Plant Cell, Vol. 10, 557-570, April 1998, Copyright © 1998, American Society of Plant Physiologists
Uncoupling PR Gene Expression from NPR1 and Bacterial Resistance: Characterization of the Dominant Arabidopsis cpr 6-1 Mutant
Joseph D. Clarkea,
Yidong Liub,
Daniel F. Klessigb, and
Xinnian Donga
a Developmental, Cell, and Molecular Biology Group, Department of Botany, Box 91000, Duke University, Durham, North Carolina 27708-1000
b Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, P.O. Box 759, Piscataway, New Jersey 08855
Correspondence to:
Xinnian Dong, xdong{at}acpub.duke.edu (E-mail), 919-613-8177 (fax).
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ABSTRACT |
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In Arabidopsis, NPR1 mediates the salicylic acid (SA)–induced expression of pathogenesis-related (PR) genes and systemic acquired resistance (SAR). Here, we report the identification of another component, CPR 6, that may function with NPR1 in regulating PR gene expression. The dominant CPR 6-1 mutant expresses the SA/NPR1–regulated PR genes (PR-1, BGL 2, and PR-5) and displays enhanced resistance to Pseudomonas syringae pv maculicola ES4326 and Peronospora parasitica Noco2 in the absence of SAR induction. cpr 6-1–induced PR gene expression is not suppressed in the cpr 6-1 npr1-1 double mutant but is suppressed when SA is removed by salicylate hydroxylase. Thus, constitutive PR gene expression in cpr 6-1 requires SA but not NPR1. In addition, resistance to P. s. maculicola ES4326 is suppressed in the cpr 6-1 npr1-1 double mutant, despite expression of PR-1, BGL 2, and PR-5. Resistance to P. s. maculicola ES4326 must therefore be accomplished through unidentified antibacterial gene products that are regulated through NPR1. These results show that CPR 6 is an important regulator of multiple signal transduction pathways involved in plant defense.
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INTRODUCTION |
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Pathogens annually cause billions of dollars in damage to crops worldwide (Baker et al. 1997 
). Consequently, an increasing amount of research has been dedicated to developing novel methods for controlling plant diseases. Such studies have centered on the plant's innate ability to resist pathogen invasion in an effort to buttress the plant's own defenses to counter pathogen attacks (Staskawicz et al. 1995 ; Baker et al. 1997 ). One such defense mechanism under study is known as systemic acquired resistance (SAR; reviewed in Ryals et al. 1996 ). SAR is defined as a generalized defense response, which is often induced by avirulent pathogens and provides enhanced resistance to a broad spectrum of virulent pathogens (Chester 1933 ; Ross 1961 ; Kuc 1982 ; Ryals et al. 1994 ). Avirulent pathogens carry an avirulence (avr) gene whose product can be recognized by the product of a corresponding resistance (R) gene carried by plants. Such recognition triggers both a programmed cell death response, known as the hypersensitive response (HR), around the point of pathogen infection and release of a systemic SAR-inducing signal (Hammond-Kosack and Jones 1996 ). After a rapid, localized HR, the elevated state of resistance associated with SAR is effective throughout the plant for a period of time ranging from several days to a few weeks (Van Loon and Van Kammen 1970 ; Malamy et al. 1990 ; Ward et al. 1991 ; Yalpani et al. 1991 ; Uknes et al. 1992 ).
Salicylic acid (SA) is an integral signaling component of SAR (Gaffney et al. 1993 ). During an SAR response, endogenous levels of SA increase dramatically throughout the plant (Malamy et al. 1990 , Malamy et al. 1992 ; Metraux et al. 1990 ; Rasmussen et al. 1991 ; Yalpani et al. 1991 ; Enyedi et al. 1992 ; Uknes et al. 1993 ). Exogenous application of SA (White 1979 ) or the SA analogs 2,6-dichloroisonicotinic acid (INA; Metraux et al. 1991 ) and benzo(1,2,3)thiadiazole-7-carbothioc acid S-methyl ester (Gorlach et al. 1996 ) can induce SAR. Removal of SA results in plants that are unable to establish an SAR response and are supersusceptible to pathogen infection (Gaffney et al. 1993 ; Delaney et al. 1994 ). Coinciding with the onset of SAR is the transcriptional activation of the pathogenesis-related (PR) genes. These genes encode proteins that exhibit antimicrobial activities (Ward et al. 1991 ).
In Arabidopsis, expression of PR-1, ß-1,3-glucanase (BGL2), and PR-5 has been shown to be tightly correlated with resistance to virulent bacterial, fungal, and oomycete pathogens; therefore, these genes are used as molecular markers for SAR (Uknes et al. 1992 ). To identify genes that regulate SAR, we conducted a genetic screen in Arabidopsis for mutants that have an altered SAR response. We isolated the npr1 mutant (for nonexpresser of PR genes; Cao et al. 1994 ; also called nim1 and sai1; Delaney et al. 1995 ; Shah et al. 1997 ) that blocks the SA-induced expression of PR-1, BGL2, and PR-5 and SA-induced resistance to virulent pathogens. NPR1 was recently cloned using a map-based approach (Cao et al. 1997 ; Ryals et al. 1997 ). In addition to npr1, which is nonresponsive to SAR induction, we isolated several cpr mutants (for constitutive expresser of PR genes) that have elevated levels of PR gene expression and enhanced pathogen resistance in the absence of SAR induction (Bowling et al. 1994 , Bowling et al. 1997 ).
By using genetic epistasis analysis, we recently demonstrated that cpr5, which is a cpr mutant unique for its formation of spontaneous lesions and defective trichome development, confers not only NPR1-dependent resistance to the virulent bacterial pathogen Pseudomonas syringae pv maculicola ES4326 but also NPR1-independent resistance to the virulent oomycete pathogen Peronospora parasitica Noco2 (Bowling et al. 1997 ). In the cpr5 npr1-1 double mutant, expression of PR-1, BGL2, and PR-5 is suppressed, whereas resistance to P. parasitica Noco2 is not, indicating that CPR5 regulates the expression of additional genes that contribute to oomycete resistance in an NPR1-independent manner.
Genetic and biochemical evidence has been accumulating recently in support of the existence of SA/NPR1–independent resistance pathways. Pieterse et al. 1996 demonstrated that resistance to Fusarium oxysporum induced by the root-colonizing biocontrol bacterium Pseudomonas fluo-rescens WCS417r is independent of SA and PR gene expression. More evidence has come from studies of induction pathways leading to the accumulation of defensin and thionin class peptides (Florack and Stiekema 1994 ; Broekaert et al. 1995 ; Terras et al. 1995 ), especially defensin PDF1.2 (Penninckx et al. 1996 ) and thionin Thi2.1 (Epple et al. 1995 ), which have been shown to be pathogen inducible. In Arabidopsis, PDF1.2 is induced by the avirulent pathogen Alternaria brassicicola (Penninckx et al. 1996 ), and Thi2.1 is induced by the pathogen F. o. f sp matthiolae (Epple et al. 1995 , Epple et al. 1997 ). Although both PDF1.2 and Thi2.1 seem to be regulated through the plant hormone jasmonic acid (JA), their expression was shown to be nonresponsive to SA or INA induction (Epple et al. 1995 ; Penninckx et al. 1996 ).
These experiments show that the SA- and JA-induced resistance responses in Arabidopsis are regulated by distinct pathways. However, other experiments show that these separate defense-related responses may have synergistic or antagonistic effects toward each other. In rice, INA was shown to elevate JA and the expression of JA-regulated genes (Schweizer et al. 1997 ); in barley, INA was found to induce the expression of thionin (Wasternack et al. 1994 ). On the other hand, antagonistic mechanisms between signaling pathways may play a role in prioritizing a plant's response to different challenges. For example, in tomato, the accumulation of SA resulting from pathogen infection suppresses the JA-regulated expression of the herbivore-deterring proteinase inhibitor pathway (Doherty et al. 1988 ; Pena-Cortes et al. 1993 ; Doares et al. 1995 ). In addition, it has been shown in tobacco that the expression of defense-related genes induced by Erwinia carotovora is suppressed by the addition of SA (Vidal et al. 1997 ). Epistasis studies will be an important tool for distinguishing between different resistance responses and determining their points of interaction. By examining the phenotypes of double mutants that affect different pathways, the complex interplay between these pathways can be elucidated.
Here, we report the characterization of a dominant mutation, cpr 6-1, of Arabidopsis. cpr 6-1 plants have elevated levels of SA, constitutive expression of the NPR1-dependent PR genes (PR-1, BGL2, and PR-5) and the NPR1-independent defense genes PDF1.2 and Thi2.1, and enhanced resistance to P. s. maculicola ES4326 and P. parasitica Noco2. Epistasis analysis shows that PR gene expression in the cpr 6-1 mutant requires SA but is independent of NPR1. We believe that the CPR 6 protein regulates the expression of multiple defense genes and functions with NPR1 in regulating PR gene expression.
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RESULTS |
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Characterization of the cpr 6-1 Mutant
The Arabidopsis cpr 6-1 mutant was identified by using a screen for plants displaying constitutive expression of a reporter gene containing the BGL2 promoter fused to the ß-glucuronidase (GUS) coding region. This screen was designed to isolate mutants in the regulatory components of SAR. The details of this screen have been described previously (Bowling et al. 1994 ). All of the following experiments were conducted with a cpr 6-1 mutant line that was backcrossed twice. Crossing cpr 6-1 with a wild-type plant containing the BGL2–GUS reporter gene revealed reporter gene expression in the F1 generation, implying that cpr 6-1 is a dominant mutation. In the F2 population, reporter gene expression was present in 69 of 98 seedlings (three of four), verifying that the cpr 6-1 mutation is inherited as a single dominant locus (
2 = 1.1; 0.05 < P < 0.1). Because cpr 6-1 is in the Columbia (Col) ecotype, crosses into the Landsberg erecta (Ler) ecotype were performed to determine the map position for the cpr 6-1 locus. By using codominant cleaved amplified polymorphic sequences (CAPS; Konieczny and Ausubel 1993 ) and simple sequence-length polymorphisms (SSLPs; Bell and Ecker 1994 ), cpr 6-1 was mapped to chromosome 1 in the ~3-centimorgan (cM) interval defined by nga280 (1.9 cM) and g4026 (1.5 cM). RPP7, an R gene in the Ler ecotype that provides enhanced resistance to P. parasitica, also maps to this region (Crute et al. 1993 ).
Expression of the BGL2–GUS reporter gene in the cpr 6-1 mutant is influenced by the developmental stage of the plant. Figure 1A shows the expression pattern of the reporter gene in the cpr 6-1 mutant grown on Murashige and Skoog (MS) medium (Murashige and Skoog 1962 ). Reporter gene expression was first detected 1 week after germination, primarily at the root–hypocotyl juncture. In the second week, expression was found throughout the hypocotyl, most prominently at the node between the hypocotyl and cotyledons. By the third and fourth weeks, expression spread from the cotyledons to the stem, petioles, and older leaves.
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Figure 1.
Developmental Expression of BGL2–GUS Reporter Gene and Morphological Phenotypes of cpr 6-1.
(A) Expression of the BGL2–GUS reporter gene in cpr 6-1 plants is linked to the developmental stage of the plant. Plants from 1 to 4 weeks of age were infiltrated with X-gluc to stain for expression of the BGL2–GUS reporter gene, according to Jefferson et al. 1987 . The age of the plant in weeks (w) is denoted at upper left, and the magnification of the image is denoted at lower right.
(B) A comparison between cpr 6-1 (cpr 6) and BGL2–GUS plants (W.T.), showing the semidominant morphological phenotype associated with the cpr 6-1 mutation. The plants were photographed at 5 weeks of age.
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Another developmental characteristic of the cpr 6-1 mutant, as shown in Figure 1B, is a loss of apical dominance and a reduction in overall plant size. cpr 6-1 plants typically have five to 10 bolts that emerge from the rosette soon after the primary bolt. All of the bolts tend to achieve the same height, which is approximately one-third the height of a wild-type plant, giving the cpr 6-1 mutant a bushy appearance. The cotyledons of cpr 6-1 senesce 5 to 7 days before the wild type does, and cpr 6-1 plants flower a week later than do wild-type plants. The morphological phenotypes of cpr 6-1 appear to be semidominant (Figure 1B). The cpr 6-1 heterozygote is larger than the cpr 6-1 homozygote but is still only one-half to three-quarters the height of wild-type plants. These morphological characteristics continue to cosegregate with the cpr phenotype after the sixth backcross.
The Arabidopsis PR-1, BGL2, and PR-5 genes are used as molecular markers for SA/NPR1–dependent SAR (Uknes et al. 1992 ), whereas PDF1.2 and Thi2.1 represent SA/NPR1–independent pathogen-responsive genes (Epple et al. 1995 ; Penninckx et al. 1996 ). We performed RNA gel blot analysis to identify the defense-related genes expressed in cpr 6-1 plants. INA was used as an inducer of the PR genes, and rose bengal (RB) was used as an inducer of PDF1.2 (Penninckx et al. 1996 ). We found that the cpr 6-1 mutant constitutively expresses these SA/NPR1–dependent and SA/NPR1–independent genes, as represented in Figure 2 by PR-1 and PDF1.2. PR-1 gene expression in cpr 6-1 can be slightly induced by the application of INA (Figure 2A) and SA (data not shown). Interestingly, the application of INA seems to suppress the constitutive expression of PDF1.2 (Figure 2A) and Thi2.1 (data not shown) in the cpr 6-1 mutant. The application of RB did not appear to increase PDF1.2 or affect PR-1 gene expression in cpr 6-1. In the cpr 6-1 heterozygote, the expression levels of PR-1 and PDF1.2 (as well as BGL2, PR-5, and Thi2.1; data not shown) are the same as those of the cpr 6-1 homozygote, further confirming that cpr 6-1 is a dominant mutation (Figure 2A).
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Figure 2.
PR-1 and PDF1.2 Gene Expression in cpr 6-1, cpr 6-1 x Wild Type F1, and cpr 6-1 x nahG F1 Plants.
(A) Expression of PR-1 and PDF1.2 genes in wild-type, cpr 6-1 heterozygous, and cpr 6-1 homozygous plants.
(B) Expression of PR-1 and PDF1.2 genes in nahG, cpr 6-1, and cpr 6-1 x nahG F1 plants.
The table below each RNA gel blot describes the fold induction of gene expression for each sample relative to that of the wild type. The values have been corrected based on the loading control and normalized to the wild type. PR-1 and PDF1.2 gene-specific probes were used for RNA gel blot analysis, and the 18S ribosomal subunit gene-specific probe was used as a loading control. RNA samples were extracted from 4-week-old plants grown on soil with or without treatment with 0.65 mM INA or 2 mM rose bengal (RB), as described by Penninckx et al. 1996 , 3 days before tissue collection. W.T., BGL2–GUS transgenic line; cpr 6, cpr 6-1 mutant line; nahG, transgenic line containing the nahG gene encoding salicylate hydroxylase.
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Constitutive expression of these defense-related genes suggests that cpr 6-1 plants may have enhanced resistance to pathogen infection. To determine the level of resistance bestowed by the cpr 6-1 mutation, we tested the growth of the virulent bacterial pathogen P. s. maculicola ES4326 in cpr 6-1 and wild-type plants. As shown in Figure 3A, cpr 6-1 plants exhibited enhanced resistance to P. s. maculicola ES4326 3 days after pathogen infection. The level of bacterial growth was 10 times less than that in wild-type plants and 12 times greater than that in INA-treated wild-type plants. Application of INA to cpr 6-1 further reduced the growth of P. s. maculicola ES4326 to the level found in INA-treated wild-type plants (data not shown). Resistance to the virulent oomycete pathogen P. parasitica Noco2 was also tested in the cpr 6-1 mutant. As shown in Figure 3B, cpr 6-1 plants have enhanced resistance to P. parasitica Noco2, displaying a reduced number of conidiosporangia 1 week after inoculation with oomycete spores. Application of INA to cpr 6-1 further increased the resistance to the level of INA-treated wild-type plants (data not shown). We also examined pathogen resistance in the cpr 6-1 heterozygote. The cpr 6-1 heterozygote exhibits the same degree of resistance to P. s. maculicola ES4326 and P. parasitica Noco2 as does the cpr 6-1 homozygote (Figure 3A and Figure 3B). Therefore, we conclude that cpr 6-1 is a dominant mutation with respect to defense gene expression and pathogen resistance, despite the semidominant morphological phenotypes.
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Figure 3.
Effects of cpr 6-1 on Pathogen Growth in cpr 6-1 Homozygous, cpr 6-1 Heterozygous, and cpr 6-1 npr1-1 Double Mutant Plants.
(A) and (C) Growth of P. s. maculicola ES4326. Plants were infected by injecting a P. s. maculicola ES4326 bacterial suspension of 10 mM MgCl2 at an OD600 reading of 0.001 into the abaxial surface of the leaf with a syringe. Samples were collected by using a hole punch to obtain a leaf disc at time points 0, 1, 2, and 3 days after infection. Eight samples were collected from each genotype at each time point, except on day 0, when only four samples were collected. Error bars represent 95% confidence limits of log-transformed data (Sokal and Rohlf 1981 ). These experiments were replicated three times with similar results. (A) shows the level of P. s. maculicola ES4326 growth in wild-type, cpr 6-1 homozygous, and cpr 6-1 heterozygous plants. (C) shows the level of P. s. maculicola ES4326 growth in wild-type, cpr 6-1, npr1-1, and cpr 6-1 npr1-1 plants.
(B) and (D) Disease rating of P. parasitica Noco2 infection. Plants were infected by spraying a conidiospore suspension (3 x 104 spores per mL) onto 3-week-old plants. Disease severity was scored 7 days after infection by counting the number of conidiophores on 25 plants for each genotype. Disease ratings are as follows: 0, no conidiophores on the plant; 1, no more than five conidiophores per infected leaf; 2, six to 20 conidiophores on a few infected leaves; 3, six to 20 conidiophores on most infected leaves; 4, more than five conidiophores on all infected leaves; 5,  20 conidiophores on all infected leaves. These experiments were replicated three times with similar results. (B) provides disease ratings for wild-type, cpr 6-1 homozygous, and cpr 6-1 heterozygous plants. (D) provides disease ratings for wild-type, cpr 6-1, npr1-1, and cpr 6-1 npr1-1 plants. The data were analyzed using Mann-Whitney U tests (Sokal and Rohlf 1981 ). cfu, colony-forming unit; W.T., BGL2–GUS transgenic line; cpr 6, cpr 6-1 mutant line; npr1, npr1-1 mutant line; W.T.+INA denotes treatment of plants with 0.65 mM INA 72 hr before infection.
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Epistasis Analysis of cpr 6-1
To place cpr 6-1 in the SAR signaling cascade and compare it with other mutants with constitutive SAR expression, such as the cpr5 (Bowling et al. 1997 ), lsd (for lesion-simulating disease; Dietrich et al. 1994 ; Weymann et al. 1995 ; Hunt et al. 1997 ), and acd2 (for accelerated cell death; Greenberg et al. 1994 ) mutants, we examined cpr 6-1 for HR-like lesions by using various approaches described previously (Bowling et al. 1997 ). We found no macroscopic or microscopic lesions in cpr 6-1 and therefore conclude that cpr 6-1 should probably be placed downstream of the HR (data not shown).
To determine the position of cpr 6-1 relative to SA, we measured the amount of endogenous SA and salicylate glucoside (SAG) in cpr 6-1. As shown in Figure 4, levels of free SA and SAG in the cpr 6-1 mutant were seven and nine times higher, respectively, than those found in the uninduced wild-type plants. Interestingly, the amount of free SA accumulation in cpr 6-1 plants is similar to the amount of SA accumulation found in wild-type plants that have been challenged with an avirulent pathogen, and the level of SAG is threefold higher than that in an induced wild-type plant. These levels are significantly lower than is the increase (up to 30-fold) of SA and SAG accumulation found in cpr1, cpr5, lsd6, and lsd7 (Uknes et al. 1993 ; Bowling et al. 1994 , Bowling et al. 1997 ; Weymann et al. 1995 ).
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Figure 4.
SA and SAG Levels in Wild-Type and cpr 6-1 Plants.
(A) Free SA.
(B) Sugar-conjugated SA (SAG).
Leaves from 4-week-old plants grown on soil were collected and analyzed by HPLC for free SA and SAG content. The values presented are the average of five replicates ±SE (micrograms of SA per gram leaf fresh weight). W.T., BGL2–GUS transgenic line; cpr 6, cpr 6-1 mutant line; W.T.+ Psm ES4326 avr, BGL2–GUS transgenic line infected with the P. s. maculicola ES4326 carrying the avrRpt2 gene 2 days before sample collection.
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To define further the role of SA in cpr 6-1 plants, we performed RNA gel blot analysis with crosses made between cpr 6-1 and a transgenic plant containing the nahG gene of Pseudomonas putida (Bowling et al. 1994 ). nahG encodes a salicylate hydroxylase that converts SA to catechol (You et al. 1991 ). Gel blot analysis was used to determine whether constitutive PR gene expression in the cpr 6-1 mutation is dependent on SA. Because both cpr 6-1 and nahG are dominant, analysis was conducted in the F1 generation. The results clearly show that PR-1 gene expression is suppressed in the F1 progeny of the cross between cpr 6-1 and nahG (Figure 2B). The same is also true for BGL2 and PR-5 (data not shown). We also found that PDF1.2 (Figure 2B) and Thi2.1 (data not shown) expression in cpr 6-1 is not suppressed by the removal of endogenous SA. On the contrary, expression of PDF1.2 and Thi2.1 is suppressed by the exogenous addition of INA. From these data, it is apparent that cpr 6-1 requires SA for PR-1, BGL2, and PR-5 gene expression but not for PDF1.2 or Thi2.1 gene expression. In fact, the SA analog, INA, seems to negatively affect the expression of both PDF1.2 (Figure 2A and Figure 2B) and Thi2.1 (data not shown) in cpr 6-1.
Because cpr 6-1 elevates PR gene expression, whereas npr1-1 abolishes PR gene expression, we decided to generate the cpr 6-1 npr1-1 double mutant to determine their hierarchy in the signaling pathway. To interpret the data obtained from the epistasis analysis accurately, it is critical that the loss-of-function mutant has null phenotypes. We chose npr1-1 because it shows no detectable PR gene expression and pathogen resistance in response to SA or INA treatment (Cao et al. 1994 ; Bowling et al. 1997 ). Crosses were performed between cpr 6-1 and npr1-1, and the resulting progeny were screened in the F2 generation. Because of the ~20-cM linkage between cpr 6-1 and npr1-1, we expected to detect one cpr 6-1 npr1-1 recombinant in 98 F2 progeny. npr1-1 is recessive; therefore, we facilitated identification of the cpr 6-1 npr1-1 double mutant by exploiting npr1's unique SA-intolerant phenotype. When grown on 0.5 mM SA, npr1 plants bleach and do not develop beyond the cotyledon stage (Cao et al. 1994 , Cao et al. 1997 ).
We began the screen for the cpr 6-1 npr1-1 double mutant by growing the F2 progeny on SA plates. In the preliminary analysis of the F2 progeny, one of four F2 plants bleached, indicating that cpr 6-1 does not affect the expression of this npr1 phenotype. All bleaching F2 plants were rescued by transferring them to SA-free plates and then to soil, where F3 seeds were collected from individual plants. Seeds from 178 of these F3 lines, which were presumed homozygous for npr1-1, were grown on soil and scored for the cpr 6-1 phenotype. We found that two of the 178 lines were segregating for the cpr 6-1 phenotype, implying that the parental lines were heterozygous for the cpr 6-1 mutation. From these two F3 populations, F4 seeds were collected only from those plants that were phenotypically homozygous for cpr 6-1. Because the npr1-1 mutation abolishes an NlaIII restriction site (Cao et al. 1997 ), restriction digestion analysis of the npr1-1 gene region amplified by polymerase chain reaction (PCR) was performed with the potential double mutants to confirm that these lines are homozygous for the npr1-1 mutation. The homozygosity of cpr 6-1 in the double mutant was confirmed by backcrossing the cpr 6-1 npr1-1 double mutant to BGL2–GUS wild-type plants and scoring for the presence of cpr 6-1–induced reporter gene expression in all F1 progeny.
The cpr 6-1 npr1-1 double mutant phenotypically resembles cpr 6-1 in size and development but has shorter siliques and bleaches in the stems approaching the inflorescence (data not shown). Unlike the cpr5 npr1-1 double mutant (Bowling et al. 1997 ), the cpr 6-1 npr1-1 double mutant does not bleach in the leaves. The pattern of reporter gene expression in the double mutant resembles the cpr 6-1 expression patterns in the hypocotyl, stem, and petioles but is fainter in the older leaves (data not shown).
RNA gel blot analysis was conducted with 3-week-old cpr 6-1 npr1-1 seedlings to determine whether npr1-1 suppresses cpr 6-1–induced expression of the PR genes. We were surprised to find that cpr 6-1 expresses PR-1, BGL2, and PR-5 independently of the npr1-1 mutation, as shown in Figure 5. This experiment was repeated two additional times with plants at different developmental stages. Although slight variations in expression levels were observed between experiments, significant levels of PR gene expression were consistently detected in the cpr 6-1 npr1-1 double mutant. In addition, the NPR1-independent defense-related genes PDF1.2 and Thi2.1 are also expressed in the double mutant (data not shown).
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Figure 5.
Expression of PR Genes in the cpr 6-1 npr1-1 Double Mutant.
PR-1, BGL2, and PR-5 gene-specific probes were used for RNA gel blot analysis of the indicated plants, with the UBQ5 gene-specific probe being used as a loading control. The table below the RNA gel blot describes the fold induction for each sample. The values have been corrected based on the loading control and normalized to that of the wild type. RNA samples were extracted from 2-week-old plants grown on MS media or MS media with 0.1 mM INA (+INA). W.T., BGL2–GUS transgenic line; cpr 6, cp r 6-1 mutant line; npr1, npr1-1 mutant line.
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Because cpr 6-1 npr1-1 mutant plants express PR-1, BGL2, PR-5, PDF1.2, and Thi2.1, we speculated that the double mutant would be resistant to P. s. maculicola ES4326 and P. parasitica Noco2. We first tested the double mutant for resistance to the oomycete pathogen P. parasitica Noco2 and found that it was indeed resistant to this virulent pathogen (Figure 3D). We then examined the double mutant for resistance to the bacterial pathogen P. s. maculicola ES4326 and found that the resistance to P. s. maculicola ES4326 conferred by cpr 6-1 was abolished by the presence of npr1-1 (Figure 3C). In both cases, the addition of INA did not enhance resistance to pathogen infection (data not shown).
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DISCUSSION |
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cpr 6-1 Is a Dominant Mutant That Induces Multiple Defense Pathways
We have identified and characterized cpr 6-1 as a dominant mutant that constitutively expresses the defense-related genes PR-1, BGL2, PR-5, PDF1.2, and Thi2.1, that has elevated levels of SA and SAG, and that exhibits enhanced resistance to the virulent bacterial pathogen P. s. maculicola ES4326 and the virulent oomycete pathogen P. parasitica Noco2. The levels of defense-related gene expression and pathogen resistance are equivalent in cpr 6-1 heterozygous and homozygous plants (Figure 2A, Figure 3A, and Figure 3B). Therefore, we speculate that CPR 6 is probably a positive regulator of SAR that activates defense gene expression in response to an internal signal. Without a loss-of-function allele in the gene, however, we cannot rule out the possibility that the cpr 6-1 phenotype results from a dominant loss-of-function mutation in a negative SAR regulator. Although one copy of cpr 6-1 is sufficient to activate SAR (dominance), two copies of cpr 6-1 amplify the plant's mutant phenotypic appearance (semidominance). This implies that CPR 6 may have a dual role in SAR and development. This is not entirely unexpected, because we have previously reported that the cpr5 mutant is defective in trichome development (Bowling et al. 1997 ). The morphological phenotypes are unlikely to result from a second, tightly linked mutation because they cosegregate with the cpr phenotypes through six rounds of backcrosses.
Like cpr5, cpr 6-1 expresses the defense genes PR-1, BGL2, PR-5, PDF1.2, and Thi2.1, but without detectable lesions. Also similar to the cpr5 npr1-1 double mutant, cpr 6-1 npr1-1 plants are resistant to P. parasitica Noco2. Therefore, the NPR1-independent resistance to P. parasitica Noco2 initially characterized in the cpr5 npr1-1 double mutant is genetically reproducible in the absence of lesions in the cpr 6-1 npr1-1 double mutant. These data also suggest that the NPR1-dependent and NPR1-independent pathways share common regulatory elements and that the activation of the pathways are not dependent on the formation of lesions. CPR 6 may therefore be a prominent regulator of multiple defense pathways in Arabidopsis.
PR Gene Expression in cpr 6-1 Plants Requires SA but Is Independent of NPR1
To define where CPR 6 acts in the SAR signaling cascade, we analyzed the F1 cross between cpr 6-1 and nahG and constructed the cpr 6-1 npr1-1 double mutant. We found that expression of PR-1, BGL2, and PR-5 in cpr 6-1 plants requires SA but does not require a functional NPR1 protein. These data argue against placing CPR 6 upstream of NPR1 in the SAR signal cascade, as shown in Figure 6A, because PR gene expression, although requiring SA, occurs independently of NPR1. CPR 6 cannot be directly downstream of NPR1, as shown in Figure 6B, because the addition of SA to npr1-1 CPR 6 plants does not rescue the npr1-1 mutant phenotypes, namely, the lack of SA-induced PR gene expression and resistance. The simplest interpretation of the data we have accumulated is that CPR 6 requires activation by SA and interaction with NPR1 to induce PR gene expression, as shown in Figure 6C. This suggests a role for NPR1 as a modifier of CPR 6. Because the cpr 6-1 mutation results in activation of multiple defense signaling pathways, we speculate that CPR 6 is directed toward transcription of the PR genes by SA and NPR1, whereas other factors may direct CPR 6 toward transcriptional activation of the SA/NPR1–independent PDF1.2 and Thi2.1 genes. Evidence in favor of this interpretation of the data is the finding that addition of INA to cpr 6-1 suppresses expression of PDF1.2 (Figure 2A and Figure 2B) and Thi2.1 (data not shown). INA may sequester CPR 6 (the mutant protein) from activating the SA/NPR1–independent pathway. Supporting this argument is the repeated observation that in the cpr 6-1 background, the addition of INA also reduces the expression levels of BGL2 (Figure 5), which has been shown also to be induced by pathways other than the NPR1 pathway (Bowling et al. 1997 ).
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Figure 6.
Possible Epistatic Relations between SA, CPR 6, and NPR1.
(A) CPR 6 could be downstream of SA and upstream of NPR1; however, this possibility does not account for the NPR1-independent PR gene expression in cpr 6-1.
(B) CPR 6 could be downstream of NPR1 and require activation by SA; however, the lack of PR gene expression in npr1-1 mutants after SA treatment precludes this possibility.
(C) CPR 6 could require activation by SA and interaction with NPR1 in PR gene regulation.
(D) CPR 6 could stimulate both SA accumulation and an unknown, independent signal. Both would then act together in some way to induce PR gene expression that is independent of NPR1.
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An alternative interpretation to the epistasis data is that PR gene induction occurs via two parallel pathways represented by CPR 6 and NPR1. If true, cpr 6 would require SA and an additional signal to induce PR gene expression, as shown in Figure 6D. The absence of this unidentified signal in npr1-1 CPR 6 plants prevents expression of the PR genes after SA treatment. Evidence in favor of this interpretation of the data is that in the npr1 mutants, even though SA-induced PR gene expression is completely abolished, significant amounts of PR gene expression were still detected when P. s. maculicola ES4326 was used as an inducer (Glazebrook et al. 1996 ). Perhaps this unidentified second signal is produced as a result of P. s. maculicola ES4326 infection. However, it must be noted that the expression of these PR genes is not sufficient to establish resistance to P. s. maculicola ES4326 in npr1 plants, even in the presence of SA (Cao et al. 1994 ; Glazebrook et al. 1996 ).
The Mechanism by Which cpr 6-1 Accumulates SA
The cpr 6-1 mutant has elevated levels of endogenous SA and requires SA for PR gene expression. We propose two possible mechanisms to explain the observed elevation of SA in cpr 6-1 that can be reconciled with NPR1-independent expression of the PR genes. The first mechanism suggests that the cpr 6-1 mutation affects a function or a component upstream of SA synthesis that stimulates both SA accumulation and an independent signal, which then work together to induce PR gene expression (Figure 6D). Although this hypothesis adequately explains elevated SA levels in cpr 6-1, an unidentified signal has to be introduced to account for the fact that SA alone does not induce PR gene expression in npr1-1 CPR 6 plants. This model also assumes the existence of an SA-responsive pathway leading to PR gene expression that is completely independent of NPR1. However, such a pathway has not yet been reported.
The second mechanism is a simpler interpretation of the data and suggests that the cpr 6-1 mutation has dual effects downstream of SA synthesis. One effect, triggered by SA, is PR gene expression, whereas the other effect is continuation of SA synthesis, as shown in Figure 7. This suggests that PR gene expression is a consequence of SA-activated cpr 6-1 rather than a consequence of cpr 6-1–activated SA accumulation. This also implies that there are downstream components of the cpr 6-1–induced defense responses that regulate subsequent SA accumulation. A feedback loop was previously proposed to explain the SA dependence of lesions in the lsd6 and lsd7 mutants (Weymann et al. 1995 ), but the way in which a plant maintains systemically elevated SA has not yet been defined. Our data suggest that maintenance of elevated SA may occur via CPR 6 after the initial burst of SA accumulation induced by the HR. Mechanistically, the systemic signal released after avirulent pathogen infection could stimulate SA accumulation throughout the plant and then rely on activated CPR 6 to maintain elevated SA levels after production of the systemic signal has ceased.
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Figure 7.
Proposed Model for Functions of CPR 6 and NPR1 in the Induction of Defense-Related Genes.
CPR 6 is shown to induce the expression of the PR genes in conjunction with or independent of NPR1 to confer resistance to pathogens. NPR1 is shown to regulate the expression of antibacterial genes that have not yet been identified. CPR 6 is hypothesized to regulate the accumulation of SA through an autoregulatory mechanism, and its role in PR gene regulation is shown to require SA. It is not known at this time whether cpr 6-1–induced expression of PDF1.2 and Thi2.1 is accomplished through the same pathway as cpr5 and/or JA.
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Expression of PR-1, BGL 2, and PR-5 Is Not Sufficient to Confer Resistance to P. s. maculicola ES4326
Expression of PR-1, BGL2, PR-5, PDF1.2, and Thi2.1 in the cpr 6-1 npr1-1 double mutant led us to believe that cpr 6-1 would be resistant to infections by virulent oomycete and bacterial pathogens. With respect to the virulent oomycete pathogen P. parasitica Noco2, we were correct. However, the cpr 6-1 npr1-1 double mutant is as susceptible as npr1-1 plants to the virulent bacterial pathogen P. s. maculicola ES4326, despite expression of PR-1, BGL2, and PR-5, which are the molecular markers for SAR used in Arabidopsis. In the cpr 6-1 npr1-1 double mutant, we have genetically uncoupled SAR-induced resistance to P. s. maculicola ES4326 from expression of PR-1, BGL2, and PR-5. These data demonstrate that NPR1 regulates a set of unidentified antibacterial genes and support previous observations that PR-1, BGL2, and PR-5 may be primarily antifungal peptides (Kauffmann et al. 1987 ; Woloshuk et al. 1991 ; Vigers et al. 1992 ; Neiderman et al. 1995 ; Hu and Reddy 1997 ). The bacterial resistance observed in the cpr 6-1 mutant could be stimulated by cpr 6-1 itself with a functional NPR1 protein or result from cpr 6-1–induced SA accumulation, which then activates the NPR1-regulated antibacterial genes. It is unclear at this point exactly how cpr 6-1 induces bacterial resistance. It is clear, however, that cpr 6-1 is an inducer of multiple defense pathways because it displays NPR1-independent resistance to P. parasitica Noco2 and constitutively expresses the antifungal defense genes PDF1.2 and Thi2.1 as well as the PR genes (PR-1, BGL2, and PR-5).
The Implications of Uncoupling PR Gene Expression from NPR1 and Bacterial Resistance
Based on our data, we present a model showing that CPR 6 is a regulator of multiple defense pathways. Through CPR 6, expression of the PR genes occurs either by interaction with NPR1 or via a parallel pathway. In addition, we show that induction of PR-1, BGL2, and PR-5 is not sufficient to confer resistance to the virulent bacterial pathogen P. s. maculicola ES4326 and that NPR1 must therefore regulate a set of unidentified antibacterial genes (Figure 7).
Our model suggests a possible mechanism in which the plant uses a modifier (NPR1) to direct a positive regulator of multiple resistance pathways (CPR 6) toward transcriptional activation of a specific set of genes (PR genes). Alternatively, the activation of particular resistance pathways may be redundant and rely on a specific avr–R gene interaction (reviewed in Bent 1996 ). Other studies have shown that different avr–R gene interactions can induce the expression of different genes while competing antagonistically to induce the expression of the same genes (Reuber and Ausubel 1996 ; Ritter and Dangl 1996 ). Pathogen signals may be able to specify the induction of CPR 6- or NPR1-regulated PR and other defense-related genes.
In conclusion, the regulatory activity of CPR 6 has significant implications for understanding the mechanisms of acquired resistance. By biochemically and genetically analyzing cpr 6-1 plants, we further dissected the SAR signaling pathways and demonstrated how the SA/NPR1–dependent pathway may relate to other pathways leading to acquired resistance. In Arabidopsis, SAR has been defined as an SA-dependent response that uses NPR1 to induce resistance to the pathogens P. parasitica, P. s. maculicola ES4326, P. s. pv tomato DC3000, and turnip crinkle virus along with the concurrent expression of PR-1, BGL2, and PR-5 (Lawton et al. 1996 ; Ryals et al. 1996 ). We have shown that resistance to P. parasitica Noco2 and PR gene expression can occur independently of NPR1, that expression of PR-1, BGL2, and PR-5 in the cpr 6-1 npr1-1 double mutant is not sufficient to confer resistance against P. s. maculicola ES4326, and that expression of the PR genes and the PDF1.2 and Thi2.1 genes is regulated by common genetic elements. Future studies will focus on more epistatic analyses between different acquired resistance mutants, the identification of NPR1-regulated antibacterial genes, and the cloning of CPR 6 in Arabidopsis.
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METHODS |
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Plant Growth Conditions and Isolation of cpr 6-1
Plants (Arabidopsis thaliana) were grown on soil (Metro-Mix 200; Grace-Sierra, Malpitas, CA) or on plates containing Murashige and Skoog (MS) media, as described previously (Murashige and Skoog 1962 ; Bowling et al. 1994 ). The cpr 6-1 mutant was isolated in a screen for constitutive expression of the ß-1,3-glucanase promoter–to–ß-glucuronidase (BGL2–GUS) reporter gene fusion in transgenic Columbia (Col) plants mutagenized with ethyl methanesulfonate, as described by Bowling et al. 1994 . Assays for BGL2–GUS reporter gene activity were performed as described previously (Jefferson et al. 1987 ; Cao et al. 1994 ).
RNA Analysis
Tissue samples were collected from 3-week-old seedlings grown on MS plates with or without 0.1 mM 2,6-dichloroisonicotinic acid (INA) or 0.3 mM salicylic acid (SA) and from 4-week-old plants grown in soil with or without 0.65 mM INA or 2 mM rose bengal (RB), as described by Bowling et al. 1997 . Samples were frozen in liquid nitrogen after collection, and RNA was isolated by a phenol–chloroform extraction, as previously described (Cao et al. 1994 ). The RNA concentration was determined by UV absorbence, and 5- or 10-µg samples were separated by electrophoresis through formaldehyde–agarose gels and transferred to a hybridization membrane (GeneScreen; Du Pont–New England Nuclear), as described by Ausubel et al. 1994 . 32P-labeled DNA probes for pathogenesis-related (PR) PR-1 mRNA and 18S rRNA were labeled as described by Cao et al. 1994 ; probes for BGL2, PR-5, ubiquitin (UBQ5), PDF1.2, and Thi2.1 were generated using a strand-biased polymerase chain reaction (PCR) protocol (Schowalter and Sommer 1989 ; Bowling et al. 1997 ). The templates for BGL2, PDF1.2, and Thi2.1 were generated by PCR from the plasmid containing the cDNA clone for each gene, using primers described by Rogers and Ausubel 1997 , Penninckx et al. 1996 , and Epple et al. 1995 , respectively. The template for UBQ5 was generated by PCR from Col genomic DNA, using primers described by Rogers and Ausubel 1997 . PCR amplification of the template included one cycle of 94°C for 2 min; 30 cycles of 94°C for 15 sec, 55°C for 15 sec, and 72°C for 45 sec; and finally one cycle of 72°C for 10 min. PCR cycles were the same for strand-biased probe production, as described by Bowling et al. 1997 . Hybridization and washing conditions were as previously described (Church and Gilbert 1984 ; Cao et al. 1994 ). Bands were quantified using ImageQuant and NIH Image 1.56 software (National Institutes of Health, Bethesda, MD).
Histochemistry and Microscopy
Leaf samples were taken from plants ranging in age from 1 to 4 weeks and grown on MS plates or soil. Staining was with trypan blue for dead cells, nitro blue tetrazolium for the presence of superoxides, and autofluorescence fixing solution for UV epifluorescence microscopy. Trypan blue staining was performed in accordance with previous descriptions (Keogh et al. 1980 ; Dietrich et al. 1994 ; Bowling et al. 1997 ). Nitro blue tetrazolium staining was performed as described by Jabs et al. 1996 . Samples for autofluorescence examination were prepared as described by Bowling et al. 1997 . Autofluorescence was examined as described by Dietrich et al. 1994 .
Genetic Analysis
Crosses were performed as described previously (Bowling et al. 1994 ). Backcrosses with the parental BGL2–GUS transgenic line were performed using cpr 6-1 plants as the pollen donor. The cpr 6-1 npr1-1 double mutant was generated using pollen from homozygous cpr 6-1 plants to fertilize homozygous npr1-1 plants. The mapping population was generated by using the pollen from cpr 6-1 to fertilize a transgenic line of the Landsberg erecta (Ler) ecotype transformed with the BGL2–GUS reporter gene. Successful crosses were scored based on loss of the Ler phenotype in the F1 plants. Homozygous cpr 6-1 mutants were isolated in the F2 generation based on the cpr 6-1 homozygous phenotype and were later tested for expression of the reporter gene in all of the F3 progeny.
PCR-Based Mapping
Mapping was performed by the codominant cleaved amplified polymorphic sequences (CAPS) protocol described by Konieczny and Ausubel 1993 and the single sequence-length polymorphisms (SSLPs) protocol described by Bell and Ecker 1994 . The primers and restriction enzyme for converting the g4026 restriction fragment length polymorphism marker to a CAPS marker are reported in the Arabidopsis database web site (http://genome-www.stanford.edu).
Pathogen Infections
Infections with Pseudomonas syringae pv maculicola ES4326 and Peronospora parasitica Noco2 were performed as described previously (Bowling et al. 1994 ). Plants used for the P. s. maculicola ES4326 infection were grown on soil for 4 weeks and were assayed as described by Bowling et al. 1994 . Statistical analyses were performed by Student's t tests of the differences between two means of log-transformed data (Sokal and Rohlf 1981 ). Plants infected with P. parasitica Noco2 were grown for 18 days on soil with a 12-hr photoperiod. Seven days after inoculation, the plants were scored for the presence of conidiophores by using a dissection microscope. The plants were given a disease rating of one to five, as previously described by Cao et al. 1997 , and the data were analyzed using Mann-Whitney U tests (Sokal and Rohlf 1981 ).
Measurement of SA
SA and SA glucoside measurements were performed with the leaf tissues from 4-week-old plants, as described previously (Bowling et al. 1994 ).
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ACKNOWLEDGMENTS |
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We thank Hui Cao, Mark Kinkema, and Xin Li for helpful discussions and comments on the manuscript; Scott Bowling for discussion and performing the P. parasitica Noco2 infections; and Susan Gordon for her contributions to the initial cpr mutant screen. We also thank Novartis (Basel, Switzerland) for the gift of INA and Petra Epple and Holger Bohlmann for sharing the Thi2.1 cDNA clone. This work is supported by U.S. Department of Agriculture Grant No. 95-37301-1917 to X.D., by a grant from the Monsanto Company to X.D., and National Science Foundation Grant Nos. MCB 9310371/ 9723952 to D.F.K.
Received December 1, 1997; accepted February 12, 1998.
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Reuber, T.L.,
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ABSTRACT
INTRODUCTION