Planta (2012) 236:623–633
DOI 10.1007/s00425-012-1637-7
ORIGINAL ARTICLE
Arsenal of elevated defense proteins fails to protect tomato against
Verticillium dahliae
Jane Robb • Hakeem Shittu • Kizhake V. Soman
Alexander Kurosky • Ross N. Nazar
•
Received: 2 February 2012 / Accepted: 23 March 2012 / Published online: 6 April 2012
Ó Springer-Verlag 2012
Abstract Although the hypersensitive reaction in foliar
plant diseases has been extensively described, little is clear
regarding plant defense strategies in vascular wilt diseases
affecting numerous economically important crops and
trees. We have examined global genetic responses to
Verticillium wilt in tomato (Lycopersicon esculentum
Mill.) plants differing in Ve1 resistance alleles. Unexpectedly, mRNA analyses in the susceptible plant (Ve1-) based
on the microarrays revealed a very heroic but unsuccessful
systemic response involving many known plant defense
genes. In contrast, the response is surprisingly low in plants
expressing the Ve1? R-gene and successfully resisting the
pathogen. Similarly, whole-cell protein analyses, based on
2D gel electrophoresis and mass spectrometry, demonstrate
large systemic increases in a variety of known plant
defense proteins in the stems of susceptible plants but only
modest changes in the resistant plant. Taken together, the
results indicate that the large systemic increases in plant
defense proteins do not protect the susceptible plant.
Indeed, since a number of the highly elevated proteins are
known to participate in the plant hypersensitive response as
well as natural senescence, the results suggest that some or
all of the disease symptoms, including ultimate plant death,
Electronic supplementary material The online version of this
article (doi:10.1007/s00425-012-1637-7) contains supplementary
material, which is available to authorized users.
J. Robb H. Shittu R. N. Nazar (&)
Department of Molecular and Cellular Biology,
University of Guelph, Guelph, ON N1G 2W1, Canada
e-mail: [email protected]
K. V. Soman A. Kurosky
Department of Biochemistry and Molecular Biology, University
of Texas Medical Branch, Galveston, TX 77555, USA
actually may be the result of this exaggerated plant
response.
Keywords
Lycopersicon Plant defense Vascular wilt
Abbreviations
CS cv
Craigella susceptible
CR cv
Craigella resistant
d.p.i
Days post-inoculation
HR
Hypersensitive response
IPG
Immobilized pH gradient isoelectric
focusing
MALDI-MS Matrix-assisted laser desorption/ionizationmass spectrometry
MMLV
Maloney murine leukemia virus
pAP3
32P-labeled anionic potato peroxidase
PR
Pathogenesis-related
R-gene
Resistance-gene
SAR
Systemic acquired resistance
SDS
Sodium dodecylsulphate
TVR
Tomato Verticillium response
Vd1
Verticillium dahliae, race 1
Introduction
Like animals, plants are continuously exposed to pathogens
and pests in the environment and have developed molecular defenses to protect themselves. Years of study have
demonstrated a large cadre of ‘‘defense proteins’’, which
can be induced by pathogens to control or eliminate
invading bacteria or fungi (for reviews see Van Loon 2006;
Castroverde et al. 2010). Some have general functions
(e.g., chitinase degrades fungal cell walls), some are more
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pathogen-specific (e.g., suberin-specific anionic peroxidase
for vascular pathogens) and the role of many remains
unclear (e.g., PR-1 class proteins). Much of our knowledge
about these resistance strategies comes from studying foliar
diseases, which exhibit well-defined ‘‘gene for gene’’ systems (Flor 1971) where a plant receptor binds a specific
pathogen-derived effector to initiate a defense response
(Van Ooijin et al. 2007). The process begins when a fungus
or bacterium attempts to penetrate a host cell and a necrotic
response or programmed cell death (PCD) is triggered
(Lam 2004; Mittler and Cheung 2004). Frequently called a
hypersensitive reaction (HR), in a resistant plant, this
occurs rapidly and intensely, killing the pathogen and a few
leaf cells in contact with the pathogen during the process
(Lam et al. 2001; Lam 2004). In susceptible plants, the
response is less effective, allowing both the pathogen and
the necrotizing process to spread and form a sizable lesion
that we recognize as disease. In either case, the necrotizing
process also triggers more distant responses, activating the
synthesis of defense proteins called pathogenesis-related
(PR) proteins, throughout the plant. This secondary
response, referred to as systemic acquired resistance
(SAR), is thought to confer long-term protection against a
broad spectrum of pathogens (Durrant and Dong 2004).
Some pathogens, termed systemic pathogens, colonize
plants extensively but penetrate few living cells of the host,
growing instead within the apoplastic spaces or water
conducting vessels. The fungus Verticillium dahliae (Vd),
representative of bacteria and fungi that occupy the water
conducting vessels, causes vascular wilt diseases (Pegg and
Brady 2002). It infects hundreds of economically important
crops and trees but, unfortunately, the effectiveness of
current management practices is very limited. The best
strategy is breeding for resistance; however, resistance in
most hosts is polygenic and poorly understood. Nonetheless, dominant R-genes have been identified in several
plant species including tomato, although ‘‘resistant’’ plants
contain substantial levels of pathogen and, currently, there
is no direct molecular evidence of a ‘‘gene for gene’’
relationship (for reviews see Fradin and Thomma 2006;
Thomma et al. 2011).
In tomato Verticillium resistance is controlled mainly by
the Ve-gene locus (Schaible et al. 1951). Molecular characterization has shown this locus to consist of two inverted
genes, Ve1 and Ve2, with a short intergenic region between
them (Kawchuk et al. 2001; Fradin et al. 2009). The predicted protein sequence suggests that the Ve-genes encode
R-proteins of the RLP class (for review see Van Ooijin
et al. 2007), characteristic of membrane receptors (Kawchuk
et al. 2001; Fradin et al. 2009). While still controversial
(Kawchuk et al. 2001; Fradin et al. 2009), the most recent
evidence suggests that resistance to both Verticillium
dahliae, race 1 and Verticillium albo-atrum is actually
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conferred by Ve1 alone (Fradin et al. 2009). Apparently,
susceptibility results from a single-base deletion that produces a stop codon in the Ve1--allele, severely truncating
the Ve1 protein. Currently, little is known about the actual
function or regulation of the Ve-genes.
For many years the tomato/Verticillium dahliae, race 1
pathosystem has been used extensively as a model for
studying Verticillium diseases (Pegg and Brady 2002: Chen
et al. 2004; Fradin and Thomma 2006). In tomato, the
fungus infects through the root system, entering the xylem
vessels in 2–3 days. There it proliferates and releases large
numbers of conidiospores that are carried upwards in the
transpiration stream to populate the stem and leaves,
becoming fully systemic 3–4 days post-infection (d.p.i.) in
both susceptible and resistant plants (Chen et al. 2004).
After a short period of fungal elimination the fungal population continues to grow and escalates in susceptible
plants, leading to severe wilt; the recovery fails in resistant
hosts, although the plants remain colonized to some extent.
To identify critical events in resistant and susceptible
plants we are undertaking global analyses of changes in
mRNA and protein. These studies are comparing two
near-isolines of tomato (Lycopersicon esculentum L.)
‘‘Craigella’’, which are Ve1? or Ve1-. Past studies were
conducted using a commercial TOM1 microarray (Alba
et al. 2004) and responses under various conditions were
used to prepare a customized tomato Verticillium response
(TVR) array, focused on reacting defense genes with eight
identical sub arrays that permit higher statistical certainty
(Robb et al. 2009). Here, the TVR array is used to study the
response to Vd1 colonization where the results are compared with simultaneously induced proteomic changes.
Materials and methods
Plant infection and symptom rating
The tomato (Lycopersicon esculentum Mill.) cv Craigella
plants were either resistant (CR), Ve1?/Ve1? (CRG CR
218), or susceptible (CS), Ve1-/Ve1- (CSG CR 26), to
the pathogen. The seed was obtained originally from
R. Cooper (University of Bath, UK) and the two isolines
were maintained as breeding lines at the University of
Guelph (Bishop and Cooper 1983; Gold and Robb 1995).
For the experiments, the seeds were surface sterilized,
planted in Kord cell flats and grown with 14 h light at 26 °C
and 10 h dark at 22 °C (Chen et al. 2004). The V. dahliae
Kleb., race 1 (Vd1) isolate (HES 88–156) was obtained
from the Horticultural Research Station at Simcoe, ON,
Canada. The fungus was cultured on potato dextrose agar at
24 °C for 4 weeks to prepare conidial suspensions (Pegg
and Street 1984). To infect plants, seedlings were
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inoculated at the four-leaf stage by dipping roots in gelatin
solution containing 1.0 9 107 spores/ml (Dobinson et al.
1996); the control plants were dipped in gelatin solution
only. The seedlings were replanted and maintained in
chambers as described above. Disease symptoms were
scored at 5 and 10 d.p.i. relative to the controls using a 0
(i.e., no symptoms) to 5 (i.e., plant dead) scale (Busch and
Smith 1981; Shittu et al. 2009).
Preparation of plant extracts
Extracts were prepared from the top two-thirds of the stem
or whole washed root tissue from six plants, quickly
chopped, pooled and divided into two aliquots. One aliquot
was frozen immediately in liquid nitrogen and stored at
-80 °C for protein extraction; the second was used
immediately to prepare nucleic acid. For whole-cell nucleic
acid, the aliquots (0.5 g) were ground in liquid nitrogen
and extracted with SDS/phenol (Shittu et al. 2009) and
quantified by rRNA analyses (Sambrook and Russell
2001). The protein was prepared essentially as described by
Hurkman and Tanaka (1986). Tissue aliquots (0.5 g) were
ground in liquid nitrogen and extracted twice with TRISbuffered phenol (pH 8.8). Protein in the pooled phenol
phases was precipitated at -20 °C with 0.1 M ammonium
acetate in methanol, washed with 0.1 M ammonium acetate
in methanol, twice with 80 % ethanol and finally with
70 % ethanol. Pellets were dried and stored at -80 °C.
PCR analysis of fungal colonization
Aliquots of whole-cell nucleic acid extracts were used to
determine the amount of fungal DNA by a PCR-based
assay (Hu et al. 1993; Robb and Nazar 1996).
Cytological procedures
For electron microscopy, samples of stem cross sections
were fixed in a solution of 2 % glutaraldehyde and 1.5 %
acrolein in 0.07 M phosphate buffer, pH 6.8 and post-fixed
in 1 % OsO4. Staining in uranyl acetate (0.5 %), dehydration, plastic embedding and preparation for transmission
electron microscopy (TEM) were carried out as previously
described (Street et al. 1986).
In situ hybridization was carried out with infected stems
as previously described (Robb et al. 1991). Free hand
sections were made with a sharp razor blade and stained for
light microscopy with 12 N KOH to identify the position of
the vascular bundles and sites of suberinization. For
hybridization, a 3-mm stem segment was sampled, immediately adjacent to the free hand section, and the cut surface
blotted onto a nylon membrane by applying gentle pressure. The blot itself was observed and photographed with a
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dissecting microscope as previously illustrated (Robb et al.
1991, Fig. 6c) for comparison with the adjacent free hand
section, and then hybridized with a 32P-labeled potato
peroxidase cDNA probe (pAP3) for the suberin-specific
anionic peroxidase gene.
Microarray analyses
mRNA changes were determined using either a commercial
tomato TOM1 cDNA array (Alba et al. 2004) or a custom
TVR chip (http://www.uoguelph.ca/*rnnarray) representing approximately 270 genes involved in tomato defense
responses to Vd1 (Robb et al. 2009) as previously described
(Robb et al. 2009; Shittu et al. 2009). Indirectly labeled probe
prepared from plant extracts was hybridized for 16 h at
37 °C and the scanned data were normalized with GenePix
pro 4 software (Axon Instruments, Foster City, CA, USA).
Log2 ratios and standard deviations were calculated for spots
on multiple arrays using a two sigma threshold (Savoie et al.
2003) to identify spots with significant changes. GPR files
also were imported into GeneTraffic (DUO) 3.2 microarray
management and analysis software (Iobion Informatics,
LaJolla, CA, USA) for normalization (Lowess method) and
scatter plot or heat map analyses,
RT-PCR analysis of mRNA
Individual mRNA levels were assayed by RT-PCR as
previously described (Robb et al. 2009). Aliquots
(250–500 ng) of each whole-cell nucleic acid extract,
together with 100–200 ng of gene specific reverse primer
as well as 2.5 pg of the truncated internal control RNA for
actin analysis, were denatured for 5 min at 65 °C to prepare cDNA using MMLV reverse transcriptase (Fermentas
MBI, Burlington, ON, Canada) at 37 °C for 60 min. After
heat inactivation (75 °C) for 5 min, the samples were
diluted tenfold and 5-ll aliquots were used for PCR
amplification. The products were fractionated on 2 %
agarose gels and, after staining with ethidium bromide,
images were captured and quantified using a Gel Doc 1000
and Molecular Analyst software (Bio-Rad, Hercules, CA,
USA). To quantify actin mRNA, the RT-PCR product ratio
(tomato actin mRNA product/internal control product) was
determined for each aliquot of extract and the amount of
mRNA of other genes was expressed relative to actin.
Primers used in these assays are described in the supplementary data (Table S1).
Protein analysis by 2-D gel electrophoresis
Whole-cell protein extracts were fractionated by twodimensional electrophoresis with IPG/SDS–polyacrylamide gradient gels (Straub et al. 2009). Aliquots (200 lg)
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were solubilized in destreaking buffer (GE Healthcare,
Piscataway, NJ, USA) and applied to 11 cm, pH 3-10 IPG
strips for isoelectric focusing, using an IPGphor (GE
Healthcare). The strips were applied to precast 8–16 %
Criterion (Bio-Rad Laboratories) 13.3 9 8.7 cm Tris–HCl
gels for further fractionation, fixed in 10 % methanol and
7 % acetic acid and stained with Sypro Ruby. Images were
captured with a ProExpress 2-D Proteomic Imaging System
(Perkin Elmer Life and Analytical Sciences, Waltham,
MA, USA) for quantitative analyses using Progenesis
SameSpots software v4.0 (Nonlinear USA, Durham, NC,
USA). Differences of intensities C1.5 (ANOVA P \ 0.05)
were selected for protein identification.
Protein identification by mass spectrometry
Selected spots were robotically picked (Genomic Solutions, Ann Arbor, MI, USA), trypsin digested and transferred to MALDI-MS target plates as recommended by the
manufacturer. Data were acquired with an Applied Biosystems (Foster City, CA, USA) 4800 MALDI-TOF/TOF
Proteomics Analyzer and 4000 Series Explorer (v3.6 RC1)
software as previously described (Straub et al. 2009) and
MASCOT (Matrix Science, London, UK) was used to
search plant protein databases with protein match probabilities based on expectation values and/or MASCOT
proteins scores used to identify the proteins.
Results
As might be anticipated, the initial studies with the TOM1
array (Robb et al. 2009) demonstrated clear defense gene
responses to colonization by Vd1 but, surprisingly, our
further analyses of these data combined with new experiments suggest an unanticipated and more intense response
in the susceptible line. As shown in Fig. 1a, a scatter plot,
comparing the expression of genes in the stems of infected
susceptible plants with those of the resistant isoline at
10 d.p.i., indicates widely divergent profiles of expression
changes. More importantly, many mRNAs are more elevated (i.e., upregulated) in the susceptible host; in particular, a distinct group comprising highly induced known
plant defense genes and some genes encoding proteins with
unknown functions are clearly differentiated in the plot
(large gray arrowhead). When Log2 changes of 1.25
(2.5-fold) or greater were selected using 2.5 threshhold, 88
genes were upregulated in the susceptible plants relative to
resistant plants while 45 were lower.
To examine further the differences in defense gene
expression between the CR and CS isolines, tissues sampled at an earlier stage of infection have been compared
more closely using the specialized TVR microarray. Since
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the fungus is known to be fully systemic by 4 d.p.i. (Chen
et al. 2004), Vd1-infected plants were sampled at 5 d.p.i. to
ensure an induced response and used to prepare cDNA
probes for hybridization analyses. As also shown by scatter
plot summaries (Fig. 1b, c), comparing infected plants with
the corresponding controls, the results again indicate a
dramatically greater response in the susceptible plant with
a very broad scatter of gene responses in tomato CS
(Fig. 1c) but only relatively modest changes in the CR
isoline (Fig. 1b), which expresses the Ve1-encoded protein,
leading to much reduced levels of fungus. Detailed data
used to prepare the scatter plots at 5 d.p.i. as well as
comparable data at 10 d.p.i., are available as supplementary Tables S2–5.
As summarized in the table of biological consequences
(Fig. 1d), the CS-infected plants, which displayed a very
diverse spectrum of genetic responses, had significantly
higher levels of the disease, were clearly stunted with
respect to growth and contained high and rapidly escalating
levels of fungal DNA. Perhaps more important, at 5 d.p.i.
when the pathogen had been in the stem for about 1 day,
the CS response (Fig. 1c) was already substantially greater
than in CR stems (Fig. 1b) although the amount of pathogen present in the samples was almost the same (i.e.,
38.0 ± 8.5 and 29.2 ± 6.7 ng/g plant tissue in CS and CR
plants, respectively) in both isolines. Furthermore, separate
analyses of Verticillium DNA in washed roots at the time
of initial sporulation (i.e., 3 d.p.i.) indicated much higher
levels of pathogen in the roots but, again, relatively little
difference (an average of 840 vs. 680 ng fungal DNA/g
root tissue for three biological replicates, respectively)
between susceptible and resistant plants. Taken together,
these data indicated that CS plants did detect the fungus
and, at early stages of fungal stem colonization, initiated
strong defensive responses, which apparently had little or
no impact on the pathogen and disease development. In
contrast, the presence of the Ve-resistance gene (Ve1?) in
CR plants enabled them to curtail Verticillium colonization
and symptoms, with only minimal changes in gene
expression.
The presence of dramatic changes in CS plants could
also be demonstrated using cytological approaches as
shown in Fig. 2. In situ hybridization analyses using tissue
blots of stem cross sections (Robb et al. 1991) readily
showed that the expression of the suberin-specific anionic
peroxidase gene, TMP1 (Bernards et al. 1999), was substantially higher in the vascular tissues of Vd1-infected CS
plants (Fig. 2c, left) than in infected CR (Fig. 2c, right).
The peroxidase enzyme that is encoded catalyzes the final
step in the synthesis of suberin which, as illustrated in
Fig. 2a is secreted by living cells of the xylem into adjacent
vessels (V); there it coats the cell walls (CW) of both the
water conducting pipes and the fungal cells with a thick
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627
(a)
(d)
(b)
(c)
Fig. 1 Analyses of defense gene mRNA levels in the tomato/
Verticillium interaction. Susceptible (CS) or resistant (CR) tomato
seedlings were infected by root dip with Vd1 spores and stem tissue
RNA extracts were used for microarray hybridization analyses at
10 d.p.i. using commercial (TOM1) whole-cell cDNA or at 5 d.p.i.
using a customized, defense gene focused tomato/Verticillium
response (TVR) chip as described in ‘‘Materials and methods’’;
stems were pooled in sets of six with four biological replicates. The
overall changes in mRNA levels are summarized as scattered plots.
a The analyses at 10 d.p.i., using TOM1 chips, directly compare data
from infected susceptible with resistant plants; the large arrow
indicates a previously identified (Robb et al. 2009) cluster of elevated
plant defense genes. The analyses at 5 d.p.i. using the TVR chip
compare infected resistant (b) or susceptible (c) plants with
uninfected controls. A summary of the biological parameters
observed in the present studies (four replicates) is included in the
table insert (d), including 1 disease scores ± SD, 2 plant height
(cm) ± SD and 3 the amount of Verticillium DNA (ng fungal DNA/
gm plant tissue) ± SD in infected CS and CR plants
suberin layer that appears not only to block fungal escape
(Robb et al. 1989) but also water flow (Robb et al. 1983,
1989). Initially the coating responses are thought to contribute to defense but eventually, when resistance fails in
CS plants, the coating apparently becomes systemic,
blocking the flow of water and nutrients to surrounding
tissues, and contributing to wilt and stunting (Robb and
Busch 1983; Robb et al. 1989) as noted in Fig. 1d.
Although also present in resistant plants (Fig. 2b), the
coatings are less extensive and high levels clearly are not
the basis of resistance.
Because changes in mRNA levels do not always reflect
actual levels of cellular proteins, the significance of the
changes in gene expression was examined more directly
when whole-cell protein extracts also were fractionated and
many of the individual elevated proteins were partially or
fully identified by mass spectrometry. Initially, a preliminary survey of protein changes in samples taken from 4
to 12 d.p.i. indicated little or no change at 4 d.p.i. with
increased change through 12 d.p.i. (results not shown).
Figure 3 compares two-dimensional fractionations of wholecell stem protein extracts from Vd1-infected susceptible
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Fig. 2 Vascular coating in Verticillium-infected tomato plants.
Seedlings were infected with Vd1 and 12 d.p.i. stem tissue was used
for the electron microscopy and tissue blot hybridization analyses as
described in ‘‘Materials and methods’’. The electron micrograph
(a) illustrates a vascular parenchyma cell (PC) in a susceptible stem,
secreting large amounts of coating or suberin-containing coating into
adjacent vessels or water conducting pipes (V), which entraps hyphae
and spores (Pathogen) and blocks fungal escape. The primary cell
walls and middle lamella (ML) and secondary cell walls (CW) also are
identified, ca 914,000. A comparable micrograph for an infected
resistant stem (b) is included for comparison. The stem crosssectional tissue blots (c) illustrate differences in levels of suberinspecific anionic peroxidase (TMP1) mRNA in the infected stems of
susceptible (CS) and resistant (CR) plants
(a), uninfected control (b) and infected resistant (c) plants
at 12 d.p.i. A number of elevated protein spots relative to
controls were obvious, even by casual inspection especially
in the basic range (pH 9–10). Images for replicate gels
were captured and used for quantitative analyses with
Progenesis SameSpots software, v4.0 (Nonlinear USA). As
shown in Fig. 4a, a scatter plot of data for the CS-infected
plants (Fig. 3a) versus uninfected controls (Fig. 3b) at
12 d.p.i. demonstrates that fungus is readily detected and a
strong response, with many elevated proteins, is triggered.
Furthermore, as summarized in Fig. 4c, the quantitative
analyses indicated that the intensities of at least 15 spots
were elevated twofold or more (ANOVA P \ 0.05), one
exceeding an eightfold increase relative to those from
uninfected control plants. At least one protein (spot 22) was
significantly fainter in the infected plant, apparently
reduced by the presence of pathogen. More surprising,
however, was the fact that again, as first demonstrated by
a scatter plot (Fig. 4b), quantitative analyses with
CR-infected plants (Fig. 3c) revealed a much reduced
response at the protein level, similar to observations with
mRNA expression levels (e.g., Fig. 1b). As also
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summarized in the table (Fig. 4c), the same cadre of proteins was much less elevated, the largest change (spot 2)
being somewhat more than twofold. Equally, this same
group of 22 proteins in either isoline was not significantly
elevated at early stages of stem colonization, the average
change at 4 d.p.i. being 1.1 ± 0.3 and 1.2 ± 0.1 for CS
and CR plants, respectively. Direct scatter plot comparisons at both 4 and 12 dpi are available as supplementary
data (Fig. S1), together with protein quantification data for
all the enumerated spots (Tables S6 and S7).
Using mass spectrometry and MASCOT software
(Matrix Science) to search protein databases (Straub et al.
2009), the best matches for the 22 most affected protein
spots are tabulated in Fig. 4c. Consistent with pathogen
infection, many were identified as known or similar to
known plant defense proteins; several, however, including
the most elevated or pathogen-reduced spots (spots 1 and
22, respectively) were identified as hypothetical plant
proteins. Among all the spots, at least 14 could be classified
as PR proteins or transcription factors associated with PR
proteins, e.g., TSI-1 (Castroverde et al. 2010). Of the six
proteins with the highest elevations, two are PR-10 proteins
(STH-2 a and b) and three are glucan endo-1,3-b glucosidase Bs (b-glucanases) from the PR-2 protein family. Other
PR protein families represented among the most elevated
proteins are PR-1, PR-3, and PR-9 (Van Loon 2006;
Castroverde et al. 2010). Of the representative proteins in
these PR-gene families, the b-glucanases, CHRK1, P2
(chitinase) and the peroxidases (including TMP1) are all
commonly associated with defensive host activities in both
resistant and susceptible plants (Van Loon 2006; Castroverde et al. 2010).
The data summarized in Figs. 1 and 4 (scatter plots)
demonstrate a striking and unanticipated difference in
levels of defense gene expression as observed in infected
resistant and susceptible plants but provide little information regarding the profile of changes in the course of
infection or the possibility of differences between uninfected plants. These questions were addressed further in
Figs. 5 and 6. As summarized by the scatter plots shown
in Fig. 5, defense gene mRNA levels, as measured by
microarray analyses (Fig. 5a) or cellular protein concentrations, as measured by two-dimensional gel fractionations (Fig. 5b) reveal minor or no differences between
uninfected CS and CR plants. Even the few points that
fall clearly off the diagonal in the comparison of mRNA
levels (Fig. 5a), represent less than twofold differences.
The striking difference between infected CS and CR
plants reflect differences in the hosts’ responses to the
Verticillium fungus and do not represent normal differences in the basal levels of uninfected plants as might
have been established by mutation in the Ve1 resistance
gene.
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Fig. 3 Two-dimensional IPG/
SDS gel fractionations of
cellular proteins from
Verticillium-infected tomato
plants. Susceptible (a) or
resistant (c) tomato seedlings
were infected by root dipping
with Vd1 spores and 12 d.p.i.
stem tissue protein extracts were
fractionated using twodimensional electrophoresis as
described in ‘‘Materials and
methods’’. An extract from a
susceptible control plant, root
dipped without fungal spores is
included (b). IEF in the first
dimension was conducted over a
pH range 3–10. Spots with
significant intensity differences
are ranked by the numbered
arrows; molecular sizes as
determined from markers are
indicated on the left
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(a)
(b)
Similarly, the time courses presented in Fig. 6 again are
consistent with substantially elevated defense gene
responses in the susceptible isoline. Because many defense
genes belong to large families, it frequently is difficult to
accurately match mRNA transcripts with specific protein
spots. In this study levels for two of the most affected
proteins (glucan endo-1,3-b-glucosidase and pathogenesis
related leaf protein 6), for which a match seemed reasonable, were examined in the course of Verticillium colonization. In Fig. 6a and c, levels of mRNA were assessed by
microarray and qRT-PCR. While some differences in
absolute levels were apparent, the trends that were
observed with these alternate approaches were entirely
comparable. With both analytical methods, there appeared
to be a strong induction in the susceptible plant, which
receded with time as the protein levels built up. This build
up in defense protein in the susceptible isoline was evident
when protein levels were determined at 4, 8 and 12 d.p.i.
Differences between the resistant and susceptible isolines,
which began to appear at 4 d.p.i., were elevated substantially by 12 d.p.i. (Fig. 6b, d). In this context, it may be
important to note that 4 d.p.i. is well within the critical
timeframe when the pathogen must escape from primary
spore trapping sites and resistance or susceptibility is
apparently determined (Beckman 1987). As stated earlier
for the 2D gel analyses, it is also important to note that the
(c)
actual levels of these specific plant defense proteins again
were little elevated in infected susceptible or resistant
plants relative to noninoculated controls at 4 d.p.i. Because
whole stem extracts were used in the various assays, small
early differences, important differences in scarce mRNAs
or proteins such as transcription factors and differences
localized in only a specific cell type cannot be eliminated.
Nevertheless, significant changes in the normal arsenal of
major defense proteins were only detected at later stages.
Discussion
In respect to Verticillium-induced wilt, the present study
shows that susceptible tomato plants, infected by Verticillium dahliae, race 1, readily detect the pathogen and mount
a strong defensive but ultimately unsuccessful effort
against the fungus. The striking changes in protein levels
do not simply reflect a generalized increase in metabolic
activities but are targeted specifically to defense as identified by mass spectrometry. Past studies have shown
defense processes to include the formation of structural
barriers such as protective apposition layers (Street et al.
1986) and vascular coating (Street et al. 1986; Robb et al.
1989) materials (e.g., Fig. 2a) as well as the production
of antifungal compounds such as fungal wall degrading
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(a)
(c)
(b)
Fig. 4 Analysis of affected proteins in the tomato/Verticillium
interaction. Protein extracts were prepared at 12 d.p.i., fractionated
and stained as described in Fig. 3; images were captured and
quantified using Progenesis SameSpots software and overall differences between Vd1-infected and control susceptible (CS) or resistant
(CR) tomato plants are summarized by the scatter plots, (a, b),
respectively. Individual spots for analyses of six plants (two
biological replicates) and exhibiting differences in intensity C1.5
(ANOVA P \ 0.05) were picked, trypsin digested and subjected to
analysis by mass spectrometry as described in ‘‘Materials and
methods’’. Protein IDs 1, as deduced from plant databases, are
summarized in the table insert (c) together with intensity difference
ratios 2 (infected/control) for both susceptible (CS) and resistant (CR)
plants. The protein numbers refer to the protein spots identified in
Fig. 3a
enzymes and phytoalexins (for reviews see Pegg and Brady
2002; Fradin and Thomma 2006). Many of the PR proteins
shown to be elevated here in both the microarray and
protein studies are crucial to these activities. The intense
activity exhibited cytologically by the parenchyma cells of
the xylem in the susceptible plant (Fig. 2a) is apparently
necessitated by the prolonged requirements of this
response, continuing until the plant is essentially moribund.
Unfortunately, despite this seemingly massive effort by the
host, resistance fails. In Craigella, these generalized
defense responses are not protecting the plant from
V. dahliae, race 1. In this context it is important to realize that,
once resistance has failed, the fungus will spread systemically, activating these same defense responses throughout the
susceptible plant, as illustrated in Fig. 3a. Paradoxically, as
reviewed by Beckman (1987) and Pegg and Brady (2002),
many of these same responses actually may be harmful to the
plant and possibly contribute to disease.
Despite many decades of research (for review see Pegg
1981) there is still no consensus about the physiological
and biochemical basis of Verticillium wilt symptoms (for
reviews see Pegg and Brady 2002; Fradin and Thomma
2006). One school of thought attributes the wilting and
browning associated with vascular diseases to fungal
activity during colonization, including the production of
host cell wall degrading enzymes (i.e., pectinolytic
enzymes, polygalacturonases, etc.) as well as toxins or cell
death elicitors (i.e., low or high molecular weight) that are
detrimental to plant tissues. Another school of thought sees
symptom development as an unfortunate secondary consequence of the host response as it attempts to defend itself
against the pathogen, including altered hormone metabolism (e.g., auxins, ethylene, and abscisic acid), the production of defense proteins involved in fungal cell wall
degradation (i.e., chitinase, b-1,3-glucanases), and altered
phenol metabolism (e.g., lignin, suberin). As an example of
the latter, in the context of the current study, the vascular
coating involving the up regulation of TMP1 protein
(Figs. 2, 4), which clearly is a defense response to Verticillium (Robb et al. 1989), has also been demonstrated to
123
Planta (2012) 236:623–633
(a)
631
(a)
(c)
(b)
(d)
(b)
Fig. 5 Analyses of defense gene mRNA and protein levels in
uninfected resistant and susceptible tomato plants. RNA and protein
extracts were prepared from tomato seedlings (four-leaf stage, three
plants per isoline, three biological replicates) and used for microarray
hybridization analyses of mRNA levels using TVR chips (a) or twodimensional IPG/SDS gel fractionation analyses of cellular protein
levels (b) as described in ‘‘Materials and methods’’. Levels in
uninfected resistant (CR) and susceptible (CS) plants, as quantified
using captured images and GenePix Pro 4 or Progenesis SameSpots
software, respectively, are compared as scatter plots
contribute to one aspect of symptom development, namely
water restriction and wilting of the host (Robb et al. 1983;
Robb and Busch 1983). In this respect the present results
appear to provide direct evidence for past speculation (e.g.,
Beckman 1987) that accumulating defense proteins actually may be the cause of some or all symptoms in vascular
diseases. Defense proteins have been implicated directly in
necrosis and cell death associated with the formation of
lesions, characteristic of foliar hypersensitive responses
(Lam 2004; Mittler and Cheung 2004). Further, the up
regulation of defense gene expression has been shown to
precede symptoms (i.e., leaf yellowing, necrosis and
abscission) associated with natural plant senescence
Fig. 6 Detailed analyses of representative defense gene mRNA and
protein levels in the tomato/Verticillium interaction. The susceptible
or resistant tomato seedlings were infected by root dip or dipped in
the absence of spores and stem tissue RNA or protein extracts were
prepared for microarray hybridization and qRT-PCR analyses or twodimensional IPG/SDS gel fractionations as described in ‘‘Materials
and methods’’. Levels of glucan endo-1,3-b-glucosidase (BGL2) and
pathogenesis-related leaf protein 6 (PR leaf protein 6) mRNA
(a, c) and protein (b, d), respectively, were determined using
captured images of the hybridization chips or gel fractionations. The
mRNA measurements summarize the results of two biological
experiments, four replications of each with three pooled stems per
replicate and represent the average Log2 ratio (CS-infected/CS
plants) ± SD at 5 (light) and 10 (dark shading) d.p.i. in susceptible
plants. The protein measurements summarize average normalized
volumes for susceptible (hatched) or resistant (solid columns) plants
at 4–12 d.p.i., as determined by the 2D gel analyses
(Quirino et al. 2000; Buchanan-Wollaston et al. 2003;
Mittler and Cheung 2004), which in many respects
resembles Verticillium wilt disease (Powelson and Rowe
1993). This is consistent with our observation that the
elevated accumulation of defense protein in the susceptible
isoline correlates with symptom development.
A general activation of defense proteins, such as one
might anticipate in SAR (Durrant and Dong 2004) is not
responsible for incompatibility in resistant tomatoes. Not
only does the build up occur too late but it is also actually
more predominant in the susceptible host. This is not to say
that these proteins have no role to play in controlling the
development of the interaction. Previous evidence shows
that the Verticillium population cycles in infected tomatoes
123
632
(Chen et al. 2004) suggesting that the host does control the
pathogen in both compatible and incompatible interactions,
at least to some extent, although it is never totally eliminated. In addition, tomatoes which are transgenic for
defense proteins such as acidic endochitinase, derived from
wild tomato (Lycopersicon chilense), have ‘‘improved’’
tolerance to Verticillium dahliae, races 1 and 2 (Tabaeizadeh et al. 1999). However, one must bear in mind that, in
these types of experiments, the defense gene is usually
under control of an over expression promoter (i.e., CAMV
35S), likely resulting in much higher levels of protein than
would be encountered in nature, and again symptoms are
only reduced but not eliminated.
Many studies with other types of pathogens have shown
that the expression of defense response genes is often up
regulated, sooner in incompatible interactions (Van Loon
2006; Castroverde et al. 2010) but this need not be true for
vascular pathogens. A previous study of gene expression in
the Craigella/Vd1 pathosystem has suggested that some
genes were somewhat up or down regulated earlier in the
stems of the resistant isoline compared with susceptible;
however, none of the encoded pathogenesis-related proteins
were related clearly to pathogen defense (Robb et al. 2009).
Equally, in a comparison of transcriptional responses in
plants infected with a foliar or vascular fungal pathogen
(van Esse et al. 2009), distinct expression profiles were
noted for each individual pathogen but largely overlapped
between the compatible and incompatible interactions. As
noted earlier, the protein data actually may be more significant; because of post-transcriptional and/or translational
regulatory control mechanisms, mRNAs could be under
expressed or not expressed at all. At 4 d.p.i., when Verticillium has been in the stem for only about 1 day, there were
minimal changes in protein levels in either isoline when
compared with uninfected plants. Subsequently, there was a
significant increase in defense protein levels in both CR and
CS plants by 12 d.p.i. that was substantially greater in the
compatible interaction. Regulatory proteins such as transcription factors may turn over rapidly; but, ‘‘metabolic
proteins’’ including PR proteins as well as others involved
in defense (e.g., PAL, ACC oxidase), do not. As a result it
seems highly unlikely that an early, generally greatly elevated, defense protein level in the resistant plant simply was
masked by turnover. In the case of root tissues where fungus
is present at much earlier stages, some differential changes
in H2O2 and lignin, which appear to favor the resistant
plant, have been reported (Gayoso et al. 2010) but these
essentially disappeared after 16 h, observations that are
more consistent with metabolic regulation.
Earlier studies (Bishop and Cooper 1983; Chen et al.
2004) and evidence presented here (Fig. 1) suggest that the
amount of fungus in the roots or stems of susceptible and
resistant plants are similar at initial stages of stem
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Planta (2012) 236:623–633
colonization and argue against the possibility that in the
resistant isoline, the pathogen is stopped by an early defense
mechanism in the root. Given these observations, the most
surprising finding in the present studies remains the reduced
level of both defense-related mRNAs and proteins associated with tomato stem tissue expressing the Ve1 resistance
protein even though substantial amounts of fungal hyphae
(Supplementary data, Fig. S2) are present. The studies
appear to be antithetical to the commonly held perception
that, as summarized by Fradin and Thomma (2006), the rate
and level of PR proteins and other defense-related compounds are higher in the roots and stems of resistant plants.
As indicated in Fig. 1d, presence of the dominant resistance
gene does lead to significantly reduced levels of both fungus
and disease by 10–12 d.p.i. Structural analyses at the
molecular level have suggested that this R-gene product
may be a membrane-bound receptor (Kawchuk et al. 2001;
Fradin et al. 2009) but, whatever its role, the present study
indicates it functions with little positive influence on the
general defense protein cadre. It may even down regulate or
suppress the general defense responses, possibly minimizing disease development, which ultimately can result in
plant death. The mRNA and protein analyses did not point
to any obvious causes; so, how this ‘‘smart’’ gene accomplishes its task in the absence of significant defense-related
protein changes remains an intriguing question, about the
‘‘battle’’ between plants and their vascular pathogens. Perhaps, the Ve1-gene controls the location of a particular plant
protein rather than the level or timing or activates a preexisting enzyme, or possibly the defective Ve1--allele
alters host metabolism in ways that allow it to be colonized
more effectively, for example, by early suppression of a
specific defense response (Gold and Robb 1995). What is
clear from protein analyses is that the levels and timing of
general increases in defense-related proteins in the stems of
Verticillium-infected tomato actually correlate with differences in symptom development between the compatible and
incompatible interactions, and that higher levels of these
proteins, including many PR proteins, appear not to help the
plant. Rather the results continue to entertain the possibility
that some or all of the disease symptoms, including ultimate
plant death, actually may be caused by the exaggerated but
impotent defense response in susceptible plants.
Acknowledgments Supported by NSERC, Canada (R.N.N. and
J.R.), NIH, NHLBI (A.K.) and a Canadian Commonwealth Scholarship (H.O.S.).
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