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First published online November 23, 2005; 10.1104/pp.105.067223 Plant Physiology 139:1890-1901 (2005) © 2005 American Society of Plant Biologists Pathogen-Responsive Expression of Glycosyltransferase Genes UGT73B3 and UGT73B5 Is Necessary for Resistance to Pseudomonas syringae pv tomato in Arabidopsis[W]Institut de Biotechnologie des Plantes, Centre National de la Recherche Scientifique-Université Paris-Sud, Unité Mixte de Recherche 8618, 91405 Orsay cedex, France
The genome sequencing of Arabidopsis (Arabidopsis thaliana) has revealed that secondary metabolism plant glycosyltransferases (UGTs) are encoded by an unexpectedly large multigenic family of 120 members. Very little is known about their actual function in planta, in particular during plant pathogen interactions. Among them, members of the group D are of particular interest since they are related to UGTs involved in stress-inducible responses in other plant species. We provide here a detailed analysis of the expression profiles of this group of Arabidopsis UGTs following infection with Pseudomonas syringae pv tomato or after treatment with salicylic acid, methyljasmonate, and hydrogen peroxide. Members of the group D displayed distinct induction profiles, indicating potential roles in stress or defense responses notably for UGT73B3 and UGT73B5. Analysis of UGT expression in Arabidopsis defense-signaling mutants further revealed that their induction is methyljasmonate independent, but partially salicylic acid dependent. T-DNA tagged mutants (ugt73b3 and ugt73b5) exhibited decreased resistance to P. syringae pv tomato-AvrRpm1, indicating that expression of the corresponding UGT genes is necessary during the hypersensitive response. These results emphasize the importance of plant secondary metabolite UGTs in plant-pathogen interactions and provide foundation for future understanding of the exact role of UGTs during the hypersensitive response.
Plant resistance to pathogen infection is often associated with the hypersensitive response (HR) characterized by localized cell death at the site of infection allowing the restriction of pathogen spread. Main events occurring during the HR are the rapid production of reactive oxygen species (ROS), ion fluxes across the plasma membrane, and transcriptional activation of defense genes as those involved in production of secondary metabolites like salicylic acid (SA) and phytoalexins (Hammond-Kosack and Jones, 1996  ). Secondary metabolites perform multiple functions in plants, including roles in UV protection, lignification, herbivore protection, and disease resistance (Vet and Dicke, 1992; Li et al., 1993; Dixon, 2001; Anterola and Lewis, 2002). These compounds rarely accumulate in their free form but are often conjugated to sugars, particularly Glc, through the action of glycosyltransferases (UGTs; EC 2.4.1.128; Vogt and Jones, 2000). Glc conjugation is thought to regulate bioactivity of aglycones, to enhance their solubility, to protect their reactivity toward cellular oxidases, and to alter their transport properties throughout the whole plant (Jones and Vogt, 2001).
UGTs are encoded by a ubiquitous gene family (family 1 of UGTs; Lim and Bowles, 2004
The first example of the identification of a UGT function was through the isolation of the maize bronze-1 transposon-tagging mutant, which was altered in a flavonol-3-O-glucosyltransferase gene (Dooner and Nelson, 1977 ). A clear characterization for the biological role of a UGT acting on the hydroxycoumarin scopoletin was made in tobacco by using transgenic plants harboring the corresponding tobacco glucosyltransferase (TOGT1) gene in antisense and sense orientation. Down-regulation of TOGT1 gene weakened resistance to Tobacco mosaic virus (TMV) during the HR, and the observed phenotype resulted from a decrease in the antiviral secondary metabolite scopoletin, correlated with an increase in the oxidative burst (Chong et al., 2002). Conversely, up-regulation of TOGT1 led to the overaccumulation of scopoletin and its glucoside scopolin, to the early appearance of lesions during the HR to TMV (Gachon et al., 2004a), and to increased resistance against Potato virus Y (Matros and Mock, 2004). In recent years, a number of UGTs were characterized in Arabidopsis (Gachon et al., 2005b); metabolic profiling of T-DNA insertion lines revealed that UGT73C6 and UGT78D1 encode UDP-rhamnose:flavonol-3-O-rhamnosyltransferase and UDP-Glc:flavonol-3-O-glycoside-7-O-glucosyltransferase, respectively (Jones et al., 2003). In the trp1-100 mutant, which is defective in Trp biosynthesis, a loss-of-function mutation in UGT74F2 led to the disappearance of anthranilic acid-Glc, indicating that anthranilic acid is metabolized by this UGT in this particular genetic background (Quiel and Bender, 2003). Likewise, overexpression of UGT84B1 led to the accumulation of 1-O-(indol-3-acetyl)- -D-Glc, suggesting that indole-3-acetic acid is the main substrate in vivo (Jackson et al., 2002). Recent studies provided evidence for a biological role of UGT74B1 in glucosinolate biosynthesis (Grubb et al., 2004) and showed that UGT78D2 and UGT75C1 encode flavonoid 3-O-glucosyltransferase and anthocyanin 5-O glucosyltransferase, respectively (Tohge et al., 2005).
UGT group D includes 13 members in Arabidopsis and contains a number of genes identified in other plant species as being involved in stress responses (Fig. 1). IS5a and IS10a genes from Bright-Yellow 2 tobacco cells were shown to be induced by SA and hydrogen peroxide (H2O2; Horvath and Chua, 1996
The aim of this study was to determine which UGT in the group D is the most relevant to plant defense, using the Arabidopsis-Pst interaction as a model system. We first developed an extensive gene expression analysis of 11 UGT genes after Pst infection and treatments with SA and H2O2, two key modulators of HR (Delaney et al., 1994
Group D UGTs Are Differentially Expressed during the HR to Pst-AvrRpm1
To point out candidate genes involved in resistance responses, we decided to gain insight into the transcriptional regulation of group D UGTs during the HR to Pst-AvrRpm1. We chose RT-qPCR to follow the transcriptional changes of UGT genes to ensure the highest possible specificity. The 13 UGT sequences exhibit between 50.8% and 91.3% identity within their coding sequences at the nucleotide level (Table I). Specific primer sets for each gene were carefully designed (Table II). UGT73C4 and UGT73C5 could not be unambiguously targeted with PCR primers and were not studied further. UGT expression levels were normalized over the constitutive tubuline
Figure 2 shows the time course of UGT73B3, UGT73B4, UGT73B5, UGT73C1, and PR1 expression during the compatible and incompatible interactions with Pst. PR1 is a well established marker gene for the defense responses of Arabidopsis against Pst (Uknes et al., 1992 ). Whereas it was barely expressed in leaves inoculated with the virulent bacteria Pst, it was activated to a high level 24 h after inoculation with the avirulent strain Pst-AvrRpm1. Among UGT genes, diverse patterns of expression could be observed. UGT73B3 and UGT73B5, which share 77.9% similarity at the amino acid level (Table I) but do not map on the same chromosome (Fig. 3), presented highly similar expression profiles. Transcripts of UGT73B3 accumulated early and strongly during the incompatible interaction and remained high throughout the time-course experiment. Similarly, UGT73B5 transcripts accumulated early and to higher levels than UGT73B3, peaking at 5 h after Pst-AvrRpm1 inoculation and then decreased to a steady-state level. Leaf inoculation with the isogenic virulent bacteria Pst resulted in a very low induction of UGT73B3 and UGT73B5, indicating that the observed response is specific of HR onset. UGT73B4 was expressed at a lower level compared to UGT73B5 but exhibited a similar pattern of expression in both interactions until 10 h after inoculation. Thereafter, UGT73B4 transcripts decreased to basal level whatever the interaction, whereas UGT73B5 expression remained strongly up-regulated. These two genes share 94% similarity at the amino acid level (Table I) and are mapped in tandem on the same chromosome (Fig. 3). A different picture emerged from UGT73C1 gene expression (Fig. 2). Here the levels of expression were low, and the patterns of induction were the same in both compatible and incompatible interactions. Altogether, these results clearly indicate that members of the group D, except UGT73B1, UGT73C2, and UGT73C6, are responsive to pathogen infection. They display divergent expression patterns after Pst challenge, which are summarized in Figure 3. Thus, genes such as UGT73D1 exhibited a high and persistent expression level, whereas UGT73C2 and UGT73C6 expression was barely detectable (Fig. 3).
Group D UGTs Respond Differentially to SA, MeJA, and H2O2 A kinetic analysis of UGT expression was performed after treatments of Arabidopsis leaves with 1 mM SA, MeJA, or 5 mM H2O2. Whereas PDF1.2, a marker gene of the jasmonate pathway, was induced, none of the UGT genes under investigation responded to MeJA treatment (Figs. 3 and 4A). On the contrary, accumulation of UGT73B3 and UGT73B5 transcripts was apparent at 3 h after application of SA (Fig. 4B) and rose continuously until approximately 10 h. Afterward, transcript levels decreased rapidly. This pattern of expression differed from the one observed after bacterial challenge (Fig. 2) where expression was maintained to a high level throughout the time-course experiment. Levels of PR1 transcripts were also determined during the time course. Prior to 5 h, no accumulation of PR1 transcripts was evident, the increase occurred between 10 and 15 h after SA treatment. Surprisingly, UGT73B2 was not induced by SA (Fig. 3), although this gene is the closest relative of the highly SA-responsive UGT73B3 gene. Only three genes, UGT73B3, UGT73B5, and UGT73C1, responded to H2O2 application (Fig. 3). They exhibited a biphasic accumulation of transcripts with an early induction at 3 h, followed by a decrease to the background level. Then transcript levels rose dramatically, peaking at 24 h, and declined at 48 h.
Microarray Data Mining Confirms a Stress-Dependent Induction of Group D UGTs Microarray data obtained under numerous stress conditions were retrieved from the publicly available datasets recently released by the AtGenExpress consortium (http://web.uni-frankfurt.de/fb15/botanik). Analysis of these datasets confirmed that UGT D members were induced differentially under stress conditions (Fig. 5). Data concerning Pst-AvrRpm1 versus Pst DC3000 induction were consistent with our RT-qPCR results confirming that some UGTs belonging to the group D are linked with plant defense responses and particularly UGT73B2-B3 and UGT73B5. Expression of these genes was also highly induced after infection with a biotrophic pathogen, Phytophthora infestans, and after oxidative stress caused either by paraquat application or UV treatment (Fig. 5). Interestingly, UGT73B genes were induced after wounding (Fig. 5), although none of these genes was expressed after MeJA treatment (Fig. 3).
The analysis of other microarray datasets available in Genevestigator (https://www.genevestigator.ethz.ch; Zimmermann et al., 2004 ) revealed that some members of the group D, including UGT73B3 and UGT73B5, were induced by the necrotrophic fungus Botrytis cinerea and after ozone treatment (data not shown). Furthermore, most members of group D responded to SA, whereas none of them was induced by MeJA, supporting our RT-qPCR results (data not shown).
To test whether SA is required for the induction of UGT D genes during the HR, we investigated UGT expression in plants with an altered SA-signaling pathway. NahG plants express constitutively a salicylate hydroxylase enzyme that degrades SA into catechol (Delaney et al., 1994
Organ-Specific Expression of UGTs Belonging to Group D To determine the organ-specific expression pattern of each UGT D member, RT-qPCR was performed on RNA isolated from roots, rosette leaves, stems, cauline leaves, and flowers. It revealed that UGT D members are weakly expressed in rosette leaves, stems, and cauline leaves, with the exception of UGT73C6, which exhibited a high expression in cauline leaves (Fig. 7A). UGT73B2, UGT73B3, and UGT73B5 were abundantly expressed in roots and flowers, whereas UGT73B4 and UGT73D1 transcripts were only detectable in roots. UGT73C6 was expressed in cauline leaves and flowers, and UGT73C1, UGT73C2, and UGT73C7 were undetectable in all organs except in flowers, where low levels of transcripts were found. Microarray data mining showed that UGT D members were mainly expressed in roots and reproductive organs confirming our RT-qPCR analyses (Fig. 7B). UGT73B2, UGT73B3, UGT73B4, and UGT73B5 were highly expressed in roots grown on agar plates containing Murashige and Skoog basal medium. Surprisingly, these genes were barely expressed in soil-grown roots, suggesting a specific induction of gene expression either under light exposure or through medium composition. Furthermore, the HR-responsive UGT73B3, UGT73B5, and UGT73D1 were also highly expressed in developmental cell death as shown in senescent leaves (Fig. 7B).
ugt73b3 and ugt73b5 Mutants Display Decreased Resistance to an Avirulent Strain of P. syringae
In the Salk library (http://signal.salk.edu/cgi-bin/tdnaexpress; Alonso et al., 2003
Resistance to pathogens was investigated using inoculation with Pst. After inoculation with a medium titer (105 colony forming units [cfu] mL–1) of the virulent Pst DC3000 strain, ugt73b3 and ugt73b5 mutants developed disease symptoms similar to those observed in wild-type plants (data not shown) and supported the same bacterial growth as in wild-type plants, 24 and 72 h after Pst DC3000 challenge (Fig. 8B). After inoculation with the avirulent Pst-AvrRpm1 strain, bacterial growth did not increase in wild-type plants. Conversely, bacterial population dramatically increased in ugt73b3 and ugt73b5 mutants, indicating an almost complete loss of resistance in these two genotypes.
By transferring sugars to a wide range of secondary metabolites, UGTs increase the stability and solubility of aglycones and therefore modify their bioactivity (Lim and Bowles, 2004 ). In Arabidopsis, 120 UGT genes have been recognized (Paquette et al., 2003) and classified into 14 groups on the basis of their sequence similarity (Ross et al., 2001). Even if UGT substrate specificities have been extensively studied in vitro (Lim et al., 2001, 2002), little functional information is available for the majority of the genes in this family. To extend our knowledge of the physiological role of UGTs in plant-pathogen interactions, we first analyzed the stress-responsive expression of 11 members belonging to the group D, combining RT-qPCR analyses and data mining of public microarray datasets.
Previous studies based on northern- or classical-PCR analyses have revealed that group D UGTs are induced under stress conditions in Arabidopsis and other plant species (Horvath and Chua, 1996
It is also clear that UGTs of group D are differentially regulated after inoculation with Pst-AvrRpm1 or after SA and H2O2 treatments. UGT73B3 and UGT73B5 share 77.9% similarity at the amino acid level and exhibit the same pattern of expression after pathogen challenge and signaling-molecule application (Fig. 3). However, UGT73B1, which shows a comparable level of sequence divergence with UGT73B3 and UGT73B5, does not respond to any treatment (Fig. 3). Moreover, UGT73C3 and UGT73C2, which exhibit 89.5% similarity, present totally different expression profiles. UGT73C3 is induced after Pst-AvrRpm1 infection and SA treatment in Arabidopsis leaves, whereas UGT73C2 expression is barely detectable. On the contrary, UGT73C7 and UGT73D1, which have 64.7% similarity within their coding sequences, present similar transcript profiles after Pst-AvrRpm1 inoculation and SA application (Fig. 3). Despite numerous studies aimed at deciphering the modalities of expression divergence between duplicated genes (Haberer et al., 2004
Importantly, their kinetics of expression resemble those of early induced genes such as GSTs (Lieberherr et al., 2003
Most group D UGTs were responsive to SA whereas none of them was induced after exposure to MeJA, suggesting a jasmonate-independent and SA-dependent induction of these UGTs. Likewise, the wound-inducible tomato UGT Tomato wound induced 1, which belongs to group D, was previously shown to be induced by SA but not by MeJA (O'Donnell et al., 1998
Our data further provide direct evidence of the involvement of UGTs during the HR of Arabidopsis to pathogens. Thus, ugt73b3 and ugt73b5 mutants both exhibited a loss of resistance to Pst-AvrRpm1 (Fig. 8). However, both mutants were not fully susceptible to Pst-AvrRpm1, due to either the presence of other effectors of disease resistance or additional UGT genes partially complementing UGT73B3 and UGT73B5. One question that still remains to be answered is which role UGT73B3 and UGT73B5 could play in this process. Since they are also induced during senescence and after challenge with different pathogens, we can assume that these genes are associated to the cell death process occurring during senescence and HR. Moreover, their responsiveness to H2O2 and paraquat oxidative stress suggests that they may also participate in the maintenance of cellular redox homeostasis. As GSTs, which might use stress metabolites as substrates (Edwards et al., 2000
Biological Materials and Plant Treatments Arabidopsis (Arabidopsis thaliana) ecotype Columbia (Col-0) was used throughout this study. The transgenic line harboring the NahG gene was obtained from R. Dietrich (Syngenta). The Col-0 mutant npr1-1 was provided by X. Dong (Duke University). T-DNA insertion lines ugt73b3 and ugt73b5 were obtained from the Salk institute (SALK_097487 and SALK_078055, respectively). Plants were grown in soil in individual pots under an 8 h/16 h light/dark photoperiod at 150 µE cm–2 s–1 of light intensity, at 20°C and 75% relative humidity. The virulent strain Pseudomonas syringae pv tomato DC3000 (Pst DC3000) and the avirulent strain Pst DC3000 carrying the avirulence gene AvrRpm1 (Pst-AvrRpm1) were obtained from J. Glazebrook (University of Minnesota). Six- to 7-week-old plants were used for Pst inoculations or signaling-molecule treatments. Half leaves were infiltrated using a 1-mL syringe without a needle. Bacteria were grown over night at 28°C on King B liquid medium containing the appropriate antibiotics (kanamycin, 25 µg mL–1; and rifampycin, 50 µg mL–1). They were collected by centrifugation, resuspended in water, and quantified using a spectrophotometer. Plants were infiltrated with Pst DC3000 or Pst-AvrRpm1 at the concentration of 107cfu mL–1 (A600 = 0.01). For signaling-molecule treatments, mature leaves were infiltrated with SA (1 mM in K2HPO4 5 mM pH 6.5) or H2O2 (5 mM). Control plants were infiltrated with K2HPO4 5 mM and water, respectively. For MeJA treatment, plants were kept in closed 12-L chambers with 10 µL MeJA. For each time point, one leaf of six different treated plants was harvested. The resultant six leaves were pooled and frozen in liquid nitrogen. For tissue-specific expression, roots were obtained from 4-week-old plants grown on Murashige and Skoog medium. Rosette leaves were harvested from 4-week-old plants grown in soil under an 8 h/16 h light/dark photoperiod. Plants were then transferred under a 16 h/8 h light/dark photoperiod and cauline leaves, stems, and flowers were harvested after 2 to 3 weeks.
Total RNA were extracted from ground, frozen material using Extract All mix (Eurobio) according to the manufacturer's instructions. Samples were treated with the RNase-free DNase I Amplification Grade (Invitrogen Life Technologies) for 15 min. The reaction was stopped by a phenol-chloroform extraction (Sambrook et al., 1989
Nucleotidic sequences were aligned to identify divergent regions between the most closely related genes. In this region, position of the primers were determined so that the size of the PCR product ranges between 50 and 200 bp. Oligo 4.0 software (Rychlik and Rhoads, 1989
Twenty-five-microliter reactions were prepared by mixing 10-µL samples with 12.5 µL Sybr Green Mastermix (Eurogentec), 1 µL of each primer (final concentration 300 nM), and 0.5 µL water. RT-PCR reactions were run on a GeneAmp 5700 device (Applied Biosystems). After 2 min at 50°C followed by a 10-min denaturation step at 95°C, samples were run for 40 cycles of 15 s at 95°C and 1 min at 60°C. After each run, a dissociation curve was acquired to check for amplification specificity by heating the samples from 60°C to 95°C. Data were analyzed with the GeneAmp 5700 SDS software (Applied Biosystems). Despite RNA treatment with DNase I before cDNA synthesis, contamination by genomic DNA was checked in each sample using primers annealing on Ask alpha gene intron (Table II; Charrier et al., 2002
Microarray data were obtained from the publicly available dataset released by the AtGenExpress consortium (http://web.uni-frankfurt.de/fb15/botanik). These data were generated using the Affymetrix ATH1 GeneChip probe array (Redman et al., 2004
Salk T-DNA insertion lines in the Col-0 background were screened in silico for ugt D mutants. Two lines (SALK_097487 and SALK_078055) were identified to carry a T-DNA insertion at the UGT73B3 and UGT73B5 loci, respectively. The position of the T-DNA insert was confirmed by PCR and DNA sequencing using primers LB (5'-GGACCGCTTGCTGCAACT-3') and B3F (5'-CCACATCATTCAACACGACAAG-3') for ugt73b3, and LB and B5R (5'-GACGGTTTCTTGTCTGGATTGG-3') for ugt73b5. Homozygous plants were identified by PCR, by amplification of the mutant allele using primers LB and B3F for ugt73B3, and LB and B5R for ugt73b5, and by the absence of amplification of the wild-type allele using primers B3F and B3R (5'-GATTTCGAAACTCGGATTCAGG-3') for ugt73b3 and B5F (5'-GTCTTCTTCAACGTGCACACG-3') and B5R for ugt73b5.
Wild-type plants and ugt73b3 and ugt73b5 mutants were inoculated with a medium titer (105 cfu mL–1) of Pst DC3000 or Pst-AvrRpm1. Whole leaves of 6- to 7-week-old plants were infiltrated using a 1-mL syringe without a needle. Leaf discs (0.5 cm2 each) were harvested from inoculated leaves at 24 and 72 h after infiltration. For each time point, three samples were made by pooling two leaf discs from different treated plants. Bacterial growth was assessed by homogeneizing leaf discs in 400 µL of water, plating appropriate dilutions on solid King B medium containing Kanamycin and Rifampicin and quantifying colony numbers after 2 to 3 d. Statistical analyses of the differences between two means of log-transformed data were performed according to one-tailed Student's t test.
Poppenberger et al. (2005) recently reported that UGT73C5 glucosylates brassinosteroids (Poppenberger B, Fujioka S, Soeno K, George GL, Vaistij FE, Hiranuma S, Seto H, Takatsuto S, Adam G, Yoshida S, et al [2005] The UGT73C5 of Arabidopsis thaliana glucosylates brassinosteroids. Proc Natl Acad Sci USA 102: 15253–15258). Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers At4g34138 (UGT73B1), At4g34135 (UGT73B2), At4g34131 (UGT73B3), At2g15490 (UGT73B4), At2g15480 (UGT73B5), At2g36750 (UGT73C1), At2g36760 (UGT73C2), At2g36780 (UGT73C3), At2g36770 (UGT73C4), At2g36800 (UGT73C5), At2g36790 (UGT73C6), At3g53160 (UGT73C7), and At3g53150 (UGT73D1).
We are grateful to R. Dietrich and X. Dong for providing seeds of NahG plants and npr1-1 mutants and to the AtGenExpress consortium for making their microarray data freely available. Special thanks to Annaïck Mingam and to Floriant Bellvert for helpful discussions. Received June 15, 2005; returned for revision September 28, 2005; accepted September 29, 2005.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Patrick Saindrenan (saindrenan{at}ibp.u-psud.fr).
[W] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.067223. * Corresponding author; e-mail saindrenan{at}ibp.u-psud.fr; fax 33–1–69–15–34–24.
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ABSTRACT
RESULTS
and during the HR of Arabidopsis to Pseudomonas syringae pv tomato (Pst) carrying the AvrRpm1 gene (Pst-AvrRpm1; Mazel and Levine, 2002
2 gene (TUB
