in response to infection by various strains of fun- gal pathogens [12]. These results indicate a possi-
ble role in the protection against pathogens, a role which has been confirmed by the finding that
nsLTPs isolated from seeds of radish and onion [13,14], and leaves of barley, maize [15], Arabidop-
sis, spinach [16] and sugar beet [17] displayed antipathogenic activity in vitro.
A major focus of the laboratory is to investigate the interaction between sugar beet Beta 6ulgaris
L. and the pathogen Cercospora beticola Sacc., the cause of leaf spot disease in sugar beet. Previ-
ously, a number of chitinases, glucanases and plant defensin-like proteins have been isolated and
their biological role examined [18 – 24].
Recently, the isolation of two nearly identical proteins from sugar beet showing antifungal activ-
ity in vitro against the pathogen C. beticola has been described [17]. The proteins were isolated
from the intercellular washing fluid IWF of sugar beet leaves and showed sequence homology to
plant nsLTPs. During infection, C. beticola re- mains in the extracellular environment [25] and
secreted proteins with antifungal activity could be advantageously applied against this specific patho-
gen. The isolation and characterization of a new protein, designated IWF5, from sugar beet leaves
is reported here. The protein consists of 92 amino acids and shows homology to nsLTPs including
the presence of eight conserved cysteine residues and displays strong antifungal activity in vitro
against spore cultures of C. beticola.
2. Materials and methods
