Plant Defensin Peptides have Antifungal and Antibacterial Activity Against Human and Plant Pathogens

Abstract

Plant defensins are small, cysteine-rich antimicrobial peptides. These peptides have previously been shown to primarily inhibit the growth of fungal plant pathogens. Plant defensins have a γ-core motif, defined as GXCX3-9C, which is required for their antifungal activity. To evaluate plant defensins as a potential control for a problematic agricultural disease, alfalfa crown rot, short chemically synthesized peptides containing γ-core motif sequences were screened for activity against numerous crown rot pathogens. These peptides showed both antifungal and, surprisingly, antibacterial activity. Core motif peptides from Medicago truncatula defensins (MtDef4 and MtDef5) displayed high activity against both plant and human bacterial pathogens in vitro. Full-length defensins had higher antimicrobial activity compared to the peptides containing their predictive γ-core motifs. These results show the future promise for controlling a wide array of economically important fungal and bacterial plant pathogens through the transgenic expression of a plant defensin. They also suggest that plant defensins may be an untapped reservoir for development of therapeutic compounds for combating human and animal pathogens.

Full text

Disease Control and Pest Management
Plant Defensin Peptides have Antifungal and Antibacterial Activity
Against Human and Plant Pathogens
Andrew E. Sathoff,
1,†
Siva Velivelli,
2
Dilip M. Shah,
2
and Deborah A. Samac
1,3
1
Department of Plant Pathology, 1991 Upper Buford Circle, University of Minnesota, St. Paul, MN 55108;
2
Donald Danforth Plant Science
Center, 975 North Warson Road, St. Louis, MO 63132; and
3
United States Department of Agriculture–Agricultural Research Service, Plant
Science Research Unit, 1991 Upper Buford Circle, St. Paul, MN 55108.
Accepted for publication 19 September 2018.
ABSTRACT
Plant defensins are small, cysteine-rich antimicrobial peptides. These
peptides have previously been shown to primarily inhibit the growth of
fungal plant pathogens. Plant defensins have a g-core motif, defined as
GXCX
3-9
C, which is required for their antifungal activity. To evaluate
plant defensins as a potential control for a problematic agricultural disease
(alfalfa crown rot), short, chemically synthesized peptides containing g-core
motif sequences were screened for activity against numerous crown rot
pathogens. These peptides showed both antifungal and, surprisingly,
antibacterial activity. Core motif peptides from Medicago truncatula
defensins (MtDef4 and MtDef5) displayed high activity against both
plant and human bacterial pathogens in vitro. Full-length defensins had
higher antimicrobial activity compared with the peptides containing
their predictive g-core motifs. These results show the future promise for
controlling a wide array of economically important fungal and bacterial
plant pathogens through the transgenic expression of a plant defensin.
They also suggest that plant defensins may be an untapped reservoir
for development of therapeutic compounds for combating human and
animal pathogens.
Peptides and small proteins with antimicrobial activity have been
identified in a wide array of organisms (Dias and Franco 2015).
Because they are found in vertebrates, invertebrates, plants, and
fungi, they may constitute an ancient, conserved line of defense
against pathogen invasion that predates the divergence in eukary-
otes (Carvalho and Gomes 2009). Plant defensins are cysteine-rich,
cationic antimicrobial peptides of 45 to 54 amino acid residues.
These peptides have a highly conserved three-dimensional structure
consisting of one a-helix and three antiparallel b-strands that are
connected by four disulfide bonds, forming a cysteine-stabilized ab
(CSab) motif (De Coninck et al. 2013; Francisco and Georgina
2017; Lay and Anderson 2005; Vriens et al. 2014). The structure
of each plant defensin has a functionally important g-core mo-
tif, GXCX
3-9
C, which alone can confer antimicrobial activity
(Sagaram et al. 2011; Yount and Yeaman 2004). Despite their
structural uniformity, plant defensins exhibit very low sequence
similarity outside the eight distinctively conserved cysteines
(Thomma et al. 2002; van der Weerden and Anderson 2013). This
divergence in primary sequences may account for different biolog-
ical functions attributed to plant defensins, including antifungal
and antibacterial activity, pollen tube guidance, and roles in plant
development (Carvalho and Gomes 2009).
Many plant defensins exhibit potent activity in vitro, inhibiting
the growth of fungi and oomycetes at micromolar concentrations.
However, they differ considerably in the spectrum of organisms
inhibited and modes of action (MOA). The initial studies aimed at
revealing MOA of plant defensins identified interactions with
fungal-specific membrane components (Thevissen et al. 1997,
2000, 2004). Defensins were shown to permeabilize fungal plasma
membranes, induce Ca
2+
influx, and disrupt a Ca
2+
gradient es-
sential for polar growth of hyphal tips (Thevissen et al. 1996,
1997, 1999). Some defensins bind with high affinity to specific
sphingolipids present in the fungal cell wall or plasma membrane of
their target fungi (Aerts et al. 2008; Thevissen et al. 2005, 2007).
During the last few years, several antifungal plant defensins,
including Psd1 from Pisum sativum, NaD1 from Nicotiana alata,
TPP3 from Solanum lycopersicum, NsD7 from N. suaveolens, and
MtDef4 and MtDef5 from Medicago truncatula, have been shown
to gain entry into fungal cells and interact with bioactive plasma
membrane resident phospholipids, cause membrane permeabiliza-
tion, and induce fungal cell death (Aerts et al. 2008; Lobo et al.
2007; Parisi et al. in press; Thevissen et al. 2004). It has been
proposed that these peptides have multiple targets in fungal cells.
Thus, in addition to disrupting the plasma membrane, these peptides
likely bind to intracellular targets, induce production of reactive
oxygen species, and inhibit cell division.
Although several plant defensins have been extensively studied
for their antifungal activity, fewer defensins with antibacterial
activity have been reported (van der Weerden and Anderson 2013).
For example, Cp-thionin II from cowpea (Franco et al. 2006),
DmAMP1 from Dahlia merckii, CtAMP1 from Clitoria ternatea,
AhAMP1 from Aesculus hippocastanum (Osborn et al. 1995),
ZmESR-6 from maize (Baland´
ın et al. 2005), fabatin from broad
been (Zhang and Lewis 1997), and SOD2 and SOD7 from spinach
(Segura et al. 1998) have been reported to exhibit antibacterial
activity against a range of Gram-positive and Gram-negative bac-
terial pathogens. Among antibacterial defensins, SOD2 and SOD7
from spinach have been demonstrated to confer resistance to Asi-
atic citrus canker and huanglongbing caused by Xanthomonas
citri subsp. citri and ‘Candidatus Liberibacter’ spp., respectively, in
transgenic citrus (Stover et al. 2013). The MOA of antibacterial
plant defensins have yet to be deciphered in detail.
Alfalfa (M. sativa), a perennial plant in the legume family, is
among the most valuable crops in the United States, with a direct
value of over $8 billion annually. Alfalfa production is essential for
sustaining the dairy industry, which used an estimated 14 million
tons of alfalfa in 2016. Plant pathogens and nematodes that infect
alfalfa account for an estimated $400 million in economic losses
annually (Leath et al. 1988). Crown rot, caused by a complex of
soil microbes, is one of the most important alfalfa diseases across
the United States. The organisms causing crown rot can differ
substantially by geographic location. Some of the most common
pathogenic fungal species are Phoma medicaginis, Rhizoctonia
†
Corresponding author: Andrew Sathoff; E-mail: satho002@umn.edu
©2019 The American Phytopathological Society
402 PHYTOPATHOLOGY
Phytopathology •2019 •109:402-408 •https://doi.org/10.1094/PHYTO-09-18-0331-R

solani, Fusarium oxysporum, F. roseum, and F. solani (Turner and
Van Alfen 1983; Uddin and Knous 1991; Wilcoxson et al. 1977).
Bacteria (Clavibacter insidiosus and Pseudomonas spp.) and
oomycetes (Pythium spp.) are also associated with the disease
complex. Another economically important alfalfa disease, Apha-
nomyces root rot caused by Aphanomyces euteiches, often accom-
panies crown rot in soil with poor drainage. Crown rot occurs to
some extent in every alfalfa stand that is over 1 year old and is the
major source of stand decline and yield loss.
Breeding for resistance has been successfully employed to
manage several alfalfa diseases but resistance has not been
identified for developing crown rot resistant cultivars. Fungicides
with the required persistent root and crown activity are not
available. Lack of cultural and chemical management practices
for this disease severely limits alfalfa production. Thus, there is an
immediate need for development of innovative methods to manage
crown rot for enhanced alfalfa persistence and yields.
We have recently reported the antibacterial activity of a bidomain
defensin, MtDef5, from M. truncatula, a model plant species
closely related to alfalfa, against the Gram-negative bacterial
pathogen X. campestris. MtDef5 permeabilizes the plasma mem-
brane and translocates into the cells of this bacterial pathogen. In
vitro, it also binds to DNA (Velivelli et al. 2018). In this study, we
identified plant defensin peptides that inhibit the in vitro growth of
alfalfa crown rot pathogens. We also discovered that they inhibit the
growth of several human bacterial pathogens. Synthetic g-core
motifs were used to initially screen for activity against numer-
ous fungal and bacterial pathogens. The g-core motifs from
M.truncatula had the greatest antagonistic biological activity
against the evaluated pathogens. However, the corresponding full-
length defensins displayed enhanced activity compared with the
g-core motifs. These results not only indicate that transgenic ex-
pression of plant defensins in alfalfa has the potential to provide
improved resistance to crown rot disease in alfalfa but also that plant
defensins and short peptides derived from them may be a valuable
resource for the development of therapeutic compounds with novel
modes of action to combat human pathogens.
MATERIALS AND METHODS
Pathogen cultures and growth media. All fungal pathogen
strains were isolated from infected alfalfa plants collected in
Minnesota from commercial production fields and deposited in the
University of Minnesota Mycological Culture Collection. The fungal
strains F. oxysporum f. sp. medicaginis 7F-3, F. oxysporum f. sp.
medicaginis 31F-3, F. solani,F. tricinctum,F. incarnatum,F. redolens,
Colletotrichum trifolii WS-5, C. trifolii FG-1, P. medicaginis STC,
and P. medicaginis WS-2 were grown on potato dextrose agar
(Difco, Sparks, MD) at 25°C. After 1 to 2 weeks of culture growth,
conidia were harvested by washing the plates with sterile water.
The spore suspensions were filtered and spore densities were
determined microscopically using a hemocytometer.
A. euteiches MF-1 (race 1) and A. euteiches MER4 (race 2) were
grown on corn meal agar (Difco) at 25°C for 1 week. Agar disks
(7 mm in diameter) from the margin of the A. euteiches colonized
medium were cultured in liquid peptone glucose (PG) medium
containing peptone at 20 g/liter and glucose at 5 g/liter at 20°C for
24 h. To trigger zoospore production, PG medium was removed and
agar disks were washed with sterile spring water at 0, 1, 2, 4, and 6 h
after PG media removal by resuspending in 100 ml of sterile spring
water. The final resuspension had just enough volume to immerse
the disks in approximately 15 ml of sterile spring water. Zoospores
were harvested 18 h after the final resuspension. Spore densities
were determined microscopically using a hemocytometer.
From glycerol stocks, the bacterial strains Pseudomonas syringae
pv. syringae ALF3, X. alfalfae subsp. alfalfae F3, Escherichia coli
DH5a,Sinorhizobium meliloti 102F51, Clavibacter insidiosus R1-
3, and C. insidiosus R1-1 were cultured on nutrient broth yeast
extract (NBY) agar at 30°C. After 2 days of growth, the bacterial
cells were harvested by flooding the plates with sterile water. The
American Type Culture Collection reference strains of human
pathogenic bacteria P. aeruginosa PAO1, Serratia marcescens,
Enterobacter aerogenes, and Enterococcus casseliflavus were
obtained from Dr. Foster-Hartnett at the University of Minnesota
and were cultured on Luria-Bertani (LB) agar (Difco) at 37°C. After
1 day of growth, the bacterial cells were harvested by flooding the
plates with sterile water.
Defensin peptide synthesis. The g-core motif peptides
derived from plant defensins MsDef1, MtDef4, MtDef5, RsAFP-
2, and So-D2 (Gao et al. 2000; Islam et al. 2017; Sagaram et al.
2011; Segura et al. 1998; Terras et al. 1992) (Table 1) were
chemically synthesized and purified by high-performance liquid
chromatography (LifeTein, Somerset, NJ).
Full-length clones encoding MtDef4 and MtDef5 were expressed
in Pichia pastoris, and the peptides were purified as previously
described (Islam et al. 2017; Spelbrink et al. 2004). Defensins were
lyophilized and resuspended in nuclease-free water. The concen-
tration of each defensin was determined by NanoDrop spectropho-
tometry. Approximately 3 mg of purified protein was collected from
1 liter of P. pastoris culture expressing the defensin.
In vitro defensin antifungal activity determination. A
microplate reader assay adapted from Broekaert et al. (1990) us-
ing absorption as a measure for fungal growth was utilized to
monitor growth inhibition by the g-core motif peptides and full-
length defensins. Flat, clear-bottom, 96-well microplates (Corning,
Corning, NY) were used with each well containing half-strength
potato dextrose broth (Difco), approximately 2,000 spores, and a
defensin peptide at concentrations of 0, 5, 10, 15, or 30 µg/ml in
a total volume of 100 µl. Samples were assayed in triplicate. The
microplates were shaken on an orbital shaker and spores were
allowed to sediment for 30 min before absorbance was measured.
The absorbance of the wells was measured at 595 nm on a Synergy
H1 microplate reader (BioTek, Winooski, VT). Further absorbance
measurements were carried out after 24- and 48-h incubation
periods at 25°C. To quantify fungal growth, the initial absorbance
measurement was subtracted from the final absorbance measure-
ment at 48 h. The changes in absorbance were averaged across
the three replications and a dose-response curve was created by
performing a regression using Microsoft Excel 2016. The amount of
defensin needed to inhibit growth of the fungal pathogens strains by
50% (IC
50
) was calculated from dose response curves as previously
described (Terras et al. 1992). This assay was repeated three times
for each fungal pathogen. The IC
50
values are presented as mean ±
standard error from the three experiments.
Antibacterial activity screen. Cell suspensions were diluted
with sterile water to an optical density at 600 nm (OD
600
) of 0.1.
Plates of NBY were spread with 100 µl of bacteria to create a
bacterial lawn. The plates were dried for 10 min before placing
sterile filter paper disks containing 30, 10, 5, or 0 µg of defensin
peptide onto plates. Each bacterial lawn had 12 filter paper disks (3
disks of each defensin concentration), and this experiment was
repeated three times on separate NBY plates. The bacterial plates
were incubated for 1 or 2 days at 25°C. If zones of bacterial growth
inhibition were observed, the defensin was considered to have
antibacterial activity. The diameter of zones of inhibition was
TABLE 1. Amino acid sequences of g-core motif (bold) and C-terminal region
(italics) of plant defensins tested in vitro
Defensin Amino acid sequence
MsDef1 GRCRDDFRCWCTKRC
MtDef4 GRCRGFRRRCFCTTHC
MtDef5 GACHRQGFGFACFCYKKC
RsAFP-2 GSCNYVFPAHKCICYFP
So-D2 GDCKGIRRRCMCSKPL
Vol. 109, No. 3, 2019 403

measured, and the average area of the zones of inhibition was
determined using the formula area =pr
2
.
In vitro defensin antibacterial activity determination. To
quantify antibacterial activity, a spread-plate assay was used for
both full-length defensins and g-core defensin peptides and was
repeated three times for each bacterial pathogen. As was done in our
initial antibacterial screen, bacterial lawns of Pseudomonas
syringae pv. syringae and X.alfalfae subsp. alfalfae were grown
on NBY plates for 2 days. The human pathogens S. marcescens,
Enterobacter aerogenes, and P. aeruginosa were grown on LB
plates for 1 day. The alfalfa bacterial wilt pathogen C. insidiosus
was grown on NBY for 1 week. The plates were flooded with sterile
water, and bacteria were harvested by rubbing with a sterile rubber
policeman. Cultures were diluted with sterile water to an OD
600
of
0.1. In microcentrifuge tubes, 200 µl of bacteria was incubated at
30°C with shaking for 3 h with various concentrations of a defensin
peptide (0, 2.5, 5, 10, 15, or 30 µg/ml). After the peptide treatment,
10-fold serial dilutions were made, and 100 µl were plated in
triplicate onto NBY plates. CFU were counted for P. syringae pv.
syringae and X. alfalfae subsp. alfalfae after incubation for 2 days at
30°C; for S. marcescens, E. aerogenes,Enterococcus casseliflavus,
and P. aeruginosa incubated 1 day at 37°C; and for C. insidiosus
incubated for 7 days at 25°C. Regression of the average CFU were
across experimental replications, whereas the defensin concentra-
tion was used to create a dose-response curve using Microsoft Excel
2016. From these curves, the IC
50
was calculated. The IC
50
values
are presented as mean ±standard error from three experiments.
RESULTS
Antifungal activity. The g-core motif peptides derived from
MsDef1, MtDef4, MtDef5, RsAFP-2, and So-D2 (Table 1) dem-
onstrated antifungal activity at micromolar concentrations (Table
2). Overall, the g-core motif of MtDef4 exhibited a wider spectrum
of antifungal activity than the corresponding motif from other
defensins tested. In particular, MtDef4 inhibited the growth of
P. medicaginis and F. solani, with IC
50
values of 5.3 to 7.3 and
6.0 µM, respectively. This peptide inhibited spore germination as
well as germ tube elongation and mycelial growth of both fungi but
did not result in morphological changes of spores or hyphae (Fig. 1).
The g-core motif of MtDef5 also inhibited the growth of
P. medicaginis but had enhanced inhibition of F. solani, with an
IC
50
value of 4.1 µM, compared with the core motif of MtDef4.
None of the g-core motif peptides demonstrated activity against the
oomycete pathogen A. euteiches, the fungal pathogen Colleto-
trichum trifolii, or, surprisingly, either F. redolens or F. incarnatum.
Because the g-core motifs of MtDef4 and MtDef5 showed the
greatest antifungal activity against alfalfa crown rot fungal
pathogens, the corresponding full-length defensin peptides were
evaluated for activity. Full-length MtDef4 and MtDef5 had greater
inhibitory activity than their corresponding g-core peptides against
P. medicaginis and F. oxysporum f. sp. medicaginis, with IC
50
values as low as 300 and 700 nM, respectively. The full-length
defensins also caused inhibition of spore germination and mycelial
growth (Fig. 2). Like the g-core motifs, the full-length defensins
also failed to inhibit the growth of A. euteiches and C. trifolii
(Table 2). These results indicate that the g-core motif peptides
may be used to predict the relative antifungal activity of the
corresponding full-length defensins, though additional compari-
sons need to be performed to see if this trend holds for other
defensins.
Antibacterial activity. Considering that plant defensins rarely
display antibacterial activity, a somewhat qualitative screen for
biological activity was first utilized. The g-core defensin peptides
were initially screened for antibacterial activity on a bacterial lawn
by measuring zones of inhibition resulting from defensin peptides
spotted onto filter paper disks. The g-core defensin peptides
inhibited the growth of Escherichia coli,P. syringae pv. syringae,
Fig. 1. The g-core motif peptide from MtDef4 inhibited growth of alfalfa
fungal crown rot pathogens in vitro. Spores of Phoma medicaginis were grown
for 24 h at 25°C in potato dextrose broth (PDB) culture medium in A, the
absence or B, presence of g-core MtDef4 at 30 µg/ml. Spores of Fusarium
oxysporum f. sp. medicaginis were grown for 24 h at 25°C in PDB culture
medium in C, the absence or D, presence of g-core MtDef4 at 30 µg/ml.
TABLE 2. Activity of the g-core motif defensin peptide constructs and full-length defensin peptides against fungal and oomycete alfalfa crown rot pathogens
a
Defensin
Fusarium oxysporum
f. sp. medicaginis Phoma medicaginis Colletotrichum trifolii
Aphanomyces
euteiches
7F-3 31F-3 STC WS-2 FG-1 WS-5 Race 1 Race 2 F. solani F. tricinctum F. redolens F. incarnatum
Core MsDef1 NA NA 12.7 ±1.1 14.8 ±1.0 NA NA NA NA NA NA NA NA
Core MtDef4 7.1 ±0.8 6.9 ±0.8 7.3 ±0.7 5.3 ±0.7 NA NA NA NA 6.0 ±1.0 14.7 ±1.3 NA NA
Core MtDef5 NA NA 19.5 ±1.2 8.5 ±1.0 NA NA NA NA 4.1 ±0.5 NA NA NA
Core RsAFP2 NA NA NA NA NA NA NA NA NA 5.3 ±0.5 NA NA
Core So-D2 33.1 ±1.9 NA 6.4 ±0.6 6.1 ±0.6 NA NA NA NA 13.8 ±0.9 NA NA NA
MtDef4 0.7 ±0.1 1.9 ±0.1 0.3 ±0.1 2.6 ±0.1 NA NA NA NA ND ND ND ND
MtDef5 0.8 ±0.1 1.3 ±0.1 1.5 ±0.1 1.6 ±0.1 NA NA NA NA ND ND ND ND
a
The mean amount of defensin needed to inhibit growth of the fungal pathogens strains by 50% (µM) values are reported ±standard error of three independent
experiments (n=3). NA indicates that the defensins at a concentration of 30 µg/ml showed no biological activity against the pathogens and ND indicates no data.
404 PHYTOPATHOLOGY

Sinorhizobium meliloti,orX. alfalfae subsp. alfalfae to varying
degrees (Fig. 3). The g-core peptide from MtDef4 displayed the
greatest antibacterial activity of the g-core motif peptides tested.
However, neither MtDef4 nor MtDef5 g-core motifs inhibited the
growth of the beneficial, nitrogen-fixing microsymbiont S. meliloti.
Overall, the g-core peptides displayed the greatest growth inhibition
against E. coli using the filter paper disk assay.
Using a more quantitative spread-plate assay, the antibacterial
activity of the g-core motifs from MsDef1, MtDef4, MtDef5, and
So-D2 against the bacterial plant pathogens were evaluated and
found to exhibit antibacterial activity at micromolar concentrations.
The MtDef4 and MtDef5 g-core motifs inhibited the growth of
P. syringae pv. syringae with IC
50
values of 3.4 and 4.5 µM,
respectively (Table 3). Notably, the MtDef4 g-core motif peptide
but not the MtDef5 g-core peptide displayed antibacterial activity
against X. alfalfae subsp. alfalfae.
The full-length defensins MtDef4 and MtDef5 were also
characterized for antibacterial activity using the spread-plate
method. In addition to the previously tested Gram-negative bacte-
ria, antibacterial activity against a Gram-positive alfalfa bacterial
wilt bacterium, Clavibacter insidiosus, was evaluated. The antibac-
terial activity of full-length MtDef4 and MtDef5 was enhanced as
compared with their corresponding g-core motifs. MtDef4 and
MtDef5 had IC
50
values at nanomolar concentrations. In accordance
with the g-core motif results, MtDef4 and MtDef5 exhibited high
activity against P. s y r i n g ae pv. syringae,withIC
50
values of 400 and
100 nM, respectively (Table 3). MtDef4 was most active against
C. insidiosus,withanIC
50
value of 100 nM. Again, MtDef5
displayed no antibacterial activity against X. alfalfae subsp. alfalfae,
which further supports the predictive capacity of the g-core motif
peptides. The broad antibacterial activity of MtDef4 and MtDef5
against plant bacterial pathogens led us to conduct antibacterial tests
against human pathogens using the spread-plate assay. MtDef4 and
MtDef5 g-core peptides displayed low IC
50
values against the
majority of human bacterial pathogens tested, with Enterobacter
aerogenes being the most sensitive to both MtDef4 and MtDef5
(Table 4). No antibacterial activity was seen against the Gram-
positive bacterium Enterococcus casseliflavus.
DISCUSSION
Plant defensins are well known to have activity against plant
fungal pathogens, inhibiting in vitro growth as well as reducing
damage from fungal diseases when expressed in heterologous plant
hosts. We tested plant defensin peptides against diverse pathogens
and found that they displayed broad inhibitory activity against plant
fungal pathogens causing alfalfa crown rot disease along with,
remarkably, both human and plant bacterial pathogens. In addition
to displaying extensive antifungal activity, MtDef4 had strong and
broad-spectrum antibacterial activity with nanomolar IC
50
values
against both Gram-positive and Gram-negative bacteria (Table 3).
There are few reports that cite antibacterial activity of plant
defensins because, traditionally, they havebeen regarded to possess
solely antifungal activity (Fujimura et al. 2003; Guill´
en-Chable
et al. 2017; Segura et al. 1998; Velivelli et al. 2018). Spinach
defensin (So-D2) displayed high activity against both Clavibacter
michiganensis subsp. sepedonicus and Ralstonia solanacearum,
which correspond to Gram-positive and Gram-negative bacteria,
but So-D2 displayed limited activity against fungal pathogens
(Segura et al. 1998). MtDef4, MtDef5, and MsDef1 are noteworthy
defensins because they exhibit potency against Gram-positive,
Gram-negative, and fungal pathogens. Our report demonstrates that
extensively studied plant defensins can have high antibacterial
activity against human and plant pathogens, which was previously
overlooked. This suggests that other well-characterized plant
defensins should be retested for antibacterial activity.
Of the fungi tested, none of the plant defensins inhibited
Colletotrichum trifolii,F. redolens,orF. incarnatum (Table 2).
Defensin antimicrobial specificity is commonly observed. For
example, RsAFP2 demonstrated extensive antifungal activity but
displayed no activity against either Sclerotinia sclerotiorum or
Rhizoctonia solani (Terras et al. 1992). This pathogen specificity
could occur due to diverse modes of action, resulting from the rich
diversity of the primary amino acid sequences of plant defensins.
RsAFP-1 and RsAFP-2 differ from each other by only 2 amino acids
in the primary structure but exhibit a striking difference in their
antimicrobial activity (Terras et al. 1992). The antifungal modes of
action of MsDef1, MtDef4, MtDef5, and RsAFP-2 all differ but
their molecular modes of action all involve interactions with fungal
membrane components (Cools et al. 2017; Islam et al. 2017; Parisi
et al. in press). These resistant fungal pathogens could have
structural differences in their membranes that inhibit defensin
recognition.
The M. truncatula defensins that we tested against alfalfa crown
rot pathogens and human bacterial pathogens are among the well-
characterized plant defensins and have potent activity against other
plant pathogens (Mun
˜oz et al. 2014; Sagaram et al. 2011, 2013).
When expressed in Arabidopsis and tomato, they give strong
protection against virulent fungal pathogens and protect the plants
from disease (Abdallah et al. 2010; Kaur et al. 2012; Sharma et al.
2017). However, the broad antibacterial activity of these defensins
had not been previously characterized. In contrast to the antifungal
modes of action, there is no proposed antibacterial mode of action
for any plant defensin. Both the full-length MtDef5 and its g-core
motif failed to inhibit the growth of X. alfalfae subsp. alfalfae
(Table 3), which was surprising because MtDef5 was previously
shown to be active against X. campestris (Velivelli et al. 2018). The
resistance of X. alfalfae subsp. alfalfae to MtDef5 may be due to the
presence of a homolog of MtDef5 in alfalfa (Scc4a34_1890, 78.8%
Fig. 2. Full-length MtDef4 inhibited growth of alfalfa fungal crown rot
pathogens in vitro. Spores of Phoma medicaginis were grown for 48 h at 25°C
in potato dextrose broth (PDB) culture medium in A, the absence or B,
presence of full-length MtDef4 at 30 µg/ml. Spores of Fusarium oxysporum f.
sp. medicaginis were grown for 48 h at 25°C in PDB culture medium in C, the
absence or D, presence of full-length MtDef4 at 30 µg/ml.
Vol. 109, No. 3, 2019 405

sequence identity). X. alfalfae subsp. alfalfae, a common alfalfa
pathogen, could have become resistant to the MtDef5 homolog in
alfalfa. Therefore, X. alfalfae subsp. alfalfae would be considered
an adapted pathogen that has overcome the antibacterial activity of
MtDef5. Plant defensins may be an excellent source for antibiotic
development because human bacterial pathogens would be
considered nonadapted to plant-derived defensins.
Currently, there are limited control and management strategies for
the alfalfa crown rot disease complex. Alfalfa crown rot is ubiquitous
and leads to stand decline, which leads to financial losses for the
growers. This disease complex poses a complex problem. Pathogens
gain entry into the crown through cut stems and mechanical damage
to the root and crown that occur during the multiple foliage harvests
throughout the year. Chemical control does not have the necessary
persistence because pathogens decay the crowns during a period of
months or years, predisposing them to winterkill and eventually
killing the plant. Breeding efforts to increase quantitative resistance
have made only minor progress (Miller-Garvin and Viands 1994).
This report establishes plant defensins as potential agents for
enhancing resistance to alfalfa crown rot, and possibly other diseases,
through genetic modification.
Obtaining functional defensin peptides through heterologous
expression can be a fastidious process. Amino acid substitutions,
improper folding, and incorrect disulfide bridge formation all
inhibit the biological activity of plant defensins (Vriens et al. 2014).
Eukaryotic expression systems such as the often-utilized Pichia
pastoris expression system can create constructs with the proper
structure, disulfide bonds, and posttranslational modifications but
the experimental setup can be difficult. Specialized Escherichia
coli-based bacterial expression systems can generate copious
Fig. 3. Antibacterial activity of plant defensin g-core motif peptides. On bacterial lawns of Escherichia coli,Pseudomonas syringae pv. syringae,Sinorhizobium
meliloti,orXanthomonas alfalfae subsp. alfalfae, the area of the zone of inhibition was measured around blank filter paper disks spotted with varying
concentrations of the g-core defensin peptides. The g-core defensin peptides tested were A, MtDef4; B, MtDef5; C, MsDef1; and D, RsAFP2. Bars represent means
and error bars indicate standard error (n=9).
TABLE 3. Activity of the g-core motif defensin peptides and full-length
defensin peptides against bacterial alfalfa crown rot pathogens
a
Defensin
Xanthomonas alfalfae
subsp. alfalfae
Pseudomonas
syringae pv.
syringae
Clavibacter
insidiosus
Core MtDef4 11.4 ±0.2 3.4 ±0.4 ND
Core MtDef5 NA 4.5 ±0.5 ND
Core So-D2 19.3 ±2.2 25.9 ±1.2 ND
Core MsDef1 7.9 ±0.7 8.8 ±1.1 ND
MtDef4 0.6 ±0.04 0.4 ±0.05 0.1 ±0.01
MtDef5 NA 0.1 ±0.01 NA
a
The mean amount of defensin needed to inhibit growth of the bacterial
pathogens strains by 50% (µM) values are reported ±standard error of three
independent experiments (n=3). NA indicates that the defensins at a
concentration of 30 µg/ml showed no biological activity against the
pathogens and ND indicates no data.
406 PHYTOPATHOLOGY

amounts of defensin peptides but these peptides have low biological
activity due to problematic structural integrity (Lacerda et al. 2014).
We have shown that truncated defensin peptides containing the
g-core motif can be chemically synthesized and may mimic the
relative antimicrobial activity of the full-length defensins (Tables 2
and 3). This warrants the further investigation of the predictive
capabilities of g-core motif defensin peptides from species other
than M. truncatula. In combination with the described microplate
and spread-plate methods, g-core motif peptides could be used to
quickly screen defensins for antimicrobial activity, which would
greatly simplify and expedite defensin bioassays.
In this report, we characterized the in vitro antifungal and
antibacterial activity of plant defensins against alfalfa crown rot
pathogens and human bacterial pathogens. Full-length defensins
were shown to have antimicrobial activity against both fungal and
bacterial pathogens at nanomolar concentrations. These experi-
ments show the previously overlooked high biological activity of
plant defensins against bacterial pathogens. Additionally, these
results indicate that the g-core motif peptide may be used to predict
the relative biological activity of the full-length defensin. Spe-
cifically, MtDef4 and MtDef5 were identified as ideal candidates
for transgenic expression in alfalfa due to their broad-spectrum
and strong antimicrobial activity. Transgenic expression of these
defensins could be utilized to implement an ecofriendly, protein-
based strategy that could provide alfalfa with enhanced resistance
against crown rot and growers with reciprocal gains in yield.
ACKNOWLEDGMENTS
We thank D. Foster-Hartnett, University of Minnesota, for providing the
American Type Culture Collection bacterial cultures. This article is a joint
contribution from the Plant Science Research Unit, United States
Department of Agriculture (USDA) Agricultural Research Service, and
the Minnesota Agricultural Experiment Station. USDA is an equal op-
portunity provider and employer. Mention of any trade names or com-
mercial products in this article is solely for the purpose of providing specific
information and does not imply recommendation or endorsement by the
U. S. Department of Agriculture.
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a
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