Expression of 35S::Pto Globally Activates Defense-Related Genes in Tomato Plants

Abstract

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 pathogenesis-related (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 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 non-transgenic 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 majority 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.

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).

Table I.

cDNA clones isolated from 35S∷Pto plants

GenBank Accession No.	Clone	Classification Based on Homology to GenBank Sequences	E Value	Tomato EST	35S∷Pto Induction		Bacterial Induction
					Ia	IIb	I	II
Oxidative burst/antioxidant
BG351996	8	NP_002486 human NADH dehydrogenase	6e-19	AW219329	+c	+
BG351997	12	P25890 pea (Pisum sativum) catalase	3e-17	BE461524	+	++c	+	++c
BG351998	20	P55312 potato (Solanum tuberosum) catalase	7e-96	AW216974	+		+c
BG351999	568	P30264 tomato (Lycopersicon esculentum) catalase isozyme 1	Identical	AW033487	+c	+c	−
BG352000	43	P29795 tomato photosystem II oxygen-evolving complex protein 2	3e-16	AW217737	+
BG352001	534	S20935 tobacco (Nicotiana tabacum) photosystem II oxygen-evolving complex protein 2	3e-06	AW442912	+		−	−
BG352002	315	AAD35009 Arabidopsis thioredoxin	3e-54	AI486521	+c		−
BG352003	486	AAC97494 tomato annexin p34	Identical	AW219769	+	+c	++c	+c
BG352004	533	AAB52954 cotton (Gossypium hirsutum) ascorbate peroxidase	3e-40	AW622838	+c		−	−
Defense-related proteins
BG352005	60	S22531 tobacco prb-1b	6e-49	AW217013	++
BG352006	502	S22531 tobacco prb-1b	2e-15	AW625666	+
BG352007	255	2624502 tomato PR1	2e-41	AW034667	+
BG352008	454	P04284 tomato PR1 (P6)	Identical	AI899427
BG352009	561	Q04108 tomato PR1 (P4)	Identical	AW034882	++		++c
BG352010	248	AAC69757 maize (Zea mays) β-1,3-1,4-glucanase	0.005	No	++c		+	++
BG352011	307	S44365 tomato β-1,3-glucanase	3e-12	AI780513	++		+c	+++
BG352012	440	Q01413 tomato β-1,3-glucanase (GluB)	Identical	AW217195	++c		++c	+c
BG352013	106	Q05538 tomato basic endochitinase	2e-11	AW216897
BG352014	243	CAA30142 potato endochitinase	3e-65	AW216454	+
BG352015	480	P29060 tobacco class III acidic chitinase	3e-44	AW030575	+	++c	++c	+++
BG352016	718	AAF29391 Arabidopsis basic endochitinase class I	7e-73	AW933508	−c		−	−
BG352017	540	P32045 tomato PR4	Identical	AW442631	+		+c
BG352018	569	AAF13707Fragaria × ananassa osmotin-like protein	1e-25	AI483135	+		−
BG352019	503	P17642 potato PR protein STH-2 (PR10)	5e-35	BE450364	+		++	+++
BG352020	616	CAA75803 tomato SA induced (PR10)	2e-63	AI489282	+			+
BG352021	554	CAA59472Catharanthus roseus hybrid proline-rich protein	1e-15	AW626221	+		+++c	++
BG352022	14	T07013 tomato Gly-rich cell wall protein	Identical	AW041743	+c	++	+c	++
BG352023	301	AAF68391 maize hypersensitive induced (prohibitin, stomatin)	5e-64	AW036075	+		++c	++c
Proteases
BG352024	271	AAA50755Alnus glutinosa Cys proteinase	6e-17	AI775536	+	++c	++c	+++c
BG352025	441	BAB08420 Arabidopsis cell division protein FtsH protease-like protein	8e-22	No			−	−c
BG352026	728	CAA71234 tomato subtilisin-like protease	Identical	AI779080	++
Stress related
BG352027	50	Q43513 tomato MT II	Identical	AW096671	+		−
BG352028	414	S50752 potato proton ATPase	2e-59	AI895441	+c		−
BG352029	465	AF261139 tomato dehydration-induced protein ERD15	Identical	AW738420	+		+c
Lipid metabolism
BG352030	27	AAF23458 pepper (Capsicum annum) lipid transfer protein	1e-44	AI778965	+
BG352031	101	JQ2343 Zinnia elegans lipid transfer protein	4e-21	AW625628	++
BG352032	267	AAC49278 Arabidopsis sterol delta-7 reductase	1e-70	AW033361	+		+	+
BG352033	398	CAA64414 tomato lipid desaturase	3e-50	AI774470	+c		+++c	+++c
BG352034	487	P04634 rat (Rattus norvegicus) triacylglycerol lipase	0.007	AW651434	+c		+c
Protein protein interactions/signal transduction
BG352035	285	NP_003738 human TRF1-interacting ankyrin-related ADP-ribose polymerase	8e-12	AW615864	+	+	+c	+
BG352036	659	NP_003738 human TRF1-interacting ankyrin-related ADP-ribose polymerase	6e-06	AW615864
BG352037	539	AAC62877 WD repeat of Arabidopsis translation initiation protein	2e-61	AI483225	+		+	+
BG352038	576	P42652 tomato 14-3-3 protein	Identical	AW622689	+		+	+
BG352039	664	CAB07805 tobacco protein phosphatase 1	1e-24	AW219414	+		+c	+
Gene expression
BG352040	452	AAD10626Lolium temulentum MADS box protein 2	4e-29	AW626110	+c		+c	+
BG352041	521	NP_002083 human G-rich RNA sequence binding factor 1	2e-11	AW738391	+		+	+
BG352042	541	AAC61751Trypanosoma cruzi poly-zinc finger protein 1	4e-21	AW094020	+c		+	+
Protein targeting
BG352043	632	CAA91162 spinach (Spinacia oleracea) secY protein homolog	9e-65	AW037812	−
Ribosomal proteins
BG352044	9	CAB65281 alfalfa (Medicago sativa) ribosomal L3	8e-65	AW220096	+
BG352045	291	P46222Drosophila melanogaster ribosomal L11	6e-30		+
BG352046	722	227228 Mouse ribosomal L28	2e-07	No	+		+c
Others
BG352047	93	AAA80594 potato chlorophyll a/b-binding protein	4e-13	AW944784	−
BG352048	276	CAA05979Lupinus albus ADP/ATP carrier	9e-27	No	+		+
BG352049	317	Q43794 tobacco glutamyl tRNA synthetase	7e-33	AW031320	−		−
BG352050	415	AAF14680 Arabidopsis DnaJ domain	5e-13	AI776948	+c		++c
BG352051	558	CAA06156 maize cytochrome P450 monooxygenase	7e-35	AW219708	+		+	−
BG352052	565	CAA47373 tobacco (Nicotiana sylvestris) Gln synthetase	7e-50	AI778053	+		−
BG352053	585	P16048 pea Gly cleavage system protein H	1e-33	AW093614	−		−
BG352054	626	AAA78277 tobacco rubisco activase	1e-45	BE432926	−
BG352055	642	004350 Arabidopsis tubulin-specific chaperone A	1e-35	AW094598	+
BG352056	697	BAA77604 tobacco (Nicotiana paniculata) plastidic aldolase	1e-45	BE354659	−		++c
Novel proteins
BG352057	309	CAB41935 Arabidopsis putative protein	9e-07	BE460000	−			+
BG352058	416	AAB61506.1 Arabidopsis putative protein	1e-26	No	+c		+c	−
BG352059	417	CAA22991.1 Arabidopsis putative protein	2e-16	BE400935	++
BG352060	522	CAA18744 Arabidopsis putative protein	3e-49	AW398892, L. pennellii	+c		−	−c
BG352061	552	AAF02150 Arabidopsis putative protein	4e-46	AI777238	+		−
BG352062	562	CAA22991 Arabidopsis putative protein	5e-30	AW625429	+		+
BG352063	606	AAB61480 Arabidopsis putative protein	6e-28	AW441993	−		+c
No matches
BG352064	272	No match	–	No	+			+
BG352065	277	No match	–	AW442444	−		+
BG352066	320	No match	–	AW649771	−c		−c	−
BG352067	407	No match	–	BE459012	+c		+c	+
BG352068	412	No match	–	No	+			−
BG352069	493	No match	–	BG132519	++			−
BG352070	528	No match	–	AW443952	+		−	−
BG352071	557	No match	–	No	−			−
BG352072	560	No match	–	AW930949	+
BG352073	567	No match	–	No	+			−
BG352074	577	No match	–	No	+			−
BG352075	603	No match	–	No			−	−
BG352076	636	No match	–	BE459012	+			−
BG352077	656	No match	–	AI989142	−

−, 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.

^a Expression determined by “reverse northern.” 

^b Expression determined by northern analysis. 

^c Results obtained from at least two independent experiments. 

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.

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.

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 significant 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.

Genes Induced by Bacterial Inoculation

We tested whether the clones from our cDNA library are relevant to defense responses in non-transgenic 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).

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 μg 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).

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 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).

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.

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 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.

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.

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 defense-related 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 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 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), ω-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 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 ΔoxyR 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, β-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 corresponding 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 well-expanded 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 TA-cloning 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 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 μg 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.