Plant Cell, Vol. 11, 2419-2428, December 1999, Copyright © 1999, American Society of Plant Physiologists
Arabidopsis PAD3, a Gene Required for Camalexin Biosynthesis, Encodes a Putative Cytochrome P450 Monooxygenase
Nan Zhoua,b,
Tina L. Tootlea, and
Jane Glazebrooka
a Center for Agricultural Biotechnology, University of Maryland Biotechnology Institute, College Park, Maryland 20742
b Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742
Correspondence to:
Jane Glazebrook, Current address: Novartis Agricultural Discovery Institute, 3115 Merryfield Row, San Diego, CA 92121-1102., jane.glazebrook{at}nadii.novartis.com (E-mail), 858-812-1105 (fax)
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ABSTRACT |
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Phytoalexins are low molecular weight antimicrobial compounds that are synthesized in response to pathogen attack. The phytoalexin camalexin, an indole derivative, is produced by Arabidopsis in response to infection with the bacterial pathogen Pseudomonas syringae. The phytoalexin deficient 3 (pad3) mutation, which causes a defect in camalexin production, has no effect on resistance to P. syringae but compromises resistance to the fungal pathogen Alternaria brassicicola. We have now isolated PAD3 by map-based cloning. The predicted PAD3 protein appears to be a cytochrome P450 monooxygenase, similar to those from maize that catalyze synthesis of the indole-derived secondary metabolite 2,4-dihydroxy-1,4-benzoxazin-3-one. The expression of PAD3 is tightly correlated with camalexin synthesis and is regulated by PAD4 and PAD1. On the basis of these findings, we conclude that PAD3 almost certainly encodes an enzyme required for camalexin biosynthesis. Moreover, these results strongly support the idea that camalexin does not play a major role in plant resistance to P. syringae infection, although it is involved in resistance to a fungal pathogen.
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INTRODUCTION |
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Plants have evolved a battery of biochemical and molecular weapons to defend themselves against invasion by microbial pathogens. Pathogen invasion triggers activation of defense responses, including the synthesis of reactive oxygen species and signal molecules such as salicylic acid (SA), accumulation of antimicrobial metabolites, and expression of many defense-related genes, such as those encoding pathogenesis-related (PR) proteins. In some instances, a potential pathogen may trigger a form of strong resistance called gene-for-gene resistance, which is mediated by the specific recognition of a pathogen avirulence gene product by the corresponding plant resistance gene. In such cases, plant defense responses are activated rapidly, disease does not ensue, and the pathogen is termed avirulent. Gene-for-gene resistance is not triggered by virulent pathogens, however, with the result that defense responses to these pathogens are activated more slowly; consequently, the responses are less effective, and disease ensues. During the past few years, much attention has been given to elucidating the mechanisms by which defense responses are activated and inhibit pathogen growth (Hammond-Kosack and Jones 1996 
; Glazebrook et al. 1997a ). Arabidopsis is an ideal model system for such studies, and many mutations that affect a wide variety of specific defense responses have been identified in that species (Glazebrook et al. 1997a ).
Phytoalexins are low molecular weight antimicrobial metabolites produced by plants in response to pathogen attack (Paxton 1981 ). The chemical structures of phytoalexins vary among different plant families and include flavonoids, terpenoids, and indoles (Darvill and Albersheim 1984 ). The antimicrobial properties of phytoalexins suggest their potential function in the host defense machinery. Despite the fact that phytoalexins have been studied extensively for many years, little direct evidence indicates whether they make important contributions to plant defenses against particular pathogens. The only phytoalexin that has been detected in Arabidopsis is an indole derivative called camalexin (3-thiazol-2'-yl-indole) (Tsuji et al. 1992 ). Accumulation of camalexin was found in tissue exposed to infection by either avirulent or virulent strains of the bacterium Pseudomonas syringae (Tsuji et al. 1992 ; Glazebrook and Ausubel 1994 ) and after inoculation with the fungus Cochliobolus carbonum (Glazebrook et al. 1997b ). In vitro studies demonstrated that camalexin inhibited bacterial and fungal growth (Jejelowo et al. 1991 ; Tsuji et al. 1992 ; Rogers et al. 1996 ).
Very little is known about camalexin biosynthesis. Analysis of camalexin accumulation in tryptophan-deficient Arabidopsis mutants and the incorporation of radiolabeled compounds into camalexin suggested that the camalexin biosynthetic pathway originates with an intermediate of the tryptophan pathway that lies between anthranilate and indole (Tsuji et al. 1993 ). Recent studies have revealed that indole is the precursor to camalexin and that the thiazole ring of camalexin is derived from cysteine (Zook and Hammerschmidt 1997 ; Zook 1998 ).
The Arabidopsis phytoalexin deficient (pad) mutants are defective in phytoalexin production and show specific alterations in their responses to a number of pathogens (Glazebrook and Ausubel 1994 ; Glazebrook et al. 1996 , Glazebrook et al. 1997b ; Zhou et al. 1998 ). Mutations in pad1, pad2, and pad4 but not in pad3 or pad5 show significantly enhanced susceptibility to virulent P. syringae strains (Glazebrook and Ausubel 1994 ; Glazebrook et al. 1997b ). The pad4 mutation causes plants to become more susceptible than wild-type plants to normally incompatible isolates of the oomycete Peronospora parasitica (Glazebrook et al. 1997b ) and the fungus Erisyphe orontii (Reuber et al. 1998 ). Moreover, pad3 plants are considerably more susceptible to the fungus Alternaria brassicicola than are wild-type plants (Thomma et al. 1998 ).
We have proposed a model to explain the phenotypes of pad mutants (Glazebrook and Ausubel 1994 ; Glazebrook et al. 1997b ; Zhou et al. 1998 ). Mutations in PAD3 or PAD5 cause a defect only in camalexin synthesis, and camalexin does not play a major role in resistance to P. syringae. The other PAD genes encode regulatory factors that control camalexin synthesis as well as other defense responses. It is these other defense responses that contribute to plant resistance to P. syringae. In support of this model, we found that PAD4 affects regulation of camalexin synthesis and the pathogenesis-related gene PR-1 (Zhou et al. 1998 ). Activation of camalexin synthesis and PR-1 expression requires the signal molecule SA. SA is sufficient for PR-1 expression and is necessary, but not sufficient, for camalexin synthesis (Ryals et al. 1996 ; Zhao and Last 1996 ; Zhou et al. 1998 ). PAD4 seems to act upstream of SA in the activation of defense responses to virulent P. syringae strains, but it is not required for responses to isogenic avirulent strains, such as those carrying the avirulence gene avrRpt2 (Zhou et al. 1998 ).
Our model predicts that PAD3 encodes a biosynthetic enzyme or a regulatory factor that affects only camalexin synthesis and other defense responses that do not restrict growth of P. syringae. In this study, we isolated PAD3 by using a map-based cloning approach and found that it encodes a putative cytochrome P450 monooxygenase similar to four maize gene products required for biosynthesis of the indole-derived metabolites 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) (Frey et al. 1997 ). PAD3 expression is pathogen inducible and requires PAD4 and PAD1. Our results strongly suggest that PAD3 is an enzyme involved in camalexin biosynthesis. Therefore, camalexin does not seem to be a crucial factor in limiting growth of P. syringae but is required for resistance to A. brassicicola.
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RESULTS |
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Map-Based Cloning of PAD3
To gain insight into the role of PAD3 in camalexin synthesis, we decided to isolate PAD3. The only known pad3 alleles were induced by ethyl methanesulfonate (Glazebrook and Ausubel 1994 ; Glazebrook et al. 1997b ), so we used a map-based approach to clone the gene. GLABROUS1 (GL1) was known to lie close to the PAD3 locus on chromosome 3 (Glazebrook and Ausubel 1994 ). To map pad3, we crossed pad3-2 gl1-1 Columbia plants and wild-type plants of the polymorphic accession Landsberg erecta. In the F2 generation, homozygous gl-1 plants were scored for pad3-2 homozygosity by using a camalexin assay. PAD3 was mapped by using the cleaved amplified polymorphic sequences (CAPS) mapping technique (Konieczny and Ausubel 1993 ) to measure the frequency of recombination between PAD3 and nearby markers. Figure 1 shows that PAD3 was found between g4711 (4.5% recombination) and GL1 (3.5% recombination) on chromosome 3.
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Figure 1.
Map of the PAD3 Region on Chromosome 3.
Thirty-three recombination events were detected between marker GL1 and PAD3 among 948 chromosomes examined, and 31 recombination events were detected between marker g4711 and PAD3 among 694 chromosomes examined, indicating that PAD3 is between these two markers, 3.5 centimorgans from GL1 and 4.5 centimorgans from g4711. Among g4711 recombinants, there were three recombination events between PAD3 and GAPA. Among 16 recombinants for marker mi287, all were recombinant at GL1, whereas none was recombinant at GAPA. Therefore, PAD3 was placed between GAPA and mi287. The 19 F3 families that were recombinant at GAPA or mi287 were scored for marker mi178, and one recombinant, an mi287 recombinant, was detected; therefore, PAD3 must lie between GAPA and mi178 on YAC clones CIC5H4 and CIC6B11 and on one of the BAC clones T7C18 or T11A23. Cosmids that complement or fail to complement pad3 are labeled (+) and (-), respectively. The position of PAD3 is indicated by the dashed lines. The cosmid pZN22 is shown in boldface.
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A physical map of this region was used to identify yeast artificial chromosomes (YACs) containing PAD3 (Camilleri et al. 1998 ). Scoring the g4711 and GL1 recombinants with the CAPS marker GAPA and with the restriction fragment length polymorphism markers mi287 and mi178 showed that PAD3 resides on two YACs, CIC5H4 and CIC6B11 (Figure 1). The Texas A&M University bacterial artificial chromosome (BAC) library (Choi et al. 1995 ) was screened by filter hybridization to identify BACs that hybridize with mi178 and CIC11D3R (Figure 1). Two BACs, T7C18 and T11A23, were found to span the interval from CIC11D3R to mi178. A cosmid library was constructed from the yeast strain carrying CIC6B11. BACs T7C18 and T11A23 were labeled and used as hybridization probes to identify cosmids corresponding to the BACs. These cosmids were arranged into a contig by using hybridization to identify overlapping cosmids.
Cosmids containing PAD3 were identified by transforming pad3-2 plants with various cosmids and testing the transformants for camalexin synthesis after infection with P. s. pv maculicola ES4326. Six cosmids complemented and 13 did not. Common to all six complementing cosmids was a section of Arabidopsis DNA of ~11 kb (Figure 1). Consequently, this is the section that must contain PAD3.
PAD3 Encodes a Putative Cytochrome P450 Monooxygenase
The nucleotide sequence of the Arabidopsis DNA in one of the cosmids, pZN22, was determined. A BLAST (Altschul et al. 1990 ) search of the GenBank database revealed that part of the sequence was similar to those of cytochrome P450 monooxygenases. To determine whether this sequence was derived from the PAD3 gene, we amplified the cytochrome P450-like sequence from wild-type, pad3-1, and pad3-2 plants. The DNA sequences of the amplified fragments were determined and compared to identify any mutations in the pad3-1 and pad3-2 plants. As shown in Figure 2, both pad3-1 and pad3-2 plants have mutations in this region. In pad3-1 plants, a single nucleotide deletion causes a frameshift and early stop codon in the predicted open reading frame, whereas in pad3-2 plants, there is a G-to-A point mutation in the predicted open reading frame (Figure 2). DNA fragments corresponding to the entire complementing region of pZN22 were amplified from wild-type, pad3-1, and pad3-2 plants. DNA sequencing of these fragments demonstrated that no other nucleotide changes were present in this region in pad3-1 or pad3-2 plants. Therefore, we concluded that the gene encoding the putative cytochrome P450 monooxygenase must be PAD3.
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Figure 2.
Nucleotide Sequence and Predicted Amino Acid Composition of PAD3.
A 98-bp intron is shown in lowercase letters. The proline-rich and heme binding motifs are shown in boldface and underlined. The mutations in pad3-1 (a single nucleotide deletion) and pad3-2 (G-to-A transition) are indicated by the filled and open arrowheads, respectively. The sense and antisense primer sequences used for making the PAD3 hybridization probe are underlined.
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The genomic sequence was used to isolate a PAD3 cDNA clone from wild-type plants by rapid amplification of cDNA ends–polymerase chain reaction (RACE-PCR). DNA sequencing of the cDNA clone revealed a 1.7-kb full-length mRNA (Figure 2). Comparison of the cDNA sequence and the genomic sequence revealed the presence of one intron in the PAD3 gene. The encoded protein, which consists of 490 amino acid residues, has a proline-rich motif near the N terminus and a heme binding motif near the C terminus (Figure 2), structures characteristic of all eukaryotic cytochrome P450 monooxygenases. The pad3-1 mutation causes a translation stop before the heme binding site, whereas the pad3-2 mutation causes substitution of glutamic acid for glycine at amino acid 176. The results of the DNA gel blot analysis shown in Figure 3 demonstrate that PAD3 is a single-copy gene.
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Figure 3.
PAD3 Is a Single-Copy Gene.
Approximately 1.5 µg of digested DNA from Arabidopsis ecotype Columbia was loaded in each lane and hybridized with the PAD3 probe. Lengths of fragments (in kilobases) are shown at left. D, DraI; E, EcoRI; H, HindIII; N, NdeI.
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The sequence that we identified as PAD3 (GenBank accession number
AB016889) was assigned the name CYP71B15 by D. Nelson (University of Tennessee, Memphis). CYP71 is a large family of plant cytochrome P450 monooxygenases. Typically, all members of a CYP family share at least 40% amino acid sequence identity and are divided into subfamilies (A, B, C, and so forth) composed of members with greater sequence identity (Chapple 1998 ). For historical reasons, the large CYP71 family is broader than this, and the identity between members of different subfamilies can be as low as 30%. PAD3 shares 47 to 61% amino acid sequence identity with five Arabidopsis cytochrome P450 monooxygenases of unknown function: CYP71B2, CYP71B3, CYP71B4, CYP71B5, and CYP71B7. The only members of the CYP71 family for which in vivo functions are known are CYP71E from Sorghum bicolor, which converts (Z)-p-hydroxyphenylacetaldoxime to p-hydroxymandelonitrile in the dhurrin biosynthetic pathway (Bak et al. 1998 ), and CYP71C1, CYP71C2, CYP71C3, and CYP71C4 from maize (encoded by Bx4, Bx3, Bx5, and Bx2, respectively), which synthesize DIBOA from indole (Frey et al. 1997 ). PAD3 is 39% identical to CYP71E and 31 to 34% identical to the maize enzymes.
Figure 4 shows an alignment of the structures of PAD3 and the CYP71C proteins. The similarity between the CYP71C enzymes and PAD3 probably results from a common affinity for indoles, because both camalexin and DIBOA are derived from indole. Given these sequence similarities between PAD3 and the cytochrome P450 monooxygenases, we concluded that PAD3 is almost certainly an enzyme required for camalexin biosynthesis.
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Figure 4.
Amino Acid Sequence Alignment of PAD3 and Other Cytochrome P450 Monooxygenases.
CYP71C1, CYP71C2, CYP71C3, and CYP71C4 are encoded by the maize genes Bx4, Bx3, Bx5, and Bx2, respectively (Frey et al. 1997 ). The proline-rich motif near the N terminus and the heme binding motif near the C terminus are underlined. Residues conserved in all protein sequences are in boldface. Multiple sequence alignment was performed with PILEUP (version 9.1; Genetics Computer Group, Madison, WI). Dots represent gaps introduced into the sequence to maximize the sequence similarities. The symbol ~ marks the ends of protein sequences.
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PAD3 Expression Is Induced by P. s. maculicola ES4326 Infection and SA Treatment
If PAD3 encodes an enzyme for camalexin biosynthesis, then the expression of PAD3 might be induced by stimuli that trigger camalexin accumulation. To test this idea, we used RNA gel blot hybridization to examine PAD3 expression in plants challenged with P. s. maculicola ES4326. Figure 5 shows a time course of the amounts of PAD3 mRNA in infected tissue. In response to infection, PAD3 mRNA was induced by 12 hr after infection (Figure 5) and reached a maximum by 36 hr after infection (data not shown). PAD3 was also induced in plants infected with avirulent bacteria. As shown in Figure 5, PAD3 mRNA started to accumulate in amounts exceeding that in the uninfected control plants by 3 hr after infection and reached a peak by 12 hr after infection. In previous work, we found that camalexin accumulated within 12 hr after infection by avirulent bacteria, whereas it accumulated more slowly in response to virulent bacteria, reaching noticeable amounts between 12 and 24 hr after infection (Zhou et al. 1998 ). The observation that accumulation of PAD3 transcripts precedes camalexin accumulation after infection with either virulent or avirulent strains is consistent with the idea that PAD3 plays a role in camalexin synthesis.
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Figure 5.
PAD3 Expression Is Induced by P. s. maculicola ES4326 Infection.
Total RNA was extracted from tissue inoculated with P. s. maculicola ES4326 (vir) or P. s. maculicola ES4326 carrying avrRpt2 (avr), or mock-inoculated with 10 mM MgSO4 (mock), at the indicated times after inoculation. An 18S RNA probe was used as a control for equal loading.
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Because SA is necessary but not sufficient for camalexin synthesis, we tested the effect of SA treatment on PAD3 expression. As Figure 6 shows, the amounts of PAD3 mRNA increased rapidly in response to SA treatment, demonstrating that SA is sufficient for PAD3 expression, even though it is not sufficient for activation of camalexin synthesis. Camalexin synthesis must be therefore subject to additional control beyond regulation of PAD3 expression. Certain mutations that cause formation of spontaneous lesions resembling disease lesions, such as the accelerated cell death (acd2-2) mutation, also cause high SA concentrations and camalexin synthesis in the tissue with lesions (Greenberg et al. 1994 ). We found that PAD3 was also expressed in acd2-2 plant tissue with lesions (Figure 6).
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Figure 6.
PAD3 Expression Is Induced by SA Treatment and by the acd2-2 Mutation.
RNA was prepared from wild-type Columbia plants sprayed with 5 mM SA in 0.02% (v/v) Silwet L-77 at various times after treatment (five lanes at left) and from leaves with lesions from 7-week-old acd2-2 plants (two lanes at right).
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Jasmonic acid (JA) is sufficient to activate phytoalexin synthesis in some plants, but it does not activate camalexin synthesis in Arabidopsis (Thomma et al. 1999 ). Nevertheless, because JA signaling might be required, we investigated the possibility. Plants homozygous for the coronatine insensitive1 (coi1) mutation are insensitive to JA and are male sterile (Feys et al. 1994 ). Camalexin concentrations in a population segregating for coi1 homozygotes and in wild-type plants were determined 34 hr after infection with P. s. maculicola ES4326. Camalexin concentrations (ng/cm2; means ±SD of three to five replicate samples) were as follows: wild-type, 375 ± 93; coi1 homozygotes (male-sterile plants from the segregating population), 87 ± 19; and COI1 homozygotes and COI1/coi1 heterozygotes (fertile plants from the segregating population), 489 ± 136. The decreased concentrations of camalexin in coi1 plants are consistent with the idea that JA plays a role in the activation of camalexin synthesis in response to infection by P. s. maculicola ES4326.
PAD3 Expression Is Reduced in pad1 and pad4 Mutants
Among the five identified PAD genes, one (PAD4) is known to have a regulatory effect on camalexin synthesis (Zhou et al. 1998 ). Mutations affecting regulation of camalexin synthesis might cause defects in pathogen-induced PAD3 expression. To test for such defects, we examined the amount of PAD3 mRNA in P. s. maculicola ES4326–infected leaves in pad mutants and wild-type plants. As shown in Figure 7A, we observed a defect in accumulation of PAD3 transcripts in pad3-1 plants. This could result from the premature stop codon in the coding sequence, causing instability of the transcribed RNA (Culbertson 1999 ). Interestingly, after infection by virulent P. s. maculicola ES4326, the amount of PAD3 mRNA expressed in the pad1 and pad4 mutants was much less than in the wild-type plants (Figure 7A). However, this decrease was not observed in pad4-1 plants inoculated with avirulent bacteria (Figure 7B), in agreement with our previous findings that the pad4 mutation does not affect camalexin synthesis in P. s. maculicola plants infected by avrRpt2-containing strains (Zhou et al. 1998 ). On the basis of these results, PAD1 and PAD4 most likely contribute to control of camalexin synthesis by mediating increased amounts of PAD3 mRNA in response to infection with P. s. maculicola ES4326.
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Figure 7.
PAD3 Expression in pad Mutants.
(A) PAD3 mRNA accumulation in pad and wild-type plants. Leaf samples were collected 8 hr after infection with virulent P. s. maculicola ES4326. Uninfected tissue of wild-type plants was used as the control.
(B) PAD3 expression in the pad4-1 mutant compared with wild-type (WT) plants in response to avirulent or virulent bacteria. Total RNA was extracted from tissue inoculated with P. s. maculicola ES4326 (vir) or P. s. maculicola ES4326 carrying avrRpt2 (avr) at the indicated times after infection.
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DISCUSSION |
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Sequence analysis of PAD3 revealed that it encodes a putative cytochrome P450 monooxygenase. Cytochrome P450 monooxygenases make up a superfamily of heme-thiolate proteins that are found in various organisms, including bacteria, plants, and mammals. Most of these proteins catalyze NADPH- and O2-dependent hydroxylation reactions (Chapple 1998 ). Plant cytochrome P450 monooxygenases participate in myriad biochemical pathways, including biosynthesis of phenylpropanoids, alkaloids, terpenoids, lipids, and plant growth regulators, such as gibberellins, JA, and brassinosteroids (Chapple 1998 ). We found that PAD3 is closely related to the Arabidopsis cytochrome P450 monooxygenases CYP71B2 through CYP71B5 and CYP71B7. However, no biochemical functions have been reported for these proteins.
PAD3 shares 31 to 34% amino acid sequence identity with the maize cytochrome P450 monooxygenase proteins encoded by Bx2 (CYP71C4), Bx3 (CYP71C2), Bx4 (CYP71C1), and Bx5 (CYP71C3) (Figure 4). Expression of these genes in yeast revealed that each of them catalyzes one of the oxidation steps between indole and DIBOA in the pathway of DIMBOA biosynthesis (Frey et al. 1997 ). DIMBOA and its precursor, DIBOA, are believed to be involved in pathogen defense and disease resistance in maize (Niemeyer 1988 ). The Bx1 gene encodes a tryptophan synthase 
homolog that appears to provide indole for DIBOA synthesis (Frey et al. 1997 ; Melanson et al. 1997 ). Presumably, this activity is needed because in plants, tryptophan synthase and ß are tightly associated, and indole is immediately converted to tryptophan without being released from the enzyme complex. Together, these five Bx genes constitute a branch pathway that converts indole to DIBOA rather than tryptophan (Frey et al. 1997 ). Like DIBOA, camalexin is also an indole derivative; therefore, the sequence similarity between the DIBOA cytochrome P450 monooxygenases and PAD3 strongly suggests that PAD3 functions in camalexin biosynthesis. Indole is a precursor to camalexin, and an Arabidopsis sequence with 80% sequence identity to Arabidopsis tryptophan synthase has been identified; therefore, Arabidopsis is also likely to have a Bx1-like activity that provides free indole for camalexin synthesis (Zook 1998 ). Similar pathways for synthesis of secondary metabolites with protective roles may have evolved in both Arabidopsis and maize.
Most cytochrome P450 monooxygenases catalyze O2- and NADPH-dependent hydroxylation reactions. Camalexin does not contain any oxygen atoms, so the role for a cytochrome P450 enzyme in camalexin synthesis is not immediately obvious. However, the finding that indole and cysteine are precursors to camalexin (Zook and Hammerschmidt 1997 ; Zook 1998 ) is consistent with a proposed pathway for camalexin biosynthesis beginning with the condensation of indole-3-carboxaldehyde and cysteine (Browne et al. 1991 ; Zook 1998 ). The cytochrome P450 monooxygenase encoded by PAD3 could be involved in the synthesis of indole-3-carboxaldehyde from indole.
In addition to camalexin synthesis, the tryptophan pathway leads to the biosynthesis of many secondary metabolites, including the auxin indole-3-acetic acid and indole glucosinolates (Radwanski and Last 1995 ). The importance of these secondary metabolites in plants suggests the need for regulatory mechanisms that control the metabolic flow and production of precursors in the tryptophan pathway to accommodate the biosynthesis of these diverse secondary metabolites. Zhao and Last 1996 pointed out that the amounts of the enzymes participating in tryptophan biosynthesis and camalexin synthesis are coordinately regulated. Infection by a pathogen and elicitor treatment induced both activity by the tryptophan pathway enzymes and accumulation of camalexin (Zhao and Last 1996 ). On the other hand, because mutations in the trp genes did not cause major changes in camalexin accumulation (Zhao and Last 1996 ), flux through the tryptophan biosynthetic pathway is apparently not rate limiting for camalexin synthesis.
We have shown that the accumulation of PAD3 transcripts is correlated with and precedes camalexin accumulation after infection with P. s. maculicola ES4326 or ES4326 carrying avrRpt2 and is correlated with camalexin synthesis in acd2-2 plants. However, SA treatment, although sufficient for activating expression of PAD3, is not sufficient for activating synthesis of camalexin. Apparently, camalexin synthesis is regulated partly by controlling the expression of a biosynthetic enzyme and partly by an unknown mechanism.
The idea that camalexin synthesis is controlled at the mRNA level is supported by the observation that the accumulation of PAD3 mRNA in response to virulent P. s. maculicola ES4326 is greatly decreased in pad1 and pad4 mutants—an indication that PAD3 expression is regulated by PAD1 and PAD4. Our finding that PAD3 expression requires PAD1 and PAD4 is consistent with our model, because it predicts that PAD1 and PAD4 should control camalexin synthesis as well as other defense responses. Previous work has shown that PAD4 encodes a regulatory factor that is required to promote SA synthesis in response to P. s. maculicola ES4326 infection (Zhou et al. 1998 ). We have now shown that PAD3 expression is enhanced by SA treatment. Therefore, the requirement of PAD4 for PAD3 induction and camalexin synthesis is expected, and the action of PAD4 is likely to be mediated by SA.
Our finding that PAD1 is required for PAD3 expression indicates that PAD1 has a role in regulating the expression of defense genes. PR-1 is expressed normally in pad1 mutants (J. Glazebrook, unpublished data); therefore, PAD1 is unlikely to affect SA signaling. The effect of PAD1 on camalexin synthesis could conceivably be mediated by the JA-dependent signaling pathway, however. This pathway (Creelman and Mullet 1997 ), which leads to induction of the plant defensin gene PDF1.2, is important for resistance to certain fungal and oomycete pathogens (Penninckx et al. 1996 ; Bowling et al. 1997 ; Thomma et al. 1998 ). In pad1 plants, expression of PDF1.2 in response to A. brassicicola infection was diminished (Thomma et al. 1999 ). Interestingly, coi1 mutants showed a defect in camalexin production in response to P. s. maculicola ES4326 infection, consistent with the idea that activation of camalexin biosynthesis requires components of the JA signaling pathway. Curiously, coi1 did not block camalexin synthesis in response to A. brassicicola infection (Thomma et al. 1999 ), suggesting that camalexin synthesis may be regulated by different mechanisms in response to different pathogens. Clearly, many more experiments are needed to explore the possible role of JA in activating camalexin synthesis.
Because PAD3 is expressed normally in pad2 and pad5 mutants, the defects in camalexin synthesis in these plants must have some other cause; for instance, PAD2 or PAD5 may regulate genes encoding other camalexin biosynthetic enzymes. Based on the enhanced susceptibility of pad2 mutants to P. s. maculicola ES4326, our model predicts that PAD2 affects a regulatory factor influencing camalexin synthesis and some other defense response that limits growth of P. s. maculicola ES4326. If so, the regulatory effect must occur by a mechanism other than control of PAD3 expression. The pad5 mutation does not affect P. s. maculicola ES4326 growth; therefore, we predict that PAD5 encodes another biosynthetic enzyme or affects regulation of camalexin synthesis without affecting defense responses required for limiting P. s. maculicola ES4326 growth. Cloning and characterization of other PAD genes should help resolve these issues.
The facts that PAD3 almost certainly encodes an enzyme for camalexin biosynthesis and that pad3 mutations do not cause disease susceptibility to the bacterial pathogen P. syringae (Glazebrook and Ausubel 1994 ) imply that camalexin does not play a major role in plant resistance to bacterial infection. In support of this idea, the concentration of camalexin that is toxic to P. syringae is much higher than the concentration in infected plants, and camalexin-resistant mutants of P. s. maculicola ES4326 are not more virulent than wild-type bacteria (Rogers et al. 1996 ). Although it is possible that other host defense responses are enhanced in pad3 mutants and that this compensatory stimulation of alternate defense responses may diminish the impact of restricted phytoalexin synthesis in the mutants, thus far no such compensatory defense responses have been found.
The enhanced sensitivity of pad3 to the fungal pathogen A. brassicicola suggests a role for camalexin in resistance to some fungal pathogens (Thomma et al. 1998 ). Consistent with our conclusion that PAD3 encodes a biosynthetic enzyme, the expression of PR-1, PR-4, and PDF1.2 in response to A. brassicicola was unaffected in pad3 plants (Thomma et al. 1999 ). Consequently, the only defect in pad3 plants probably involves camalexin synthesis. Therefore, camalexin seems to be an important factor contributing to resistance to A. brassicicola. Alterations in camalexin concentrations might have dramatic effects on other fungal pathogens as well.
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METHODS |
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Plants and Growth Conditions
Plants (Arabidopsis thaliana ecotype Columbia) with the pad3-1, pad3-2, and acd2-2 mutations have been described previously (Glazebrook and Ausubel 1994 ; Greenberg et al. 1994 ; Glazebrook et al. 1997b ). Plants were grown in pots in Metro-Mix 200 soil (Scotts-Sierra Horticultural Products, Marysville, OH) either in a plant room (23 ± 2°C at 15 to 50% humidity and 60 µmol m-2 sec-1 fluorescent illumination) or in a growth chamber (22 ± 2°C at 85% relative humidity and 100 µmol m-2 sec-1 fluorescent illumination) on a 12-hr-light/12-hr-dark cycle. Fully expanded leaves of 4-week-old plants were used for all experiments.
Inoculations with Bacteria
Pseudomonas syringae pv maculicola ES4326 has been described previously (Dong et al. 1991 ). The avirulence gene avrRpt2 was carried on plasmid pLH12, as described previously (Dong et al. 1991 ; Whalen et al. 1991 ). P. syringae strains were grown in King's B medium supplemented with appropriate antibiotics (Glazebrook and Ausubel 1994 ). Bacteria were infiltrated into Arabidopsis plants, as described previously (Glazebrook and Ausubel 1994 ). Unless stated otherwise, for camalexin assays, the bacterial dose for P. syringae strains was 3 x 104 colony-forming units per cm2 leaf area (equivalent to OD600 = 0.006). For experiments involving extraction of total RNA from infected leaves, strains P. s. maculicola ES4326 and P. s. maculicola ES4326 carrying avrRpt2 were introduced at a dose of 104 colony-forming units per cm2 leaf area (equivalent to OD600 = 0.002).
Camalexin Determination
Camalexin assays were performed as described previously (Glazebrook and Ausubel 1994 ).
Preparation of DNA
Plant genomic DNA samples were isolated from frozen leaf tissues, according to standard procedures (Dellaporta et al. 1983 ). Yeast strains carrying appropriate yeast artificial chromosome (YAC) clones were grown in 5 mL of AHC medium (6.7 g/L yeast nitrogen base with amino acids or ammonium sulfate, 10 g/L acid-hydrolyzed casein, 20 g/L glucose, and 20 µg/mL adenine sulfate) at 30°C until late-log phase. After being washed with 1 M sorbitol, the cell pellet was resuspended in a solution of 1 M sorbitol, 10 mM EDTA, 100 mM sodium citrate, pH 5.8, and 30 mM ß-mercaptoethanol and then incubated with 10 mg/mL lyticase at 30°C for 1 hr. DNA was extracted in a 300-µL solution containing 50 mM Tris, 20 mM EDTA, pH 7.5, and 1% SDS at 65°C for 30 min. After the addition of 250 µL of 5 M potassium acetate and incubation on ice for 30 min, DNA in the supernatant was precipitated in 500 µL of isopropanol, treated with RNaseA, extracted with 50:50 (v/v) phenol:chloroform, and precipitated with ethanol. The final yield of yeast DNA was ~1 µg/mL. This procedure was scaled up to obtain large quantities of yeast DNA for subcloning. Bacterial artificial chromosome (BAC) DNA was purified by using the protocol provided by the Arabidopsis Biological Resource Center (Columbus, OH).
Construction of the Cosmid Library
Yeast DNA from YAC clone CIC6B11 was partially digested with Taq I and fractionated on a sucrose gradient. The fraction containing 19- to 22-kb fragments was ligated with ClaI-digested pCLD04541 (Bent et al. 1994 ) and packaged by using Gigapack II Gold Packaging Extract (Stratagene, La Jolla, CA).
Generation of Probes from YAC and BAC Ends
To obtain the right end (CIC11D3R) from YAC CIC11D3, we digested ~200 ng of yeast DNA with HincII and treated it with the Klenow fragment of DNA polymerase I followed by T4 DNA ligase. Inverse polymerase chain reaction (PCR) was conducted by using primers that hybridize with the pYAC4 vector (GenBank accession number
U01086): P8, 5'-TCTGGGAAGTGAATGGAG-3'; and P13, 5'-TGGGCTGCTTCCTAATGCA-3'. BAC ends from T7C18 and T11A23 were obtained by using a modified version of the adapter ligation protocol (Siebert et al. 1995 ). Probes were labeled with 11-digoxygenin-dUTP, according to the instructions of the supplier (Boehringer Mannheim).
Plant Transformation
Plasmids were introduced into Agrobacterium tumefaciens GV3101 pMP90 (Koncz and Schell 1986 ) by triparental mating in the presence of the helper strain of Escherichia coli MM294 pRK2013. Plants were transformed by the method of Bechtold et al. 1993 , except that 0.005% Silwet L-77 (Lehle Seeds, Round Rock, TX) was added to the bacterial suspension and the vacuum step was omitted. Transformants were selected on plates containing 0.5 x Murashige and Skoog salts (Life Technologies, Gaithersburg, MD), 1 x Gamborg's B5 vitamins (Sigma), and 50 µg/mL kanamycin.
DNA Sequencing
Cosmid DNA containing PAD3 was digested with HindIII and subcloned in Bluescript II SK+ (Stratagene). DNA sequencing was performed by using standard dye-terminator sequencing procedures and automated sequencers (models 373 and 377; Applied Biosystems, Foster City, CA).
Isolation of cDNA
The PAD3 cDNA was obtained by using a Marathon cDNA amplification kit (Clontech, Palo Alto, CA) and mRNA prepared from wild-type plants infected with P. s. maculicola ES4326. The gene-specific primer for 5' rapid amplification of cDNA ends (RACE)–PCR was 5'-GCTCTCTCTTCCAGGCTTAAGATGCTCG-3'; for 3' RACE-PCR, it was 5'-CTGAGTTTGCTACGAGACAATCTCCGGTG-3'. A full-length double-stranded cDNA was obtained by performing end-to-end amplification with primer pairs complementary to the ends of 5' and 3' RACE products: 5'-CCTTCGAAATATAAGCTTCCTCCGGGTCC-3' and 5'-GGCTTCCTCCTGCTTCGCCAATCCCCAATC-3'.
RNA Gel Blot Analysis
RNA gel blots with 5 µg of total RNA per lane were prepared as described previously (Zhou et al. 1998 ). The PAD3 probe was made from pZN22 cosmid DNA by PCR amplification with sense primer 5'-CCGGTGAATCTTGAGAGAGCC-3' and antisense primer 5'-GATCAGCTCGGTCATTCCCC-3'; PCR was used to label the antisense single-strand DNA with digoxygenin-11-dUTP. Blots were stripped and reprobed with the 18S rRNA probe to assess equal loading of RNA samples (Zhou et al. 1998 ).
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ACKNOWLEDGMENTS |
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We thank David Bouchez (INRA, Versailles, France) for kindly providing the physical map of chromosome 3 before publication, John Turner (University of East Anglia, UK) for the gift of coi1 seed, Willem Broekaert (Katholieke Universiteit Leuven, Belgium) for communication of unpublished data, David Nelson (University of Tennessee, Memphis) for help with cytochrome P450 monooxygenase classification, and Clint Chapple (Purdue University, West Lafayette, IN) and Willem Broekaert for helpful discussions and critical reading of the manuscript. We also thank the Arabidopsis Biological Resource Center for providing BAC library filters, YAC and BAC clones, and restriction fragment length polymorphism markers. This work was supported by start-up funding awarded to J.G. by the University of Maryland Biotechnology Institute and by Grant No. MCB 9723493 from the National Science Foundation to J.G.
Received July 14, 1999; accepted October 6, 1999.
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ABSTRACT
INTRODUCTION
