The Plant Journal (2003) 36, 390±400
doi: 10.1046/j.1365-313X.2003.01886.x
Overexpression of a cell wall glycoprotein in Fusarium
oxysporum increases virulence and resistance to a
plant PR-5 protein
Meena L. Narasimhan1,,y, Hyeseung Lee1,y, Barbara Damsz1, Narendra K. Singh2, Jose I. Ibeas1, Tracie K. Matsumoto1,
Charles P. Woloshuk3 and Ray A. Bressan1
1
Center for Plant Environmental Stress Physiology, 625 Agriculture Mall Drive, Purdue University, West Lafayette,
IN 47907-2010, USA,
2
Department of Biological Sciences, 101 Rouse Life Sciences Building, Auburn University, Auburn, AL 36849-5407,
USA, and
3
Department of Botany and Plant Pathology, 915 West State Street, Purdue University, West Lafayette, IN 47907-2054,
USA
Received 9 June 2003; revised 24 July 2003; accepted 8 August 2003.
For correspondence (fax ‡1 765 494 0391; e-mail [email protected]).
y
These two authors contributed equally to this work.
Summary
Fusarium oxysporum f. sp. nicotianae is a causal agent for vascular wilt disease in tobacco. It is sensitive to
osmotin, a tobacco pathogenesis-related protein (PR-5) that is implicated in plant defense against phytopathogenic fungi. We show that osmotin susceptibility of F. oxysporum f. sp. nicotianae was reduced by overexpression of the heterologous cell wall glycoprotein Saccharomyces cerevisiae protein containing inverted
repeats (PIR2), a member of the PIR family of fungal cell wall glycoproteins that protect S. cerevisiae from the
toxic action of osmotin. S. cerevisiae PIR2 was targeted to the cell wall of F. oxysporum. Disease severity and
fungal growth were increased in tobacco seedlings inoculated with F. oxysporum transformed with PIR2
compared to seedlings infected with untransformed F. oxysporum or that transformed with vector, although
accumulation of transcript and protein of defense genes was similar. The results show that fungal cell wall
components can increase resistance to plant defense proteins and affect virulence.
Keywords: cell wall, Fusarium, pathogenesis, plant defense, thaumatin-like.
Introduction
Pathogenesis-related (PR) proteins of plants are de®ned as
proteins that accumulate intra- or extracellularly under
pathological conditions (van Loon and van Strien, 1999).
Their importance to plant defense has been inferred
because they are induced in response to pathogen attack
or pathogen-derived elicitors, and their accumulation is
often associated with incompatibility. Many proteins have
antimicrobial activity in vitro, and overexpression of one
or more PR proteins in planta has been shown to protect
against disease symptom development (Yun et al., 1997a).
Much less is known about microbial factors that contribute
to plant disease because of their effect on the activity of
plant PR proteins.
In the case of plant±fungus interactions, the fungal cell
wall has a role in protecting the fungus from the deleterious
action of plant PR proteins (Veronese et al., 2003). This is
390
re¯ected in the fact that b-glucanases and chitinases, i.e.
enzymes that hydrolyze fungal cell wall polymers, are
represented among PR protein families. In the case of
the PR-5 protein family, whose members are structurally
related to thaumatin, some members have b-glucanase
activity in vitro (Grenier et al., 1999). However, it has not
been demonstrated that this activity is related to their
mechanism of antifungal action. Other PR-5 family members have no known enzymatic activity. Osmotin is a
tobacco PR-5 protein of the latter class. It accumulates in
tobacco cells in response to pathogen attack and osmotic
stress, and is thought to prevent further pathogen attack or
limit spread of disease (Liu et al., 1994; Singh et al., 1987).
Osmotin has wide-spectrum antifungal activity in vitro
(Abad et al., 1996), and overexpression in planta delays
onset of disease symptoms (Liu et al., 1994). Studies using
ß 2003 Blackwell Publishing Ltd
F. oxysporum resistance to a plant PR-5 protein
Saccharomyces cerevisiae as a model fungal target have
shown that osmotin induces fungal apoptosis, and that speci®c fungal cell wall components greatly in¯uence osmotin
antifungal action (Ibeas et al., 2000, 2001; Narasimhan
et al., 2001; Yun et al., 1997b).
The cell wall of the budding yeast S. cerevisiae is composed of glycoproteins that are covalently linked to or
entrapped in a network of the polysaccharides, glucans
(b-1,3-linked and b-1,6-linked) and chitin (Kapteyn et al.,
1999a). In S. cerevisiae, there are three classes of cell wall
glycoproteins (CWPs; Kapteyn et al., 1999a). Those extractable by detergent are presumed to be entrapped in the
wall. Glycoproteins that cannot be extracted by detergent
fall into two categories: glycosylphosphatidylinositol (GPI)CWPs and PIR-CWPs. GPI-CWPs are covalently linked at
their C-terminus, via the remnant of a GPI anchor, to b-1,6glucan and thereby to b-1,3-glucan. The GPI-CWPs possess
an N-terminal signal sequence, contain internal repeats, a
C-terminal GPI-anchor addition sequence and several putative N- and O-glycosylation sites. PIR-CWPs (PIR1/CCW6,
PIR2/CCW7/HSP150, PIR3/CCW8, and PIR4/CIS3/CCW11)
are a family of functionally equivalent and structurally
related CWPs (Moukadiri et al., 1999; Mrsa and Tanner,
1999; Mrsa et al., 1997; Toh-e et al., 1993; Yun et al.,
1997b). PIR is an acronym for protein containing internal
repeats, and the PIR proteins contain one or more repeats of
an internal sequence. They are highly O-glycosylated and
attached to cell wall b-1,3-glucan through an O-glycosidic
linkage (Kapteyn et al., 1999a; Mrsa et al., 1997; Russo
et al., 1992; Toh-e et al., 1993). PIR2 is induced by heat
shock (Russo et al., 1992). The PIR proteins contribute to
the reinforcement of the cell wall and are part of the
compensatory mechanisms that are activated when cell
wall weakening is caused by b-1,6-glucan de®ciency
(Kapteyn et al., 1999b; Mrsa and Tanner, 1999). Disruption
of three or all four PIR genes results in signi®cant weakening or altered structure of cell wall (Mrsa and Tanner, 1999)
and increased sensitivity to osmotin (Yun et al., 1997b). In
addition to PIR proteins, alkali-insoluble glucans and other
unidenti®ed cell wall components regulated by the SSD1
gene confer osmotin resistance to a susceptible yeast strain
(Ibeas et al., 2001). Mannosylphosphate modi®cation of
CWP-conjugated glycans increases osmotin sensitivity
(Ibeas et al., 2000). These cell wall components appear to
control passage of osmotin across the cell wall because
removal of the cell wall from osmotin-sensitive and -resistant strains renders spheroplasts equally osmotin sensitive
(Ibeas et al., 2000, 2001; Yun et al., 1997b).
Cell wall components of other fungi, particularly fungal
CWPs, are not well studied. It is known that cell walls of
other ascomycete fungi, including Fusarium oxysporum,
are composed of similar polymers, i.e. chitin, glucans
(b-1,3-linked and b-1,6-linked), and glycoproteins (Barran
et al., 1975; Schoffelmeer et al., 1996, 1999, 2001). Two
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 36, 390±400
391
proteins that cross-react with S. cerevisiae PIR2 antibodies
have been detected in the cell wall of Candida albicans, and
they seem to play an analogous role in compensatory
strengthening of the wall under weakening conditions
(Kandasamy et al., 2000; Kapteyn et al., 2000). QID74, a cell
wall protein of the ®lamentous fungus Trichoderma harzianum has highly conserved tandem repeats of a 59-residue
unit (Rey et al., 1998), suggesting that tandem repeated
sequences are characteristic of cell wall proteins of ®lamentous fungi as well as of S. cerevisiae. Electron microscopy and lectin binding studies indicate that the cell wall of
F. oxysporum also has an inner glucan and chitin layer
surrounded by a glycoprotein layer. A putative GPI-CWP,
fusarium extracellular matrix protein (FEM1), has been
identi®ed in F. oxysporum (Schoffelmeer et al., 2001).
Fusarium oxysporum is an osmotin-sensitive fungus that
causes vascular wilt disease in many important crops
including tobacco (Abad et al., 1996; Tjamos and Beckman,
1989). To test whether structurally related compounds
would perform analogous functions in the cell walls of
yeast and other ascomycete fungi, i.e. if CWPs could modulate susceptibility of a phytopathogen to osmotin, as in the
yeast model, we expressed S. cerevisiae PIR2 in F. oxysporum f. sp. nicotianae. PIR2 was chosen because it is
the most highly expressed member of the S. cerevisiae PIR
gene family (Yun et al., 1997b). We report that S. cerevisiae
PIR2 is correctly targeted to the F. oxysporum cell wall.
Deposition of S. cerevisiae PIR2 in the cell wall increases
both osmotin resistance and virulence of F. oxysporum.
These results demonstrate that fungal cell wall constituents
that block access of non-enzymatic plant antifungal proteins to their plasma membrane targets can in¯uence both
fungal growth and development of plant disease.
Results
Transformation of F. oxysporum with S. cerevisiae PIR2
Saccharomyces cerevisiae PIR2 was cloned into the fungal
expression vector pBARGPE1 that was designed for the
expression of open-reading frames (ORFs) from their
own initiation codons by providing a multiple cloning site
between the highly expressed Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpdA) promoter
and the A. nidulans tryptophan biosynthetic pathway gene
encoding glutamine amidotransferase, indoleglycerolphosphate synthase and phosphoribosylanthronilate isomerase
activities (trpC ) terminator (Pall and Brunelli, 1993). The
resulting plasmid was called pBARGPE1-PIR2. Although
pBARGPE1 also contains the bialaphos resistance gene
(BAR) as the fungal selection marker, direct selection of
fungal transformants using bialaphos was not successful.
Therefore, pBARGPE1-PIR2 and pBARGPE1 were introduced
392 Meena L. Narasimhan et al.
Figure 2. Fusarium oxysporum transformants expressing S. cerevisiae
PIR2 have increased osmotin resistance.
Hyphal lengths of germlings of wild-type (wt) strain, vector transformants
(22V, 23V) and PIR2 transformants (9, 14, 16, 20, and 34) were measured after
incubation of conidia (105 conidia ml 1) with the indicated concentrations of
osmotin for 20 h at room temperature. Lengths of 25 hyphae were measured
for each sample, and data of triplicate samples were averaged. Shown are
the average hyphal lengths at each osmotin concentration, expressed as a
percentage of the average hyphal length in absence of osmotin. The average
hyphal lengths SE in absence of osmotin were (in micrometers)
1.40 0.05, 1.38 0.06, 1.37 0.06, 1.28 0.06, 1.22 0.06, 1.34 0.06,
1.20 0.06, and 1.13 0.05 for strains wt, 22V, 23V, 9, 14, 16, 20, and 34,
respectively.
Figure 1. Evidence for transformation of F. oxysporum.
(a, b) Shown are blots of restriction endonuclease-digested genomic DNA
(a; 15 mg per lane) or total RNA (b; 10 mg per lane) of pBARGPE1-PIR2
transformed (9, 14, 16, 20, and 34) and wild-type (wt) F. oxysporum strains
probed with 32P-labeled PIR2 probe.
(c) Shown is a blot of restriction endonuclease-digested genomic DNA
(15 mg per lane) of pBARGPE1 transformed (22V, 23V) and wt strains probed
with 32P-labeled BAR probe. Shown are the restriction endonucleases used
for digestion.
into F. oxysporum f. sp. nicotianae along with pUCH1, a
plasmid bearing the hygromycin resistance gene, to facilitate selection of transformants. Single-spore isolates of
hygromycin-resistant pBARGPE1-PIR2 and pBARGPE1 cotransformants were checked, by PCR with gene-speci®c
primers, for the presence of insert and vector sequences
(PIR2 and BAR, respectively). Five independent pBARGPE1PIR2 transformants (PIR2 transformants) as well as two
independent pBARGPE1 transformants (vector transformants) were identi®ed by Southern blot analysis of the
PCR-positive clones using PIR2 and BAR genes as probes,
respectively (Figure 1a,c). Expression of S. cerevisiae PIR2
in the PIR2 transformants was con®rmed by detecting the
transcripts in total RNA isolated from mycelia (Figure 1b).
The PIR2 probe failed to detect any transcripts in the
untransformed F. oxysproum strain (Figure 1b) and in
the vector transformants (data not shown).
Saccharomyces cerevisiae PIR2 expression decreases
osmotin susceptibility of F. oxysporum
Conidia of the ®ve independent PIR2 transformants, two
independent vector transformants, and wild-type F. oxy-
sporum strain were germinated in the presence of various
osmotin concentrations, and the effect of osmotin on
hyphal length was determined (Figure 2). By analysis of
variance (ANOVA), at the level of a ˆ 0.05, the P-value
between the average hyphal lengths of wild-type strain
and the two vector transformants at each osmotin concentration tested was >0.05, indicating no signi®cant
difference in osmotin resistance between wild-type F. oxysporum strain and vector transformants. No statistical difference could be detected between average hyphal lengths
of PIR2 transformants and wild-type strain in absence of
osmotin. However, the P-values between average hyphal
lengths of PIR2 transformants and wild-type strain at each
osmotin concentration tested were <0.01, indicating signi®cant differences between the two groups. That is, at each
osmotin concentration tested, the average hyphal length of
the PIR2 transformants was signi®cantly greater than that
of the vector transformants or wild-type strain. The same
results, depicting the average hyphal lengths as a percentage of the average hyphal length in absence of osmotin,
are shown in Figure 2. Clearly, expression of S. cerevisiae
PIR2 in F. oxysporum rendered the fungus more resistant to
osmotin-induced inhibition of hyphal elongation.
Saccharomyces cerevisiae PIR2 is deposited on the
cell wall of F. oxysporum
The subcellular location of PIR2 was investigated using an
immunocytochemical method. The results of the immunolocalization experiments using polyclonal antibody raised
against recombinant PIR3 protein that also recognizes PIR1
and PIR2 (Yun et al., 1997b) are depicted in Figure 3.
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 36, 390±400
F. oxysporum resistance to a plant PR-5 protein
393
Figure 3. Saccharomyces cerevisiae PIR2 is targeted to the cell wall of F. oxysporum.
Ultrathin sections of wild-type (wt) strain (a,b,e,f) and a PIR2 transformant (c,d) were reacted with pre-immune (a,c,e) or anti-PIR3 (b,d,f) serum. PIR2 proteins
were detected with secondary antibodies conjugated to 10-nm gold particles, which appear as small black dots at the sites of positive reaction. The average
densities of gold particles per mm2 of cell wall in the samples (a±d) were, respectively, 194 13.3, 304 17.7, 182 13.3, and 730 41.3 (n 10 cells). CW, cell
wall; S, septum; W, Woronin body.
Proteins that cross-react speci®cally with anti-PIR3 antibody were detected on the cell wall and Woronin bodies,
in both wild-type strain and PIR2 transformant. Woronin
bodies are believed to be specialized peroxisomes that act
as septal plugs. They seem to function under cellular stress
conditions, in the regulation of cytoplasmic ¯ow and maintenance of cellular integrity (Calvo and Agut, 2000; Jedd
and Chua, 2000; Tenney et al., 2000; Wergin, 1973). Thus,
the cell wall protein(s) detected in the cell walls and Woronin bodies of wild-type strain that is serologically related
to S. cerevisiae PIR2 also behaves as a stress protein. The
average density of gold particles per micrometer square of
cell wall, after subtracting the background counts obtained
with pre-immune serum, was 110 in wild-type strain and
548 in the PIR2 transformant. Enrichment (®vefold) of PIR2
cross-reacting material in the cell wall of the PIR2 transformant indicates that the heterologous protein is correctly
targeted to the cell wall in the fungus.
Osmotin-resistant PIR2 transformants of F. oxysporum
have greater virulence
Fusarium oxysporum infection is typically studied in soilgrown plants by a root dip assay. As shown in Figure 4(a),
the pathogen causes stunting of shoot and root, as well as
chlorosis of lower leaves. Using a root dip assay in soil and
measuring total leaf area as indicator of growth, it was
observed that the mean total leaf area of plants inoculated
with the PIR2 transformants was less than that of plants
inoculated with wild-type strain and both values were signi®cantly lower than the mean total leaf area of mockß Blackwell Publishing Ltd, The Plant Journal, (2003), 36, 390±400
inoculated controls (not shown). However, the variation
between individual plants was such that the difference
between the two fungus-inoculated groups was not statistically signi®cant.
It has been reported that osmotin is induced upon fungal
infection (Liu et al., 1994) and detectable amounts accumulate in seedlings grown in closed containers such as Petri
dishes (Xu et al., 1994). As wild-type strain and PIR2 transformants differ in osmotin sensitivity, it was reasoned that
if osmotin was constitutively expressed in sterile-grown
seedlings, this could be a good system to test for differences in the infectivity between the fungal strains. Accordingly, tobacco seedlings were grown aseptically on a
cellophane membrane to the three to four-leaf stage. To
maximize fungal growth on the seedlings, the membrane
was then moved to a nutrient-free medium, and the seedlings were acclimated for 2 days before inoculation. The
results of such experiments are shown in Figures 4±6.
On the day of inoculation, as expected, osmotin gene
expression and protein were detected in root and shoot
tissues of the seedlings (Figure 5). Disease symptoms of
the F. oxysporum-inoculated seedlings were stunting of
root and shoot, and at later stages, chlorosis of lower leaves
(Figure 4b±d; data not shown). Thus, they were similar to
disease symptoms of soil-grown plants.
When low pathogen pressure was used, no difference
was observed between shoots of wild-type, vector transformant, and mock-inoculated control seedlings at 18 days
after inoculation (dai) (Figure 4b,d). There was a slight reduction in root length in wild-type and vector transformantinoculated seedlings compared to the mock-inoculated
394 Meena L. Narasimhan et al.
controls. However, the average root wet weight per seedling that accounts for both root length and the number of
roots emerging from the crown was not signi®cantly different (Figure 4c,d). Inoculation with the PIR2 transformants
caused greater stunting than that with the wild type or
vector transformants. The difference between disease
symptoms of seedlings inoculated with the PIR2 transformants and the other two groups was noticeable from about
15 dai (data not shown). The stunting was evident in the size
of leaves (Figure 4b), length of roots (Figure 4c), and in the
average shoot or root wet weight per seedling (Figure 4d).
Similar results were obtained in two more replicate experi-
Figure 5. Tobacco seedlings constitutively express osmotin at the time of
infection.
During the experiments described in Figure 4, some seedlings were harvested just before inoculation with fungus. Shown are analyses of osmotin
gene expression (a) and levels of osmotin protein (b) at this stage, in shoot
and root tissues. Analyses were performed as described in Figure 4. For
protein analysis, the lanes contained basic cellular proteins isolated from
550 mg total shoot proteins and 40 mg total root proteins.
ments. Analysis of defense gene expression at this stage
showed that the fungus induced a defense response in leaf
tissues (Figure 4e). Osmotin (PR-5), basic chitinase (PR-3),
and basic PR-1 transcripts were all more abundant in
fungus-inoculated seedlings than in mock-inoculated
seedlings. Osmotin protein levels were also elevated in
leaf tissues of fungus-inoculated seedlings compared to
mock-inoculated seedlings (Figure 4f). However, signi®cant levels of osmotin transcript, chitinase transcript, and
osmotin protein were also detected in shoots of mockinoculated seedlings. As the magnitude of the defense
response and osmotin levels were similar in both fungusinoculated samples, it was concluded that the difference
in disease symptoms elicited by the PIR2 transformant and
wild-type F. oxysporum strains could not be attributed to
protection conferred to the fungus by an altered plant
defense response.
Fusarium oxysporum is expected to penetrate through
the root and ascend into the shoot system via the vascular
system. In later stages of infection (>30 dai), the lower
leaves became chlorotic and the upper leaves remained
green in fungus-inoculated seedlings, whereas no chlorosis
Figure 4. Fusarium oxysporum transformants expressing S. cerevisiae
PIR2 have increased virulence.
(a) Shown are the symptoms, at 11 dai, of soil-grown plants mock-inoculated (mock) or inoculated with wild-type (wt) strain of F. oxysporum by a
root dip assay.
(b±d) Shown are representative shoots (b), roots (c), and average FW per
shoot or root SE (d), at 18 dai, of seedlings that were mock-inoculated
(mock), inoculated with wt strains, and strains transformed with vector (22V,
23V) or S. cerevisiae PIR2 (9, 14, 16, 20, and 34). Data were obtained from 12
to 19 seedlings inoculated with each fungal strain. Infection was performed
on seedlings grown in Petri dishes on membranes placed on 0.8% agar as
described in Experimental procedures.
(e, f) Shown are analyses of defense gene expression (e) and levels of
osmotin protein (f) at this stage, in shoot tissues. These analyses were
performed in a parallel experiment with about 120 seedlings that were either
mock-inoculated (mock), inoculated with wt strain, or the strain transformed
with S. cerevisiae PIR2 (16). Expression of osmotin (PR-5), chitinase (PR-3),
PR-1, and a±tubulin as control was analyzed by RT-PCR using gene-speci®c
primers. Osmotin levels were measured on immunoblots of the basic
cellular proteins separated by 12% SDS±PAGE. The lanes contained the
basic cellular proteins isolated by CM-Sephadex chromatography from 900,
900, and 750 mg of total cellular proteins from mock-, wt- and PIR2 transformant (16)-inoculated seedlings, respectively.
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 36, 390±400
F. oxysporum resistance to a plant PR-5 protein
395
Figure 6. Fusarium oxysporum transformants expressing S. cerevisiae PIR2 spread to a greater extent.
Infection was performed on seedlings grown in Petri dishes on membranes placed on 0.8% agar as described in Experimental procedures. Seedlings were mockinoculated (mock), inoculated with wild-type (wt) strain, and strains transformed with vector (22V, 23V) or S. cerevisiae PIR2 (9, 14, 16, 20, and 34).
(a, b) At 39 dai, the 5th and 6th leaves of each plantlet were stained with trypan blue and rated for fungal growth as: 0, no fungus; 1, some fungi on edge of leaf; 2,
upto one-third of the leaf covered with fungus; 3, one-third to two-thirds of the leaf covered with fungus; 4, whole leaf covered with fungus; 5, whole leaf covered
with fungus, but more densely than in other categories. Data were obtained from 12 to 18 seedlings inoculated with each fungal strain. The number of leaves
receiving each fungal growth rating is shown in (a). Shown in (b) are the tips of trypan blue-stained leaves to illustrate the fungal density corresponding to the
indicated fungal growth ratings. The density of fungal growth was the same for growth ratings 2, 3, and 4, although the area of the leaf that was covered with
fungus increased.
(c, d) Shown are analyses of defense gene expression (c) and levels of osmotin protein (d) at this stage of infection, in shoot tissues. These analyses were
performed in a parallel experiment with about 120 seedlings that were either mock-inoculated (mock), inoculated with wt strain, or the strain transformed with
S. cerevisiae PIR2 (16). Analyses of gene expression and osmotin levels were as described in Figure 4. For protein analysis, each lane contained the basic cellular
proteins isolated from 850 mg of total cellular proteins.
was observed in mock-inoculated seedlings (data not
shown). At 39 dai when the seedlings had eight to nine
leaves, trypan blue staining was performed on the ®fth leaf
(mostly green, with some leaves showing mild chlorosis)
and the sixth leaf (variable degrees of chlorosis) of every
plantlet to measure fungal spread. The leaves were visually
scored for presence of fungus on an arbitrary scale based
on the area of the leaf showing detectable fungus
(Figure 6a,b). No fungus was detected on mock-inoculated
seedlings. More fungus was detected on the lower (6th) leaf
than on the upper (5th) leaf in all infected seedlings as
expected, if the fungus spreads upwards from the roots.
Furthermore, whether the ®fth or the sixth leaves were
compared, more fungus was detected on leaves of seedlings infected with the PIR2 transformants than on those
infected with wild-type strain or vector transformants.
Again, analysis of defense gene expression (Figure 6c)
and osmotin levels (Figure 6d) at this stage showed that
the difference in the degree of fungal penetration could not
be attributed to protection conferred to the fungus by an
altered plant defense response. Constitutive accumulation
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 36, 390±400
of defense gene transcripts and osmotin protein was
observed in leaf tissues of mock-inoculated seedlings.
Fungal infection induced accumulation of osmotin protein
and chitinase transcripts. Osmotin and basic PR-1 gene
expression remained at constitutive levels. No signi®cant
difference was observed in defense gene expression or
osmotin levels between seedlings inoculated with wildtype strain and those inoculated with PIR2 transformant.
Thus, as expected, the PIR2 tranformants that have the
capacity for greater hyphal elongation in the presence of
osmotin than that of wild-type strain or vector transformants were able to induce more severe plant disease
symptoms and colonize to a greater extent in the seedlings.
The difference in virulence could not be attributed to an
altered plant defense response and therefore appeared to
result from the S. cerevisiae PIR2 transgene expression.
Discussion
We have shown that S. cerevisiae PIR2 is correctly targeted to the cell wall in a heterologous fungal species,
396 Meena L. Narasimhan et al.
F. oxysporum. Deposition of S. cerevisiae PIR2 in the
F. oxysporum cell wall increases both osmotin resistance
of that fungus and its virulence.
The vector transformants were indistinguishable from
wild-type strain in the osmotin-induced growth inhibition
tests and in the virulence tests (Figures 2, 4, and 6).
Although the level of PIR2 gene expression varied
(Figure 1b), the magnitude of osmotin-induced hyphal
growth inhibition was not signi®cantly different between
the PIR2 transformants at each osmotin concentration. All
PIR2 transformants were more osmotin resistant than
were the vector transformants and wild-type control
strains (Figure 2; data not shown). A similar degree of
stunting was induced in tobacco seedlings upon inoculation with each of the various PIR2 transformants, which
were all more virulent than the control strains (Figure 4;
data not shown). This lack of correlation between degree
of PIR2 gene expression and osmotin resistance or virulence could be ascribed to the fact that: (i) osmotin sensitivity of a fungus depends on the composition of the assay
medium (Abad et al., 1996; Ibeas et al., 2000); (ii) the IC50
for osmotin in in vitro assays depends on the concentration of fungal cells (M. L. Narasimhan, unpublished data);
and (iii) even at the lowest transcript level, the amount of
PIR2 protein in the cell wall may be saturating for osmotin
resistance.
Occurrence of CWPs with PIR-like structure and function
maybe fairly widespread in fungi
Saccharomyces cerevisiae PIR2 is a highly glycosylated
cell wall protein covalently linked to b-1,3-glucan that
renders yeast cell walls less permeable to osmotin and,
consequently, increases osmotin resistance of yeast cells
(Kapteyn et al., 1999a,b; Yun et al., 1997b). Conservation
of subcellular location and at least one function of S. cerevisiae PIR2 upon heterologous expression in a ®lamentous fungus suggest that secretion and perhaps
glycosylation signals are conserved between fungal species. Additionally, mechanisms for cross-linking may also
be conserved. A protein(s) that cross-reacts with S. cerevisiae PIR3 polyclonal antibody was detected by an
immunocytochemical method on cell walls of wild-type
F. oxysporum strain (Figure 3), and bands that hybridized
to the S. cerevisiae PIR2 probe were detected on Southern
blots of its genomic DNA under reduced stringency (not
shown). Heat-regulated proteins serologically related to
PIR2 have been detected in the yeasts C. albicans and
Schizosaccharomyces pombe (Russo et al., 1992). PIR-like
proteins have been localized to the cell wall of C. albicans,
and their role in compensatory strengthening of the cell
wall under b-1,6-glucan de®ciency conditions appears to
be conserved between C. albicans and S. cerevisiae
(Kapteyn et al., 1999b, 2000). The properties of the
F. oxysporum CWP(s) that is serologically related to PIR
proteins are not known. It is possible that the effects of the
S. cerevisiae PIR2 protein are a result of overexpression or
some unique property of the yeast protein. However,
serological detection of the PIR-related Fusarium protein(s) in Woronin bodies suggests a role in protection
against stress similar to that for the PIR proteins. Thus, the
occurrence of CWPs with PIR-like structure and function
maybe fairly widespread in cell walls of various fungal
species.
Pathogenesis-related proteins have a role in controlling
Fusarium wilt disease
The connection between host antimicrobial proteins/
peptides and the protection that they offer against F. oxysporum disease is tenuous at best. Many of these proteins
are induced in compatible interactions concomitantly with
the appearance of disease symptoms, some others are
induced in incompatible interactions, and some in both
(Figures 4 and 6; Benhamou et al., 1989, 1994; Krebs and
Grumet, 1993; Rep et al., 2002; Van Pelt-Heerschap and
Smit-Bakker, 1999). It has been hypothesized that the
timing of induction is important, with rapidly induced
proteins being important for containing the fungus
(Benhamou et al., 1989; Krebs and Grumet, 1993; Rep
et al., 2002). This is supported by the observation that
constitutive overexpression of a thionin protects Arabidopsis thaliana seedlings against F. oxysporum f. sp.
matthioli (Epple et al., 1997), and by the fact that pretreatment of tomato seedlings with chitosan, which
induces b-glucanase (PR-2), protects against subsequent
infection by F. oxysporum f. sp. radicis-lycopersici
(Benhamou et al., 1994; Krebs and Grumet, 1993). Our
demonstration that forti®cation of the fungal cell wall
against at least one host-defensive protein resulted in a
measurable increase in virulence of the pathogen reinforces the concept that plant PR proteins have a defensive
role against vascular wilt pathogens, particularly F. oxysporum. Speci®cally, our results demonstrate that the
antimicrobial activity of PR-5 proteins aids in controlling
colonization of host plants by F. oxysporum. This is particularly true if the protein is constitutively expressed
(Figures 4±6), and by extension, maybe true also if the
protein is induced early in the plant±pathogen interaction.
Rep et al. (2002) reported that a protein with 92% identity
to osmotin at the protein level is the only PR protein that
accumulates in xylem sap in an incompatible F. oxysporum f. sp. lycopersici±tomato interaction and earlier
than all other PR proteins in a compatible interaction. Our
data and the sensitivity of F. oxysporum f. sp. lycopersici
to osmotin (Abad et al., 1996) suggest that this PR-5
protein may have a role in controlling the extent of F. oxysporum colonization of tomato.
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 36, 390±400
F. oxysporum resistance to a plant PR-5 protein
Fungal cell wall barriers to plant PR protein action affect
progression of fungal disease in the host
Interaction of F. oxysporum with host plants is always
species speci®c. Disease symptom development always
parallels the extent of fungal colonization in compatible
interactions, as we observed, but the fungus is contained
at the site of penetration in incompatible interactions
(Figures 4 and 6; Benhamou and Garand, 2001; Benhamou
et al., 1989; Gao et al., 1995; Lafontaine and Benhamou,
1996; Mueller and Morgham, 1996). The extent of fungal
colonization is controlled by fungal factors involved in
speci®c interaction with plant gene products (Veronese
et al., 2003). Our results show that these speci®c interactions can be mediated by speci®c fungal CWPs (such as
S. cerevisiae PIR2) that increase resistance of fungi to
plant defense antimicrobial proteins (such as osmotin).
Osmotin is the only one of a battery of antifungal proteins
and peptides synthesized by the tobacco plant that are
presumed to function synergistically in defense against
pathogens (Veronese et al., 2003). The activity of the other
tobacco antimicrobial proteins and peptides against
F. oxysporum f. sp. nicotianae is not known. It is possible
that S. cervisiae PIR2 affects the ef®cacy of more than one
host defense protein, resulting in increased disease
symptom development and fungal penetration that we
observed in seedlings inoculated with PIR2 transformants.
It also remains to be proven that PIR-like proteins of the
cell walls of phytopathogenic fungi have a natural function as resistance factors or `barriers' for plant defense
proteins, as our results imply. It follows that fungal cell
wall components having a natural function as `barriers' for
several plant defense proteins must have a large effect on
the ability of the fungus to elicit disease and are potential
targets of novel antifungals. Greater knowledge of the
shared `barrier' cell wall components of fungi, their structure, the plant defense proteins that they are effective
against, and evaluation of their relevance for pathogenesis should improve our ability to protect against plant
disease.
397
DNA and RNA manipulations
pBARGPE1-PIR2 was constructed by cloning the ORF of S. cerevisiae PIR2 into the XhoI and SmaI sites of the polylinker of the
plasmid pBARGPE1 (Pall and Brunelli, 1993). The plasmid pUCH1,
containing the gene for resistance to hygromycin B, has been
described by Bej and Perlin (1989).
For isolation of genomic DNA, conidia were inoculated into PDB
(10 ml) contained in Petri dishes and allowed to grow for 2 days at
288C without shaking. Mycelia were harvested, frozen in liquid
nitrogen, and stored at 808C. Genomic DNA was isolated from the
mycelia as described by Woloshuk et al. (1995). For Southern
hybridization, aliquots of restriction enzyme-digested genomic
DNA were fractionated on a 0.8% agarose gel. The DNA was
transferred to a nitrocellulose membrane and hybridized overnight
at 428C with 32P-labeled probes in hybridization buffer containing
50% formamide (Sambrook et al., 1989). The probes were prepared by random primer labeling of the PIR2 or BAR coding
regions (Ready-to-Go labeling system, Amersham Biosciences,
Piscataway, NJ, USA). The membrane was washed at 428C, successively in 2, 1, 0.5, and 0.1 SSC containing 0.1% SDS for
20 min each and then exposed to X-ray ®lm at 808C for 18±48 h.
For isolation of fungal RNA, conidia were inoculated into PDB
containing 1 M KCl (250 ml) and incubated for 48±60 h at 288C with
shaking. Mycelia were harvested, frozen in liquid nitrogen, and
stored at 808C. Total RNA was extracted using TRIzol reagent (Life
Technologies, Gaithersburg, MD, USA) according to the manufacturer's recommendation. Plant total RNA was extracted from shoot
or root tissues using the Plant RNeasy Mini Kit (Qiagen, Chatsworth, CA, USA) according to the manufacturer's instructions. For
Northern analysis, total RNA was fractionated on 1.5% denaturing
formaldehyde gels and transferred to nitrocellulose membrane
(Sambrook et al., 1989). Hybridization and washing conditions
were as described for Southern analysis. For RT-PCR, 4 mg of total
RNA were used for ®rst-strand cDNA synthesis using Superscript II
RNaseH-Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA)
and 10% of the ®rst-strand reaction was used for PCR. The
gene-speci®c primer pairs used were: 50 -CTTCCTCCTTGCCTTGGTGACTTA-30 /50 -TTGGGTGAGCTTGACCATTAGGAC-30 for tobacco
osmotin (locus S40046); 50 -TAACCCTCACAATGCAGCTCGTAG-30 /
50 -CATTGTTGCTTCGAACCCTAGCAC-30 for tobacco basic PR-1
(locus NTPRP-1); and 50 -TGCTGGGAACTTTACTGCCTC-30 /50 -CCAAGTTTTGATTTCTTCCCATAGTC-30 for tobacco a-tubulin (locus
NTA421411). The primer pair used for amplifying tobacco class
1a basic chitinase (locus NTCHN50) has been described by Yun
et al. (1996). PCR conditions were 27 cycles of 948C for 30 sec, 508C
for 30 sec, and 728C for 1 min.
Protein analysis
Experimental procedures
Fungal strains, media, and culture conditions
The wild-type strain of F. oxysporum f. sp. nicotianae was
obtained from Dr G. Chilosi (Universita della Tuscia, Viterbo).
Potato dextrose broth (PDB) and potato dextrose agar (PDA) used
for cultivating the fungus were purchased from Sigma (St Louis,
MO, USA). YEPD (0.3% yeast extract, 1% peptone, and 2% dextrose) was used as growth medium for protoplast preparation. For
harvesting conidia, the fungi were grown on PDA plates under
alternating 12-h light/12-h dark cycles at room temperature. All
cultures were stored in 15% glycerol at 808C.
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 36, 390±400
Plant tissues (0.5 g) were homogenized in Buffer A (20 mM potassium phosphate buffer, pH 6.0) at 48C using a Tekmar tissuemizer.
After centrifugation at 12 000 g for 10 min at 48C, the supernatant
was ®ltered through one layer of miracloth. Protein content of the
clear cell-free extract was estimated with the Bio-Rad protein assay
reagent using bovine serum albumin as standard.
Cell-free extract containing upto 1 mg protein was incubated
with 1 ml of a CM-Sephadex C-25 slurry (Sigma; 50% v/v slurry in
buffer A) at room temperature for 1 h with gentle shaking. The
slurry was then poured into a disposable plastic column and
allowed to drain. The column was washed with 12 bed volumes
of buffer A and then centrifuged at 400 g for 2 min to remove all the
liquid. Basic proteins were eluted three times with buffer B (buffer
A containing 0.5 M NaCl). Buffer B was added to the dry column
398 Meena L. Narasimhan et al.
bed in 0.2-ml aliquots and allowed to soak into the column bed for
5±10 min, and the eluate was collected by centrifugation as above.
Pooled buffer B eluates were treated with 6% trichloroacetic acid
for 30 min at 48C, and the precipitated proteins were collected by
centrifugation at 12 000 g for 10 min. Trichloroacetic acid was
removed by repeated washing with diethyl ether. The dry precipitates were solubilized by boiling in SDS±PAGE sample buffer for
3 min and applied quantitatively on a 12% gel for separation by
SDS±PAGE (LaRosa et al., 1992). Immunoblot analysis was performed as described using rabbit antiosmotin as the primary
antibody and alkaline phosphatase-conjugated secondary antibody (LaRosa et al., 1992).
Transformation procedure
Fusarium oxysporum was co-transformed with pBARGPE1-PIR2
(or vector, pBARGPE1) and pUCH1 (1 : 1, w/w) essentially as
described by Shim and Woloshuk (2001), except that 20 mg of
transforming DNA was used and 0.7 M sucrose was substituted
as the osmotic stabilizer in the protoplast regeneration step.
Hyphal growth inhibition assay
Osmotin was puri®ed to apparent homogeneity from NaCladapted suspension cultures of tobacco cells as described by
Singh et al. (1987). Conidia were harvested in 2 PDB from F. oxysporum cultures grown on PDA plates containing 20% polyethylene glycol (to increase and maintain the high expression of
transgene). Each conidial suspension was ®ltered through two
layers of sterile miracloth to remove mycelial fragments, and
conidial numbers were determined using a hemocytometer. To
test the effect of osmotin, equal volumes (100 ml) of conidial
suspension (1 104 conidia) and sterile osmotin solution (or
water) were mixed in wells of a 24-multiwell tissue culture plate.
The plates were incubated at room temperature in a chamber with
alternating 12-h light/12-h dark cycles. After 20 h, a 10-ml aliquot of
the samples was examined under a Nikon Optiphot microscope
(Nippon Kogaku K.K., Tokyo, Japan) and hyphal lengths were
measured with a micrometer. Lengths of hyphae from 25 germinated conidia from each of the three replicates per treatment were
measured.
Immunoelectron microscopy
Conidia were inoculated into PDB containing 1 M KCl (50 ml) and
allowed to grow overnight at 288C with shaking. Mycelia were ®xed
and embedded as described by Mulholland et al. (1994). Immunoreactions were performed on thin sections with anti-PIR3 polyclonal antibodies essentially as described by Yun et al. (1997b). All
observations were made on an EM200 transmission electron
microscope (Philips Electronic Instruments, Mahwah, NJ, USA).
Fungal virulence test
Tobacco (Nicotiana tabacum L. cv. Wisconsin 38) seeds were
surface-sterilized with 20% bleach (containing a drop of Tween
20) for 30 min, rinsed well, and allowed to germinate under sterile
conditions in Petri dishes on two layers of Whatman No.1 ®lter
papers wetted with 0.1 Murashige and Skoog salts solution (JRH
Biosciences, Lenexa, KS, USA). Ten-day-old seedlings of uniform
size were transferred onto cellophane membrane (Bio-Rad,
Hercules, CA, USA, no. 1650963) that was placed over growth
medium (4.33 g l 1 Murashige and Skoog salts, 1% sucrose,
0.8% agar, B5 vitamins, and myo-inositol, pH 5.7), and were grown
for 10±15 days until three to four leaves appeared. The seedlings
were then transferred to nutrient-free medium (0.8% agar) by
moving the entire cellophane membrane onto a fresh Petri dish.
After a 2-day acclimation period, the population of seedlings was
carefully adjusted for uniform size by picking out extremely large
or small members. Each seedling then received an aliquot (10 ml) of
conidial suspension (1 104 conidia ml 1) pipetted on the roots.
Mock-inoculated control seedlings received water. For the root dip
assay in soil, washed roots of soil-grown seedlings at the four to
®ve-leaf stage were dipped in a conidial suspension (2 108
conidia ml 1) for 30 min prior to re-planting. Plants were maintained in a growth chamber at 238C with a 16-h light/8-h dark cycle.
Trypan blue staining was performed as described by Shipton and
Brown (1962).
Acknowledgements
We thank Dr G. Chilosi for the fungal strain. We also thank
D. Sherman and the Life Sciences Microscopy Facility, Purdue
University, for their help. This work was supported in part by
funds from the NSF Award no. 9808551-MCB. This is Purdue
University Agricultural Research Program Paper no. 16 877.
References
Abad, L.R., D'Urzo, M.P., Liu, D., Narasimhan, M.L., Reuveni, M.,
Zhu, J.K., Niu, X., Singh, N.K., Hasegawa, P.M. and Bressan, R.A.
(1996) Antifungal activity of tobacco osmotin has speci®city and
involves plasma membrane permeabilization. Plant Sci. 118,
11±23.
Barran, L.R., Schneider, E.F., Wood, P.J., Madhosingh, C. and
Miller, R.W. (1975) Cell wall of Fusarium sulphureum. Part I.
Chemical composition of the hyphal wall. Biochim. Biophys.
Acta, 392, 148±158.
Bej, A.K. and Perlin, M.H. (1989) A high ef®ciency transformation
system for the basidiomycete Ustilago violacea employing
hygromycin resistance and lithium-acetate treatment. Gene,
80, 171±176.
Benhamou, N. and Garand, C. (2001) Cytological analysis of
defense-related mechanisms induced in pea tissues in response
to colonization by nonpathogenic Fusarium oxysporum Fo47.
Phytopathology, 91, 730±740.
Benhamou, N., Grenier, J., Asselin, A. and Legrand, M. (1989)
Immunogold localization of beta glucanases in two plants
infected by vascular wilt fungi. Plant Cell, 1, 1209±1221.
Benhamou, N., Lafontaine, P.J. and Nicole, M. (1994) Induction of
systemic resistance to Fusarium crown and root rot in tomato
plants by seed treatment with chitosan. Phytopathology, 84,
1432±1444.
Calvo, M.A. and Agut, M. (2000) Observation of Woronin bodies in
Arthrinium aureum by scanning electron microscopy. Mycopathologia, 153, 137±139.
Epple, P., Apel, K. and Bohlmann, H. (1997) Overexpression of an
endogenous thionin enhances resistance of Arabidopsis against
Fusarium oxysporum. Plant Cell, 9, 509±520.
Gao, H., Beckman, C.H. and Mueller, W.C. (1995) The nature of
tolerance to Fusarium oxysporum f. sp. lycopersici in polygenically ®eld-resistant marglobe tomato plants Physiol. Mol. Plant
Pathol. 46, 401±412.
Grenier, J., Potvin, C., Trudel, J. and Asselin, A. (1999) Some
thaumatin-like proteins hydrolyse polymeric beta-1,3-glucans.
Plant J. 19, 473±480.
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 36, 390±400
F. oxysporum resistance to a plant PR-5 protein
Ibeas, J.I., Lee, H., Damsz, B., Prasad, D.T., Pardo, J.M., Hasegawa,
P.M., Bressan, R.A. and Narasimhan, M.L. (2000) Fungal cell wall
phosphomannans facilitate the toxic activity of a plant PR-5
protein. Plant J. 23, 375±383.
Ibeas, J.I., Yun, D.J., Damsz, B., Narasimhan, M.L., Uesono, Y.,
Ribas, J.C., Lee, H., Hasegawa, P.M., Bressan, R.A. and Pardo,
J.M. (2001) Resistance to the plant PR-5 protein osmotin in the
model fungus Saccharomyces cerevisiae is mediated by the
regulatory effects of SSD1 on cell wall composition. Plant J.
25, 271±280.
Jedd, G. and Chua, N.H. (2000) A new self-assembled peroxisomal
vesicle required for ef®cient resealing of the plasma membrane.
Nat. Cell Biol. 2, 226±231.
Kandasamy, R., Vediyappan, G. and Chaf®n, W.L. (2000) Evidence
for the presence of pir-like proteins in Candida albicans. FEMS
Microbiol. Lett. 186, 239±243.
Kapteyn, J.C., Van Den Ende, H. and Klis, F.M. (1999a) The contribution of cell wall proteins to the organization of the yeast cell
wall. Biochim. Biophys. Acta, 1426, 373±383.
Kapteyn, J.C., Van Egmond, P., Seivi, E., Van Den Ende, H.,
Makarow, M. and Klis, F. (1999b) The contribution of the
O-glycosylated protein Pir2p/Hsp150 to the construction of the
yeast cell wall in wild-type cells and b-1,6-glucan-de®cient
mutants. Mol. Microbiol. 31, 1835±1844.
Kapteyn, J.C., Hoyer, L.L., Hecht, J.E., Muller, W.H., Andel, A.,
Verkleij, A.J., Makarow, M., Van Den Ende, H. and Klis, F.M.
(2000) The cell wall architecture of Candida albicans wild-type
cells and cell wall-defective mutants. Mol. Microbiol. 35,
601±611.
Krebs, S.L. and Grumet, R. (1993) Characterization of celery hydrolytic enzymes induced in response to infection by Fusarium
oxysporum. Physiol. Mol. Plant Pathol. 43, 193±208.
Lafontaine, P.J. and Benhamou, N. (1996) Chitosan treatment: an
emerging strategy for enhancing resistance of greenhouse
tomato plants to infection by Fusarium oxysporum f. sp. radicis-lycopersici. Biocontrol Sci. Technol. 6, 111±124.
LaRosa, P.C., Chen, Z., Nelson, D.E., Singh, N.K., Hasegawa, P.M.
and Bressan, R.A. (1992) Osmotin gene expression is posttranscriptionally regulated. Plant Physiol. 100, 409±415.
Liu, D., Raghothama, K.G., Hasegawa, P.M. and Bressan, R.A.
(1994) Osmotin overexpression in potato delays development
of disease symptoms. Proc. Natl. Acad. Sci. USA, 91, 1888±1892.
van Loon, L.C. and van Strien, E.A. (1999) The families of pathogenrelated proteins, their activities, and comparative analysis of
PR-1 type proteins. Physiol. Mol. Plant Pathol. 55, 85±97.
Moukadiri, I., Jaffar, L. and Zueco, J. (1999) Identi®cation of two
mannoproteins released from cell walls of a Saccharomyces
cerevisiae mnn1 mnn9 double mutant by reducing agents.
J. Bacteriol. 181, 4741±4745.
Mrsa, V. and Tanner, W. (1999) Speci®c labelling of cell wall
proteins by biotinylation. Identi®cation of four covalently linked
O-mannosylated proteins of Saccharomyces cerevisiae. Yeast,
15, 813±820.
Mrsa, V., Seidl, T., Gentzsch, M. and Tanner, W. (1997) Role of
NaOH-extractable cell wall proteins Ccw5p, Ccw6p, Ccw7p and
Ccw8p (members of the pir protein family) in stability of the
Saccharomyces cerevisiae cell wall. Yeast, 13, 1145±1154.
Mueller, W.C. and Morgham, A.T. (1996) Ultrastructure of the
vascular responses of tobacco to Fusarium oxysporum f. sp.
nicotianae. Can. J. Bot. 74, 1273±1278.
Mulholland, J., Preuss, D., Moon, A., Wong, A., Drubin, D. and
Botstein, D. (1994) Ultrastructure of the yeast actin cytoskeleton
and its association with the plasma membrane. J. Cell Biol. 125,
381±391.
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 36, 390±400
399
Narasimhan, M.L., Damsz, B., Coca, M.A., Ibeas, J.I., Yun, D.J.,
Pardo, J.M., Hasegawa, P.M. and Bressan, R.A. (2001) A plant
defense response effector induces microbial apoptosis. Mol.
Cell, 8, 921±930.
Pall, M.L. and Brunelli, J.P. (1993) A series of six compact fungal
transformation vectors containing polylinkers with multiple
unique restriction sites. Fungal Genet. Newslett. 40, 59±62.
Rep, M., Dekker, H.L., Vossen, J.H., de Boer, A.D., Buckel, S.,
Houterman, P.M., Speijer, D., Back, J.W. and Cornelissen,
B.J.C. (2002) Mass spectrometric identi®cation of isoforms of
PR proteins in xylem sap of fungus-infected tomato. Plant Physiol. 130, 904±917.
Rey, M., Ohno, S., Pintor-Toro, J.A., Llobell, A. and Benitez, T.
(1998) Unexpected homology between inducible cell wall protein QID74 of ®lamentous fungi and BR3 salivary protein of the
insect Chrionomus. Proc. Natl. Acad. Sci. USA, 95, 6212±6216.
Russo, P., Kalkkinen, N., Sareneva, H., Paakkola, J. and Makarow,
M. (1992) A heat shock gene from Saccharomyces cerevisiae
encoding a secretory glycoprotein. Proc. Natl. Acad. Sci. USA,
89, 3671±3675.
Sambrook, J., Fritsch, E.F. and Maniatis, T.A. (1989) Molecular
Cloning: A Laboratory Manual. NY: Cold Spring Harbor Laboratory.
Schoffelmeer, E.A.M., Kapteyn, J.C., Montijn, R.C., Cornelissen,
B.C. and Klis, F.M. (1996) Glucosylation of fungal cell wall
proteins as a potential target for novel antifungal agents. In
Modern Fungicides and Antifungal Compounds (Lyr, H., Russel,
P.E. and Sisler, H.D., eds). UK: Intercept, pp. 157±162.
Schoffelmeer, E.A.M., Klis, F.M., Sietsma, J.H. and Cornelissen,
B.J.C. (1999) The cell wall of Fusarium oxysporum. Fungal
Genet. Biol. 27, 275±282.
Schoffelmeer, E.A.M., Vossen, J.H., van Doorn, A.A., Cornelissen,
B.J.C. and Haring, M.A. (2001) FEM1, a Fusarium oxysporum
glycoprotein that is covalently linked to the cell wall matrix and
is conserved in ®lamentous fungi. Mol. Genet. Genomics, 265,
143±152.
Shim, W.B. and Woloshuk, C.P. (2001) Regulation of fumonisin B1
biosynthesis and conidiation in Fusarium verticillioides by a
cyclin-like (C-type) gene, FCC1. Appl. Environ. Microbiol. 67,
1607±1612.
Shipton, W.A. and Brown, J.F. (1962) A whole-leaf clearing and
staining technique to demonstrate host-pathogen relationships
of wheat stem rust. Phytopathology, 52, 1313.
Singh, N.K., Bracker, C.A., Hasegawa, P.M., Handa, A.K., Buckel,
S., Hermodson, M.A., Pfankoch, E., Regnier, F.E. and Bressan,
R.A. (1987) Characterization of osmotin. A thaumatin-like protein
associated with osmotic adaptation in plant cells. Plant Physiol.
85, 529±536.
Tenney, K., Hunt, I., Sweigard, J., Pounder, J.I., McClain, C.,
Bowman, E.J. and Bowman, B.J. (2000) Hex-1, a gene unique
to ®lamentous fungi, encodes the major protein of the Woronin
body and functions as a plug for septal pores. Fungal Genet.
Biol. 31, 205±217.
Tjamos, E.C. and Beckman, C.H. (1989) Vascular wilt diseases of
plants: basic studies and control. In NATO ASI Series H: Cell
Biology, Vol. 28 (Tjamos, E.C., Beckman, C.H., eds) Berlin:
Springer±Verlag.
Toh-e, A., Yasunaga, S., Nisogi, H., Tanaka, K., Oguchi, T. and
Matsui, Y. (1993) Three yeast genes, PIR1, PIR2 and PIR3, containing internal tandem repeats, are related to each other, and
PIR1 and PIR2 are required for tolerance to heat shock. Yeast, 9,
481±494.
Van Pelt-Heerschap, H. and Smit-Bakker, O. (1999) Analysis of
defense-related proteins in stem tissue of carnation inoculated
400 Meena L. Narasimhan et al.
with a virulent and avirulent race of Fusarium oxysporum f. sp.
dianthi. Eur. J. Plant Pathol. 105, 681±691.
Veronese, P., Ruiz, M.T., Coca, M.A. et al. (2003) In defense against
pathogens. Both plant sentinels and foot soldiers need to know
the enemy. Plant Physiol. 131, 1580±1590.
Wergin, W.P. (1973) Development of Woronin bodies from microbies in Fusarium oxysporum f. sp. lycopersici. Protoplasma, 76,
249±260.
Woloshuk, C.P., Yousibova, G.L., Rollins, J.A., Bhatnagar, D. and
Payne, G.A. (1995) Molecular characterization of the a¯-1
locus in Aspergillus ¯avus. Appl. Environ. Microbiol. 61,
3019±3023.
Xu, Y., Chang, P.F.L., Liu, D., Narasimhan, M.L., Raghothama, K.G.,
Hasegawa, P.M. and Bressan, R.A. (1994) Plant defense genes
are synergistically induced by ethylene and methyl jasmonate.
Plant Cell, 6, 1077±1085.
Yun, D.J., D'Urzo, M.P., Abad, L., Takeda, S., Salzman, R., Chen, Z.,
Lee, H., Hasegawa, P.M. and Bressan, R.A. (1996) Novel osmotically induced antifungal chitinases and bacterial expression of
an active recombinant isoform. Plant Physiol. 111, 1219±1225.
Yun, D.J., Bressan, R.A. and Hasegawa, P.M. (1997a) Plant antifungal proteins. In Plant Breeding Reviews, Vol. 14 (Janick, J.,
ed.). New York: John Wiley, pp. 39±88.
Yun, D.J., Zhao, Y., Pardo, J.M., Narasimhan, M.L., Damsz, B., Lee,
H., Abad, L.R., D'Urzo, M.P., Hasegawa, P.M. and Bressan, R.A.
(1997b) Stress proteins on the yeast cell surface determine
resistance to osmotin, a plant antifungal protein. Proc. Natl.
Acad. Sci. USA, 94, 7082±7087.
ß Blackwell Publishing Ltd, The Plant Journal, (2003), 36, 390±400