Research ArticleResearch Article

Uncoupling PR Gene Expression from NPR1 and Bacterial Resistance: Characterization of the Dominant Arabidopsis cpr6-1 Mutant

Joseph D. Clarke, Yidong Liu, Daniel F. Klessig and Xinnian Dong
The Plant Cell April 1998, 10 (4) 557-569; DOI: https://doi.org/10.1105/tpc.10.4.557

Abstract

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, CPR6, that may function with NPR1 in regulating PR gene expression. The dominant CPR6-1 mutant expresses the SA/NPR1–regulated PR genes (PR-1, BGL2, and PR-5) and displays enhanced resistance to Pseudomonas syringae pv maculicola ES4326 and Peronospora parasitica Noco2 in the absence of SAR induction. cpr6-1–induced PR gene expression is not suppressed in the cpr6-1 npr1-1 double mutant but is suppressed when SA is removed by salicylate hydroxylase. Thus, constitutive PR gene expression in cpr6-1 requires SA but not NPR1. In addition, resistance to P. s. maculicola ES4326 is suppressed in the cpr6-1 npr1-1 double mutant, despite expression of PR-1, BGL2, 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 CPR6 is an important regulator of multiple signal transduction pathways involved in plant defense.

INTRODUCTION

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, 1992; Métraux 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; Métraux et al., 1991) and benzo(1,2,3)thiadiazole-7-carbothioc acid S-methyl ester (Görlach 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, β-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, 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 fluorescens 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, 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; Peña-Cortés 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, cpr6-1, of Arabidopsis. cpr6-1 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 cpr6-1 mutant requires SA but is independent of NPR1. We believe that the CPR6 protein regulates the expression of multiple defense genes and functions with NPR1 in regulating PR gene expression.

RESULTS

Characterization of the cpr6-1 Mutant

The Arabidopsis cpr6-1 mutant was identified by using a screen for plants displaying constitutive expression of a reporter gene containing the BGL2 promoter fused to the β-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 cpr6-1 mutant line that was back-crossed twice. Crossing cpr6-1 with a wild-type plant containing the BGL2–GUS reporter gene revealed reporter gene expression in the F1 generation, implying that cpr6-1 is a dominant mutation. In the F2 population, reporter gene expression was present in 69 of 98 seedlings (three of four), verifying that the cpr6-1 mutation is inherited as a single dominant locus (χ2 = 1.1; 0.05 < P < 0.1). Because cpr6-1 is in the Columbia (Col) ecotype, crosses into the Landsberg erecta (Ler) ecotype were performed to determine the map position for the cpr6-1 locus. By using codominant cleaved amplified polymorphic sequences (CAPS; Konieczny and Ausubel, 1993) and simple sequence-length polymorphisms (SSLPs; Bell and Ecker, 1994), cpr6-1 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 cpr6-1 mutant is influenced by the developmental stage of the plant. Figure 1A shows the expression pattern of the reporter gene in the cpr6-1 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.

Figure 1.
Figure 1.

Developmental Expression of BGL2–GUS Reporter Gene and Morphological Phenotypes of cpr6-1.

(A) Expression of the BGL2–GUS reporter gene in cpr6-1 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 cpr6-1 (cpr6) and BGL2–GUS plants (W.T.), showing the semidominant morphological phenotype associated with the cpr6-1 mutation. The plants were photographed at 5 weeks of age.

Another developmental characteristic of the cpr6-1 mutant, as shown in Figure 1B, is a loss of apical dominance and a reduction in overall plant size. cpr6-1 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 cpr6-1 mutant a bushy appearance. The cotyledons of cpr6-1 senesce 5 to 7 days before the wild type does, and cpr6-1 plants flower a week later than do wild-type plants. The morphological phenotypes of cpr6-1 appear to be semidominant (Figure 1B). The cpr6-1 heterozygote is larger than the cpr6-1 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 cpr6-1 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 cpr6-1 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 cpr6-1 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 cpr6-1 mutant. The application of RB did not appear to increase PDF1.2 or affect PR-1 gene expression in cpr6-1. In the cpr6-1 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 cpr6-1 homozygote, further confirming that cpr6-1 is a dominant mutation (Figure 2A).

Constitutive expression of these defense-related genes suggests that cpr6-1 plants may have enhanced resistance to pathogen infection. To determine the level of resistance bestowed by the cpr6-1 mutation, we tested the growth of the virulent bacterial pathogen P. s. maculicola ES4326 in cpr6-1 and wild-type plants. As shown in Figure 3A, cpr6-1 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 cpr6-1 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 cpr6-1 mutant. As shown in Figure 3B, cpr6-1 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 cpr6-1 further increased the resistance to the level of INA-treated wild-type plants (data not shown). We also examined pathogen resistance in the cpr6-1 heterozygote. The cpr6-1 heterozygote exhibits the same degree of resistance to P. s. maculicola ES4326 and P. parasitica Noco2 as does the cpr6-1 homozygote (Figures 3A and 3B). Therefore, we conclude that cpr6-1 is a dominant mutation with respect to defense gene expression and pathogen resistance, despite the semidominant morphological phenotypes.

Figure 2.
Figure 2.

PR-1 and PDF1.2 Gene Expression in cpr6-1, cpr6-1 × Wild Type F1, and cpr6-1 × nahG F1 Plants.

(A) Expression of PR-1 and PDF1.2 genes in wild-type, cpr6-1 heterozygous, and cpr6-1 homozygous plants.

(B) Expression of PR-1 and PDF1.2 genes in nahG, cpr6-1, and cpr6-1 × 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; cpr6, cpr6-1 mutant line; nahG, transgenic line containing the nahG gene encoding salicylate hydroxylase.

Epistasis Analysis of cpr6-1

To place cpr6-1 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 cpr6-1 for HR-like lesions by using various approaches described previously (Bowling et al., 1997). We found no macroscopic or microscopic lesions in cpr6-1 and therefore conclude that cpr6-1 should probably be placed downstream of the HR (data not shown).

To determine the position of cpr6-1 relative to SA, we measured the amount of endogenous SA and salicylate glucoside (SAG) in cpr6-1. As shown in Figure 4, levels of free SA and SAG in the cpr6-1 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 cpr6-1 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, 1997; Weymann et al., 1995).

To define further the role of SA in cpr6-1 plants, we performed RNA gel blot analysis with crosses made between cpr6-1 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 cpr6-1 mutation is dependent on SA. Because both cpr6-1 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 cpr6-1 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 cpr6-1 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 cpr6-1 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 (Figures 2A and 2B) and Thi2.1 (data not shown) in cpr6-1.

Figure 3.
Figure 3.

Effects of cpr6-1 on Pathogen Growth in cpr6-1 Homozygous, cpr6-1 Heterozygous, and cpr6-1 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, cpr6-1 homozygous, and cpr6-1 heterozygous plants. (C) shows the level of P. s. maculicola ES4326 growth in wild-type, cpr6-1, npr1-1, and cpr6-1 npr1-1 plants.

(B) and (D) Disease rating of P. parasitica Noco2 infection. Plants were infected by spraying a conidiospore suspension (3 × 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, cpr6-1 homozygous, and cpr6-1 heterozygous plants. (D) provides disease ratings for wild-type, cpr6-1, npr1-1, and cpr6-1 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; cpr6, cpr6-1 mutant line; npr1, npr1-1 mutant line; W.T.+INA denotes treatment of plants with 0.65 mM INA 72 hr before infection.

Because cpr6-1 elevates PR gene expression, whereas npr1-1 abolishes PR gene expression, we decided to generate the cpr6-1 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 cpr6-1 and npr1-1, and the resulting progeny were screened in the F2 generation. Because of the ~20-cM linkage between cpr6-1 and npr1-1, we expected to detect one cpr6-1 npr1-1 recombinant in 98 F2 progeny. npr1-1 is recessive; therefore, we facilitated identification of the cpr6-1 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, 1997).

Figure 4.
Figure 4.

SA and SAG Levels in Wild-Type and cpr6-1 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; cpr6, cpr6-1 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.

We began the screen for the cpr6-1 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 cpr6-1 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 cpr6-1 phenotype. We found that two of the 178 lines were segregating for the cpr6-1 phenotype, implying that the parental lines were heterozygous for the cpr6-1 mutation. From these two F3 populations, F4 seeds were collected only from those plants that were phenotypically homozygous for cpr6-1. 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 cpr6-1 in the double mutant was confirmed by backcrossing the cpr6-1 npr1-1 double mutant to BGL2–GUS wild-type plants and scoring for the presence of cpr6-1–induced reporter gene expression in all F1 progeny.

The cpr6-1 npr1-1 double mutant phenotypically resembles cpr6-1 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 cpr6-1 npr1-1 double mutant does not bleach in the leaves. The pattern of reporter gene expression in the double mutant resembles the cpr6-1 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 cpr6-1 npr1-1 seedlings to determine whether npr1-1 suppresses cpr6-1–induced expression of the PR genes. We were surprised to find that cpr6-1 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 cpr6-1 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).

Figure 5.
Figure 5.

Expression of PR Genes in the cpr6-1 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; cpr6, cpr6-1 mutant line; npr1, npr1-1 mutant line.

Because cpr6-1 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 cpr6-1 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).

DISCUSSION

cpr6-1 Is a Dominant Mutant That Induces Multiple Defense Pathways

We have identified and characterized cpr6-1 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 cpr6-1 heterozygous and homozygous plants (Figures 2A, 3A, and 3B). Therefore, we speculate that CPR6 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 cpr6-1 phenotype results from a dominant loss-of-function mutation in a negative SAR regulator. Although one copy of cpr6-1 is sufficient to activate SAR (dominance), two copies of cpr6-1 amplify the plant's mutant phenotypic appearance (semidominance). This implies that CPR6 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 back-crosses.

Like cpr5, cpr6-1 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, cpr6-1 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 cpr6-1 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. CPR6 may therefore be a prominent regulator of multiple defense pathways in Arabidopsis.

PR Gene Expression in cpr6-1 Plants Requires SA but Is Independent of NPR1

To define where CPR6 acts in the SAR signaling cascade, we analyzed the F1 cross between cpr6-1 and nahG and constructed the cpr6-1 npr1-1 double mutant. We found that expression of PR-1, BGL2, and PR-5 in cpr6-1 plants requires SA but does not require a functional NPR1 protein. These data argue against placing CPR6 upstream of NPR1 in the SAR signal cascade, as shown in Figure 6A, because PR gene expression, although requiring SA, occurs independently of NPR1. CPR6 cannot be directly downstream of NPR1, as shown in Figure 6B, because the addition of SA to npr1-1 CPR6 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 CPR6 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 CPR6. Because the cpr6-1 mutation results in activation of multiple defense signaling pathways, we speculate that CPR6 is directed toward transcription of the PR genes by SA and NPR1, whereas other factors may direct CPR6 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 cpr6-1 suppresses expression of PDF1.2 (Figures 2A and 2B) and Thi2.1 (data not shown). INA may sequester CPR6 (the mutant protein) from activating the SA/NPR1–independent pathway. Supporting this argument is the repeated observation that in the cpr6-1 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).

An alternative interpretation to the epistasis data is that PR gene induction occurs via two parallel pathways represented by CPR6 and NPR1. If true, cpr6 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 CPR6 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).

Figure 6.
Figure 6.

Possible Epistatic Relations between SA, CPR6, and NPR1.

(A) CPR6 could be downstream of SA and upstream of NPR1; however, this possibility does not account for the NPR1-independent PR gene expression in cpr6-1.

(B) CPR6 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) CPR6 could require activation by SA and interaction with NPR1 in PR gene regulation.

(D) CPR6 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.

The Mechanism by Which cpr6-1 Accumulates SA

The cpr6-1 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 cpr6-1 that can be reconciled with NPR1-independent expression of the PR genes. The first mechanism suggests that the cpr6-1 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 cpr6-1, an unidentified signal has to be introduced to account for the fact that SA alone does not induce PR gene expression in npr1-1 CPR6 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 cpr6-1 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 cpr6-1 rather than a consequence of cpr6-1–activated SA accumulation. This also implies that there are downstream components of the cpr6-1–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 CPR6 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 CPR6 to maintain elevated SA levels after production of the systemic signal has ceased.

Expression of PR-1, BGL2, 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 cpr6-1 npr1-1 double mutant led us to believe that cpr6-1 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 cpr6-1 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 cpr6-1 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 cpr6-1 mutant could be stimulated by cpr6-1 itself with a functional NPR1 protein or result from cpr6-1–induced SA accumulation, which then activates the NPR1-regulated antibacterial genes. It is unclear at this point exactly how cpr6-1 induces bacterial resistance. It is clear, however, that cpr6-1 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 CPR6 is a regulator of multiple defense pathways. Through CPR6, 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).

Figure 7.
Figure 7.

Proposed Model for Functions of CPR6 and NPR1 in the Induction of Defense-Related Genes.

CPR6 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. CPR6 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.

Our model suggests a possible mechanism in which the plant uses a modifier (NPR1) to direct a positive regulator of multiple resistance pathways (CPR6) 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 CPR6 has significant implications for understanding the mechanisms of acquired resistance. By biochemically and genetically analyzing cpr6-1 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 cpr6-1 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 CPR6 in Arabidopsis.

METHODS

Plant Growth Conditions and Isolation of cpr6-1

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 cpr6-1 mutant was isolated in a screen for constitutive expression of the β-1,3-glucanase promoter–to–β-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 (Gene-Screen; 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 cpr6-1 plants as the pollen donor. The cpr6-1 npr1-1 double mutant was generated using pollen from homozygous cpr6-1 plants to fertilize homozygous npr1-1 plants. The mapping population was generated by using the pollen from cpr6-1 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 cpr6-1 mutants were isolated in the F2 generation based on the cpr6-1 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).

ACKNOWLEDGMENTS

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.
  • Published April 1, 1998.

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