Download Constitutive expression of Vitis vinifera thaumatin

CSIRO PUBLISHING
Functional Plant Biology, 2003, 30, 1105–1115
www.publish.csiro.au/journals/fpb
Constitutive expression of Vitis vinifera thaumatin-like protein
after in vitro selection and its role in anthracnose resistance
Subramanian JayasankarA,B, Zhijian LiA and Dennis J. GrayA,C
AMid-Florida
Research and Education Center, Institute of Food Agricultural Sciences University of Florida,
2725 Binion Road, Apopka, FL 32703, USA.
BCurrent address: Department of Plant Agriculture — Vineland Campus, University of Guelph,
4890 Victoria Avenue N, PO Box 7000, Vineland Station, Ontario, Canada L0R 2E0.
CCorresponding author; email: [email protected]
Abstract. Anthracnose-resistant grapevine (Vitis vinifera L. cv. Chardonnay) plants were regenerated from
embryogenic cultures that had been subjected to in vitro selection with culture filtrate of Elsinoe ampelina (de Bary)
Shear. Three secreted proteins differentially expressed by in vitro-selected embryogenic cultures and regenerated
plants were identified. An 8-kDa protein was identified as a lipid-transfer protein (LTP) by N-terminal amino acid
sequence comparison. Two other differentially expressed proteins, with estimated molecular weights of 21.6 and
22 kDa, immuno-reacted with antiserum raised against a thaumatin-like protein (TLP) protein from pinto bean
(Phaseolus vulgaris L.). N-terminal amino acid sequencing of the 21.6-kDa protein showed a high degree of
homology to V. vinifera thaumatin-like protein 2 (VVTL-2 = grapevine osmotin; Acc no. CAA71883), and that of
the 22-kDa protein was homologous to V. vinifera thaumatin-like protein 1 (VVTL-1; AAB61590). Interestingly,
both VVTL-1 and VVTL-2 are pathogenesis-related (PR) proteins, belonging to the PR-5 group. Protein produced
from the cloned grapevine VVTL-1 gene significantly inhibited E. ampelina spore germination and hyphal growth
in vitro. Plants regenerated from in vitro-selected cultures similarly inhibited fungal growth in vivo. Enhanced
expression of antifungal VVTL-1 in anthracnose resistant grapevine strongly suggests that it plays an important
role, either alone or in conjunction with other PR proteins, by suppressing pathogen growth.
Keywords: Chardonnay, disease resistance, Elsinoe ampelina, lipid-transfer protein.
Introduction
The capacity of plants to counter the attack of pathogenic
fungi depends, in part, on their ability to trigger defence
mechanisms. Such defence mechanisms include elicitation
of active oxygen species, biosynthesis of phytoalexins,
strengthening of barriers to invasion, hypersensitive
responses, and induction of pathogenesis-related (PR) genes
(Fritig et al. 1998; Dempsey et al. 1999).
Induction of PR genes has been associated with incompatibility and overexpression of one or more PR proteins can
delay disease development (Hammond-Kosack and Jones
1996). PR proteins are induced intra- and extracellularly by
pathogens, chemical elicitors or, in some instances, environmental stresses. PR proteins are separated into several
groups, based primarily upon sequence homology, but
electrophoretic mobility and functional characteristics also
are considered (van Loon et al. 1994). Proteins with close
sequence homologies to PR proteins have been added to
respective PR groups without proof of inducibility; these are
sometimes referred to as PR-like until functionality is
determined (van Loon et al. 1994). The PR-5 protein group
was established based upon sequence homology to thaumatin. Several PR-5 proteins have antifungal properties
(Roberts and Selitrennikoff 1990; Vigers et al. 1992; Cheong
et al. 1997). For example, osmotin, a tobacco (Nicotiana
tabacum L.) PR-5 protein has antifungal activity in vitro and
in vivo (Abad et al. 1996). Because some PR-5 proteins were
discovered to be basic, the group was subdivided into the
acidic thaumatin-like proteins (TLPs) and basic osmotin-like
proteins (Kombrink and Somssich 1997).
Thaumatin-like proteins are expressed in different
tissues, such as pistils, fruits and seeds (Neale et al. 1990),
associated with fruit ripening in banana, cherry and tomato
(Vu and Huynh 1994; Fils-Lycaon et al. 1996; Barre et al.
2000). In addition, TLPs are induced by viral, bacterial and
fungal infection. For example, in tobacco, infection with
Abbreviations used: ECP, extracellular protein; ICF, intercellular fluid; LTP, lipid transfer protein; PEM, proembryogenic mass; PR, pathogenesis
related; SEM, scanning electron microscopy; TLP, thaumatin-like protein; VVTL, Vitis vinifera thaumatin-like protein.
© CSIRO 2003
10.1071/FP03066
1445-4408/03/111105
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tobacco mosaic virus resulted in increased accumulation of
TLP within 6–8 d (Pierpoint 1986; Pierpoint et al. 1987).
The timing, localization and specificity of expression
strongly suggest that TLPs function in microbial defence
(Vigers et al. 1992; Velazhahan et al. 1999). Two TLPs
(VVTL-1 and VVTL-2) were described from grapevine
(Tattersall et al. 1997; Jacobs et al. 1999). VVTL-1 was
identified as a developmentally-regulated, fruit-specific
protein (Tattersall et al. 1997). VVTL-2, expressed in leaves
and fruits, previously was named grapevine osmotin
(Loulakakis 1997; Salzman et al. 1998), but was renamed
when it was determined to be acidic. VVTL-2 shows
considerable homology to VVTL-1, but is more responsive
to biotic and abiotic stresses (Jacobs et al. 1999). Antifungal
properties of VVTL-1 have not been determined, whereas
purified VVTL-2 inhibited fungal growth in the presence of
sugars (Salzman et al. 1998).
The phenomenon of phenotypic changes induced through
the process of cell culture is well documented and termed
‘somaclonal variation’ (Larkin and Scowcroft 1981). Exposure of cell cultures to various toxins from disease-causing
organisms led to disease-resistant plants (Daub 1986),
presumably by the same mechanism. Induction of PR
proteins, such as chitinases (PR-3) and glucanases (PR-2),
has been demonstrated after challenge with fungal or
bacterial culture filtrates in several cell culture systems
including grapevine (Gentile et al. 1993; Busam et al. 1997;
Jayasankar and Litz 1998; Jayasankar et al. 2000). However,
PR-5 proteins induced by pathogen challenge have not been
identified. Earlier we demonstrated that in vitro selection of
grapevine embryogenic cultures with Elsinoe ampelina
(de Bary) Shear (causal agent of grapevine anthracnose
disease) culture filtrate resulted in resistant phenotypes that
overexpressed chitinase (Jayasankar et al. 2000). Here we
show that VVTL-1 and VVTL-2 are differentially expressed
in these in vitro-selected grapevine embryogenic cultures as
well as plants regenerated from them. We also demonstrate
that purified, recombinant VVTL-1 protein inhibits spore
germination and hyphal growth of E. ampelina. This is the
first demonstration that grapevine TLPs possess antifungal
activity, strengthening the evidence that they play a role in
plant defence mechanisms.
Materials and methods
In vitro selection, culture establishment and plant regeneration
Suspension culture initiation, somatic embryogenesis and plant
regeneration of Vitis vinifera L. cv. Chardonnay were described
previously (Jayasankar et al. 1999). In vitro selection was carried out
as described by Jayasankar and Litz (1998). Log-phase
proembryogenic masses (PEM) cultures were passed through a
960-µm sieve to generate a synchronized culture. Synchronized PEMs
were selected in vitro against a 40% (v/v) culture filtrate-containing
medium as described previously (Jayasankar et al. 2000). The culture
filtrate was obtained by growing a virulent strain of
Elsinoe ampelina (de Bary) Shear for 3 weeks in Czapek–Dox broth.
S. Jayasankar et al.
Cell-free extract was collected and stored at 20°C until further use.
Selection was carried out for four or five cycles, each cycle lasting for
10 d. After the fourth and fifth cycles (40 or 50 d total exposure),
putative resistant cultures were proliferated in suspension culture
medium and established as ‘resistant culture 1’ (RC1) and ‘resistant
culture 2’ (RC2), respectively. Somatic embryogenesis was achieved
by culturing selected PEMs in auxin-free suspension culture medium
and resulting somatic embryos were germinated on solid medium.
Regenerated plants were acclimatized in potting mixture and
established in a greenhouse. A set of non-selected PEMs were cultured
in a similar way; the non-selected cultures and plants regenerated from
them served as experimental controls.
Extraction and quantification of extracellular proteins
Extracellular proteins (ECP) were precipitated by the method of
Gavish et al. (1992). Spent medium was collected in sterile flasks
during subculture and filtered through a double layer KimwipeTM to
eliminate any cellular debris. ECP was precipitated from the filtered
medium by adding three volumes of ice-cold 95% ethanol and kept
overnight at –20°C. Proteins were pelleted by centrifugation,
concentrated by vacuum and re-suspended in sterile distilled water.
Protein quantification was accomplished by the Bradford protein assay
with bovine serum albumin as a standard. Protein samples were stored
at –20°C until further use.
Extraction of intercellular fluids and protein concentration
Intercellular fluid (ICF) was extracted as described by Ignatius et al.
(1994) with minor modifications. Fully expanded, flaccid leaves were
collected from greenhouse-grown plants early in the morning. The leaves
were washed thoroughly with distilled water and blot-dried. Lamina
were cut into 2-cm-wide strips and vacuum infiltrated for 15 min in
a buffer containing 100 mM TRIS–HCl, (pH 6.8), 2.0 mM CaCl2,
10 mM EDTA, 50 mM β–mercaptoethanol and 0.5 M sucrose, at a ratio
of 10 mL per g of leaf tissue. After infiltration, leaf strips were gently
blotted and rolled to fit into 0.5 mL centrifuge tubes (without caps)
with a 1-mm-diameter hole at the bottom. Only two or three strips were
placed in each microfuge tube. These tubes were then placed so that
they fitted into 1.5-mL centrifuge tubes. The apparatus was centrifuged
at 3140 g for 15 min at room temperature. ICF was collected as dense
drops in the 1.5 mL centrifuge tubes. The ICF was diluted with four
volumes of distilled water and proteins were precipitated with three
volumes of ice-cold 95% ethanol overnight at 0°C. Protein pellets were
collected by centrifugation, concentrated by vacuum and re-suspended
in sterile distilled water. Protein quantification was accomplished as
described above.
Electrophoresis of proteins
SDS–PAGE with 1-mm-thick mini gels was carried out as described by
Laemmli (1970). Proteins were diluted with an equal volume of
SDS–PAGE buffer (Sigma, St Louis, MO) and diluted samples were
boiled for 5 min and then cooled. Samples were centrifuged at 5590 g
for 5 min at room temperature to remove insoluble particles. Total
protein (10 µg) was loaded onto each lane and electrophoresed for
approximately 80 min at 200 V. The gels were then silver-stained using
SilverSnapTM (Pierce, Rockford, IL).
Immuno-detection of proteins
After SDS–PAGE, proteins were transferred to ImmunoBlotTM PVDF
membranes (Bio-Rad, Almeda, CA) in a mini trans-blotter according to
manufacturer’s instructions. Transferred proteins were probed with a
PR-5 antiserum raised against pinto bean thaumatin-like protein
(kindly provided by Dr OP Sehgal, University of Missouri, Columbia,
MO) at a dilution of 1 :500 for 2 h at room temperature with gentle
Constitutive expression of Vitis vinifera thaumatin-like protein
shaking. Colour development was carried out with a Opti-4Cn kit
(Bio-Rad), according to manufacturer’s instructions.
N-terminal amino acid sequencing
For N-terminal amino acid sequencing, proteins were transferred to an
ImmunoBlotTM PVDF membrane with a buffer lacking glycine. After
transfer, proteins were stained with Coomassie blue (Sigma).
Appropriate bands were identified on the basis of molecular weight and
excised. N-terminal amino acid sequence determination was
accomplished by the automated Edman degradation method in the
Protein Chemistry Core Laboratory (University of Florida,
Gainesville, FL) with a protein sequencer (Model 494HT, Applied
Biosystems, Foster City, CA). Phenylthiohydantoin amino acid
derivatives were detected by a 120A analyser used in conjunction with
the sequencer. Amino acid sequences were compared with those in the
database by BLAST (Altschul et al. 1997).
Cloning and sequencing VVTL-1
Total genomic DNA was isolated from immature leaf tissues of both
selected and non-selected plants of ‘Chardonnay’, according to a modified procedure of Lodhi et al. (1994). Two oligonucleotide primers were
designed based on the sequence information from the database for
VVTL-1 (AF003007, Tattersall et al. 1997). The forward primer GTL-51
contained 5′-ATGGATCCAACATCAAAATGCGCTTCACCACCA-3′
and the reverse primer GTL32 contained 5′-TGTCTAGAGTTCCAACTCTCAAGGGCAAAACGTGACCTTG-3′. Polymerase chain
reactions (PCR) were performed using a reaction mixture containing
1 µg template DNA, 20 pmol of each primer, 200 µM dNTP, 2 mM MgCl2,
1× Mg-free reaction buffer and 2 units Taq polymerase in a total volume
of 80 µL. Cycling conditions were as follows: 95°C for 3 min followed
by 1 min each of 93°C, 55°C and 72°C for a total of 35 cycles. The
resulting PCR products were separated by agarose gel electrophoresis.
The amplified DNA fragments were isolated from gel blocks, purified
and then cloned into pUC18 cloning vector (Amersham–Pharmacia
Biotech, Piscataway, NJ) according to the manufacturer’s instructions.
Cloned DNA fragments from both selected and non-selected plants were
subsequently sequenced and analysed with the Vector NTI DNA analysis
program (InforMax Inc., Frederick, MD).
Construction of the VVTL-1 transfer vector and recombinant virus
To generate the viral transfer plasmid, PCR was used to add BamHI
(5′) and NotI (3′) restriction enzyme sites, a putative viral ribosomebinding site, and a C-terminal hexa-histidine sequence to the cloned
VVTL-1 gene sequence. The PCR product was directly subcloned into
the baculovirus transfer vector pVL1393 (PharMingen, San Diego,
CA) and the sequence verified by double stranded sequencing using
primers based on the polyhedrin promoter and 3′ flanking sequences.
Liposome-mediated co-transfection (Invitrogen, Carlsbad, CA) of
Spodoptera frugiperda J.E. Smith (Sf9) cells was performed with
2.5 µg of the recombinant transfer plasmid and 0.5 µg of linearized
viral DNA. After 5 d the resultant virus was collected and a portion
used to infect Sf9 cells to generate an amplified viral stock. The virus
was titered by plaque assay (O'Reilly et al. 1994) and used for
expression of recombinant VVTL-1.
Expression of VVTL-1 in insect cells
The Trichoplusia ni Hübner cell line BTI-TN-5B1-4 (Invitrogen) was
grown in spinner flasks at 27°C in Ex-Cell 400 (JRH Biosciences,
Lenexa, KS) to a density of 2 × 106 cells per mL. The cells were
collected by centrifugation and infected with a multiplicity of infection
(MOI) of five. The infected cells were tested at 24 h intervals for the
expression of VVTL-1 by binding to Ni-NTA (Qiagen, Valencia, CA)
and analysis by SDS–PAGE. The culture supernatants were collected
by centrifugation when expression was determined to be maximal
(72 h post-infection).
Functional Plant Biology
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Purification of recombinant VVTL-1
The culture supernatants were concentrated by ultrafiltration and the
buffer exchanged to 20 mM TRIS, 100 mM NaCl, 10 mM imidazole,
0.01% Nonidet P-40 (NP-40), pH 8.0. After binding to Ni-NTA for 2 h
at 4°C, the matrix was collected by centrifugation, transferred to a
column, and washed with 10 column volumes of the same buffer. The
bound protein was eluted with six column volumes of 20 mM TRIS,
100 mM NaCl, 250 mM imidazole, pH 8.0. The eluted protein was
concentrated by ultrafiltration.
In vitro spore germination assay
Spores were collected from a culture plate of E. ampelina and
suspended in 1 mL Czapek-Dox broth. After estimating the spore
density, a stock suspension of 500 000 spores per mL in sterile distilled
water was made. Spore germination was tested in 96-well microwell
plates by diluting 100 µL of spore suspension with 100 µL of sterile
distilled water per well. To test for the effect of VVTL-1 on spore
germination, recombinant protein was dissolved into the sterile
distilled water before spore dilution to yield final amounts of 20, 40
and 80 µg per 200 µL. Each treatment and a no-protein control was
replicated three times (i.e. three wells). The microwell plate was sealed
with ParafilmTM and incubated at room temperature. Spores that
exhibited a visible germ tube were counted as positive for germination.
Spore germination was observed at 24, 48 and 72 h after inoculation
and data on germination was collected and averaged from five distinct
zones in each well during each count. The relative amount of hyphal
growth was rated subjectively.
In vitro leaf bioassay and scanning electron microscopy (SEM)
Young, fully expanded leaves (typically the third or fourth leaf from the
shoot tip) of selected and non-selected greenhouse-grown plants were
excised carefully with a sterile scalpel and gently rinsed with distilled
water. After blot-drying the excess water, leaves were placed in a sterile
Petri dish (100 × 15 mm), with the cut end of the petiole resting on a
sterile KimwipeTM (Fisher Scientific, Savannah, GA) saturated with
distilled water. E. ampelina spores were collected from an actively
growing culture and suspended at a density of 10 6 spores per mL
(adjusted using a hemacytometer) in sterile water. Spore suspension
(100 µL) was gently placed on three spots per leaf. Five leaves each
from selected and non-selected plants were tested. The Petri dishes
with inoculated leaves were carefully sealed with ParafilmTM and
incubated at 25 ± 2°C with a 16 h photoperiod. For SEM, spore
suspensions were placed on leaves of selected and non-selected plants
for 48 h. Leaf discs from inoculated spots were processed and
examined with a scanning electron microscope as described previously
(Jayasankar et al. 2002).
Results
Grapevine embryogenic cultures were subjected to recurrent
in vitro selection with E. ampelina culture filtrate and
surviving embryogenic cultures were used to regenerate
plants. These selected embryogenic cultures and plants were
compared with a set of non-selected cultures and plants
regenerated at the same time. Embryogenic cultures that
survived in vitro selection and plants regenerated from them
are hereafter referred to as ‘resistant cultures’ and ‘resistant
plants’, respectively.
In order to understand the molecular basis of the
resistance, we profiled proteins secreted from resistant
cultures and compared them with non-selected control.
Polypeptides with molecular masses of 8, 21.6 and 22 kDa
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Functional Plant Biology
S. Jayasankar et al.
were predominant proteins differentially expressed by resistant cultures. In order to ascertain whether these elicited
proteins also expressed in planta, similar profiling of
proteins in the ICF of leaves from resistant plants was
performed. All three proteins were differentially expressed
in the ICF of resistant plants as well (Fig. 1).
Molecular identity of differentially expressed proteins using
N-terminal amino acid sequencing
A BLAST search of the 21 amino acid residues from the
8-kDa protein N-terminus revealed that this sequence
exhibits a high homology to non-specific lipid transfer
proteins (nsLTP). Among the nsLTPs that showed high
similarity was a 9-kDa protein identified from grapevine
somatic embryos previously as LTP P4 (Coutos-Thevenot
et al. 1993). The 8-kDa protein exhibited 75% sequence
similarity with a 9-kDa nsLTP from grapevine berries
(Salzman et al. 1998). The molecular mass and sequence
identifies the 8-kDa protein as a nsLTP (Fig. 2A).
The 21.6-kDa protein secreted by resistant cultures and
regenerated plants had identical N-terminal sequences. This
sequence had a high homology with grapevine osmotin from
‘Sultania’ (Loulakakis 1997) and ‘Concord’ (Salzman et al.
1998), which is very similar, but not identical, to V. vinifera
M
1
* **
2
3
4
5
6
7
22
21.6
8
Fig. 1. Extracellular protein profile of intercellular fluid (ICF) from
regenerated plants and in vitro-selected embryogenic cultures. Proteins
were separated by SDS–PAGE on a 12% mini-gel. (Lanes: M, marker;
1, ICF from non-selected plants; 2 and 3, ICF of regenerated plants
from in vitro selected line RC1; 4 and 5, ICF of regenerated plants
from in vitro-selected line RC2; 6, secreted proteins from embryogenic
cultures RC1; 7, secreted proteins from embryogenic cultures RC2).
Asterisks indicate a lightly-stained 22-kDa band in the control and two
distinct 21.6- and 22-kDa bands in the selected plants and embryogenic
cultures.
thaumatin-like protein (VVTL-1) (Fig. 2B). The amino acid
sequences of the 22-kDa protein from resistant cultures and
regenerated plants were identical and also exhibited a very
high similarity to that of VVTL-1 from grapevine berries
(Tattersall et al. 1997). In addition, the 22-kDa protein
exhibited a very high N-terminal sequence homology with
several other TLPs including two from tobacco, E22 and E2
(Fig. 2C). The sequence homology implies that the 22-kDa
protein is VVTL-1. To further verify the molecular identity
of these two proteins, we carried out an immunoblot assay of
secreted proteins from the spent medium of embryogenic
cultures and ICF of leaves, using pinto bean PR-5 antibody
as a probe. The antibody reacted with both 21.6- and 22-kDa
proteins (Figs 3A, B). Therefore, our results show that both
21.6- and 22-kDa peptides are PR-5 proteins that are
differentially and constitutively expressed by resistant cultures and plants as secreted proteins.
Effect of recombinant VVTL-1 on E. ampelina spore
germination
The entire VVTL-1 gene was cloned from genomic DNA of
resistant plants by PCR. The recombinant protein was
produced in a baculovirus expression system that allowed
recovery of 2.0 mg of purified VVTL-1 from 4 L of
transfected cultures. The purified protein was subsequently
tested for antifungal activity. Spore germination of
E. ampelina was significantly inhibited at three VVTL-1
concentrations and two exposure periods, except for the
lowest concentration (20 µg) at 48 h (Table 1). For example,
more spores had germinated at 24 h in untreated control
compared with spores treated with 20, 40 or 80 µg of
VVTL-1 (Table 1). Even after 48 h, the single dose of
VVTL-1 at 40 or 80 µg significantly inhibited spore
germination. Similarly, spores kept for 24 or 48 h in
untreated control conditions had longer germ tubes than
those incubated for the same periods with 40 or 80 µg of
VVTL-1 (Fig. 4). Among treatments, there was a gradual
increase in spore germination and growth over time; this
may have been due to protein degradation occurring over the
course of the incubation period. This is illustrated in Table 1
and Fig. 4, which shows that the percentage of germination
increased from 7.9 to 41.2% from 24 to 48 h in spores
treated with 80 µg VVTL-1, and germ tubes became longer;
however, germination and growth still was less than in the
untreated control. At 72 h, germ tube length in the 40 µg
treatment was similar to that of the control. The shorter germ
tubes in VVTL-1 treatments correlated with respective
germination data; this indicated that the protein had the
effect of delaying germination and/or retarding growth.
Because the germination percentage did not increase appreciably in the untreated control after the first count, it appears
that 24 h is sufficient for E. ampelina spores to germinate
under favourable conditions. Thus, the significant reduction
in spore germination and germ tube length at 24 h and later
Constitutive expression of Vitis vinifera thaumatin-like protein
Functional Plant Biology
1109
A
Present study
8 kDa protein
nsLTP - P4
nsLTP
Sorghum LTP - 2
Rice LTP - 1
T V T X G O V A S A V G P X I S Y L O
T V T C
T I T C
X V T C
X I T C
G O V A S A L S P C I D Y L O
G O V A S A L S P I I N Y L O
G O V S S A I G P C L S Y X X
G O V N S A V G P C L T Y A R
B
Present study
21.6 kDa protein
VVOsm
A T F N I
Q N K G G Y T V
A T F N I Q N H G
G G Y T V
A T F N I Q N H C P
Y T V
A T F D I
L N K X T
Y T V X
VVTL1
A T F D I
L N K C T
Y T V W A
Tobacco PR5 E22
A T F D I
V N K C T
Y T V W A
Tobacco PR5 E2
A T F D I
V N O C T
Y T V W A
GO
C
Present study
22 kDa protein
VVOsm
GO
A
A T F N I Q N H G G Y T V W A
A T F N I Q N H C P Y T V W A
Fig. 2. N-terminal amino acid sequence comparison of differentially expressed extracellular proteins. (A) 8-kDa protein from embryogenic
cultures. This protein had high sequence homology with non-specific lipid transfer proteins (nsLTP): Vitis nsLTP P4 (SwissProt [SP] accession
number P 80274), Vitis nsLTP (Salzman et al. 1998), Sorghum nsLTP (SP accession number Q 43194), rice nsLTP (SP accession number P 23096).
(B) 21.6-kDa protein from in vitro-selected embryogenic cultures and plants had high homology with grapevine osmotins: VVOsm (SP accession
number Y 10992), and grape osmotin (GO; Salzman et al. 1998). (C) 22-kDa ICF protein from in vitro-selected embryogenic cultures and plants.
This protein had a high homology with several thaumatin-like proteins (TLPs): VVTL-1 (GenBank accession number AF 003007), Tobacco
TLP-E22 (SP accession number P 13046), Tobacco TLP-E2 (SP accession number P 07052), VVOsm and GO. Identical amino acids are shaded
dark and similar amino acids are shaded light. X denotes unidentified amino acid residues.
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Functional Plant Biology
S. Jayasankar et al.
suggests that VVTL-1 plays a pivotal role in inhibiting
E. ampelina.
Plants regenerated from in vitro selected cultures show
enhanced resistance to E. ampelina
The antifungal activity of VVTL-1 prompted us to explore
whether plants regenerated from in vitro selected lines also
exhibited resistance to E. ampelina. Inoculation with
E. ampelina spores resulted in diagnostic necrotic spots with
greyish centers on leaves from non-selected control plants
within 5 d (Fig. 5A, left). However, plants regenerated from
in vitro-selected lines had fewer symptoms at 5 d postinoculation (Fig. 5A, right).
1
2
Discussion
3
A
22
21.6
1
2
To further analyse the nature of observed resistance we
performed scanning electron microscopy (SEM) on inoculated leaves from resistant and control plants. SEM analysis
showed widespread spore germination and hyphal growth on
leaves from non-selected control plants (Fig. 5B). In these
leaves, most spores formed germ tubes, which developed
into hyphae. At infection sites, the surface wax layer was
degraded, suggesting that hyphae had the ability to freely
penetrate the outer epidermis. In contrast, spore germination
was greatly reduced on leaves of resistant plants (Fig. 5C)
and very few spores had germ tubes. The majority of spores
remained ungerminated and tumid, indicating that hyphal
growth from germ tubes was arrested. In addition, the wax
layer of resistant plant leaves remained intact.
3
B
22
21.6
Fig. 3. (A) Immuno-detection with PR-5 antiserum of secreted
proteins from embryogenic cultures. (Lanes: 1, non-selected control;
2, secreted proteins from RC1; 3, secreted proteins from RC2).
(B) Immuno-detection with PR-5 antiserum of secreted proteins in ICF
of regenerated plants (Lanes: 1, non-selected control; 2, in vitroselected line RC1; 3, in vitro-selected line RC2).
We reported previously that grapevine plants regenerated
after in vitro selection with E. ampelina culture filtrate had a
high degree of anthracnose resistance and that a chitinase
(PR-1 or PR-2) was constitutively expressed (Jayasankar
et al. 2000). We now demonstrate that resistance is accompanied by expression of three additional proteins, including
an nsLTP and two PR-5 proteins. One PR-5 protein,
VVTL-1, is shown here to have antifungal properties.
Lipid-transfer proteins belong to a multigene family of
cysteine-rich antimicrobial peptides that are present in many
species (Garcia-Olmedo et al. 1995; Broekaert et al. 1997).
Recently, nsLTPs were grouped into the PR-14 family of
proteins (Van Loon and Van Strien 1999). In grapevine,
LTPs occurred in somatic embryos as secreted proteins, and
in fruits (Coutos-Thevenot et al. 1993; Salzman et al. 1998).
Based upon deduced amino acid sequence, it is likely that
the LTP expressed after in vitro selection is the same as
previously reported. In contrast to other plant nsLTPs,
grapevine LTP was not considered to be antifungal per se
(Salzman et al. 1998). However, it is possible that nsLTP
may have a synergistic effect with PR proteins in
anthracnose resistance, since all have membrane permeating
capacity.
Originally, VVTL-2 was identified as grapevine osmotin
based on its nucleotide sequence (Loulakakis 1997;
Table 1.
Effect of VVTL-1 on germination of E. ampelina
spores
Means were separated by Duncan’s multiple range test. Means within
each column followed by the same letter are not significantly different
at P=0.05
VVTL-1 Treatments
(Fg/200:l)
Control
20
40
80
Spore germination
after 24h (%)
Spore germination
after 48h (%)
90.7 ± 1.94a
62.0 ± 3.68b
41.5 ± 3.75c
7.9 ± 3.21d
91.4 ± 3.58a
83.9 ± 5.50a
64.8 ± 6.54b
41.2 ± 4.77c
Constitutive expression of Vitis vinifera thaumatin-like protein
Functional Plant Biology
Salzman et al. 1998). Osmotins are basic proteins having a
C-terminal extension that targets them to the vacuole
(Kombrink and Somssich 1997). However, Jacobs et al.
(1999) placed it into the TLP subgroup of PR-5 and renamed
it VVTL-2 because, although the predicted protein product
of the cloned gene corresponded to osmotin, it had an acidic
pI with no vacuole-targeting C-terminal extension. In the
present study, the 21.6-kDa protein expressed by resistant
cultures and regenerated plants exhibited a high N-terminal
sequence homology to VVTL-2 (Loulakakis 1997); it was
extracellular and thus, presumably acidic. Hence, the
21.6-kDa protein was identified as VVTL-2 as reported
earlier (Jacobs et al. 1999; Davies and Robinson 2000).
Induction of PR proteins following challenge with fungal
or bacterial culture filtrate was considered to be epigenetic
because PR protein expression returned to low levels after
the challenge was withdrawn (Punja and Zhang 1993).
However, mango embryogenic cultures produced the PR
proteins chitinase and glucanase several months after
1111
in vitro selection with fungal culture filtrate, indicating that
a constitutive change in expression had occurred
(Jayasankar and Litz 1998). Prior to Jayasankar et al. (2000)
and the present report, only transient expression of PR
proteins was identified in non-transgenic grapevine. For
example PR-3 chitinase was transiently expressed in cell
cultures and plants after treatment with various elicitors
(Busam et al. 1997). Selection of embryogenic cells with
toxic filtrates can cause somaclonal variation (Larkin and
Scowcroft, 1981) and the plants regenerated from such
resistant cells then express changed phenotype(s) as
described by Daub (1986); this provides an explanation for
the constitutive expression of PR-5 proteins and nsLTP for
over 3 years to date.
Most PR-5 proteins, including the original ‘thaumatin’,
were identified only in reproductive organs, such as floral
and fruit tissues and in seeds. In grapevine, expression of
VVTL-1 (Tattersall et al. 1997) and VVTL-2 (Loulakakis
1997; Salzman et al. 1998), was reported to be fruit
VVTL-1 (µg)
0
40
80
24
48
72
Incubation time (h)
Fig. 4. Effect of 0, 40 and 80 µg of VVTL-1 protein (per 200 µL total volume) on germination of E. ampelina spores and subsequent germ tube
growth over time (refer to Table 1 for numerical data on these treatments). At 24 h, germination is inhibited in both 40 and 80 µg treatments (note
the longer germ tubes and greater number of germinants in the 0 µg VVTL-1 control). After 48 h, germ tube growth and number of germinants are
less at 80 µg compared with 0 and 40 µg. After 72 h, germination increased at the 80 µg treatment (possibly due to protein degradation), but growth
was less than at 40 µg treatment, which, in turn, was less than at 0 µg.
1112
Functional Plant Biology
ripening-specific and developmentally controlled. VVTL-2
exhibited antifungal properties, especially in the presence of
high concentrations of glucose, indicating it to be a defence
protein, active in berries (Salzman et al. 1998). Based on its
appearance in ripening berries, VVTL-1 was considered to
function in disease resistance (Tattersall et al. 1997) but its
antifungal properties remained unknown. In studies using
other species, the actual induction of PR-5 proteins was
thought to be caused by various elicitors and signal
molecules (Xu et al. 1994) and enhanced expression
occurred predominantly in leaves (Cornelissen et al. 1986;
Vigers et al. 1991; Regalado and Ricardo 1996). In this
study the antifungal nature of VVTL-1 and its enhanced
expression in vegetative tissues of grapevine is
S. Jayasankar et al.
demonstrated, strongly suggesting the role of this PR-5
protein in anthracnose resistance.
Programmed expression and accumulation of PR-5 proteins in ICF during fruit ripening was suggested to be a preemptive defence response to fungal pathogenesis, especially
since fruit tissues accumulate high concentrations of sugars
and hence are highly vulnerable to pathogen attack
(Velazhahan et al. 1999). Correlation between accumulation
of PR-5 proteins during a specific stage of fruit ripening and
onset of resistance to powdery mildew was established
(Tattersall et al. 1997; Salzman et al. 1998). Furthermore,
Jacobs et al. (1999) showed that genes encoding two PR-5
proteins were induced upon infection by powdery mildew,
both in leaves and berries of grapevine. However, actual
A
B
C
Fig. 5. Inoculation of in vitro-selected and non-selected V. vinifera cv. Chardonnay leaves with E. ampelina. (A) Non-selected (left) and in vitroselected (right) leaves 5 d after inoculation with spore suspension. Note severe and mild anthracnose symptoms on non-selected leaves v. in vitroselected, respectively. (B) SEM of a non-selected leaf inoculation site. Note that many spores have collapsed owing to successful germination and
germ tube growth. More germ tubes are present; they have branched and appear to have attached to the leaf surface by development of appressoria.
(C) SEM of an in vitro-selected leaf inoculation site. Note that E. ampelina spores remain tumid and most have not germinated. Germ tubes, when
present, do not branch nor do they possess appressoria.
Constitutive expression of Vitis vinifera thaumatin-like protein
inhibition of powdery mildew by PR-5 proteins was not
substantiated. Accumulation of sugars in concert with
elevated levels of PR-5 proteins may provide resistance to
powdery mildew in berries of resistant plants produced from
in vitro selection, as suggested by Salzman et al. (1998).
However, our resistant plants cannot yet be evaluated for
differences in PR-5 protein content in berries because they
require at least two more years to flower and fruit.
Inability of E. ampelina spores to germinate and colonize
leaves of resistant plants indicates the presence of an
inhibitory substance. Restricted hyphal growth on the leaf
surface is similar to that described for other examples of
plant resistance (e.g. Hoch et al. 1987; Podila et al. 1993;
Scholthof 2001) and has been attributed to several factors,
including physical barriers (cuticular waxes and plant
surface topography) and chemical signals (secondary
metabolites and defence proteins). In the present study, the
response of E. ampelina spores on leaves of plants from
in vitro selection was strikingly similar to that exhibited
when they were incubated in vitro with VVTL-1 protein
(i.e. spores remained tumid, germination was reduced and
germ tube growth was restricted — compare Fig. 5C with
Fig. 4, VVTL-1 at 80 µg and 48 h), strongly suggesting that
VVTL-1 is a key factor in providing resistance to
E. ampelina, both in vitro and in vivo.
In conclusion, we have demonstrated that recurrent
in vitro selection of grapevine PEMs has resulted in the
expression of several proteins. That these proteins were
activated at the cellular level, but continued to express in
regenerated resistant plants for three years to date suggests
that the response is due to a stable genetic change.
Molecular mechanisms such as demethylation and differential activation of promoter(s) leading to altered gene
expression can occur as a result of selection pressure with
fungal culture filtrate (Jayasankar and Litz 1998), resulting
in somaclonal variants with enhanced protein levels as
demonstrated here. In contrast to earlier studies, we have
demonstrated that PR-5 proteins can be constitutively
expressed throughout grapevine tissues after in vitro selection and that at least one of them (VVTL-1) is antifungal.
Resistance to fungal diseases in transgenic grapevines
expressing PR proteins is an encouraging step
(Yamamoto et al. 2000; Gray et al. 2002). However, since
in vitro selection is not encumbered by intellectual property
issues or subject to societal concerns currently affecting
transgenic plant development, this non-transgenic approach
of constitutively expressing native defence proteins offers an
attractive alternative method of producing genetically
improved varieties.
Acknowledgments
We thank Dr OP Sehgal, Dept of Plant Pathology, University
of Missouri, Columbia, MO, for the generous gift of
antiserum; Dr S Muthukrishnan, Department of Bio-
Functional Plant Biology
1113
chemistry, Kansas State University, Manhattan, KS, and
Dr Senthil K Ramu, Department of Genetics, Harvard
Medical School, Boston, MA, for critical reading of the
manuscript; Dr Nancy Denslow and Scott McClung, PCCL,
University of Florida, Gainesville performed N-terminal
amino acid sequencing; Dr Peter Snow of Protein Expression Center, Beckman Institute, California Institute of
Technology assisted in the expression and purification of
VVTL-1. We acknowledge the excellent technical assistance
of Marilyn Van Aman, Karen Kelley, Ajitharani Ravindran,
Nancy Barnett and Sivagama Sundhari. This research was
supported by the Florida Agricultural Experiment Station
and a grant from The Florida Department of Agriculture and
Consumer Services’ Viticulture Trust Fund and approved for
publication as Journal Series No. R-08969.
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Manuscript received 10 April 2003, accepted 11 September 2003
http://www.publish.csiro.au/journals/fpb