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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2006 Dec 5;103(50):19200–19205. doi: 10.1073/pnas.0607279103

RAR1, a central player in plant immunity, is targeted by Pseudomonas syringae effector AvrB

Yulei Shang *, Xinyan Li *,†, Haitao Cui *, Ping He †, Roger Thilmony ‡, Satya Chintamanani †, Julie Zwiesler-Vollick ‡, Suresh Gopalan ‡, Xiaoyan Tang †, Jian-Min Zhou *,§
PMCID: PMC1748199  PMID: 17148606

Abstract

Pathogenic bacterial effectors suppress pathogen-associated molecular pattern (PAMP)-triggered host immunity, thereby promoting parasitism. In the presence of cognate resistance genes, it is proposed that plants detect the virulence activity of bacterial effectors and trigger a defense response, referred to here as effector-triggered immunity (ETI). However, the link between effector virulence and ETI at the molecular level is unknown. Here, we show that the Pseudomonas syringae effector AvrB suppresses PAMP-triggered immunity (PTI) through RAR1, a cochaperone of HSP90 required for ETI. AvrB expressed in plants lacking the cognate resistance gene RPM1 suppresses cell wall defense induced by the flagellar peptide flg22, a well known PAMP, and promotes the growth of nonpathogenic bacteria in a RAR1-dependent manner. rar1 mutants display enhanced cell wall defense in response to flg22, indicating that RAR1 negatively regulates PTI. Furthermore, coimmunoprecipitation experiments indicated that RAR1 and AvrB interact in the plant. The results demonstrate that RAR1 molecularly links PTI, effector virulence, and ETI. The study supports that both pathogen virulence and plant disease resistance have evolved around PTI.

Keywords: bacterial virulence, disease resistance, innate immunity, type III effector

Microbe-derived molecules such as bacterial flagellin and lipopolysaccharides, collectively called pathogen-associated molecular patterns (PAMPs) or microbe-associated molecular patterns (MAMPs; ref. 1), trigger immunity in both animals and plants. Similar to many Gram-negative bacterial pathogens, Pseudomonas syringae uses the type III secretion system to deliver effector proteins into host cells to promote parasitism (2). Emerging evidence indicates that many of the effectors function by actively suppressing PAMP-triggered immunity (PTI; refs. 3 and 4).

Some of the effectors are recognized by host surveillance systems and trigger a strong resistance when their cognate resistance genes are present (2, 4). Often, this so-called “gene-for-gene resistance” or effector-triggered immunity (ETI; ref. 4) is activated by an indirect interaction between the resistance protein and the cognate effector protein (5). Three proteins, HSP90, RAR1, and SGT1, play an important role in ETI by regulating the stability of NB-LRR resistance proteins (6–11), but they are not known for a role in PTI regulation. It is thought that the plant resistance gene products somehow sense the virulence activity of these effectors, rather than the effectors themselves, which in turn activates resistance. Supporting this hypothesis, several host proteins have been shown to interact with both effector and resistance proteins and are required for ETI (12–16). However, a role of these proteins in effector-mediated virulence function remains to be demonstrated.

The P. syringae effector protein AvrB enhances virulence on soybean and Arabidopsis plants lacking cognate resistance genes but triggers ETI on soybean and Arabidopsis plants carrying the resistance genes (17). The virulence function of AvrB is expressed as increased bacterial growth in soybean plants and leaf chlorosis in Arabidopsis plants. The virulence and ETI activity of AvrB have the same structural requirements, suggesting that the virulence function and ETI are intimately connected (17, 18). Therefore, host proteins required for AvrB virulence function may provide a molecular link between effector virulence function and ETI.

Here we show that AvrB inhibits PTI through RAR1, a HSP90 cochaperone required for disease resistance gene functions. When expressed in plants, AvrB suppresses plant defenses and enhances bacterial growth in a RAR1- and jasmonate (JA) pathway-dependent manner. Furthermore, rar1 mutants exhibit an enhanced cell wall defense response to flg22, indicating that RAR1 is a negative regulator of basal defense and that RAR1 plays a central role in both PTI and ETI.

Results

RAR1 Is Required for the Induction of a JA-Response Gene by P. syringae(avrB).

We have shown previously that the AvrB effector delivered by P. syringae bacteria induces Arabidopsis RAP2.6 gene expression in the absence of the cognate resistance gene RPM1 (19), although the presence of RPM1 further enhances this induction (data not shown). The induction requires COI1, an F-box protein essential for JA signaling (20). To identify additional host factors required for AvrB function, we sought to screen for Arabidopsis mutants that failed to induce RAP2.6 in response to AvrB. A RAP2.6-LUC reporter line (19) was mutagenized by ethane methyl sulfonate, and 16,000 M2 plants derived from 9,000 M2 families were screened by infiltrating P. syringae DC3000 (avrB) bacteria into leaves. Plants displaying reduced RAP2.6-LUC expression (reduced responsiveness to avrB;rrb) were selected and further confirmed in the M3 generation. Seven rrb mutants were identified. One mutant, rrb3, displaying a loss of avrB-specific induction of RAP2.6-LUC was characterized in detail. Fig. 1A shows that RAP2.6-LUC reporter activity was strongly activated by DC3000 (avrB) in the wild-type transgenic line 6 h after inoculation. In contrast, luciferase (LUC) activity was reduced by 3-fold in the rrb3 mutant, a level identical to plants treated with DC3000 lacking avrB (Fig. 1B), indicating that the rrb3 mutant was rendered insensitive to avrB. RNA blot analysis showed that the endogenous RAP2.6 transcript level following DC3000 (avrB) infiltration was similarly reduced in the rrb3 mutant [supporting information (SI) Supporting Text and SI Fig. 7 ]. However, RAP2.6-LUC expression in response to DC3000 lacking avrB was not altered in rrb3 (Fig. 1B), indicating an AvrB-specific defect. Both the wild-type RAP2.6-LUC transgenic plants and rrb3 contain RPM1, the cognate resistance gene for avrB. We therefore tested whether the rrb3 mutation impacts the effector-triggered resistance. When inoculated with DC3000 (avrB) at a high dose, the rrb3 mutant showed a delayed hypertensitive response (HR) (SI Supporting Text, Fig. 8 and SI Table 1). Bacterial growth assay indicated that the rrb3 was completely susceptible to DC3000 (avrB) (SI Fig. 9). Genetic analysis indicated that rrb3 the phenotype was caused by a single recessive mutation. The rrb3 mutation was mapped to chromosome 5 between BACs K10D11 and MIO24, a 46-kb interval containing RAR1 (data not shown). Complementation tests indicated that rrb3 is a rar1 allele, because rrb3 × rar1–20 F1 plants displayed a delayed HR and reduced RAP2.6-LUC expression (SI Table 1) in response to DC3000 (avrB). Sequencing analysis indicated that the rrb3 mutant carried a point mutation in RAR1 that led to a H217Y substitution. Transformation of the wild-type RAR1 gene into rrb3 restored the normal HR induction (data not shown). The rrb3 mutation reduced the level of RAR1 protein accumulation in plants (SI Fig. 10). Twenty-eight rar1 alleles had been reported before this study (21); we therefore renamed rrb3 as rar1–29. H217 is a highly conserved residue located in the CHORD II domain of the RAR1 family proteins. The CHORD II domain is known to be required for interaction with SGT1 but not HSP90 (6). Indeed, the yeast two-hybrid experiment indicated that the rar1–29 mutant was unable to interact with SGT1b but interacted normally with HSP90.1 (Fig. 1C).

Fig. 1.

Fig. 1.

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rrb3 does not respond to AvrB delivered by P. syringae. (A and B) RAP2.6-LUC reporter activity of WT transgenic plants and rrb3 in response to DC3000 (avrB) (A) or DC3000 (B). The RAP2.6-LUC assay on rrb3 was performed four times with similar results. (C) Yeast two-hybrid assay for rar1–29 interaction with SGT1b and HSP90. (Upper) X-Gal assay. At least six individual colonies from each transformation were tested for β-galactosidase activities on X-Gal plates, and two representative clones are shown. (Lower) Western blot for RAR1-HA and rar1–29-HA protein detected with an anti-HA monoclonal antibodies.

Expression of AvrB in Plants Enhances Disease Susceptibility and Causes Chlorosis.

When delivered by P. syringae, AvrB confers virulence on soybean plants lacking the cognate resistance gene Rpg1 (22). A similar virulence function was not detected in Arabidopsis when delivered by P. syringae bacteria. However, when directly expressed in plants lacking the RPM1 gene, AvrB induces leaf chlorosis that is reminiscent of disease symptoms, suggesting a role of AvrB in virulence (23, 24). We similarly introduced the AvrB transgene into Nd-0, an ecotype lacking the cognate resistance gene RPM1, by using the dexamethasone (Dex)-inducible system (25). Fig. 2A shows that Dex treatment induces AvrB protein accumulation in the plant. Four days after Dex treatment, AvrB-expressing leaves developed chlorosis, whereas the nontransgenic wild-type plants showed no symptoms (Fig. 2B). All seven independent transgenic lines displayed identical phenotype (data not shown). We further tested whether the chlorosis phenotype is associated with enhanced susceptibility by examining bacterial growth of a nonpathogenic strain lacking the hrpL gene that is required for the expression of type III genes and coronatine biosynthetic genes in the bacterium (26). Plants expressing AvrB enhanced the growth of the DC3000 hrpL mutant bacteria by up to 50-fold (Fig. 2C). These results support that AvrB can act as virulence factor to promote bacterial colonization in plants lacking RPM1.

Fig. 2.

Fig. 2.

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Overexpression of AvrB induces chlorosis and enables nonpathogenic P. syringae mutant growth. (A) Induced accumulation of AvrB protein (arrow) after Dex treatment. Wild-type (Nd-0) and AvrB transgenic plants were sprayed with 30 μM Dex and harvested after 24 h for protein extraction. (B) Leaf chlorosis 4 days after daily spraying with 30 μM Dex or H2O. All seven independent AvrB transgenic lines tested showed similar phenotypes. (C) DC3000 hrpL mutant bacterial population in wild-type (Nd-0) and AvrB transgenic plants treated with H2O or Dex. The bacterial growth assay was repeated twice with similar results.

The AvrB-Dependent Susceptibility Is COI1-Dependent.

To determine whether the enhanced susceptibility phenotype caused by AvrB overexpression is relevant to the activity of AvrB delivered by P. syringae, we tested whether they require the same genetic components in plants. Because COI1 is required for P. syringae (avrB)-induced RAP2.6 expression (19), we crossed the AvrB-expressing plants with the coi1–1 mutant to construct F2 plants with COI1/COI1,rpm1/rpm1,AvrB/± and coi1–1/coi1–1,rpm1/rpm1,AvrB/± genotypes. Treatment of these plants with Dex induced chlorosis in both genotypes (Fig. 3A), indicating that the chlorosis was independent of COI1. The Dex-treated plants were also inoculated with the DC3000 hrpL mutant bacteria. Fig. 3B shows that bacterial growth was enhanced in Dex-treated COI1/ COI1,rpm1/rpm1,AvrB/± plants but not coi1–1/coi1–1,rpm1/rpm1,AvrB/± plants, suggesting that AvrB enhances bacterial growth by activating of the JA pathway. These results indicate that the AvrB virulence function is mediated, at least in part, by the JA signaling pathway.

Fig. 3.

Fig. 3.

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COI1 is required for AvrB-mediated bacterial growth. (A) AvrB-induced chlorosis. Photographs were taken 4 days after spray of Dex or H2O. (B) DC3000 hrpL mutant bacterial growth in COI1/COI1,rpm1/rpm1,AvrB/± (COI1) and coi1–1/coi1–1,rpm1/rpm1,AvrB/± (coi1) plants pretreated with H2O or Dex. Two independent experiments were done with similar results.

RAR1 Is Required for AvrB-Dependent Susceptibility and Chlorosis.

Because the rar1–29 mutant was isolated from plants carrying RPM1, and RAR1 is required for RPM1 stability, the observed phenotype could result from reduced RPM1 protein in the rar1–29 mutant. We therefore sought to determine whether RAR1 is required for AvrB-enhanced susceptibility in plants lacking RPM1 by crossing rar1–29 to the AvrB-transgenic plants. Homozygous F4 lines of RAR1/RAR1,rpm1/rpm1,AvrB/AvrB and rar1–29/rar1–29,rpm1/rpm1,AvrB/AvrB were generated and tested for chlorosis and enhanced susceptibility phenotypes associated with AvrB expression. As shown in Fig. 4A, Dex-induced chlorosis was observed in the RAR1/RAR1,rpm1/rpm1,AvrB/AvrB genotype but not the rar1–29/rar1–29,rpm1/rpm1,AvrB/AvrB genotype. Similarly, AvrB expression promoted DC3000 hrpL mutant bacterial growth only in RAR1 plants but not in rar1–29 plants (Fig. 4B).

Fig. 4.

Fig. 4.

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RAR1 is required for AvrB-induced leaf chlorosis and disease susceptibility. (A) AvrB-induced chlorosis. Photographs were taken 4 days after Dex or H2O treatment. (B) DC3000 hrpL mutant bacterial growth in RAR1/RAR1,rpm1/rpm1,AvrB/AvrB (RAR1) and rar1–29/rar1–29,rpm1/rpm1,AvrB/AvrB (rar1–29) plants pretreated with H2O or Dex. The experiments were repeated four times with similar results.

Because rar1–29 does not eliminate RAR1 protein accumulation, we also tested whether rar1–20, which lacks the entire RAR1 locus (21), abolished virulence activity associated with AvrB expression. rar1–20 was crossed to the AvrB transgenic plants to construct rar1–20/rar1–20,rpm1/rpm1,AvrB/± plants. As shown in SI Fig. 11, rar1–20 completely abolished the ability of AvrB to enhance hrpL mutant bacterial growth in plants. Together, these results demonstrate that RAR1 is required for the virulence function induced by AvrB overexpression.

AvrB Suppresses PAMP-Induced Cell Wall Defense Through RAR1.

Suppression of PTI is critical for bacterial virulence (3, 4). More than a dozen P. syringae effectors have been shown to suppress PAMP-induced defenses (27–30). Treatment of plants with the flg22 peptide, a well known PAMP derived from bacterial flagellin, induces callose deposition, a cell-wall-based defense required for resistance to Pseudomonas bacteria (29, 31). We therefore tested whether AvrB overexpression suppressed the flg22-induced callose deposition. Indeed, although RAR1/RAR1,rpm1/rpm1,AvrB/AvrB plants treated with flg22 developed numerous callose deposits, a prespray of Dex reduced the flg22-induced callose deposition by ≈80% (Fig. 5A). In contrast, Dex treatment had no effect on callose deposition in the rar1–29/rar1–29,rpm1/rpm1,AvrB/AvrB genotype, indicating that RAR1 is required for the suppression of flg22-induced callose deposition by AvrB.

Fig. 5.

Fig. 5.

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RAR1 negatively regulates flg22-induced callose deposition. (A) RAR1 is required for AvrB-mediated suppression of callose deposition. Leaves from RAR1/RAR1,rpm1/rpm1,AvrB/AvrB (RAR1) and rar1–29/rar1–29,rpm1/rpm1,AvrB/AvrB (rar1–29) plants were sprayed with Dex or H2O for 24 h before being induced with 50 μM flg22 for 12 h. Callose deposition was examined 12 h later. (B) RAR1 attenuates flg22-induced callose deposition. RAR1 and rar1–20 plants were treated with 2 μM flg22 for the indicated hours before staining for callose. The number of callose deposits represents an average of four microscopic fields of 0.1 mm2 obtained from four different leaves. Error bars represent standard error. Experiments were repeated four times with similar results.

RAR1 Is a Negative Regulator of PAMP-Induced Basal Defense.

Interestingly, we reproducibly observed less callose deposition in RAR1/RAR1,rpm1/rpm1,AvrB/AvrB plants compared with rar1–29/rar1–29,rpm1/rpm1,AvrB/AvrB in the absence of Dex treatment (Fig. 5A), suggesting a negative role of RAR1 in flg22-induced basal defense in the absence of effectors. To further test this possibility, we compared rar1–20 and wild-type Col-0 plants, neither plant carries the AvrB transgene, for flg22-induced callose deposition. As shown in Fig. 5B, the wild-type plants reproducibly showed fewer callose deposits than did rar1–20 plants. The effect of the rar1–20 mutation was most prominent when plants were induced with a low concentration of flg22 (2 μM) and examined at early hours after flg22 treatment. Together, these results indicate that RAR1 negatively regulates flg22-induced callose deposition.

RAR1 and AvrB Are in the Same Complex.

Yeast two-hybrid assays failed to detect an interaction between AvrB and RAR1 (data not shown). To determine whether AvrB is capable of interacting with RAR1 in vivo, we generated additional transgenic lines carrying AvrB-3xFLAG under the control of an estrogen-inducible promoter in the rpm1 background (32, 33). Estradiol-induced expression of AvrB-3xFLAG resulted in typical leaf chlorosis (data not shown). Fig. 6 shows that RAR1 and AvrB-3xFLAG were coprecipitated by an anti-FLAG monoclonal antibody in extracts from AvrB-3xFLAG transgenic but not nontransgenic plants. This result indicates that AvrB and RAR1 are present in the same protein complex.

Fig. 6.

Fig. 6.

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RAR1 and AvrB interact in plants. Nontransgenic rpm1 plants (−) and AvrB-3xFLAG transgenic rpm1 plants (+) were sprayed with estradiol to induce protein expression. Coimmunoprecipitation was carried out with an anti-FLAG monoclonal antibody. The immunocomplex was subjected to Western blot analyses by using anti-RAR1 and -FLAG antibodies.

Because AvrB is known to interact with RIN4 (12), we asked whether RAR1 and RIN4 are in the same protein complex. A HA-tagged RIN4 was overexpressed in protoplasts (in the rpm1 mutant background). Coimmunoprecipitation failed to detect RAR1 in the HA-RIN4 immunocomplex even when RAR1 was overexpressed, whereas AvrB-FLAG was successfully detected in the HA-RIN4 complex when coexpressed (SI Supporting TextFig. 12), suggesting that RIN4 and RAR1 exist in different protein complexes.

Discussion

RAR1, together with SGT1 and HSP90, is known to play a key role in ETI to diverse pathogens (6–8, 21, 34–39). A role of these proteins in bacterial effector virulence function has not been reported. Our results show that RAR1 negatively regulates basal defense and is required for AvrB-mediated suppression of basal defense, providing a molecular link between the effector-mediated suppression of basal defense and R gene-mediated disease resistance.

RAR1 is known to act as a cochaperone of HSP90 to stabilize certain NB-LRR resistance proteins, including RPM1, the cognate resistance protein of AvrB (7–11, 21). Perhaps RAR1 also assists the accumulation of proteins that negatively regulate PAMP-induced defenses. Because RAR1 is required for all aspects of AvrB-induced phenotypes, whereas COI1 is required only for the enhanced bacterial growth in the plant, it is most likely that RAR1 acts upstream of COI1. It is not clear how RAR1 mediates the activation of JA signaling after AvrB expression. One scenario is that RAR1 does so through the interaction with SGT1. Consistent with this speculation, rar1–29 is unable to interact with SGT1b. Although we have not tested whether it is required for AvrB virulence, SGT1b is known to play a role in JA signaling (40).

Although enhanced bacterial growth is observed only when AvrB is expressed in plants, two lines of evidence indicate that our findings are relevant to the AvrB function in the natural setting. Both the P. syringae (avrB)-induced RAP2.6 gene expression and the AvrB-mediated bacterial growth required COI1 (Fig. 3B; ref. 3). Similarly, RAR1 is required for both P. syringae (avrB)-induced RAP2.6 gene expression and the AvrB transgene-dependent susceptibility and defense suppression (Figs. 1A and 4). These results support that the AvrB transgene-dependent susceptibility is intrinsically linked to the virulence function of the P. syringae-delivered AvrB.

The previously identified effector target proteins are required for ETI (13–16) but not the effector virulence function. For example, the RPM1-interacting protein RIN4 is an intensively studied effector target that mediates both RPM1 and RPS2 resistance by interacting with their cognate effectors AvrRpm1, AvrB and AvrRpt2. Similar to RAR1, the RIN4 protein also acts as a negative regulator that attenuates PAMP-induced callose deposition in the absence of the effectors (29). Both AvrRpm1 and AvrRpt2 were shown to suppress PTI. AvrRpt2 is a cysteine protease that cleaves RIN4 (13, 14, 16), whereas AvrRpm1 and AvrB cause the phosphorylation of RIN4 through an unknown mechanism (12). However, it remains unexplained why the degradation of RIN4 by AvrRpt2 does not enhance PTI in plants lacking the cognate resistance gene RPS2. In fact, RIN4 is not required for the virulence function of AvrRpt2 and AvrRpm1 (29) and leaf chlorosis caused by AvrB overexpression (41). It is suggested that other proteins associated with RIN4 might be required for the AvrRpt2 and AvrRpm1 virulence functions (29).

It is likely that RAR1 is targeted by AvrB to suppress PTI. Although the yeast two-hybrid experiments failed to detect an interaction of RAR1 with AvrB, coimmunoprecipitation detected an AvrB–RAR1 interaction. Perhaps the interaction is indirect or stabilized by a plant-specific protein. The interaction does not appear to involve RIN4, because we were not able to detect an interaction between RIN4 and RAR1. Nonetheless, it is significant that both RAR1 and RIN4 negatively regulate PTI and are targeted, directly or indirectly, by AvrB. Therefore, host proteins that negatively regulate PTI may be an Achilles' heel in innate immunity that is actively exploited by bacterial effectors. That RIN4 and RAR1 are required for the function of multiple resistance proteins suggests that these negative regulators of PTI are common “guardees” of resistance proteins (SI Fig. 13). It is possible that resistance proteins are recruited to a protein complex containing negative regulators of the PAMP signaling pathway such as RIN4 and RAR1 to monitor effectors that suppress PTI. This enables the rapid activation of effector-triggered defenses when PAMP-triggered resistance is inhibited by the virulence factors.

Materials and Methods

Mutagenesis, Mutant Screening, and Map-Based Cloning.

Seeds of a homozygous RAP2.6-LUC transgenic line (19) were mutagenized with 0.3% (wt/vol) EMS (Sigma–Aldrich, St. Louis, MO) for 8 h and grown to maturity in 30 plant pools, and M2 seeds were harvested. A total of 300 pools of M2 seeds representing 9,000 M2 families were obtained. Approximately 16,000 M2 plants derived from all of the 300 pools were individually infiltrated with 2 × 106 cfu/ml DC3000 (avrB) bacteria, and inoculated leaves were removed 6 h later for the reporter assay (19). Plants with reduced LUC activity were identified as putative mutants and confirmed in the M3 generation. All experiments involving rrb3 were carried out with a back-crossed line.

For mapping the rrb3 gene, the rrb3 mutant was crossed with the Nossen ecotype, and 5-week-old F2 plants were inoculated with DC3000 (avrB) at 108 cfu/ml. Plants displaying delayed HR were scored as mutants. Simple sequence-length polymorphism (SSLP), insertion/deletion (InDel), and in-house-developed cleared amplified polymorphic sequence (CAPS) or SNP markers were used in fine mapping as described previously (42).

CCD Imaging and Luciferase Activity Assay.

Four- to 6-week-old plants were infiltrated with 2 × 106 cfu/ml DC3000 (avrB) containing 0.02 mM luciferin. The inoculated leaves were then collected at different time points and sprayed with 1 mM luciferin containing 0.01% Triton X-100. The leaves were kept in the dark for 6 min before luminescence images were captured. Quantitative LUC assay was performed as described (19).

Production of AvrB Transgenic Plants.

A FLAG-tagged version of AvrB was PCR amplified from pCPP2306 (23) by using the following primers: 5′-cgggatccccatgggctgcgtctcgtcaaaaagcac-3′ and 5′-gctctagatcacttgtcatcgtcgtccttgtag-3′. The AvrB fragment was ligated into pTA7002 (25) that had been digested with XhoI and then blunt ended. A resulting clone containing AvrB under the control of the pTA70002 Dex-inducible promoter was transformed into Arabidopsis thaliana Nd-0 plants (rpm1-null; ref. 33), as described (43). Seven independent AvrB transformants were analyzed, and all exhibited characteristics similar to the results reported here.

Western Blot Analysis.

Anti-RAR1 antiserum was raised in rabbits by using full-length recombinant RAR1 protein as antigen as described (36). Anti-AvrB antibodies were a gift from Alan Collmer (Cornell University, Ithaca, NY). Total protein was extracted from 5-week-old plants in a 1× PBS buffer containing 10 mg/ml leupeptin, 1 mM PMSF, 2 mM EDTA, 1× proteinase inhibitor mixture (Roche, Basel, Switzerland), and 1% Triton X-100. For AvrB protein detection, plants were sprayed with 30 μM Dex (Sigma) for 24 h before protein extraction. Thirty-microgram protein samples were electrophoresed through a 12% or 15% SDS/PAGE. Protein was electrotransferred to an Immobilon P membrane (Millipore, Bedford, MA). Immunodetection was performed with a 1:2,500 dilution of anti-RAR1 antibodies or a 1:10,000 dilution of anti-AvrB antibodies. The blot was then hybridized with a goat anti-rabbit or goat anti-mouse HRP-conjugated secondary antibody (Sigma) and visualized with ECL Western blotting detection reagents (Amersham, Piscataway, NJ), following the manufacturer's instructions.

Construction of rar1–29/rar1–29,rpm1/rpm1,AvrB/AvrB; rar1–20/rar1–20,rpm1/rpm1,AvrB/± and coi1/coi1,rpm1/rpm1,AvrB/± Plants.

To construct rar1–29/rar1–29,rpm1/rpm1,AvrB/AvrB and RAR1/RAR1,rpm1/rpm1,AvrB/AvrB plants, the Nd (AvrB) transgenic line 1 was crossed with the rar1–29 plants (Col-0 background). The SSLP marker K17N15–19K (primers 5′-gactagagagtaagaacatgactc-3′ and 5′-aagtcgaatcgttcacgcaataag-3′) closely linked to the RAR1 locus was used to identify the respective genotypes at the RAR1 locus. Homozygous F4 plants were used for experiments. rar1–20 (21) and coi1–1 (20) mutants (Col-0 background) were similarly crossed with Nd(AvrB). rar1–20/rar1–20,rpm1/rpm1,AvrB/± and RAR1/±,rpm1/rpm1,AvrB/± plants and COI1/COI1,rpm1/rpm1,AvrB/± and coi1–1/coi1–1,rpm1/rpm1,AvrB/± plants were identified from F2 plants by PCR. The genotype at the COI1 locus was identified by using a CAPS maker as described (20). Primers 5′-atcttcaagtctcaaagtgtgc-3′ and 5′-gattccacaagataacttgaagc-3′ were used to determine the genotype at the RPM1 locus (Nd lacks RPM1). Plants carrying the AvrB transgene were confirmed with AvrB-specific primers 5′-atcaatgcttaattggtgcagc-3′ and 5′-atcagaatctagcaagcttctg-3′. All of the plants carry a chromosome segment from the Nd ecotype and thus are rpm1-null.

Bacterial Growth Assay.

Five-week-old plants were sprayed daily with a 30 μM Dex solution containing 0.02% Silwet L-77 (Osi Specialties, Friendship, WV). DC3000 hrpL mutant (26) bacterial suspension was infiltrated at 105 cfu/ml into leaves 2 days after the first Dex treatment. Leaf bacterial number was counted at the indicated time points. Each data point consisted of at least six replicates.

Callose Staining.

To visualize callose deposition, 5-week-old Arabidopsis leaves were untreated or pretreated by spraying 50 μM Dex or distilled water 24 h before infiltration of flg22 at the indicated concentrations. Whole leaves were harvested at the indicated times, cleared, stained with aniline blue (27), and mounted in 50% glycerol, and fluorescence from callose was visualized with an epifluorescence microscope under UV light. For each treatment, four leaves were examined, and one microscopic field per leaf that best represented the overall staining of the leaf was used to calculate the number of callose deposits per field of 0.1 mm2 with Image J software (www.uhnresearch.ca/wcif/imagej).

Coimmunoprecipitation.

Coimmunoprecipitation experiment was done as described (12). AvrB-3xFLAG transgenic and nontransgenic rpm1 plants (33) were sprayed with 30 μM estradiol (Sigma) before protein extraction. The immune complex was precipitated with an agarose-conjugated monoclonal anti-FLAG antibody (Sigma). The presence of RAR1 and AvrB-FLAG in the complex was detected by using Western blotting.

Yeast Two-Hybrid Assay.

The RAR1, rar1–29, SGT1b, and HSP90.1 coding sequences were amplified from total cDNA of Arabidopsis Col-0 wild-type (RAR1, SGT1b, and HSP90.1) or rar1–29 plants by using gene specific primers 5′-aactctgaattcatggaagtaggatctgca-3′ and 5′-aatctcgagctttgaatcgaaaatctcagg-3′ (RAR1 and rar1–29), 5′-gaattccctctgaaagaatcaatgg-3′ and 5′-ctcgagagatcaatactcccacttc-3′ (SGT1b), and 5′-gaattcctaaagttcgttgcgatgg-3′ and 5′-ctcgagcttcatctcttagtcgac-3′ (HSP90.1). PCR products were digested with EcoRI and XhoI and inserted into pJG4–5 (RAR1 and rar1–29) or pEG202 (SGT1b and HSP90.1). The constructs were sequence-verified and cotransformed in pairs into the EGY48 yeast strain containing pSH18–34. At least six individual colonies from each transformation were tested for β-galactosidase activities on X-Gal plates following the protocol described (44).

To determine whether the RAR1 and rar1–29 proteins accumulated to similar levels in yeast, total yeast protein was extracted by boiling equal amounts of yeast cells in 2× Laemmli sample buffer. The total protein was separated by 10% SDS/PAGE gel and transferred to immobilon membrane (Millipore). The membrane was then hybridized with monoclonal mouse anti-HA antibody (Sigma) and detected with the HRP-conjugated goat anti-mouse antibodies (Sigma) and ECL regents (Amersham, Piscataway, NJ).

Supplementary Material

Supporting Information
pnas_0607279103_index.html (8KB, html)

Acknowledgments

We thank Alan Collmer for anti-AvrB antibodies, Xing Wang Deng (Yale University, New Haven, CT) and Yan Guo (National Institute of Biological Sciences, Beijing, China) for plasmids, Frank White and ShengYang He for critical reading of the manuscript, and Liqin Fu for technical assistance with microscopy. J.-M.Z. was supported by a grant from the Chinese Ministry of Science and Technology (2003-AA210080). R.T. and J.Z.-V. were supported in part by a U.S. Department of Energy grant awarded to Sheng Yang He (DE-FG02–91ER20021).

Abbreviations

PAMPs

pathogen-associated molecular patterns

JA

jasmonates

PTI

PAMP-triggered immunity

ETI

effector-triggered immunity

Dex

dexamethasone

LUC

luciferase.

Footnotes

This article is a PNAS direct submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0607279103/DC1.

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