Download Expression of 35S::Pto Globally Activates

Expression of 35S::Pto Globally Activates Defense-Related
Genes in Tomato Plants1
Fangming Xiao, Xiaoyan Tang, and Jian-Min Zhou*
Department of Plant Pathology, Kansas State University, Manhattan, Kansas 66506
The tomato (Lycopersicon esculentum) resistance gene Pto confers resistance to the bacterial pathogen Pseudomonas syringae pv
tomato carrying the avirulent gene avrPto. Overexpressing Pto under the control of the cauliflower mosaic virus 35S promoter
constitutively activates defense responses in the absence of pathogen infection and nonspecifically enhances disease
resistance. To elucidate the mechanisms underlying this resistance, we isolated cDNAs corresponding to transcripts that
accumulated in 35S::Pto plants. By using suppression subtractive hybridization, we isolated 82 unique cDNA clones, most
of which corresponded to differentially expressed transcripts. Most of the genes examined were also induced by pathogen
inoculation. Sequence analysis showed that a large number of genes encode defense-related proteins, and most had not been
previously isolated from tomato. The isolated cDNAs also include those with a putative role in the oxidative burst,
proteolysis, the hypersensitive response, signal transduction, and a number of genes with unknown functions. The isolation
of these cDNAs of diverse functions will assist in the characterization of defense pathways activated during disease
resistance.
Induced plant resistance to pathogens involves a
complex array of biochemical and structural alterations in the plant cell. Induced expression of a large
number of defense-related genes is essential for
plants to counter pathogen infections. This has been
appreciated since the identification of pathogenesisrelated (PR) proteins in virus-infected plants (Van
Loon and Van Kammen, 1970). Many defense-related
genes encode proteins possessing antifungal or antibacterial activities or enzymes that catalyze defense
metabolites (Bowles, 1990). Others encode regulatory
proteins important for defense signal transductions
(Eulgem et al., 1999; Glazebrook, 1999). The isolation and characterization of these genes are essential
for our understanding of plant disease resistance
mechanisms.
The tomato (Lycopersicon esculentum) disease resistance gene Pto confers gene-for-gene resistance to the
bacterial pathogen Pseudomonas syringae pv tomato
(avrPto). When constitutively expressed under the
control of the CaMV 35S promoter, Pto induces the
development of spontaneous microscopic lesions and
PR gene expression in tomato plants in the absence of
pathogen infections (Tang et al., 1999). The 35S::Pto
plants display broad resistance to both bacterial and
fungal pathogens. To facilitate the study of mechanisms underlying lesion formation and disease resistance, we have used suppression subtractive hybridization (SSH; Diatchenko et al., 1996) to isolate
1
This work was supported by the National Science Foundation
(grant no. MCB9808701 to J.-M.Z.) and by the U.S. Department of
Agriculture (grant no. 9802511 to X.T.). This is Kansas Agricultural
Experimental Station contribution no. 01–338 –J.
* Corresponding author; e-mail [email protected]; fax
785–532–5692.
tomato cDNA corresponding to 35S::Pto-induced
transcripts. Characterization of 82 unique cDNA
clones indicated that a large number of genes belonging to diverse pathways are induced in 35S::Pto
plants. It is notable that there were a large number of
genes encoding PR proteins, proteins with a putative
role in hypersensitive reaction (HR)/lesion development, and a number of proteins related to the oxidative burst, proteolysis, signal transduction, and lipid
metabolism. Furthermore, 10 cDNA clones have no
match with the tomato expressed sequence tag (EST)
database, which contains 107,000 entries as of February 2001. Consistent with the role of Pto in disease
resistance, many of these genes induced by the
35S::Pto transgene were also induced by pathogen
inoculation. The isolation of these cDNA clones that
are related to a diverse array of functions should
assist in the characterization of defense pathways
regulated by Pto.
RESULTS
Characterization of the Subtracted cDNA Library
To isolate cDNA for transcripts that accumulated
in 35S::Pto transgenic plants, an SSH library (Diatchenko et al., 1996) was made with the tester cDNA
from 35S::Pto line 48 (Tang et al., 1999), which consistently produces uniform microscopic lesions on
leaves, and driver cDNA from isogenic nontransgenic tomato cv Money Maker plants. The library comprised approximately 2,000 clones, the majority of which carried fragments of 300 to 1,000 bp.
Northern-blot analysis was conducted with six randomly selected clones from the cDNA library (Fig. 1).
Transcripts corresponding to all six clones were more
abundant in the 35S::Pto line, indicating that the ma-
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1637
Xiao et al.
nificant similarity with genes encoding known proteins. These were placed into several functional categories including plant defense, oxidative burst,
proteolysis, signal transduction, gene expression and
regulation, lipid metabolism, stress response, ribosomal function, and others. Seven clones shared homology only with those encoding proteins of unknown functions. Fourteen clones did not match any
protein sequences in the database. Furthermore, 18
clones were novel tomato cDNAs that did not have a
match with the tomato EST database or previously
reported tomato sequences.
Figure 1. RNA expression of randomly selected clones from the SSH
cDNA library confirms differential expression in 35S::Pto-transgenic
plants. Ten micrograms of total RNA from tomato cv Money Maker
plants with (⫹) or without (⫺) the 35S::Pto transgene was separated
in a denaturing agarose gel, and duplicated RNA blots were hybridized to radiolabeled cDNA for each clone. Ethidium bromide staining from one of the duplicates indicates equal loading of the RNA
(rRNA).
jority of clones in the library represent transcripts
with increased abundance in plants that overexpress
Pto. Initial sequencing of the library showed that
several cDNA clones were highly redundant in the
library. These encode catalases, a Gly-rich cell wall
protein, and several members of the PR1 family of
proteins. These cDNA clones, together with several
known tomato PR gene cDNA clones (PR1a1, PR1b1,
GluB, Osmotin, and Chia; cited in Tang et al., 1999),
were used as probes in colony hybridization in the
subsequent experiments, and non-hybridizing clones
were sequenced. This effectively removed the majority of redundant clones except for clone 440 that
encodes GluB (Table I). A total of 190 cDNAs were
sequenced. CLUSTALW analysis (Thompson et al.,
1994) showed that they belonged to 82 nonredundant
cDNA fragments.
The clones were characterized by “reverse northern” analysis (see “Materials and Methods”). Southern blots containing PCR products of the cDNA
clones were probed with radiolabeled cDNA probes
made from RNA extracted from non-transgenic tomato leaves or the 35S::Pto tomato leaves. Of 77
clones tested, 64 showed increased transcript expression in 35S::Pto plants (Table I). A representative
example of the hybridization is shown in Figure 2.
The remaining five clones were either not tested or
tested with no informative results. The tight correlation with results from northern analysis (Fig. 1 and
Table I) validates the “reverse northern” experiments.
We used the BLASTX program to search the GenBank database for proteins that are homologous to
those encoded by the 82 clones (Table I). In a similar
manner, the BLASTN program was used to search for
tomato EST sequences identical to these cDNAs. The
clones were grouped according to functions of the
putative protein products. Sixty-two clones had sig1638
Genes Induced by Bacterial Inoculation
We tested whether the clones from our cDNA library are relevant to defense responses in nontransgenic plants. Reverse northern analysis indicated that 35 of the 52 clones examined showed
increased accumulation when tomato cv RioGrande
PtoR plants that carry the resistance gene Pto were
inoculated with P. syringae pv tomato carrying the
corresponding avirulence gene avrPto. Northern
analysis was conducted for 40 clones to further determine the bacterial inducibility. Twenty-four clones
exhibited increased transcripts when plants were infiltrated with bacteria, whereas the remaining clones
showed either no bacterial induction or did not yield
detectable signals in the northern analysis (Table I).
Figure 3 shows northern analysis of four clones that
were tested repeatedly and showed early induction
of transcripts following bacterial inoculation (1–3 h).
Involvement of Salicylic Acid (SA) in Gene Regulation
SA is globally involved in defense responses during plant-pathogen interactions (Ryals et al., 1996).
35S::Pto transgenic tomato plants accumulate high
levels of SA and exhibit increased resistance to both
bacterial and fungal pathogens (Tang et al., 1999). To
test if any genes described here are induced by an
SA-dependent pathway, we crossed transgenic tomato plants carrying the bacterial nahG gene, which
encodes a salicylate hydroxylase, to the 35S::Pto
transgenic tomato plants (Brading et al., 2000) and
examined gene expression in F1 plants. The hemizygous nahG/35S::Pto plants were indistinguishable
from the hemizygous 35S::Pto plants in the expression of spontaneous lesions, and all plants displayed
resistance to both the virulent strain and avirulent
strain (avrPto) of P. syringae pv tomato (J. Zhou and X.
Tang, unpublished data). Reverse northern hybridizations of nylon filter arrays containing the 82 cDNA
clones showed that majority of the clones hybridized
equally with the cDNA probe generated from
35S::Pto plants and that from 35S::Pto/nahG plants.
However, four clones (14, 267, 554, and 561) showed
reduced signal when probed with cDNA derived
from 35S::Pto/nahG plants (data not shown). These
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Plant Physiol. Vol. 126, 2001
Global Defense Gene Expression in Tomato
Table I. cDNA clones isolated from 35S⬋Pto plants
⫺, No induction; ⫹, 2- to 4-fold induction; ⫹⫹, 5- to 9-fold induction; ⫹⫹⫹, greater than 10-fold induction; blank, not tested. Relative signal
on the x-ray film was quantified by densitometry and normalized to constitutive controls (see “Materials and Methods”) before fold induction was
calculated.
GenBank Accession No.
Oxidative burst/antioxidant
BG351996
Clone
8
BG351997
12
BG351998
20
BG351999
568
BG352000
43
BG352001
534
BG352002
315
BG352003
BG352004
486
533
Defense-related proteins
BG352005
BG352006
BG352007
BG352008
BG352009
BG352010
60
502
255
454
561
248
BG352011
BG352012
307
440
BG352013
106
BG352014
BG352015
243
480
BG352016
718
BG352017
BG352018
540
569
BG352019
503
BG352020
616
BG352021
554
BG352022
14
Classification Based on Homology to
GenBank Sequences
NP_002486 human NADH dehydrogenase
P25890 pea (Pisum sativum) catalase
P55312 potato (Solanum tuberosum) catalase
P30264 tomato (Lycopersicon esculentum) catalase isozyme 1
P29795 tomato photosystem II
oxygen-evolving complex protein 2
S20935 tobacco (Nicotiana tabacum) photosystem II oxygenevolving complex protein 2
AAD35009 Arabidopsis thioredoxin
AAC97494 tomato annexin p34
AAB52954 cotton (Gossypium
hirsutum) ascorbate peroxidase
S22531 tobacco prb-1b
S22531 tobacco prb-1b
2624502 tomato PR1
P04284 tomato PR1 (P6)
Q04108 tomato PR1 (P4)
AAC69757 maize (Zea mays)
␤-1,3-1,4-glucanase
S44365 tomato ␤-1,3-glucanase
Q01413 tomato ␤-1,3-glucanase
(GluB)
Q05538 tomato basic endochitinase
CAA30142 potato endochitinase
P29060 tobacco class III acidic
chitinase
AAF29391 Arabidopsis basic endochitinase class I
P32045 tomato PR4
AAF13707 Fragaria ⫻ ananassa
osmotin-like protein
P17642 potato PR protein STH-2
(PR10)
CAA75803 tomato SA induced
(PR10)
CAA59472 Catharanthus roseus
hybrid proline-rich protein
T07013 tomato Gly-rich cell wall
protein
E Value
Tomato EST
35S⬋Pto
Induction
Ia
IIb
Bacterial
Induction
I
II
⫹
⫹⫹c
6e-19
AW219329
⫹c
⫹
3e-17
BE461524
⫹
⫹⫹c
7e-96
AW216974
⫹
Identical
AW033487
⫹c
3e-16
AW217737
⫹
3e-06
AW442912
⫹
⫺
3e-54
AI486521
⫹c
⫺
Identical
3e-40
AW219769
AW622838
⫹
⫹c
6e-49
2e-15
2e-41
Identical
Identical
0.005
AW217013
AW625666
AW034667
AI899427
AW034882
No
⫹⫹
⫹
⫹
3e-12
Identical
AI780513
AW217195
2e-11
AW216897
3e-65
3e-44
AW216454
AW030575
⫹
⫹
7e-73
AW933508
Identical
1e-25
⫹c
⫹c
⫺
⫺
⫹⫹c
⫺
⫹c
⫺
⫹⫹
⫹⫹c
⫹⫹c
⫹
⫹⫹
⫹⫹
⫹⫹c
⫹c
⫹⫹c
⫹⫹⫹
⫹c
⫹⫹c
⫹⫹⫹
⫺c
⫺
⫺
AW442631
AI483135
⫹
⫹
⫹c
⫺
5e-35
BE450364
⫹
⫹⫹
2e-63
AI489282
⫹
1e-15
AW626221
⫹
Identical
AW041743
⫹c
⫹c
⫹⫹c
⫹⫹⫹
⫹
⫹⫹
⫹⫹⫹c
⫹⫹
⫹c
⫹⫹
(Table continues.)
Plant Physiol. Vol. 126, 2001
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Copyright © 2001 American Society of Plant Biologists. All rights reserved.
1639
Xiao et al.
Table I. (Continued from previous page).
GenBank Accession No.
Clone
Classification Based on Homology to
GenBank Sequences
E Value
Tomato EST
35S⬋Pto
Induction
Ia
Bacterial
Induction
IIb
I
II
⫹⫹c
⫹⫹c
⫹⫹c
⫹⫹⫹c
⫺
⫺c
BG352023
301
AAF68391 maize hypersensitive
induced (prohibitin, stomatin)
5e-64
AW036075
⫹
Proteases
BG352024
271
6e-17
AI775536
⫹
BG352025
441
8e-22
No
BG352026
728
AAA50755 Alnus glutinosa Cys
proteinase
BAB08420 Arabidopsis cell division protein FtsH protease-like
protein
CAA71234 tomato subtilisin-like
protease
Identical
AI779080
⫹⫹
Stress related
BG352027
BG352028
BG352029
50
414
465
Identical
2e-59
Identical
AW096671
AI895441
AW738420
⫹
⫹c
⫹
1e-44
AI778965
⫹
4e-21
AW625628
⫹⫹
1e-70
AW033361
⫹
⫹
⫹
3e-50
AI774470
⫹c
⫹⫹⫹c
⫹⫹⫹c
0.007
AW651434
⫹c
⫹c
8e-12
AW615864
⫹
6e-06
AW615864
2e-61
AI483225
Identical
1e-24
Lipid metabolism
BG352030
27
BG352031
101
BG352032
267
BG352033
398
BG352034
487
Protein protein interactions/
signal transduction
BG352035
285
Q43513 tomato MT II
S50752 potato proton ATPase
AF261139 tomato dehydrationinduced protein ERD15
AAF23458 pepper (Capsicum annum) lipid transfer protein
JQ2343 Zinnia elegans lipid transfer protein
AAC49278 Arabidopsis sterol
delta-7 reductase
CAA64414 tomato lipid desaturase
P04634 rat (Rattus norvegicus)
triacylglycerol lipase
NP_003738 human TRF1interacting ankyrin-related ADPribose polymerase
NP_003738 human TRF1interacting ankyrin-related ADPribose polymerase
AAC62877 WD repeat of Arabidopsis translation initiation protein
P42652 tomato 14-3-3 protein
CAB07805 tobacco protein phosphatase 1
⫹
⫹
⫹
⫹
AW622689
AW219414
⫹
⫹
⫹
⫹c
⫹
⫹
4e-29
AW626110
⫹c
⫹c
⫹
2e-11
AW738391
⫹
⫹
⫹
4e-21
AW094020
⫹c
⫹
⫹
659
BG352037
539
BG352038
BG352039
576
664
Gene expression
BG352040
452
BG352041
521
BG352042
541
Protein targeting
BG352043
632
CAA91162 spinach (Spinacia oleracea) secY protein homolog
9e-65
AW037812
⫺
9
CAB65281 alfalfa (Medicago sativa) ribosomal L3
P46222 Drosophila melanogaster
ribosomal L11
227228 Mouse ribosomal L28
8e-65
AW220096
⫹
Ribosomal proteins
BG352044
BG352045
291
BG352046
722
1640
⫺
⫺
⫹c
⫹c
BG352036
AAD10626 Lolium temulentum
MADS box protein 2
NP_002083 human G-rich RNA
sequence binding factor 1
AAC61751 Trypanosoma cruzi
poly-zinc finger protein 1
⫹⫹c
⫹
6e-30
2e-07
⫹
No
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Copyright © 2001 American Society of Plant Biologists. All rights reserved.
⫹
⫹c
(Table continues.)
Plant Physiol. Vol. 126, 2001
Global Defense Gene Expression in Tomato
Table I. (Continued from facing page).
GenBank Accession No.
Clone
Others
BG352047
93
BG352048
276
BG352049
317
BG352050
415
BG352051
558
BG352052
565
BG352053
585
BG352054
626
BG352055
642
BG352056
697
Novel proteins
BG352057
309
BG352058
416
BG352059
417
BG352060
522
BG352061
552
BG352062
562
BG352063
606
No matches
BG352064
BG352065
BG352066
BG352067
BG352068
BG352069
BG352070
BG352071
BG352072
BG352073
BG352074
BG352075
BG352076
BG352077
272
277
320
407
412
493
528
557
560
567
577
603
636
656
Classification Based on Homology to
GenBank Sequences
AAA80594 potato chlorophyll
a/b-binding protein
CAA05979 Lupinus albus ADP/
ATP carrier
Q43794 tobacco glutamyl tRNA
synthetase
AAF14680 Arabidopsis DnaJ domain
CAA06156 maize cytochrome
P450 monooxygenase
CAA47373 tobacco (Nicotiana
sylvestris) Gln synthetase
P16048 pea Gly cleavage system
protein H
AAA78277 tobacco rubisco activase
004350 Arabidopsis tubulinspecific chaperone A
BAA77604 tobacco (Nicotiana
paniculata) plastidic aldolase
CAB41935 Arabidopsis putative
protein
AAB61506.1 Arabidopsis putative
protein
CAA22991.1 Arabidopsis putative
protein
CAA18744 Arabidopsis putative
protein
AAF02150 Arabidopsis putative
protein
CAA22991 Arabidopsis putative
protein
AAB61480 Arabidopsis putative
protein
No
No
No
No
No
No
No
No
No
No
No
No
No
No
match
match
match
match
match
match
match
match
match
match
match
match
match
match
a
Expression determined by “reverse northern.”
independent experiments.
b
35S⬋Pto
Induction
Tomato EST
Ia
IIb
Bacterial
Induction
I
II
4e-13
AW944784
⫺
9e-27
No
⫹
⫹
7e-33
AW031320
⫺
⫺
5e-13
AI776948
⫹c
⫹⫹c
7e-35
AW219708
⫹
⫹
7e-50
AI778053
⫹
⫺
1e-33
AW093614
⫺
⫺
1e-45
BE432926
⫺
1e-35
AW094598
⫹
1e-45
BE354659
⫺
9e-07
BE460000
⫺
1e-26
No
⫹c
2e-16
BE400935
⫹⫹
3e-49
4e-46
AW398892,
L. pennellii
AI777238
5e-30
⫺
⫹⫹c
⫹
⫹c
⫺
⫹c
⫺
⫺c
⫹
⫺
AW625429
⫹
⫹
6e-28
AW441993
⫺
⫹c
–
–
–
–
–
–
–
–
–
–
–
–
–
–
No
AW442444
AW649771
BE459012
No
BG132519
AW443952
No
AW930949
No
No
No
BE459012
AI989142
⫹
⫺
⫺c
⫹c
⫹
⫹⫹
⫹
⫺
⫹
⫹
⫹
Expression determined by northern analysis.
clones encode a Gly-rich cell wall protein, sterol reductase, Pro-rich protein, and PR1a1, respectively.
Northern-blot analysis confirmed that the expression
of the four genes in 35S::Pto plants was reduced by
the presence of the nahG transgene (Fig. 4).
Plant Physiol. Vol. 126, 2001
E Value
⫹
⫺
c
⫹
⫺c
⫹c
⫺
⫺
⫹
⫺
⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
Results obtained from at least two
We further examined the involvement of SA in the
P. syringae-induced expression of two genes (clones
271 and 398) that were induced early by bacterial
inoculation. Repeated northern analyses indicated
that transcripts of clones 271, which encodes a Cys
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1641
Xiao et al.
Figure 2. Reverse northern analysis of the subtracted library. PCR-amplified cDNA inserts were separated on agarose gels
and transferred to nylon filters. Lane a contained the cDNA insert of clone 93 that encodes a chlorophyll a/b-binding protein.
Lanes b through t contained cDNA inserts from 19 randomly selected clones. The duplicated filters contained equal amounts
of the PCR products and were hybridized with cDNA probes synthesized from non-transgenic tomato cv Money Maker (MM)
or tomato cv 35S::Pto transgenic mRNA.
protease, were induced more strongly following bacterial inoculation in nahG plants (Fig. 5), suggesting a
negative role of SA in the expression of this gene. In
contrast, the expression of transcripts of clone 398,
encoding a fatty acid desaturase, was not significantly affected by nahG (Fig. 5), indicating that its
induction is independent of SA accumulation.
DISCUSSION
In this report, we describe the isolation and characterization of a significant number of cDNA clones
corresponding to genes expressed at a elevated level
in 35S::Pto plants. Two lines of evidence indicate that
the majority of these genes are bona fide defenserelated genes. First, a large number of clones (19)
encode proteins with an apparent role in plant defense against pathogens. Considering that the cDNA
library had been prescreened with six PR gene probes
prior to sequencing, the actual number of clones
coding for antimicrobial proteins could be larger
than 20. This is a clear indication that the library is
highly enriched for genes involved in defense responses. In addition, most of the clones that showed
Figure 3. Pathogen-induced expression of selected genes. Tomato cv
Rio Grande PtoR plants were inoculated with 106 colony forming
units (cfu) mL⫺1 P. syringae pv tomato (avrPto) or 10 mM MgCl buffer
(Mock). RNA was isolated at indicated times, and 10 ␮g RNA was
separated in a denaturing agarose gel. Duplicated RNA blots were
hybridized with indicated probes. Ethidium bromide staining from
one of the duplicates indicates equal loading of the RNA (rRNA).
1642
an increased expression in the 35S::Pto plants appeared to be induced by pathogen inoculation.
Most clones in the “defense-related protein” category had a match with known proteins that encode
structural proteins for plant defense. These include
16 clones encoding various PR proteins that may play
a direct role in inhibiting pathogens. Except for
clones 60 and 502, which appear to be two fragments
of the same gene for a PRb-1b-like protein, the remaining clones encode 14 distinct PR proteins (clones
255, 454, 561, 248, 307, 440, 480, 540, 569, 718, 243, 106,
503, and 616). Proteins encoded by clones 14 and 554
are cell wall proteins that probably are involved in
cell wall fortification in plants. In addition, lipid
transfer proteins encoded by clones 27 and 101 in the
“lipid metabolism” category may also play a direct
role in defense. It has been reported that lipid transfer proteins possess antibacterial activities (Caaveiro
et al., 1997). Clone 301 encodes a protein similar to
maize HR-associated protein. This protein is also
similar to the tobacco NG8 that has been implicated
to play a role in tobacco mosaic virus-induced HR
(Karrer et al., 1998). An Arabidopsis homolog (accession no. 7269612) accumulates its transcripts in the
mpk4 mutant that exhibits constitutively activated
defense responses (Petersen et al., 2000). It appears
Figure 4. Effects of nahG transgene on the 35S::Pto-induced expression of genes. Homozygous 35S::Pto plants (line 48) were crossed to
homozygous nahG plants to produce 35S::Pto/nahG hemizygous
plants. As a control, homozygous 35S::Pto plants were crossed to
non-transgenic tomato cv Money Maker plants to produce hemizygous 35S::Pto plants. RNA from the hemizygous plants was separated
in a denaturing agarose gel, and duplicated RNA blots were hybridized with the indicated cDNA probes.
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Plant Physiol. Vol. 126, 2001
Global Defense Gene Expression in Tomato
Figure 5. Effects of nahG on pathogen-induced expression of clones
271 and 398. Non-transgenic tomato cv Money Maker or tomato cv
Money Maker plants containing the nahG transgene were inoculated
with 106 cfu mL⫺1 P. syringae pv tomato, and RNA was isolated at
the indicated times. Duplicated RNA blots were hybridized with the
indicated cDNA probes.
that this class of proteins maybe involved in HR
development in a variety of plants.
In addition to antimicrobial proteins, several
classes of proteins encoded by the cDNA clones are
potentially important for plant defense. Three clones
encode proteases. It is notable that the Cys protease
encoded by clone 271 and the FtsH protease encoded
by clone 441 may be related to programmed cell
death. Caspases, a group of Cys proteases, are important components in animal programmed cell
death pathway (Green, 2000). In plants, inhibitors of
Cys proteases can inhibit cell death triggered by avirulent P. syringae bacteria (Solomon et al., 1999). The
Xanthomonas campestris Avr protein AvrBsT shares
homology with Cys proteases, and mutation of the
protease catalytic site of AvrBsT eliminates its ability
to activate the hypersensitive response in plants
(Orth et al., 2000). Clone 271 is also similar to Arabidosis SAG12 that is expressed during leaf senescence,
a cell death program invoked during normal development of plants (Gan and Amasino, 1995). Clone
441 is similar to the FtsH class of metalloproteases
that is conserved in both prokaryotes and eukaryotes. In tobacco, a chloroplast FtsH protein has
been shown to be a negative regulator of tobacco
mosaic virus-induced HR (Seo et al., 2000). Overexpression of this gene in tobacco attenuates the HR.
Clone 728 encodes a subtilisin-like Ser protease that
is induced by pathogen infection (P69B; Tornero et
al., 1996, 1997). It will be important to determine if
any of these genes are involved in programmed cell
death during plant disease resistance.
A group of proteins encoded by the cDNA clones
are related to lipid metabolism. These include lipid
transfer proteins (clones 27 and 101), microsomal
lipase (clone 487), ␻-6 fatty acid desaturase (clone
398), and sterol delta-7 reductase (clone 267). In parsley (Petroselinum crispum), the treatment of a peptide
elicitor from Phytophthora sojae greatly alters fatty
acid profiles of the plant cell. This correlates with the
elicitor-induced expression of transcripts of several
fatty acid desaturases (Trezzini et al., 1993; Kirsch et
Plant Physiol. Vol. 126, 2001
al., 1997). Some of these events maybe involved in the
accumulation of jasmonic acids that are important
defense hormones. EDS1 and PAD4, two proteins
that function in disease resistance gene-mediated
pathways, are homologous to lipases, and their transcripts are induced upon pathogen infection (Falk et
al., 1999; Jirage et al., 1999). The accumulation of
transcripts related to lipid metabolism in 35S::Pto
plants reinforces the significance of lipids in plant
defense.
Several clones encode proteins involved in the generation or scavenging of oxidative stress. The NADH
dehydrogenase encoded by clone 8 may have a role
in generating reactive oxygen species, whereas the
catalases encoded by clones 12, 20, and 568 may help
the host cell to cope with reactive oxygen species
accumulated during defense reactions. The transcripts represented by clones 8, 12, 20, and 568 accumulated in the 35S::Pto plants. At least those represented by clones 12 and 20 were induced by bacterial
infection. Clone 486 encoding annexin was also induced by both pathogen and the 35S::Pto transgene.
The expression of annexin also may play a role in
scavenging reactive oxygen species. An Arabidopsis
annexin-like protein has been shown to possess peroxidase activity, and the expression of its cDNA restored H2O2 tolerance to the Escherichia coli ⌬oxyR
mutant that is unable to express catalase in response
to oxidative stress (Gidrol et al., 1996). It is interesting that the Arabidosis Annexin-like gene is induced
by H2O2 and SA, suggesting a role in defense responses. In contrast to clones 12, 20, and 486, clone
533, which encodes an ascorbate peroxidase, was
expressed at a higher level in 35S::Pto plants but was
not induced by pathogen infection. The accumulation
of the ascorbate peroxidase transcripts maybe an indirect adaptive response to the reactive oxygen species accumulated during the lesion development of
the 35S::Pto plants rather than a defense response
activated by Pto.
The cDNA clones identified in this report represent
genes that are induced by pathogen via a variety of
signaling pathways. For example, clones 271, 301,
398, and 486 were induced early (1–3 h) after P.
syringae inoculation, whereas clones 12, 14, 307, and
480, encoding catalase, Gly-rich cell wall protein,
␤-1,3-glucanase, and class III acidic chitinase, respectively, are induced late (12 h) after bacterial inoculation (data not shown). A detailed study of two early
induced genes showed that the pathogen induction
of RNA corresponding to clone 271 appeared to be
regulated negatively by SA. This could be a result of
the antagonistic interaction between SA and ethylene. In fact, the pathogen-induced expression of transcripts corresponding to clone 271 is delayed in never
ripe plants that are insensitive to ethylene (data not
shown). In contrast, the transcripts corresponding to
clones 301 and 398 did not appear to be affected by
nahG. In addition, the induction of genes correspond-
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Copyright © 2001 American Society of Plant Biologists. All rights reserved.
1643
Xiao et al.
ing to clones 14, 267, 554, and 561, encoding Gly-rich
cell wall protein, sterol reductase, Pro-rich cell wall
protein, and PR1, respectively, by the 35S::Pto transgene required SA signaling. The availability of a
large number of genes that are differentially regulated by distinct defense pathways and encode proteins with diverse functions provides an excellent
tool to study the cell death and disease resistance
mechanisms in tomato plants.
MATERIALS AND METHODS
Plants and Inoculation
Tomato (Lycopersicon esculentum) cultivars used were:
Rio Grande PtoR, which carries a native Pto gene; Money
Maker, which contains no Pto gene; a transgenic line carrying nahG (Money Maker background; Brading et al.,
2000); and 35S::Pto transgenic line 48 (Money Maker background; Tang et al., 1999). Plants were grown in the greenhouse at 28°C (day) and 20°C (night). Healthy and wellexpanded leaves from 6-week-old plants were used for
experiments.
Pseudomonas syringae pv tomato strain T1 (avrPto) was
grown in King’s B (King et al., 1954) medium containing 50
mg L⫺1 rifampicin and 50 mg L⫺1 kanamycin. For inoculation, overnight bacterial culture was washed twice with
10 mm MgCl2, and the density was determined by turbidity
at 600 nm. Bacteria were diluted to 106 cfu mL⫺1 in 10 mm
MgCl2 plus 0.04% (v/v) silwet l-77 (Osi, Danbury, CT) for
vacuum infiltration of six-week-old plants. Well-expanded
leaf tissue was collected at different time points after inoculation for RNA isolation. The 0-h sample was harvested
immediately prior to vacuum infiltration.
Suppression Subtractive Cloning
A cDNA subtraction kit (PCR-Select; CLONTECH, Palo
Alto, CA) was used for isolating cDNA clones from 35S::Pto
plants. The subtractive cDNA library was constructed by
following the user manual with slight modification. The
cDNA from the 35S::Pto line was used as tester, and the
cDNA from the non-transgenic tomato cv Money Maker
plants was used as driver. The driver/tester ratio was
increased to 4-fold over the suggested ratio. The subtracted
cDNA was fractionated through an agarose gel and fragments greater than 300 bp were inserted into the TAcloning vector (CLONTECH). To eliminate the common PR
genes and redundant clones from the subtractive library, a
pool of cDNA clones for five PR genes (PR1a1, PR1b1, GluB,
Osmotin, and Chia; Tang et al., 1999) and other redundant
clones from the library were used as probes to hybridize to
the nylon membrane containing clones from the cDNA
library. Single-pass sequencing with the T7 primer was
carried out for non-hybridizing clones.
Reverse Northern and Northern-Blot Analysis
cDNA inserts were individually PCR amplified from the
plasmid with T3 and T7 primers, separated in an agarose
1644
gel, and transferred to nylon membranes. The DNA blot
contained cDNA inserts from the library and a chlorophyll
a/b-binding protein gene (clone 93) or an actin cDNA
(accession no. AW737353) as a constitutive control. The
actin cDNA was PCR amplified from tomato cDNA with
the following primers: 5⬘-GAAGAAGAAGAAAGAGSGCTTTTC-3⬘ and 5⬘-AGCCTGAATAGCAACATACATAG-3⬘.
Duplicated filters were hybridized with 32P-labeled, oligo
(dT)-primed cDNA probes derived from uninoculated tomato cv Money Maker or tomato cv 35S::Pto plants, or
probes from uninoculated or bacterial-inoculated tomato
cv RioGrande PtoR plants. Each cDNA probe was synthesized from 1 ␮g mRNA by using a cDNA synthesis kit (Life
Technologies, Rockville, MD). The mRNA was removed by
RNaseH treatment. RNA gel-blot analysis was carried out
as described (Goldsbrough et al., 1990; Tang et al., 1999).
Relative signal on the x-ray film was quantified by densitometry and normalized to constitutive controls before fold
induction was calculated.
ACKNOWLEDGMENTS
We thank Drs. Scot Hulbert, Jyoti Shah, Randall Warren,
and Venkatappa Thara for critical review of the manuscripts. We are also grateful to Venkatappa Thara for sharing RNA and Jennifer Foltz for digital art work.
Received March 5, 2001; returned for revision April 27,
2001; accepted May 14, 2001.
LITERATURE CITED
Bowles DJ (1990) Defense-related proteins in higher plants.
Annu Rev Biochem 59: 873–903
Brading PA, Hammond-Kosack KE, Parr A, Jones JD
(2000) Salicylic acid is not required for Cf-2- and Cf-9dependent resistance of tomato to Cladosporium fulvum.
Plant J 23: 305–318
Caaveiro JM, Molina A, Gonzalez-Manas JM, RodriguezPalenzuela P, Garcia-Olmedo F, Goni FM (1997) Differential effects of five types of antipathogenic plant peptides on model membranes. FEBS Lett 410: 338–342
Diatchenko L, Lau Y-FC, Campbell AP, Chenchik A, Moqadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov E et al. (1996) Suppression subtractive
hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc
Natl Acad Sci USA 93: 6025–6030
Eulgem T, Rushton PJ, Schmelzer E, Hahlbrock K, Somssich IE (1999) Early nuclear events in plant defense signalling: rapid gene activation by WRKY transcription
factors. EMBO J 18: 4689–4699
Falk A, Feys BJ, Frost LN, Jones JD, Daniels MJ, Parker JE
(1999) EDS1, an essential component of R gene-mediated
disease resistance in Arabidopsis has homology to eukaryotic lipases. Proc Natl Acad Sci USA 96: 3292–3297
Gan S, Amasino RM (1995) Inhibition of leaf senescence by
autoregulated production of cytokinin. Science 270:
1986–1988
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2001 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 126, 2001
Global Defense Gene Expression in Tomato
Gidrol X, Sabelli PA, Fern YS, Kush AK (1996) Annexinlike protein from Arabidopsis thaliana rescues delta oxyR
mutant of Escherichia coli from H2O2 stress. Proc Natl
Acad Sci USA 93: 11268–11273
Glazebrook J (1999) Genes controlling expression of defense responses in Arabidopsis. Curr Opin Plant Biol 2:
280–286
Goldsbrough PB, Hatch EM, Huang B, Kosinski WG,
Dyre WE, Herrman KM, Weller SC (1990) Gene amplification in glyphosate-tolerant tobacco cells. Plant Sci 72:
53–62
Green DR (2000) Apoptotic pathways: paper wraps stone
blunts scissors. Cell 102: 1–4
Jirage D, Tootle TL, Reuber TL, Frost LN, Feys BJ, Parker
JE, Ausubel FM, Glazebrook J (1999) Arabidopsis thaliana
PAD4 encodes a lipase-like gene that is important for
salicylic acid signaling. Proc Natl Acad Sci USA. 96:
13583–13588
Karrer EE, Beachy RN, Holt CA (1998) Cloning of tobacco
genes that elicit the hypersensitive response. Plant Mol
Biol 36: 681–690
King EO, Ward MK, Raney DE (1954) Two simple media
for the demonstration of pyocyanin and fluorescin. J Lab
Clin Med 44: 301–307
Kirsch C, Takamiya-Wik M, Reinold S, Hahlbrock K,
Somssich IE (1997) Rapid, transient, and highly localized
induction of plastidial omega-3 fatty acid desaturase
mRNA at fungal infection sites in Petroselinum crispum.
Proc Natl Acad Sci USA 94: 2079–2084
Orth K, Xu Z, Mudgett MB, Bao ZQ, Palmer LE, Bliska JB,
Mangel WF, Staskawicz B, Dixon JE (2000) Disruption
of signaling by Yersinia effector YopJ, a ubiquitin-like
protein protease. Science 290: 1594–1597
Petersen M, Brodersen P, Naested H, Andreasson E,
Lindhart U, Johansen B, Nielsen HB, Lacy M, Austin
MJ, Parker JE et al. (2000) Arabidopsis MAP kinase 4
negatively regulates systemic acquired resistance. Cell
103: 1111–1120
Plant Physiol. Vol. 126, 2001
Ryals JA, Neuenschwander UH, Willits MG, Molina A,
Steiner H-Y, Hunt MD (1996) Systemic acquired resistance. Plant Cell 8: 1809–1819
Seo S, Okamoto M, Iwai T, Iwano M, Fukui K, Isogai A,
Nakajima N, Ohashi Y (2000) Reduced levels of chloroplast FtsH protein in tobacco mosaic virus-infected tobacco leaves accelerate the hypersensitive reaction. Plant
Cell 12: 917–932
Solomon M, Belenghi B, Delledonne M, Menachem E,
Levine A (1999) The involvement of cysteine proteases
and protease inhibitor genes in the regulation of programmed cell death in plants. Plant Cell 11: 431–444
Tang X, Xie M, Kim YJ, Zhou J, Klessig DF, Martin GB
(1999) Overexpression of Pto activates defense responses
and confers broad resistance. Plant Cell 11: 15–29
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL
W: improving the sensitivity of progressive multiple
sequence alignment through sequence weighting,
positions-specific gap penalties and weight matrix
choice. Nucleic Acids Res 22: 4673–4680
Tornero P, Conejero V, Vera P (1996) Primary structure
and expression of a pathogen-induced protease (PR-P69)
in tomato plants: similarity of functional domains to
subtilisin-like endoproteases. Proc Natl Acad Sci USA 93:
6332–6337
Tornero P, Conejero V, Vera P (1997) Identification of a
new pathogen-induced member of the subtilisin-like
processing protease family from plants. J Biol Chem 272:
14412–14419
Trezzini GF, Horrichs A, Somssich IE (1993) Isolation of
putative defense-related genes from Arabidopsis thaliana
and expression in fungal elicitor-treated cells. Plant Mol
Biol 21: 385–389
Van Loon LC, Van Kammen A (1970) Polyacrylamide disc
electrophoresis of the soluble leaf proteins from Nicotiana
tabacum var. “Samsun” and “Samsun NN”: II. Changes in
protein constitution after infection with tobacco mosaic
virus. Virology 40: 199–211
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2001 American Society of Plant Biologists. All rights reserved.
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