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Plant Cell. Feb 2001; 13(2): 437–447.
PMCID: PMC102253

Arabidopsis NHO1 Is Required for General Resistance against Pseudomonas Bacteria

Abstract

Nonhost interactions are prevalent between plants and specialized phytopathogens. Although it has great potential for providing crop plants with durable resistance, nonhost resistance is poorly understood. Here, we show that nonhost resistance is controlled, at least in part, by general resistance. Arabidopsis plants are resistant to the nonhost pathogen Pseudomonas syringae pv phaseolicola NPS3121 and completely arrest bacterial multiplication in the plant. Ten Arabidopsis mutants were isolated that were compromised in nonhost (nho) resistance to P. s. phaseolicola. Among these, nho1 is caused by a single recessive mutation that defines a novel gene. nho1 is defective in nonspecific resistance to Pseudomonas bacteria, because it also supported the growth of P. s. tabaci and P. fluorescens bacteria, both of which are nonpathogenic on Arabidopsis. In addition, the nho1 mutation also compromised resistance mediated by RPS2, RPS4, RPS5, and RPM1. Interestingly, the nho1 mutation had no effect on the growth of the virulent bacteria P. s. maculicola ES4326 and P. s. tomato DC3000, but it partially restored the in planta growth of the DC3000 hrpS mutant bacteria. Thus, the virulent bacteria appear to evade or suppress NHO1-mediated resistance by means of an Hrp-dependent virulence mechanism.

INTRODUCTION

Plants interact with a wide array of microbes in the environment. However, a given plant species is resistant to most phytopathogens, and this phenomenon is referred to as nonhost resistance (Heath, 1987, 1996; Staskawicz et al., 1995). Nonhost resistance is pathovar/formae speciale specific or species specific, in contrast with the widely studied race- or cultivar-specific resistance that is mediated by resistance (R) gene–avirulence (avr) gene interactions. Considerable progress has been made in our understanding of Ravr interactions (Staskawicz et al., 1995; Martin, 1999). However, these advances do not readily explain why a pathogen fully virulent on one plant species is nonpathogenic on others. Nonhost resistance has been difficult to characterize due to the lack of a genetic system.

Pathogens possess basic compatibility even on a nonhost plant. For example, fungal pathogens are capable of forming an appressorium, penetrating nonhost plant tissues, and occasionally developing a haustorium (Heath, 1979, 1987). Phytophthora megasperma f sp glycinea (now renamed P. sojae) is a nonhost pathogen on parsley that normally causes a nonhost hypersensitive response (HR). However, parsley plants kept at 100% humidity after inoculation were completely susceptible to the otherwise incompatible fungus and allowed sporulation (Jahnen and Hahlbrock, 1988). Basic compatibility also is evident during the interaction of a bacterial pathogen and a nonhost plant. The expression of hrp genes (required for the HR on nonhost plants and for pathogenicity on host plants) that encode the type III secretion system and avr genes in bacterial pathogens often depends on signals from the host plant (Brito et al., 1999). This process apparently occurs normally when the bacterial pathogen is in contact with the nonhost plant cell. An avr gene in the bacterium can be recognized by a cognate R gene in the nonhost plant and can elicit the HR (Yu et al., 1993). This suggests that the bacterial pathogen is capable of sensing the contact with nonhost plant cells and delivering its effectors to the plant cell via the type III secretion system. The basic compatibility associated with the many potential pathogens reinforces the importance of plant defense mechanisms in nonhost interactions.

Heath (1987)(1996) proposed that nonhost resistance is a result of general resistance that is parasite nonspecific. The so-called general resistance assumes that plants are commonly equipped to recognize potential parasites in a nonspecific manner. This recognition leads to a defense reaction that limits the growth of pathogens. A successful parasite must suppress, avoid, or tolerate the general resistance by acquiring matching pathogenicity factors.

Consistent with the general defense hypothesis, plants possess highly sensitive receptors for bacterial and fungal elicitors (Boller, 1995). These elicitors are pathovar or formae speciale nonspecific and induce rapid plant defense responses at very low concentrations. The general elicitors characterized to date include oligosaccharides, peptides, and glycoproteins. A recent addition is the flagellin protein from a wide range of eubacteria (Felix et al., 1999). A 22–amino acid peptide from the conserved domain in flagellin proteins is a highly potent elicitor for the induction of oxidative burst, alkalinization of cultures, accumulation of callose, and expression of pathogenesis-related (PR) genes (Gomez-Gomez et al., 1999). Arabidopsis flagellin sensing (fls) mutants have been isolated. Map-based cloning enabled the isolation of a putative flagellin receptor gene with striking similarity to the rice resistance gene Xa21 (Gomez-Gomez and Boller, 2000). However, it remains to be determined if flagellin sensing is required for resistance to any phytopathogens.

Early experiments suggested that the general defense is responsible, at least in part, for nonhost resistance. For example, treatment of nonhost plants with heat shock or protein synthesis inhibitors before the inoculation of several rust fungi enhanced hyphal growth and haustorium development (Heath, 1979). However, genetic evidence is scarce for a role of plant defense in limiting nonhost pathogen growth. Recent studies show that mutant plants defective in preformed or induced defense mechanisms can develop severe diseases upon infection by weak fungal pathogens or fungal pathogens that normally infect closely related plant species (Parker et al., 1996; Knoester et al., 1998; Multani et al., 1998; Vijayan et al., 1998; Papadopoulou et al., 1999; Thomma et al., 1999). Although the parasite–plant interactions in these examples are not always viewed as nonhost interactions, they do demonstrate the importance of race/cultivar-nonspecific resistance that is not determined by gene-for-gene interaction. Similar data do not exist for nonhost resistance to bacterial pathogens.

Here, we describe the Arabidopsis–Pseudomonas syringae pv phaseolicola interaction as a model genetics system to study plant defense mechanisms involved in nonhost interactions. We show that general resistance plays an important role in limiting the growth of P. s. phaseolicola on Arabidopsis and that nonhost resistance is amenable to genetic studies.

RESULTS

Reaction of Different Arabidopsis Ecotypes to P. s. phaseolicola

P. s. phaseolicola causes halo blight in bean but incites no disease on cruciferous plants, including Arabidopsis (Yu et al., 1993). To test the potential pathogenicity of P. s. phaseolicola strain NPS3121 on Arabidopsis, we inoculated NPS3121 at various concentrations on 17 accessions of Arabidopsis (see Methods). No accession showed any disease symptoms when inoculated with [less-than-or-eq, slant]106 colony-forming units (cfu)/mL of bacteria. We also examined bacterial population in the plant. NPS3121 was unable to grow to a significant level in any of the Arabidopsis ecotypes tested. Four days after inoculation, bacterial populations either decreased onefold or increased less than fourfold. Similar results were obtained with repeated experiments. These findings support the idea that Arabidopsis is a nonhost species to P. s. phaseolicola.

Bacterial pathogens often induce the HR in nonhost plants. However, Arabidopsis ecotype Columbia (Col-0) plants do not react with an HR to NPS3121 (Yu et al., 1993). Similarly, we found that none of the 17 ecotypes tested developed an HR after NPS3121 inoculation (at 108 cfu/mL).

Active Defense in Arabidopsis Is Involved in Nonhost Resistance to P. s. phaseolicola

We next examined the expression of PR1 and GST1 in plants challenged with NPS3121 as an indication of active defense during a nonhost interaction. Inoculation of NPS3121 on Col-0 plants induced the expression of PR1 and GST1 (Figure 1A). The induction of PR1 is abolished by the nahG transgene. Plants carrying nahG, a bacterial gene that encodes salicylic acid hydroxylase, are unable to accumulate salicylic acid in response to pathogen infection (Ryals et al., 1996). These results indicated that active defense was activated during the nonhost interaction.

Figure 1.
Active Defense Is Required for the Nonhost Resistance of Arabidopsis to P. s. phaseolicola NPS3121.

We hypothesize that plant defense mechanisms are responsible, at least in part, for the absence of P. s. phaseolicola multiplication in Arabidopsis. To test this notion further, we challenged Arabidopsis plants carrying the nahG transgene with NPS3121 and measured bacterial growth (Figure 1B). Four days after inoculation, the bacterial number decreased slightly in the wild-type Col-0 plants. In contrast, the bacterial number increased ~50-fold in Col-0 (nahG) plants. In addition, leaves of the nahG plants showed water-soaked lesions 5 days after inoculation. The results indicate that the salicylate-mediated defense pathway plays a profound role in the nonhost resistance against P. s. phaseolicola. More importantly, they indicate that screening for Arabidopsis mutants with compromised resistance to P. s. phaseolicola is feasible.

Isolation of nho Mutants

An initial screen based on the appearance of lesions or chlorosis upon P. s. phaseolicola inoculation failed because the observed symptoms did not correlate with the bacterial number in planta. We revised the strategy by monitoring leaf bacterial populations. We introduced the uidA gene (β-glucuronidase [GUS]) under the control of the lacZ promoter into NPS3121 (see Methods). Four days after bacterial inoculation, leaf discs were excised, incubated with 4-methylumbelliferyl β-d-glucuronide, and visualized under UV light. Plants with strong fluorescence were selected as putative mutants that supported bacterial growth. Putative mutants then were verified by standard bacterial growth assays.

Approximately 18,000 ethyl methanesulfonate–mutagenized M2 plants (Col-0 background) constituting 12,000 families were screened. A total of 216 putative mutants were identified. Among these, 10 lines consistently supported more NPS3121 growth in the M3 and M4 generations. Four days after inoculation, bacterial populations in these mutants increased 10- to 50-fold compared with those in wild-type plants (Figure 2).

Figure 2.
nho Mutants Support the Growth of P. s. phaseolicola NPS3121 Bacteria.

nho Mutations Do Not Enhance Disease Susceptibility to the Virulent Bacterium P. s. maculicola ES4326

Our mutant screening strategy was similar to that for the eds (for enhanced disease susceptibility) mutants (Glazebrook et al., 1996). Most eds mutants were isolated based on enhanced disease susceptibility to the virulent bacterial strain P. s. maculicola ES4326. However, these mutants do not exhibit compromised resistance to P. s. phaseolicola (Glazebrook et al., 1996; Rogers and Ausubel, 1997; Volko et al., 1998). We determined the growth of P. s. maculicola ES4326 on seven nho mutants. All seven nho mutants supported the same level of bacterial growth as did the wild-type plant in repeated experiments (Figure 3). These results indicate that the nho and eds mutant screens revealed distinct classes of mutants.

Figure 3.
nho Mutations Do Not Enhance Susceptibility to the Virulent Bacterium P. s. maculicola ES4326.

nho Mutants Support Hrp-Independent Bacterial Growth

We tested whether the virulence of NPS3121 on the nho mutants was Hrp dependent. The P. s. phaseolicola NPS4000 strain carries a Tn5 insertion in the hrpA gene that encodes a pilus structural protein essential for the type III secretion system (Lindgren et al., 1986; Wei et al., 2000). All nho mutants tested allowed more NPS4000 growth compared with Col-0 plants (Figure 4). The level of NPS4000 growth on nho plants was similar to that of the wild-type NPS3121 strain. These results indicate that the enhanced growth of NPS3121 on nho mutants was Hrp independent. They also suggest that the resistance defined by nho mutants did not require an Hrp-dependent signal.

Figure 4.
The Growth of P. s. phaseolicola in the nho Mutant Occurs without a Functional hrpA Gene.

nho1 Defines a Novel Gene for Disease Resistance

Among all mutants, nho1409, hereafter referred to as nho1, showed the strongest phenotype and thus was chosen for detailed analysis. The nho1 mutant consistently supported more bacterial growth than any other mutants. Four days after infiltration with P. s. phaseolicola at 106 cfu/mL, necrosis was observed frequently in some leaves of nho1, but not wild-type plants. In the remaining experiments, a lower bacterial concentration (104 cfu/mL) was used because this simplified the bacterial growth assay. nho1 mutant consistently showed 10- to 100-fold higher bacterial populations compared with wild-type plants when inoculated with either 104 or 106 cfu/mL bacteria. Genetic analysis of the backcrossed F1 and F2 plants indicated that nho1 is a single recessive mutation (Table 1). The analysis of the backcrossed F2 progenies also indicated that the nho1 mutation was not associated with any morphological phenotype.

Table 1.
Genetic Analysis of nho1

A mapping population was made by crossing the nho1 mutant with the Landsberg erecta (Ler) ecotype. F2 plants were infiltrated with NPS3121 bacteria (104 cfu/mL), and leaf bacteria were measured 4 days after inoculation. Initial mapping by using 109 F2 plants and 28 microsatellite markers (Bell and Ecker, 1994) that cover the five Arabidopsis chromosomes placed NHO1 to the bottom of chromosome 1. Further mapping was performed by using four microsatellite markers spanning the last 2.5 Mb of chromosome 1. Analysis of 350 chromosomes identified 15 recombinant chromosomes between nho1 and a marker derived from bacterial artificial chromosome (BAC) F22P28 (position 26,700 bp), five recombinant chromosomes for a marker derived from BAC T30F21 (position 28,500 kb), three recombinant chromosomes for a marker from BAC F18B13 (position 28,970 kb), and two recombinant chromosomes for a marker derived from BAC F23A5 (position 29,200 kb). F23A5 is the telomeric end of chromosome 1. Our current mapping data do not resolve whether NHO1 is located on F23A5 or between F23A5 and F18B13. Nevertheless, the results suggested that NHO1 was located on the telomeric side of the F18B13 marker that was only 270 kb from the telomere (chromosome 1 has a total of 29,240 kb).

nho1 Is Compromised in General Resistance

The susceptibility of nho1 to P. s. phaseolicola bacteria was studied in greater detail. By using a strain carrying a green fluorescent protein (GFP) construct and confocal microscopy, we observed that the NPS3121 bacteria were able to form colonies in the intercellular spaces of nho1 but not wild-type plants (data not shown). The GFP results qualitatively demonstrate that the nho1 mutant permits the multiplication of NPS3121 bacteria.

We determined whether the defect in nho1 also affected resistance to other bacteria, including P. s. tabaci and P. fluorescens. As shown in Figures 5A and 5B, leaf bacterial numbers for P. s. tabaci and P. fluorescens remained unchanged in the wild-type plant 4 days after inoculation. In contrast, bacterial numbers in nho1 leaves increased at least 100-fold compared with those in the wild-type plant.

Figure 5.
nho1 Is Compromised in Nonspecific Resistance to Pseudomonas Bacteria.

We also tested nho1 for disease resistance mediated by gene-for-gene interactions. Arabidopsis Col-0 plants carry RPS2, RPM1, RPS4, and RPS5 that confer resistance to Pseudomonas bacteria containing the avirulence genes avrRpt2, avrB, avrRPS4, and avrPphB, respectively. Although wild-type plants supported low levels of bacterial growth because of gene-for-gene resistance, nho1 plants showed diminished resistance to these avirulent strains and gave rise to significantly higher (20- to 100-fold) bacterial numbers in the leaf compared with wild-type plants (Figures 5C to 5F). The results indicate that nho1 has partially lost disease resistance mediated by multiple resistance genes. Thus, the NHO1 gene is required for nonhost resistance to Pseudomonas bacteria and gene-for-gene resistance. The reduced resistance did not seem to be associated with an alteration of HR. Inoculation of nho1 plants with high concentrations of avrB or avrRpt2 strains produced strong HR similar to that observed in wild-type plants (data not shown).

P. s. tomato Evades the NHO1-Mediated Resistance by Using an Hrp-Dependent Virulence Mechanism

nho1 supports increased bacterial growth by Pseudomonas strains that are nonpathogenic on wild-type Arabidopsis plants, but it does not display enhanced susceptibility to the virulent bacterium P. s. maculicola ES4326. To further investigate the relationship between the virulence of the pathogen and the observed general resistance in nho1, we inoculated plants with the P. s. tomato DC3000 strain that is virulent on Arabidopsis. In repeated experiments, nho1 and wild-type plants supported similar bacterial growth 4 days after inoculation (Figure 6A). Therefore, the resistance defined by NHO1, although effective at limiting growth by the nonhost bacteria, was completely ineffective against the virulent bacteria. We speculate that the virulent strains have evolved strategies to suppress or avoid the general resistance conditioned by NHO1. To determine whether these strategies are Hrp dependent, we conducted bacterial growth assays on nho1 with a DC3000 hrpS mutant (Wei et al., 2000). hrpS is a regulatory gene required for the expression of other hrp genes and many effector genes that are controlled by the hrp box in the promoter. hrpS bacteria failed to grow in wild-type plants but grew 100-fold in nho1 plants. Note that nho1 did not completely restore the virulence of the hrpS strain, because the growth of the latter in nho1 plants was 10- to 100-fold lower than the wild-type DC3000, and the hrpS strain only occasionally caused necrosis on nho1 plants when high concentration (106 cfu/mL) of bacteria was used (data not shown).

Figure 6.
nho1 Mutation Enhances the Growth of P. syringae hrp Mutants but Not of Virulent P. s. tomato DC3000.

DISCUSSION

Nonhost disease resistance is a remaining challenge in the field of plant–microbe interactions. The work described here demonstrates that it is possible to systematically study the molecular mechanisms involved in nonhost disease resistance in plants. Nonhost resistance is thought to be more durable than R gene–mediated resistance and therefore is attractive for agricultural purposes. It has been suggested that nonhost resistance is a multigenic trait and thus difficult for genetic manipulation (Heath, 1996). Our success with the isolation of 10 nho mutants with compromised resistance to P. s. phaseolicola indicates that major genetic determinants do exist in plants for nonhost resistance. Therefore, the mechanisms involved in nonhost resistance are amenable to genetic studies. Note that Arabidopsis resistance to P. s. phaseolicola occurs in the absence of the HR. The isolation of NHO genes should provide novel tools for improving disease resistance in crop plants.

The nho mutants are different from the Arabidopsis eds mutants isolated to date in that they display distinct responses to the virulent pathogen P. s. maculicola and the nonhost pathogen P. s. phaseolicola (Glazebrook et al., 1996; Glazebrook, 1999). The NHO1 gene may be a common component that also is shared by the R gene pathways. The Arabidopsis mutants eds1, ndr1, pbs2, and pbs3 also are blocked in R gene functions (Century et al., 1995; Parker et al., 1996; Warren et al., 1998). Interestingly, the eds1 mutant also supported sporulation by several Peronospora isolates and one Albugo candida isolate from the Brassica species, suggesting an effect on nonhost resistance (Parker et al., 1996). However, none of these mutants supported nonhost bacterial growth (Glazebrook, 1999). NHO1 was mapped to the bottom of chromosome 1. This region does not contain any nucleotide binding site–leucine-rich repeat sequence, nor does it carry a disease-related locus defined by known mutants. Therefore, nho1 defines a novel gene required for disease resistance. Together, these results demonstrate that novel disease resistance genes can be identified through genetic analysis of nho mutants.

It is well known that cloned avr genes from bacterial pathogens are recognized by previously unidentified R genes in nonhost plants (Whalen et al., 1988; Kobayashi et al., 1990). This has led to the hypothesis that nonhost resistance is determined by gene-for-gene interactions. In fact, the Arabidopsis R genes RPS4 and RPS5 recognize avrRps4 from P. s. pisi and avrPphB from P. s. phaseolicola, respectively (Warren et al., 1998; Gassmann et al., 1999). Both bacteria are nonhost pathogens on Arabidopsis. However, it remains to be shown whether the R genes and avr genes determine the host range of different pathovars. For example, the PT23 strain of P. s. tomato, a nonhost pathogen on soybean, carries four avr genes (avrA, avrD, avrE, and avrPto). When transformed individually into P. s. glycinea, each avr gene can induce the HR and disease resistance in soybean in a cultivar-dependent manner (Lorang et al., 1994). However, simultaneous mutation of all four avr genes by deletions or Tn insertion fails to alter the nonhost interaction either qualitatively or quantitatively (Lorang et al., 1994). Conversely, the absence of known R genes in plants does not result in susceptibility to nonhost pathogens. Although R gene–mediated resistance certainly contributes to resistance in nonhost interactions, other mechanisms in addition to gene-for-gene recognition are likely to be involved as well.

Some strains of nonhost Pseudomonas pathogens do not induce the HR in Arabidopsis (Davis et al., 1991; Yu et al., 1993), suggesting that they may not carry avr genes recognizable to Arabidopsis. However, the lack of the HR does not permit these bacteria to grow in Arabidopsis. The Arabidopsis–P. s. phaseolicola NPS3121 interaction described in this study likely involves a general resistance rather than a specific resistance mediated by R–avr recognition. First, the NPS3121 strain does not elicit the HR on Arabidopsis plants, suggesting a lack of avr genes that can be recognized by Arabidopsis. More importantly, nho mutants supported the growth of both the wild-type strain and the hrpA mutant of P. s. phaseolicola, whereas the wild-type plant did not. Thus, resistance defined by the nho mutants function in the absence of an Hrp-dependent signal. This suggests that recognition of an avr gene is not required for the nho-defined resistance, because almost all bacterial avr genes require the Hrp system to function. Furthermore, the nho1 mutant showed compromised resistance to multiple bacterial strains. These findings collectively indicate that general resistance plays an important role in the nonhost interaction between Arabidopsis and P. s. phaseolicola.

The nho1 mutant supported the growth of two hrp mutants and one nonpathogenic Pseudomonas bacterium, suggesting that nho1 plants are affected in their immunity to saprophytes. One possibility is that the mutant plants are leaking nutrients into the apoplast. However, nho1 plants did not support the growth of Escherichia coli when the latter is infiltrated into the intercellular spaces (B. Yang and J.-M. Zhou, unpublished results), arguing against the nutrient leakage hypothesis. The precise biochemical lesion awaits the isolation and characterization of the NHO1 gene.

Results with hrp mutants and virulent strains of Pseudo-monas indicate a strong interaction between NHO1-mediated general resistance and the pathogenicity of virulent bacteria. The nho1 mutation did not enhance susceptibility to the virulent bacterial strains ES4326 and DC3000. This is not because of spatial and nutrient constraints, because ES4326 was able to grow 100-fold more in nahG plants compared with Col-0 and nho1 plants. Therefore, these results indicate that the NHO1-mediated general resistance has no effect against virulent bacteria. The DC3000 hrpS mutant was completely unable to grow in the wild-type Arabidopsis plant. Surprisingly, the nho1 mutation in this plant largely restored the virulence of the DC3000 hrpS mutant. One plausible explanation for this finding is that the virulent bacteria are capable of evading or suppressing NHO1-mediated general resistance by an Hrp-dependent mechanism. This is similar to the recently discovered type III–dependent virulence function of Salmonella (Vazquez-Torres et al., 2000). The Salmonella pathogenicity island 2 (SPI2) encodes the type III secretion apparatus required for virulence. One of the virulence functions mediated by SPI2 was interference with the deposition of phagocyte NADPH oxidase into bacteria-containing vacuoles. Mutants defective in the SPI2- encoded secretion system are diminished in virulence. However, the virulence can be restored when the host cell is deficient in NADPH oxidase. The isolation of the NHO1 gene and understanding of its biochemical function should provide unique opportunities for understanding virulence mechanisms of plant bacterial pathogens.

According to the current model, the key function of the type III system is to secrete pathogenicity factors, including effector proteins that are targeted directly into host cells (Galan and Collmer, 1999). Although it is not clear if different pathovars of bacterial pathogens secrete distinct sets of effectors, it is possible that these effectors play an important role in the adaptation of bacteria to their respective host plant species. Increasing evidence from animal and plant bacterial pathogens indicates that at least some effectors secreted by the type III system interfere with host defense mechanisms. The YopJ protein secreted by the human pathogen Yersinia and the AvrBst protein secreted by the plant pathogen Xanthomonas are cysteine proteases that interfere with host defense signaling pathways by blocking ubiquitin conjugation and inhibiting mitogen-activated protein kinase kinases (Orth et al., 1999, 2000). Interestingly, YopJ homologs have been found in Salmonella, Erwinia, Pseudomonas, and Rhizobium (White et al., 2000). X. c. vesicatoria hrp mutants are unable to suppress papilla formation in the host plant cell, suggesting that pathogenicity factors secreted by the Hrp system are able to suppress defense responses in the host plant cell (Brown et al., 1995). Recently, Kunkel's group showed that the AvrRpt2 protein suppressed PR gene expression in Arabidopsis plants lacking the cognate RPS2 gene (Kloek et al., 2000). Furthermore, suppression of R–avr interactions in bean by a virulence gene has been reported for P. s. phaseolicola (Jackson et al., 1999; Tsiamis et al., 2000). Together with our results, these findings support the idea that the suppression of general resistance by bacterial effectors may play a major role in the adaptation of bacterial pathogens to their host plant species.

METHODS

Bacterial Strains and Plants

Bacterial strains used in this study include Pseudomonas syringae pv phaseolicola NPS3121 and NPS4000 (Lindgren et al., 1986), P. s. tabaci R1152 race 0 (Willis et al., 1988), P. s. maculicola ES4326 (Davis et al., 1991), P. s. tomato DC3000 (Innes et al., 1993), P. s. tomato DC3000 hrpS mutant (Wei et al., 2000), P. s. tomato DC3000 (avrRpt2; Kunkel et al., 1993), P. s. tomato DC3000 (avrRPS4; Gassmann et al., 1999), P. s. tomato DC3000 (avrPphB; Warren et al., 1998), and P. fluorescens 2-79 (Thara et al., 1999). NPS3121 (β-glucuronidase [GUS]), NPS3121 (green fluorescent protein [GFP]), and ES4326 (avrB) were constructed by transforming the bacteria with the pHM1::pBS-GUS (obtained from Frank White, Kansas State University, Manhattan, KS), pCPP3069 (carrying a red-shift S65T GFP under the control of the trp promoter; obtained from Alan Collmer, Cornell University, Ithaca, NY; Gage et al., 1996), and pVSP61::avrB (Innes et al., 1993) plasmids, respectively.

All Arabidopsis thaliana ecotypes (Columbia [Col-0], Nd-0, Landsberg erecta [Ler], Bensheim, RLD, Ws, No-0, Dijon G, S96, RLD1, Mh-0, Gre-0, C24, Kendalville, Aua/Rhon, Cvi-0, and Turk Lake) and ethyl methanesulfonate–mutagenized M2 seed (Col-0 background) were obtained from Lehle Seeds (Tucson, AZ). Col-0 (nahG) seeds (Gaffney et al., 1993) were supplied by Novartis Agricultural Business (Research Triangle Park, NC). All plants were grown in growth chambers at 20°C at night and 22°C during the day with a 10-hr/day photoperiod.

Bacterial Growth Assay

Bacterial culture was grown in King's medium B (King et al., 1954) with appropriate antibiotics, and inoculum was prepared as described (Thara et al., 1999). In earlier experiments, 5- to 6-week-old plants were syringe infiltrated at the 106 colony-forming units (cfu)/mL for bacterial growth assay. A lower concentration (104 cfu/mL) was used in the remaining experiments because it simplified the bacterial growth assay. Leaf bacterial numbers were measured by plating bacteria on King's medium B agar plates containing appropriate antibiotics. Each data point represents four to five replicates, two discs per replicate. All bacterial growth assays were repeated, and only results that were observed consistently are shown.

For confocal microscopy, plants were syringe infiltrated with NPS3121 (GFP) bacteria at a concentration of 106 cfu/mL. Leaves were examined with a confocal microscope and photographed 4 days after inoculation.

Mutant Screening and Genetic Analysis

M2 plants (18,000) were hand inoculated with NPS3121 (GUS) bacteria at a concentration of 106 cfu/mL. Four days after inoculation, leaf discs were removed from plants and incubated in a buffer containing 50 mM Na2HPO4, pH 7.0, 10 mM EDTA, 0.1% Triton X-100, 0.1% sarkosyl, 0.7 μL/mL β-mercaptoethanol, and 0.7 mg/mL 4-methylumbelliferyl β-d-glucuronide. Twelve hours after incubation, the leaf discs were visualized under UV light, and plants showing strong fluorescence were selected for bacterial growth assay. Plants that showed significant bacterial growth were verified further in the M3 and M4 generations. All results for nho1 were verified with backcrossed progeny.

Reciprocal crosses were performed between nho1 and wild-type Col-0 plants. F1 and F2 plants were hand inoculated with NPS3121 bacteria at a concentration of 104 cfu/mL. Plants were scored susceptible or resistant by using log(cfu/cm2) values >3.2 or <2.8, respectively. For mapping, Ler was used as a recipient for pollen grains from the nho1 plants. F2 plants were syringe infiltrated with NPS3121 bacteria at 104 cfu/mL and scored by bacterial growth assay. Genomic DNA was isolated from the susceptible F2 plants and analyzed with microsatellite markers covering the five Arabidopsis chromosomes (Bell and Ecker, 1994). Primers for additional microsatellite markers are given below: bacterial artificial chromosome (BAC) F28P22, 5′-TGAGCAAAAGAAACTCCAGTTG-3′ and 5′-TTGAAGACAAGCTTTGCTCAGAG-3′; BAC T30F21, 5′-GGAACAATA-ACGGTCTCAAGAATG-3′ and 5′-TATGGCTTTTCGAAAGGATCCTAG-3′; BAC F18B13, 5′-TTTCGTTCTGCTTCCGAGCTTAG-3′ and 5′-ACCTGAAGCATCGTCACATTTATG-3′; and BAC F23A5, 5′-GTAAGAACCAACCTATTTCAATCAG-3′ and 5′-GCTTTAACAGTTATT-GTAATCAGTCG-3′.

RNA Gel Blot Analysis

For RNA expression, plants were vacuum infiltrated with bacterial suspension, and leaves were collected at the times indicated for RNA isolation. Total RNA (10 μg/lane) was fractionated with formaldehyde gel, transferred to a nitrocellulose membrane, and hybridized to radiolabeled PR1 cDNA (Uknes et al., 1992) and an expressed sequence tag for GST1 (Reuber et al., 1998; GenBank accession number AA713231).

Acknowledgments

We thank Drs. Peter Lindgren, Nickolas Panopoulos, Frederick Ausubel, Brian Staskawicz, Roger Innes, Shen-Yang He, Jan Leach, Frank White, and Alan Collmer for providing bacterial strains and plasmids. We also thank Novartis for providing Col-0 (nahG) seed and the PR1 cDNA clone and the Arabidopsis Biological Resource Center (Ohio State University, Columbus) for expressed sequence tag clones. The authors are grateful to Drs. Frank White, Jan Leach, and Bing Yang for critical reading of the manuscript. This work was supported in part by a United States Department of Agriculture grant (No. 2001-35319-09847) to J.-M.Z. This is Kansas Agricultural Experimental Station contribution number 01-74-5.

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