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Plant Physiol. (1999) 119: 1447-1456
Characterization of Two Novel Type I Ribosome-Inactivating
Proteins from the Storage Roots of the Andean Crop
Mirabilis
expansa1
Jorge M. Vivanco,
Brett J. Savary, and
Hector E. Flores*
Department of Plant Pathology and Biotechnology Institute, The
Pennsylvania State University, University Park, Pennsylvania 16802 (J.M.V., H.E.F.); and United States Department of
Agriculture-Agricultural Research Service, Eastern Region Research
Center, 600 East Mermaid Lane, Wyndmoor, Pennsylvania 19038 (B.J.S.)
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ABSTRACT |
Two novel type I
ribosome-inactivating proteins (RIPs) were found in the storage roots
of Mirabilis expansa, an underutilized Andean root crop.
The two RIPs, named ME1 and ME2, were purified to homogeneity by
ammonium sulfate precipitation, cation-exchange perfusion
chromatography, and C4 reverse-phase chromatography. The
two proteins were found to be similar in size (27 and 27.5 kD) by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their
isoelectric points were determined to be greater than pH 10.0. Amino
acid N-terminal sequencing revealed that both ME1 and ME2 had conserved
residues characteristic of RIPs. Amino acid composition and
western-blot analysis further suggested a structural similarity between
ME1 and ME2. ME2 showed high similarity to the Mirabilis
jalapa antiviral protein, a type I RIP. Depurination of yeast
26S rRNA by ME1 and ME2 demonstrated their ribosome-inactivating activity. Because these two proteins were isolated from roots, their
antimicrobial activity was tested against root-rot microorganisms, among others. ME1 and ME2 were active against several fungi, including Pythium irregulare, Fusarium oxysporum
solani, Alternaria solani, Trichoderma reesei, and Trichoderma
harzianum, and an additive antifungal effect of ME1 and ME2 was
observed. Antibacterial activity of both ME1 and ME2 was observed
against Pseudomonas syringae, Agrobacterium
tumefaciens, Agrobacterium radiobacter, and
others.
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INTRODUCTION |
For the past 3 years, we have been studying the biology of
underutilized ARTCs (Flores and Flores, 1997 ; Flores et al., 1997). These species constitute an important staple for the people in the
highlands of Peru, Bolivia, and Ecuador, and they are a possible source
for novel bioactive phytochemicals and future novel food products
(Johns et al., 1982; Flores and Flores, 1997). The ARTCs have great
potential as new introduced crop species (Sperling and King, 1988), but
they have been largely overlooked and poorly studied at the biological
and biochemical levels. No fewer than nine plant families (Asteraceae,
Basellaceae, Brassicaceae, Leguminoseae, Nyctaginaceae, Oxalidaceae,
Solanaceae, Tropaeolaceae, and Umbelliferae) are represented among this
crop complex, which is well adapted to cultivation at 6,000 to 14,000 feet above sea level and includes a fascinating reservoir of
intraspecific and interspecific germ plasm biodiversity (National
Research Council, 1989). One of the most neglected ARTCs is
Mirabilis expansa, a relatively disease-resistant Andean
crop that produces edible storage roots (Angeles Millones, 1996; Franco
Pebe et al., 1996). The cultivation of this crop has been
restricted to three small regions in Peru, Ecuador, and Bolivia
(National Research Council, 1989), and its basic biology and agronomy
are virtually unknown.
Storage organs such as tubers and roots synthesize proteins, the major
function of which is to provide a store of N, S, and C (Shewry, 1995).
Sprouting of such tissues may be accompanied by general proteolysis,
during which structural and metabolic proteins are digested. In some
cases, storage proteins have also been shown to exhibit antipest and
antipathogen activity (Yeh et al., 1997). Because of the reported
disease-resistant behavior of M. expansa (Angeles Millones,
1996), we decided to focus on the isolation of potential root defense
proteins, with emphasis on RIPs. Our focus on RIPs was based on studies
of Mirabilis jalapa (MacBride, 1951), a widely known
ornamental plant also originally from South America and, like M. expansa, a member of the Nyctaginaceae. The storage roots of
M. jalapa contain an RIP (MAP) with antiviral activity
against important economically devastating viruses, such as tobacco
mosaic virus, potato virus X, potato virus Y, and viroids such as
potato spindle tuber viroid (Kubo et al., 1990; Kataoka et al., 1991,
1992; Vivanco, 1997). Root extracts of M. jalapa sprayed
over potato plants conferred stable inhibition of viral infection
(Vivanco, 1997). The viral inhibition was attributable to MAP and was
based on its RIP activity and its biochemical
stability.
RIPs are widely distributed among higher plants (Mehta and Boston,
1998). They inhibit protein synthesis by virtue of their N-glycosidase activity, selectively cleaving an adenine
residue at a conserved site of the 28S rRNA (26S rRNA in yeast), such as the adenine-4324 of rat liver 28S rRNA (Endo and Tsurigi, 1988). This cleavage prevents the binding of elongation factor 2 (Stirpe et
al., 1992), with the consequent arrest of protein synthesis. In
addition to rRNA N-glycosidase activity, many RIPs display a
variety of other biological and enzymatic activities. RIPs have broad-spectrum antiviral activity against RNA and DNA plant and animal
viruses (Batelli and Stirpe, 1995). Furthermore, some RIPs have
specific DNA-nuclease activity against supercoiled, covalently closed,
circular plasmid DNA and single-stranded phage DNA (Roncuzzi and
Gasperi-Campani, 1996). Some type I RIPs have been shown to inhibit
fungal growth (Roberts and Selitrennikoff, 1986). Likewise, RIPs
have been found to have insecticidal properties against coleopteran and
lepidopteran species (Verma and Kymar, 1979; Gatehouse et al.,
1990).
RIPs are compartmentalized in vacuoles and cell walls (Kataoka et al.,
1991), which apparently allows the ribosome-inactivating activity to be
sequestered from their own ribosomes. They may be released or induced
in response to pathogen infection or injury. This apparent defense
activity is usually coordinated with other defense proteins, such as
chitinases (Leah et al., 1991),  -1,3 glucanases (Mauch et al.,
1988a), and thaumatins (Hejgaard et al., 1991). Studies of
trichosanthin, a type I RIP isolated from the roots of
Trichosanthes kirilowii, suggest that full RIP expression is
developmentally coordinated (Savary and Flores, 1994). However, a clear
function of RIPs in plants has yet to be elucidated.
In this paper, we report the isolation and characterization of two type
I RIPs, ME1 (27.5 kD) and ME2 (27 kD), from the storage roots of
M. expansa. We also report antifungal and antibacterial activities of ME1 and ME2 against soil-borne microorganisms. Based on
our findings, we suggest that ME1 and ME2 hold promise as candidates for broad-spectrum disease protection.
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MATERIALS AND METHODS |
Plant Material and Protein Extraction from Roots
Seeds from Mirabilis expansa obtained from the
International Potato Center (Lima, Peru) were washed five times with
sterile water and germinated on filter paper in a Petri dish at room
temperature. The seeds were then transferred to pots and placed in the
greenhouse. Five months after the seeds were planted, the storage roots
were harvested and the total soluble proteins from the roots were
extracted according to the method described by Savary and Flores
(1994). Analyses were also conducted on storage roots collected by J.V. from the experimental station at the Universidad Nacional de Cajamarca (Peru). Storage roots were immersed in liquid
N2, ground to a powder, and stored at  20°C
until use. Root protein extracts were prepared by homogenizing 100 mg
of ground root tissue per 1 mL of extraction buffer (25 mM NaPO4, pH 7.0, with 250 mM NaCl, 10 mM EDTA, 10 mM thiourea, 5 mM DTT, 1 mM PMSF, and 1.5% [w/v]
polyvinylpolypyrrolidone). The solution was subsequently centrifuged
for 20 min at 10,000g. The supernatant was incubated
overnight at 4°C as a 20% (w/v) saturated ammonium sulfate solution
under constant stirring. The supernatant was collected after
centrifugation (10,000g for 20 min). Samples were dialyzed
with two changes of 20 mM Hepes buffer (pH 8.0)
containing 50 mM NaCl. Smaller sample volumes
were desalted using Econopac 10DG columns (Bio-Rad). The protein
solution was concentrated to 1 mg/mL using a Stirred Cell 8050 (Amicon,
Beverly, MA) with a YM 10 membrane. Protein concentration was
determined by the Bradford (1976) method using BSA as a standard and by
using a laser densitometer (Ultrascan XL, LKB, Bromma, Sweden) to
quantify individual proteins.
HPLC
A 4.6- × 100-mm column (1.66-mL volume) was packed with Poros 20 HS cation-exchange perfusion medium (Perkin-Elmer-Applied Biosystems)
and used in conjunction with a 600E HPLC system equipped with a
photodiode-array detector (model 990, Waters). Equilibration, loading,
and washing were carried out in 25 mM Hepes, pH 8.0, containing 50 mM NaCl. The target proteins were eluted with
a linear gradient of 30 column volumes (approximately 50 mL) from 50 to
200 mM NaCl at a flow rate of 5 mL/min. Individual peaks were collected and concentrated by ultrafiltration using a Stirred Cell
8050 (Amicon). Protein purity and peak size were confirmed by SDS-PAGE.
Reverse-phase HPLC was used to prepare proteins for N-terminal
sequencing and for antiserum production. Individual peaks collected during the cation-exchange step were further separated on a 4.6- × 100-mm column packed with Poros R2 reverse-phase perfusion medium. The
column was equilibrated with 0.1% (v/v) trifluoroacetic acid containing 10% (v/v) acetonitrile at a flow rate of 5 mL/min. Proteins
were eluted through a 50-column-volume linear gradient from 10% to
60% acetonitrile (in 0.1% trifluoroacetic acid). All chromatographic separations were performed at room temperature.
Amino Acid Analysis and N-Terminal Sequencing
Amino acid analysis and composition were determined using an
analyzer (model 420H, Perkin-Elmer-Applied Biosystems), according to
the methodology described by Tarr (1986). The
N-terminal-sequence analysis was performed on a protein sequencer
(model 477A) equipped with an analyzer (model 120A,
Perkin-Elmer-Applied Biosystems) at the Hershey Medical Center (The
Pennsylvania State University, University Park). The standard Edman
degradation procedure was used as described by Allen (1981).
Electrophoresis and Western-Blot Analysis
SDS-PAGE was performed with 13.5% or 15% (v/v) acrylamide
discontinuous gels (Laemmli, 1970) using an electrophoresis cell (Mini-Protean II, Bio-Rad), according to the manufacturer's
instructions. A low-molecular-mass (14-66 kD) protein-marker kit
(Sigma) was used to determine approximate protein sizes. Proteins were
visualized with Coomassie brilliant blue G-250 (Calbiochem, La Jolla,
CA) or zinc staining (Bio-Rad). Proteins were electroblotted to
Immobilon-P PVDF membranes (Millipore) with a Bio-Rad Mini-Trans
electrotransfer cell for 1 h at 150 V (constant
voltage), using 10 mM 3-(cyclohexylamino)propanesulfonic acid (pH 11.0 with NaOH) and 10% (v/v)
methanol-transfer buffer (LeGendre and Matsudaira,
1989). Membranes were developed with the Promega secondary
antibody-alkaline phosphatase detection system, according to the
manufacturer's instructions. An antiserum titer of 1:5000 was used for
all experiments.
IEF
The pI of purified ME1 and ME2 was estimated by IEF using an
Ampholine PAG plate (pH 3.5-9.5; Pharmacia) with high-range pI marker proteins (Pharmacia) stained with Coomassie blue.
Antibodies
Polyclonal antibodies were produced in male New Zealand White
rabbits against reverse-phase HPLC-purified ME1 and ME2. Three injections were given 20 d apart, to a total of 300 µg of each protein. The proteins were injected together with Freund's adjuvant. After clot removal, the serum was stored at 40°C. Antibodies were
further purified according to the method described by Olmsted (1986).
The procedure was carried out at the Centralized Biological Laboratories (The Pennsylvania State University).
rRNA Depurination Assay
The assay was conducted according to the method of Tumer et al.
(1997). Yeast ribosomes (50 µg) were incubated with 100 ng of ME1,
ME2, ME1 plus ME2, and PAP at 30°C for 30 min. The reaction was
stopped by the addition of 0.1% SDS. RNA was extracted from yeast
ribosomes, incubated on ice for 30 min with 1 M aniline acetate, pH 4.5, and precipitated with ethanol. RNA was electrophoresed in a 4.5% urea-polyacrylamide gel and stained with ethidium bromide.
Antimicrobial Assays
Antifungal activity of M. expansa root proteins was
demonstrated in a radial growth-inhibition assay adapted from the
method of Schlumbaum et al. (1986). A fungal plug was placed in the
center of a potato-dextrose-agar plate. Sterile paper discs were placed into punched wells in the agar around the outside of the plate. Protein solutions ranging from 0.5 to 100 µg were filter
sterilized using 0.22-µm filters (Ultrafree-MC Durapore, Millipore).
Various amounts of protein were pipetted into the wells, and
sterile buffer was used to bring the final volume to 100 µL. The
Petri plates were incubated in the dark at 23°C. Antifungal activity
was observed as a crescent-shaped zone of inhibition at the mycelial
front. The effect on fungal growth was expressed qualitatively,
according to the procedure of Schlumbaum et al. (1986).
Antibacterial activity was screened using an inhibition-halo-plate
assay. Bacterial cultures were inoculated into liquid Luria-Bertani medium and placed onto a shaker at 23°C for 2 to 3 d. The
A600 of bacterial cultures was measured and
adjusted to an optical density of 0.2 for the antibacterial
experiments. One hundred microliters of bacterial suspension was placed
onto a sterile plate containing Luria-Bertani medium and subsequently
spread over the entire surface of the plate. A sterile paper disc was placed into punched wells in the agar in the middle of the plate. Protein solutions were filter-sterilized using 0.22-µm filters (Ultrafree-MC Durapore, Millipore) and applied to the paper discs. The
Petri plates were incubated in a dark room at 23°C for 24 h.
Antibacterial activity was measured as the radius of inhibition from
the border of the protein-impregnated disc.
The following bacterial strains were obtained from a collection of
field isolates maintained in the laboratory of Dr. Leland S. Pierson,
III, at the University of Arizona in Tucson: Bacillus subtilis 613R, Bacillus thuringiensis Gnatrol,
Clavibacter michigenensis subsp. nebraskensis
CN74-1, Agrobacterium radiobacter K84, Agrobacterium tumefaciens C58, Bacillus cepacea Deny,
Escherichia coli ESS, Erwinia amylovora,
Erwinia carotovora ATCC 15713, Pseudomonas aureofaciens 30-84, Pseudomonas fluorescens 2-79, Pseudomonas syringae B, Pseudomonas syringae pv
phaseolicola, Pseudomonas putida Qd8,
Ralstonia solanacearum, Serratia marcescens,
Xanthomonas campestris pv versicatoria,
Streptomyces griseovivides Mycostop, and Rhizobium
leguminosarum. Erwinia herbicola was isolated from pea
seedling roots by Dr. Lindy Brigham at the University of Arizona. Agrobacterium rhizogenes ATCC 15834 was maintained by Dr.
Hector E. Flores at The Pennsylvania State University.
Xanthomonas campestris pv pelargonii was obtained
from the collection of Dr. Gary Moorman at The Pennsylvania State
University.
Microscopy
The inhibitory halo was observed on a stereoscopic microscope
(model SMZ-U, Nikon), and the antifungal effect was confirmed in vivo
(see Fig. 8). The effect of the basic M. expansa proteins was studied in hyphae growing on agar, using a modification of the
method described by Arlorio et al. (1992). Fungal hyphae from the
center of the actively growing plates (healthy) and from the front of
the inhibition zone adjacent to the wells (unhealthy) were mounted on
glass slides and stained with lacto-phenol blue. The samples were
destained with distilled water before viewing with bright-field
microscopy using an inverted microscope (Diaphot-TMD, Nikon).
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| Figure 8.
A, Antifungal activity of M. expansa proteins against T. reesei. Fifty
micrograms of total storage-root protein (T), ME1 and ME2 together, and
acidic (unretained) proteins (A) were applied to the discs and assayed
for antifungal activity, as described in ``Materials and Methods''.
B, Antifungal activity of the purified ME1 and ME2 against T. reesei. Numbers indicate different concentrations of ME1 and
ME2. The controls consisted of boiled M. expansa total
storage-root proteins (V) and 25 mM Hepes buffer, pH 8.0 (W).
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RESULTS |
Purification of ME1 and ME2
The root proteins of M. expansa were buffer exchanged
to pH 8.0 (see ``Materials and Methods''), which facilitates the
enrichment and separation of strongly basic proteins, such as RIPs, by
cation-exchange chromatography. Two major basic proteins, with
molecular masses of 27 and 27.5 kD, were resolved through
cation-exchange perfusion chromatography and eluted through a 200 mM NaCl step gradient. As shown in Figure 1, these two proteins eluted close to
each other, indicating a similarity in charge. Reverse-phase
chromatography was performed to further purify the proteins for amino
acid composition analysis and sequencing. This technique yielded
individual bands of essentially pure protein, as confirmed by SDS-PAGE
(Fig. 2). The two proteins were named ME1
(27.5 kD) and ME2 (27 kD). A cation-exchange perfusion chromatography
linear gradient (0-150 mM NaCl) was also used to resolve ME1 and ME2 under nondenaturing conditions, and these fractions
were used individually and in combination to determine their biological
activities. The perfusion medium greatly enhanced the speed and
resolution of our separations and enabled us to purify milligram
quantities of ME1 and ME2 in 15 min.
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| Figure 1.
Cation-exchange perfusion chromatography of the
total storage-root proteins from M. expansa. I, SDS-PAGE
of the fractions collected by cation-exchange perfusion chromatography.
II, Cation-exchange perfusion chromatogram. Samples for electrophoresis
were prepared as described in ``Materials and Methods''. Fifteen and
five micrograms were loaded onto the gel for total proteins and
fractions, respectively. Approximately 33 mg of total protein was
injected into the column, and 6.1 mg was recovered in the 200 mM NaCl fraction. This separation was repeated several
times to obtain sufficient protein separation for further
characterization.
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| Figure 2.
Isolation of ME1 and ME2 from the 200 mM cation-exchange fraction by reverse-phase column
chromatography, as described in ``Materials and Methods''. I,
SDS-PAGE of the proteins separated after the reverse-phase HPLC step.
Approximately 2 µg of protein was loaded per lane. II, Reverse-phase
perfusion chromatogram. Approximately 300 µg of protein from the 200 mM NaCl fraction was injected into the column, and 100 µg
of each fraction was collected.
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The patterns of total root-soluble proteins of M. jalapa and
M. expansa were compared by SDS-PAGE. Species-specific
patterns were observed in both species, including common and unique
major proteins. It was clear that MAP, a type I RIP from M. jalapa, migrated at the same protein range (24-29 kD) as ME1 and
ME2 from M. expansa (Fig. 3A).
MAP was purified to homogeneity and compared with ME1 and ME2. As shown
in Figure 3B, ME2 was similar in size to MAP (27 kD). Densitometric
determination of the total storage-root proteins of M. expansa revealed that ME1 and ME2 account for almost 20% of the
soluble proteins (data not shown).
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| Figure 3.
A, Comparison of the total root-protein patterns
from M. expansa and M. jalapa. The arrows
indicate ME1 (top arrow) and ME2 (bottom arrow) in M. expansa, and the asterisk indicates MAP in M. jalapa. B, Comparison of the RIP from M. jalapa
(MAP) and the two proteins isolated from M. expansa (ME1
and ME2). Samples were extracted as described in ``Materials and Methods''. About 15 and 3 µg of total and individual proteins,
respectively, were loaded.
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Characterization of ME1 and ME2
ME1 and ME2 are strongly basic proteins with pI values greater
than pH 10.0, as determined by IEF-PAGE (data not shown). Amino acid
composition analysis of ME1 and ME2 indicated a similar distribution of
amino acids, with the exception of Pro, Ala, and Phe (Table I). ME1 had more Pro and less Ala and Phe
than ME2, and the amino acid composition of ME1 and ME2 showed a strong
similarity with that of MAP. ME1 and ME2 appear to share distinctive
features among RIPs, such as low Cys and His levels and high Lys
content.
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Table I.
Amino acid composition of ME1 and ME2
Amino acid composition of ME1 and ME2, MAP (Wong et al., 1992), PAP-S
(Barbieri et al., 1982), and Petrocoptis glaucifolia
(petroglaucin) (Arias et al., 1992).
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The N-terminal amino acid sequences of ME1 and ME2 were determined and
compared with each other and with those from other RIPs (Fig.
4). A significant matching with conserved
hydrophobic amino acids was found in the N-terminal region with 11 RIPs
reported to date (Funatsu et al., 1991). Furthermore, the ME1 and ME2
amino acid residues Tyr-16 (Y) and Phe-19 (F) aligned perfectly with the consensus sequences found in the same positions for all of the
examined RIPs. Our data suggest homology between ME1 and ME2, as well
as between ME2 and MAP. Antisera were produced for each protein, from
which monospecific antibodies were prepared and used for western-blot
analysis (Olmsted, 1986). Each antiserum was reactive with both
proteins in the total root-protein extract (Fig.
5). In other experiments in which the
individual purified proteins were blotted, the antisera were
cross-reactive between proteins (data not shown), indicating structural
homology.
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| Figure 4.
Comparison of the N-terminal sequences from RIPs.
RIC, Ricin A-chain from Ricinus communis; RCA,
agglutinin from R. communis; ABR, abrin A-chain from
Abrus precatorius; LUFa and LUFb, luffin-a and luffin-b
from Luffa cylindrica; TRI, trichosanthin from T. kirilowii; MOM, momordin from Momordica
charantia; SAP, saporin from Saponaria
officinalis; and BAR, barley translation inhibitor from barley
(Funatsu et al., 1991). Shaded boxes and boldface characters
represent two amino acids that are conserved in all RIPs currently
reported. Shaded boxes and lightface characters symbolize homology
regions among ME1, ME2, and MAP. The asterisk shows the presence of
conserved amino acids with polar R groups (hydrophobic).
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| Figure 5.
Western-blot analysis of ME1 and ME2. A, SDS-PAGE
of the total storage-root proteins from M. expansa
stained with Coomassie blue. B and C, Western-blot analysis of
M. expansa total storage-root proteins using ME1
antibody (B) and ME2 antibody (C). SDS-PAGE, electrobloting, and
western-blot development were conducted as described in ``Materials and Methods''.
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rRNA Depurination Assay
RIP-mediated depurination of the large rRNA renders the RNA
sugar-phosphate backbone susceptible to hydrolysis at the depurination site (Endo and Tsurugi, 1988). When depurinated rRNA is treated with
aniline, cleavage occurs at the depurinated site and a small 367-nucleotide fragment (in the yeast 26S rRNA) is released (Stirpe et
al., 1986). Yeast ribosomes were individually incubated with ME1, ME2,
ME1 and ME2 together, and PAP, a type I RIP, as a control. RNA was
extracted, treated with aniline, and analyzed by gel electrophoresis. As shown in Figure 6, ME1 and ME2
depurinated the 26S rRNA and released the 367-nucleotide fragment
upon treatment with aniline. The combination of ME1 and ME2 also
produced the same fragment. These results demonstrate that ME1 and ME2
are RIPs.
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| Figure 6.
Depurination of yeast ribosomes in vitro.
Ribosomes were isolated and incubated with ME1 and ME2 individually,
ME1 and ME2 together, and PAP as described in ``Materials and Methods''. rRNA was extracted, treated with aniline, separated on a
4.5% urea-polyacrylamide gel, and stained with ethidium bromide. The
presence (+) or absence () of aniline is denoted. The arrow shows the
presence of the diagnostic 367-nucleotide cleavage product of rRNA.
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Antifungal Activity of ME1 and ME2
To explore the biological activity of ME1 and ME2, we used a
standard antifungal assay (Schlumbaum et al., 1986). Growth of Trichoderma harzianum treated with the total root soluble
proteins of M. expansa was inhibited, indicating antifungal
activity. As shown in Figure 7,
inhibitory activity was observed at doses as low as 6.5 µg of total
protein. The antifungal activity of ME1 and ME2 was also tested against
an array of plant fungal pathogens. As shown in Table
II, the antifungal action was more
evident against the hyphomycete fungi, such as Verticillium
dahliae, Alternaria solani, Fusarium oxysporum
solani, Fusarium proliferatum, Trichoderma reesei, and
T. harzianum. Based on these observations, we focused our
studies on T. reesei, an indicator organism commonly used to
study antimicrobial activity (Roberts and Selitrennikoff, 1986; Schlumbaum et al., 1986; Arlorio et al., 1992). A time-course experiment for antifungal activity was performed using 50 µg of total
root soluble protein, acidic root protein, and ME1 and ME2 together
(Fig. 8A). The results showed strong
antifungal activity of the total protein and the purified ME1 and ME2
for up to 112 h of incubation. The acidic proteins showed only weak
antifungal activity, but this could be the result of minor
contamination by ME1 or ME2 during the cation-exchange chromatography
separation.
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| Figure 7.
Antifungal activity of M. expansa
total storage-root proteins against T. harzianum.
Numbers indicate the concentrations (in micrograms) of proteins applied
to the discs and tested for antifungal activity, as described in
``Materials and Methods''. The controls consisted of boiled M. expansa total storage-root proteins (V) and 25 mM
Hepes buffer, pH 8.0 (W).
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Table II.
Antifungal activity of ME1 and ME2
Antifungal activity of ME1 and ME2 against various fungal pathogens.
About 50 µg of purified protein in 100 µL of 25 mM
phosphate buffer, pH 70, was used for determination of antifungal
activity. The effect on fungal growth is expressed in qualitative
terms, according to the method of Schlumbaum et al. (1986),
in which +++ stands for strong inhibition, + for just detectable
inhibition, and for no inhibition.
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T. reesei was treated with various doses of ME1 and ME2. As
little as 0.5 µg of protein showed inhibitory activity after 24 h of
incubation (data not shown). Zones of inhibition were dose dependent.
As shown in Figure 8B, the most prominent inhibition zones were
obtained with 50 and 5 µg after 80 h of incubation. Fungal inhibition
persisted after 3 weeks of incubation in the dark growth chamber at
23°C and after the plates were transferred to the cold room (4°C)
for 2 more weeks. The individual effects of ME1 and ME2 separated by
cation-exchange chromatography were also monitored against T. reesei growth, revealing an additive inhibitory effect of ME1 and
ME2 (data not shown). The inhibitory activity of ME1 was higher than
that of ME2.
Evaluation of the Effect of ME1 and ME2 on T. reesei
Hyphae Growth at the Microscopic Level
The inhibitory halo caused by ME1 and ME2 was evaluated
microscopically to analyze the antifungal effect in detail. Comparison by light microscopy of the healthy hyphae growing at the center of the
plate and the weakened hyphae at the border of the inhibition halo
showed extensive septum formation and thinning of the unhealthy hyphae
(Fig. 9). Enlarged tips were also seen on
the unhealthy hyphae, showing a marked swelling and lysis (Fig.
9).
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| Figure 9.
Microscopic analysis of the antifungal effect in
T. reesei. Comparison of T. reesei
healthy hyphae growing at the middle of the Petri dish and weakened
hyphae growing at the border of the inhibition halo.
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Antibacterial Activity of ME1 and ME2
Because the two M. expansa root proteins were isolated
from underground organs, the inhibitory activity of ME1 and ME2 was tested against 27 soil-borne bacterial species (Table
III), and inhibitory activity was found
against 8 of them. Among these bacteria there were nonpathogenic
species, such as B. subtilis, R. leguminosarum, and S. marcescens, and pathogenic species, including
P. syringae, A. tumefaciens, A. rhizogenes R100nal, X. campestris pv
versicatoria, and E. carotovora. These bacteria
include important plant pathogens that cause severe diseases, such as
wilts, crown galls, leaf spots, and soft rots. It is interesting that
S. marcescens, one of the bacteria most susceptible to ME1
and ME2, can be a human pathogen under certain conditions (von
Graevenitz, 1980). To our knowledge, this is the first time that in
vitro antibacterial activity has been reported for type I RIPs against
bacterial plant pathogens. Previous studies performed by Kataoka et al.
(1991) showed that E. coli expressing MAP displayed a
reduced growth rate, which is consistent with our antibacterial assays.
A. rhizogenes inoculations were also performed on M. expansa plants to obtain "hairy-root" cultures (data not
shown). However, no infection occurred. Thus, it is possible that
A. rhizogenes was inhibited by ME1 and ME2 activity.
View this table:
[in this window]
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Table III.
Antibacterial activity of ME1 and ME2
Antibacterial activity of ME1 and ME2 against various root-tor
bacteria. Approximately 50 µg of purified protein in 100 µL of 25 mM phosphate buffer, pH 70), was used for determination of
antibacterial activity. These experiments were repeated three times.
|
|
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DISCUSSION |
We have isolated and characterized two major basic proteins (ME1
and ME2) that constitute about 20% of the total soluble proteins from
the storage roots of M. expansa. ME1 and ME2 showed
antimicrobial activity against various fungal and bacterial species,
including some important plant pathogens. Based on amino acid
composition, N-terminal sequence analysis, and enzyme activity, we
conclude that ME1 and ME2 are type I RIPs. Both proteins share features common to RIPs, such as low Cys and His levels and high Lys content. Highly conserved hydrophobic amino acid residues were also observed at
the N-terminal regions of ME1 and ME2 (Funatsu et al., 1991), as was
the presence of conserved amino acids with polar R groups (hydrophobic), such as Met (M), Ile (I), and Val (V) (Fig. 4). It is
well established that hydrophobic residues stabilize the interaction
between nucleic acids and proteins by intercalating between bases of
DNA or RNA (Frankel et al., 1989). The molecular-weight and
N-terminal sequence similarities between ME2 and MAP suggest the
presence of RIPs among the genus Mirabilis, similar to the widespread content of RIPs in the genus Trichosanthes (Ng et
al., 1992, 1993).
RIPs are widely represented in taxonomically distinct plant families,
such as Asparagaceae, Caryophyllaceae, Cucurbitaceae, Euphorbiaceae,
Nyctaginaceae, Phytolaccaceae, and Poaceae (Mehta and Boston, 1998).
Some plant species show a higher constitutive content of RIPs than
others (Stirpe and Barbieri, 1986; Stirpe et al., 1992; Mehta and
Boston, 1998). It has been speculated that RIPs were originally used by
all plant species as a primary defense response (Stirpe et al., 1992).
ME1 and ME2 showed strong antifungal activity against an array of
pathogenic and nonpathogenic fungi, such as P. irregulare, V. dahliae, A. solani, F. oxysporum
solani, F. proliferatum, and T. reesei
(Table II); the hyphomycetes was the most susceptible fungal group. It
has been suggested that as little as one RIP molecule per cell is
capable of shutting down protein synthesis (Stirpe and Barbieri, 1986).
However, to reach the ribosomes, RIPs must penetrate the target cell.
Anatomic features such as gaps, natural openings, and damaged tissue
may facilitate RIP penetration. According to Roberts et al. (1986), a
barley RIP was able to penetrate T. reesei through gaps in
cell membranes. The antifungal activity of ME1 and ME2 on T. reesei growth could be attributable to the penetration of these
two proteins into the hyphae by the same mechanism. It has also been
suggested that some RIPs may have a role as chitinases (Remi Shih et
al., 1997). In vivo RIPs may play a synergistic role with other
defense-related proteins such as chitinases (Broekaert et al., 1989)
and -1,3 glucanases (Kombrick et al., 1988; Mauch et al., 1988b);
the latter proteins may break down fungal cell walls and thus
facilitate RIP entrance into the cell. We found that ME1 and ME2 showed
antibacterial activity against important plant pathogens. The mechanism
by which ME1 and ME2 inhibit certain bacteria remains unknown. In
addition to ME1 and ME2 antimicrobial activity, preliminary results
suggest antiviral activity associated with these two proteins (data not shown). Preventive applications of ME1 and ME2 over Gomphrena globosa leaves inhibited potato virus X infection. Nonetheless, toxicity was observed with higher concentrations of ME1 and ME2.
It is intriguing that ME1 and ME2 have an additional role as storage
proteins, because they constitute 20% of the total storage-root proteins in M. expansa. This hypothesis is in accordance
with studies of trichosanthin, a root-specific RIP from T. kirilowii, in which trichosanthin accumulation was associated with
the onset of root secondary growth (Savary and Flores, 1994). In fact,
maximum levels of trichosanthin (>25% total soluble root protein)
were observed in fully developed tuberous roots. The main storage
protein of sweet potato roots, sporamin, may function in response to
root pests as well (Yeh et al., 1997). Patatin, the major tuber storage protein of potato, has been reported to have lipid acyl hydrolase activity and to inhibit the growth of southern corn rootworm and western corn rootworm when fed to them in an artificial diet
(Strickland et al., 1995). Thus, we may speculate that more than a few
major proteins found in underground storage organs have evolved more than one function.
The majority of RIPs reported so far have been isolated from leaves,
stems, and seeds. To date, the isolation of root-specific RIPs has been
restricted to MAP, trichosanthin, and related proteins from
Cucurbitaceae species (Maragonore et al., 1985; Savary and Flores, 1994; Mehta and Boston, 1998). Preliminary investigations using
tissue printing suggest the localization of ME1 and ME2 in the
epidermis of the storage root, as well as higher concentrations at the
site of emergence of lateral roots (data not shown), consistent with
their role as pathogen/pest defense proteins. PAP, a type I RIP, and
PAP mutants have been genetically engineered into potato and tobacco
plants. The transformants have been shown to express resistance to a
broad spectrum of viruses and to fungal pathogens as well, such as
R. solani (Lodge et al., 1993; Zoubenko et al., 1997). Our
data suggest the role of ME1 and ME2 as broad-spectrum defense proteins
in M. expansa and their potential use in the development of
transgenic pathogen-resistant crops. Alternatively, their antimicrobial
and antiviral activity could be used in simple crop-protection methods
in low-input agricultural systems, such as the spraying of root
extracts on leaves of various crops to prevent or control pathogen
infection (Vivanco, 1997).
As stated above, M. expansa is an underutilized root crop,
the storage roots of which are used as a food source. Presumably, ME1
and ME2 become denatured during the cooking and roasting process, thus
allowing the root to be eaten. However, some type I RIPs, such as PAP,
are able to resist boiling without denaturing (Irvin, 1995). Our
findings thus warrant detailed studies of the potential toxicity or
beneficial use of M. expansa roots to humans (Moore, 1988).
The study of new RIPs, such as those reported in this paper, may lead
to a better understanding of their biology and diversity. Efforts are
currently under way to isolate cDNA and genomic clones for ME1 and ME2
and to define their potential role in root-pathogen interactions.
| |
FOOTNOTES |
1
This work was supported by the McKnight
Foundation.
*
Corresponding author; e-mail hef1{at}psu.edu; fax 1-814-863-7217.
Received September 3, 1998;
accepted December 21, 1998.
| |
ABBREVIATIONS |
Abbreviations:
ARTCs, Andean root and tuber crops.
MAP, Mirabilis jalapa antiviral protein.
ME1 and ME2, Mirabilis expansa proteins
1 and 2.
PAP, pokeweed antiviral protein.
RIP, ribosome-inactivating protein.
| |
ACKNOWLEDGMENTS |
We thank Drs. N. Tumer and K. Hudak for assistance with the
depurination assay. We also thank Dr. S. Kang, Ms. P. Michaels, and Ms.
D. Needle for editing suggestions in the preparation of the manuscript.
| |
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Abstract
Introduction