A family of conserved bacterial effectors inhibits salicylic acid-mediated basal immunity and promotes disease necrosis in plants

June 21, 2004
101 (26) 9927-9932

Abstract

Salicylic acid (SA)-mediated host immunity plays a central role in combating microbial pathogens in plants. Inactivation of SA-mediated immunity, therefore, would be a critical step in the evolution of a successful plant pathogen. It is known that mutations in conserved effector loci (CEL) in the plant pathogens Pseudomonas syringae (the ΔCEL mutation), Erwinia amylovora (the dspA/E mutation), and Pantoea stewartii subsp. stewartii (the wtsE mutation) exert particularly strong negative effects on bacterial virulence in their host plants by unknown mechanisms. We found that the loss of virulence in ΔCEL and dspA/E mutants was linked to their inability to suppress cell wall-based defenses and to cause normal disease necrosis in Arabidopsis and apple host plants. The ΔCEL mutant activated SA-dependent callose deposition in wild-type Arabidopsis but failed to elicit high levels of callose-associated defense in Arabidopsis plants blocked in SA accumulation or synthesis. This mutant also multiplied more aggressively in SA-deficient plants than in wild-type plants. The hopPtoM and avrE genes in the CEL of P. syringae were found to encode suppressors of this SA-dependent basal defense. The widespread conservation of the HopPtoM and AvrE families of effectors in various bacteria suggests that suppression of SA-dependent basal immunity and promotion of host cell death are important virulence strategies for bacterial infection of plants.
Plant immunity to pathogen infections is believed to have contributed to the remarkable success of land plants on earth. Accumulation of the signaling molecule salicylic acid (SA) is associated with many immune responses in plants, such as systemic acquired resistance, basal resistance, and even gene-for-gene resistance, playing a central role in plant defense to many microbial pathogens (1-4). SA-mediated immunity involves increased expression of a suite of responsive genes and subsequent, largely undefined, cellular changes that collectively inhibit pathogen multiplication. Despite the presence of powerful plant defense responses, numerous microbes have evolved the ability to colonize plants and often cause devastating diseases. Given the importance of SA-mediated plant immunity, successful pathogens are expected to have evolved virulence mechanisms to evade or suppress SA-mediated host immunity. To date, however, no known pathogen virulence mutants have been shown to be defective in evading or suppressing this important host defense.
Many Gram-negative plant and mammalian bacteria evolved an essential virulence system, the conserved type III secretion system (TTSS), to inject virulence effector proteins into the host cell, promoting disease development (5-8). TTSS genes are often clustered in a large pathogenicity island (PAI). Although direct evidence is lacking, acquisition of a TTSS PAI through horizontal gene transfer is believed to have played a particularly important role in the evolution of bacteria as aggressive pathogens of plants and animals.
Erwinia amylovora and Pseudomonas syringae are important necrosis-causing bacterial pathogens of plants. E. amylovora causes severe systemic infection in rosaceous plants, killing leaves, stems, and roots, whereas P. syringae infects a wide spectrum of plant species, most strains causing local necrotic lesions in infected tissues. Despite the differences, the two bacteria contain similar TTSS PAIs, including a partially overlapping conserved effector locus (CEL) (9, 10). The CEL of P. syringae pv. tomato (Pst) strain DC3000 contains at least four characterized effector genes [avrE, hopPtoM, hrpW, and hopPtoA1 (formerly CEL ORF5)] that are conserved among diverse P. syringae pathovars (11). Individual mutations in these effector genes do not significantly affect bacterial multiplication (12-15). Yet, deletion of these four effector genes as well as shcM (the chaperone gene for hopPtoM; ref. 12) and ORF2 (a putative chaperone gene for avrE) in the ΔCEL mutant results in drastically reduced virulence in tomato (11). The E. amylovora CEL contains dspA/E (an orthologue of avrE) and hrpW (9, 10). Mutations in the dspA/E gene abolish the pathogenicity of E. amylovora in apple (9, 10). Similarly, mutations in the dspE gene of Pantoea agglomerans pv. gypsophilae and the wtsE gene (homologous to avrE) of Pantoea stewartii subsp. stewartii result in great reductions of bacterial virulence in gypsophila and corn, respectively (16, 17). The molecular basis of the loss of virulence in these bacterial mutants is not understood.
In this study, we used a combination of cytological examination, characterization of host and bacterial mutants, and host gene expression analysis to study the functions of AvrE, HopPtoM, and DspA/E in Arabidopsis-Pst DC3000 and apple-E. amylovora interactions. We show that these conserved bacterial effectors play a dual role in the inhibition of SA-mediated basal immunity and promotion of disease necrosis.

Materials and Methods

Plant Growth and Bacteria Enumeration. Methods for plant growth and bacteria enumeration were described previously (18, 19). Unless specified, all experiments reported in this paper were performed at least three times with similar results.
Callose Staining. Callose staining was performed 7-9 h after bacterial inoculation as described previously, except that no dexamethasone was used (18, 20). Leaves were examined with a Zeiss Axiophot D-7082 Photomicroscope with an A3 fluorescence cube. The number of callose depositions was determined with imagepro plus software (Media Cybernetics, Silver Spring, MD). The values provided are the averages and standard deviations of more than four independent leaves for each treatment.
Bacterial Mutants and Recombinant Plasmids. The following wild-type (WT) and mutant bacterial strains were used: Pst DC3000 (WT; provided by Diane Cuppels, Southern Crop Protection and Food Research Centre, London ON, Canada), E. amylovora CFBP1430 (WT; ref. 10), Pst DC3000 ΔCEL mutant (11), Pst DC3000 hrpA- mutant (21), and E. amylovora dspA/E- mutant (10). Construction of pORF43, which carries hopPtoM-shcM in pUCP19, was described previously (12). pEF carries a 6,329-bp AflII(blunted)-KpnI fragment, which contains avrE-ORF2, in the BamHI(blunted)-KpnI sites of pDSK519-PavrPto. pDSK519-PavrPto is a pDSK519 (22) derivative containing the promoter region of avrPtoDC3000. The AvrPto promoter was amplified by using Pst DC3000 genomic DNA, primer A (5′-CTGCATGC-(SphI site)GGTCCAGGAAATGAATACCCAG-3′), and primer B (5-ATGGATCC(BamHI)CATATG(NdeI)TATACCCTCTTTAGTACAG-3′), and was cloned into the SphI-BamHI sites of pDSK519. In pEF, avrE expression was controlled by its native promoter, and ORF2 expression was controlled by the heterologous avrPto promoter. pW5 carries a 4,294-bp StuI(blunted)-BamHI fragment, which contains hrpW-hopPtoA1, in the PstI(blunted)-BamHI sites of pDSK519.
RNA Blot Analysis. Total RNA was extracted from leaves by using the RNAgents isolation kit and following the manufacturer's instructions (Promega). Total RNA was fractionated on formaldehyde gels, blotted onto nylon membranes, and hybridized with radioactively labeled probes as previously described (23).

Results

CEL-Encoded Effectors and DspA/E Are Required for Disease Necrosis in Compatible Arabidopsis-Pst DC3000 and Apple-E. amylovora Interactions. Recent studies suggested that several type III effectors promote disease by suppressing the hypersensitive response (HR), a rapid cell death response mainly associated with host-range restriction of pathogens (24-31). HR cell death occurs much faster than disease-associated cell death in the same plant. If DspA/E and CEL-encoded effectors functioned as HR suppressors, we would expect to observe a more rapid, HR-like cell death response in host leaves infiltrated with a high concentration (OD600 = 0.2) of the dspA/E or ΔCEL mutant compared with leaves infiltrated with the same concentration of the WT bacteria. However, neither the dspA/E mutant nor the Pst DC3000 CEL mutant elicited an accelerated cell death in leaves of apple or Arabidopsis, respectively. On the contrary, both mutants caused much-delayed cell death, as indicated by slower tissue necrosis, compared with their parental WT bacteria, even at this high bacterial concentration (Fig. 1). This result shows that CEL-encoded effectors and DspA/E are not involved in the suppression of an HR. On the contrary, DspA/E and CEL-encoded effectors are required for the development of host cell death that is associated with disease necrosis.
Fig. 1.
Responses of apple (cultivar Jonathan, Left) and Arabidopsis thaliana (ecotype Col-0 gl1, Right) to infiltration of high concentrations of bacteria suspensions (OD600 of 0.2). Apple leaves inoculated with WT E. amylovora (Ea) showed disease necrosis (brown color) 17-18 h after inoculation, whereas apple leaves inoculated with the dspA/E mutant did not show any necrosis in two experiments and a delayed, very spotty necrosis at 24 h in one experiment. Arabidopsis leaves inoculated with DC3000 collapsed (shrunken appearance in this picture) 22-24 h after inoculation, whereas Arabidopsis leaves inoculated with the ΔCEL mutant showed delayed disease necrosis at 28-30 h after inoculation. Pictures were taken at 24 h after inoculation.
Increased Multiplication of the ΔCEL Mutant in Arabidopsis Plants Affected in SA Accumulation or Synthesis. We next examined the growth of the ΔCEL mutant in several Arabidopsis genotypes that are defective in host-defense pathways. We found that the ΔCEL mutant consistently multiplied to a higher level and caused more pronounced chlorosis and necrosis in NahG plants than in WT Col-0 plants (Fig. 2 A and C). NahG plants cannot accumulate SA, owing to the degradation of SA by nahG-encoded SA hydroxylase (32). The ΔCEL mutant also multiplied more efficiently in the eds5 mutant, which is defective in the synthesis of SA upon pathogen infection (33), but the increase was modest compared with that in NahG plants (Fig. 2B). Recent studies suggest that eds5 and NahG plants may be affected in overlapping but distinct pools of SA (34) and that catechol, the product of NahG-mediated degradation of SA, may partially contribute to the loss of nonhost plant resistance (35). These observations may explain the more significant increase of ΔCEL mutant growth in NahG plants. In contrast, no significant increase in ΔCEL mutant growth was detected in ein2 plants, which are defective in ethylene-mediated host defenses (36), compared with that in Col-0 plants (data not shown). This result suggests that, unlike the SA pathway, the ethylene-response pathway is not involved in CEL effector-mediated suppression of host defenses.
Fig. 2.
Enhanced growth of the ΔCEL mutant in SA-impaired plants. (A) DC3000 growth in Col-0 (black bars) and NahG (light gray bars) plants. ΔCEL mutant growth in Col-0 (dark gray bars) and NahG (white bars) plants. cfu, Colony-forming units. (B) DC3000 growth in WT (black bars) and eds5 (light gray bars) plants. ΔCEL mutant growth in WT (dark gray bars) and eds5 (white bars) plants. (C) Disease symptoms of Col-0 and NahG leaves infected by DC3000 or the ΔCEL mutant (OD600 = 0.002). Arrows indicate inoculated leaves.
ΔCEL and dspA/E Mutants Activate Host Cell Wall-Based Defense. Even though Arabidopsis leaves inoculated with the ΔCEL mutant bore few macroscopic disease symptoms, we found, by cytological examination, that these leaves had a much higher level (≈10-fold) of defense-associated callose deposition in the host cell wall than did leaves inoculated with Pst DC3000 (Fig. 3A). Similarly, we found that apple leaves inoculated with the dspA/E mutant had a much higher level (≈10-fold) of callose deposition in the host cell wall than did those inoculated with the WT bacteria (Fig. 3B). Thus, mutations in effector genes within the CELs of two different pathogens, Pst DC3000 and E. amylovora, result in greatly reduced virulence. This reduction in virulence correlated with a severe impairment in their ability to suppress host cell wall-based defense and to promote disease necrosis.
Fig. 3.
Callose deposition in Arabidopsis and apple leaves. (A) Portions of Arabidopsis Col-0 gl1 leaves stained for callose deposits (white dots in these images) after inoculation with WT DC3000 or the ΔCEL mutant. (B) Portions of WT apple leaves stained to show callose deposition after inoculation with WT Erwinia amylovora (Ea) or the dspA/E mutant. Below leaf images, average numbers of callose deposits per field of view (0.9 mm2) are displayed with standard deviations as errors. (Scale bar, 100 μm.)
The ΔCEL Mutant Is Unable to Suppress SA-Dependent Basal Immunity. A previous study showed that the Pst DC3000 hrcC mutant, which cannot secrete any of the >40 effectors, activated cell wall-based callose deposition in Arabidopsis (18). Furthermore, this cell wall defense was effectively suppressed by transgenic expression of the DC3000 effector AvrPto. Global Arabidopsis gene expression analysis suggested that the hrcC mutant-induced and AvrPto-suppressed cell wall defense was SA-independent (18).
To characterize the nature of cell wall-based basal immunity elicited by the ΔCEL mutant, we examined the SA-dependence of cell wall defenses activated by hrp/hrc and ΔCEL mutants in Arabidopsis plants. We found that the hrpA mutant elicited very high levels of callose deposition in Col-0, NahG, and eds5 leaves (Fig. 4 A and D), confirming the SA-independent nature of cell wall-based immunity elicited by hrp/hrc mutants. In contrast, the ΔCEL mutant elicited strong callose deposition only in Col-0 leaves, not in NahG (Fig. 4B) or eds5 mutant (Fig. 4E) plants. It is interesting to note that in Col-0 leaves, the ΔCEL mutant elicited consistently less callose deposition than the hrpA mutant (Fig. 4 G and H), suggesting that AvrPto and other effectors in the ΔCEL mutant may still be partially repressing callose-associated defense. As a control, WT Pst DC3000 suppressed callose deposition in the host cell wall irrespective of host genotypes, giving similarly low levels of callose deposition in leaves of Col-0, NahG, and eds5 (Fig. 4 C, F, G, and H). Taken together, these results clearly demonstrate that the ΔCEL mutant is not able to suppress SA-mediated cell wall defense, which is distinct from hrp/hrc mutant-activated and AvrPto-suppressed SA-independent cell wall defense.
Fig. 4.
Callose deposition in Arabidopsis leaves. (A-C) Portions of Arabidopsis Col-0 and NahG leaves stained to show callose deposition after inoculation with the hrpA mutant (A), the ΔCEL mutant (B), and WT DC3000 (C). (D-F) Portions of Arabidopsis Col-0 and eds5 leaves stained for callose after inoculation with the hrpA mutant (D), the ΔCEL mutant (E), and WT DC3000 (F). (G and H) Average numbers of callose depositions per field of view (0.9 mm2) with standard deviations displayed as errors. (Scale bar, 100 μm.)
CEL-Encoded Effectors Suppress SA-Dependent Basal Immunity Without Affecting the Expression of Known SA-Responsive Defense Genes. Genome-wide microarray analysis was conducted to further characterize SA-dependent activation and inactivation of cell wall-based immunity in Arabidopsis by WT Pst DC3000 and the ΔCEL mutant (see Supporting Materials and Methods, which is published as supporting information on the PNAS web site). We used the 22,500-element ATH1 Arabidopsis GeneChip (Affymetrix, Santa Clara, CA), which contains nearly all of the Arabidopsis genes in the genome. Remarkably, the strong activation and repression of SA-dependent cell wall immunity by the ΔCEL mutant and Pst DC3000, respectively, were not accompanied by strong activation and repression of well-known SA-responsive genes, such as PR (pathogenesis-related) genes. None of the known SA-responsive genes was expressed at a significantly higher level in ΔCEL mutant-inoculated leaves than in Pst DC3000-inoculated leaves (Fig. 8, which is published as supporting information on the PNAS web site). We also conducted Northern blot analysis of leaves inoculated with 108 cfu/ml bacteria (100 times higher than concentrations used for the microarray experiments to minimize the bacterial population differences). Again, no significant difference in PR-1 or PAD4 gene expression was observed (Fig. 5). Thus, CEL effector-mediated inactivation of the SA-dependent cell wall-based immunity does not involve changes of classical SA-responsive gene expression. However, because the entire SA regulon in Arabidopsis has not been defined yet, we cannot exclude the possibility that CEL-encoded effectors modulate expression of some yetto-be-identified SA-regulated genes.
Fig. 5.
RNA gel blot analysis of PR-1 and PAD4 expression 3, 6, and 9 h after infiltration of 1 × 108 cfu/ml ΔCEL mutant (ΔCEL) and DC3000 (WT). Below each autoradiogram is a picture of the ethidium bromide-stained 28S ribosomal RNA.
HopPtoM and AvrE Are Suppressors of SA-Dependent Basal Immunity. In contrast to the single effector mutation in the E. amylovora dspA/E mutant (10), the Pst DC3000 ΔCEL mutant lacks four known effector genes, avrE, hopPtoM, hrpW, and hopPtoA1 (11). To identify the specific effector(s) involved in the suppression of SA-dependent cell wall defense in Arabidopsis, we carried out subclone-based complementation experiments. Two independent subclones, pEF, which contains avrE-ORF2, and pORF43, which contains hopPtoM-shcM, partially or completely restored the ability of the ΔCEL mutant to multiply in WT Col-0 gl1 plants (Fig. 6 A and B). pORF43 also fully restored the ability of the ΔCEL mutant to cause disease necrosis and chlorosis. In contrast, a subclone containing the hrpW and hopPtoA1 genes neither increased the multiplication nor restored the ability to cause disease symptoms of the ΔCEL mutant in Col-0 gl1 plants (data not shown).
Fig. 6.
Complementation of the ΔCEL mutant. (A) Bacterial populations in Col-0 gl1 plants inoculated with DC3000 (black bars), the ΔCEL mutant (white bars), and the ΔCEL mutant complemented with pORF43 (light gray bars). (B) Bacterial populations in young leaves of Col-0 gl1 plants inoculated with DC3000 (black bars), the ΔCEL mutant (white bars), and the ΔCEL mutant complemented with pEF (light gray bars). (C) Callose deposits in portions of Arabidopsis Col-0 gl1 leaves inoculated with DC3000, the ΔCEL mutant, and the ΔCEL mutant containing pORF43. (D) Average numbers of callose deposits per field of view with standard deviations displayed as errors. (E) Callose deposits in portions of Arabidopsis Col-0 gl1 leaves inoculated with DC3000, the ΔCEL mutant, and the ΔCEL mutant containing pEF. (F) Average numbers of callose deposits per field of view with standard deviations displayed as errors.
pEF and pORF43 also partially or completely complemented the ΔCEL mutant for suppression of callose-associated host cell wall defense (Fig. 6 C-F). In the chromosome, avrE and ORF2 are encoded by two different, but adjacent, operons with opposite transcriptional directions (11). We had to use the heterologous avrPto promoter to drive the expression of ORF2 from one direction and the native avrE promoter to drive the expression of avrE from the other direction in pEF (see Materials and Methods). The incomplete complementation of the CEL mutation by the avrE-ORF2 subclone may therefore be attributed to the use of the heterologous avrPto promoter. In contrast, the hopPto-shcM genes are encoded by the same operon and we used their native promoter to drive the expression of both genes.

Discussion

Type III effectors secreted through the type III secretion system are now considered to be the primary virulence factors in many plant pathogenic bacteria. Genomic analysis shows that P. syringae carries a remarkable repertoire of type III effectors, with the current estimate of at least 40 effectors in strain DC3000 alone (37, 38). However, mutations in most effector genes do not show a large effect on bacterial virulence, presumably because of the functional subtlety and redundancy of most effectors when delivered by bacteria. In contrast to most effectors, mutations in the HopPtoM/AvrE families of effector genes often give a drastic virulence effect (9-11, 16, 17, 39), strongly suggesting that the functions of these particular effectors are not only important but also distinct from the majority of other effectors produced by the same bacterium. The strong virulence loss of the bacterial mutants defective in these effectors also provides a rare opportunity for studies using appropriate bacterial and host mutants in the context of native host-bacteria interactions. We show here that the severe virulence defect of the Pst DC3000 ΔCEL mutant is caused by the inability of this mutant to promote disease necrosis and to suppress the SA-dependent host cell wall defense in Arabidopsis. We show that the dspA/E mutant of E. amylovora, like the ΔCEL mutant of Pst DC3000, activates a strong cell wall-based response in host leaves, whereas the WT bacteria effectively suppress this defense. Even though we could not determine whether DspA/E-suppressed cell wall defense in apple leaves is also dependent on SA because of the inherent experimental limitations in the apple-E. amylovora system, the previously demonstrated functional cross-complementation of DspA/E and AvrE (9) strongly suggests that DspA/E also targets SA-dependent cell wall defense in apple.
We found that both avrE-ORF2 and hopPtoM-shcM can partially or completely complement the growth defect of the ΔCEL mutant in Arabidopsis. This result suggests that these two effector-chaperone pairs (AvrE-ORF2 and HopPtoM-ShcM), which do not share any sequence similarities, are at least partially functionally redundant in the Arabidopsis-Pst DC3000 interaction. This functional redundancy in Pst DC3000 may explain the previously observed lack of a strong bacterial growth phenotype in tomato plants of either the avrE mutant or the hopPtoM mutant alone (12, 13), although there may also be tomato- or Arabidopsis-specific reactions. For example, even though the hopPtoM mutant is not affected in bacterial multiplication in tomato, it is reduced in causing disease symptoms in this host (12). On the other hand, the drastic effect of the dspA/E mutation in E. amylovora can now be explained by the apparent lack of a functionally redundant effector, hopPtoM, within the CEL of E. amylovora.
A very recent study suggests that AvrRpt2 from Pst strain JL1065, which presumably also carries a CEL similar to that of Pst DC3000, might function downstream or independently of SA to promote virulence in the Arabidopsis-Pst DC3000 interaction (40). We therefore tested the ability of AvrRpt2 to complement the Pst DC3000 ΔCEL mutant in rps2- Arabidopsis plants (41). However, AvrRpt2 did not increase the ability of the ΔCEL mutant to multiply or to cause disease symptoms in rps2- Arabidopsis plants (data not shown), suggesting that the host target(s) of AvrRpt2 is different from those of HopPtoM and AvrE. Interestingly, the general virulence function of AvrRpt2 appears to be independent of the ability of AvrRtp2 to suppress the RPM1-dependent HR in Arabidopsis and may involve modulation of the auxin-response pathway (40).
Our demonstration of the HopPtoM/AvrE-mediated suppression of the SA-dependent basal immunity and promotion of disease necrosis likely has broad implications for our understanding of bacterial pathogenesis in diverse plants. This is because effectors of the HopPtoM/AvrE family appear to be among the most widespread in plant-pathogenic bacteria. For example, avrE-homologous genes are found in several plant pathogenic bacteria (Table 1, which is published as supporting information on the PNAS web site), whereas hopPtoM is apparently found in all examined P. syringae pathovars (11). The widespread presence of the HopPtoM and AvrE families of effectors in many pathogens therefore suggests a common need of plant-pathogenic bacteria to target conserved host pathways. Results presented here suggest that SA-dependent basal immunity and disease necrosis are two conserved host pathways targeted by potentially all members of the HopPtoM and AvrE families in different bacteria.
The requirement of the HopPtoM and AvrE families of effectors for promoting disease necrosis during pathogenesis (Fig. 1) is worthy of further discussion. Two previous studies show that inhibiting host cell death negatively affects the pathogenesis of Pseudomonas syringae pv. maculicola ES4326 and Pst T1 in Arabidopsis and tomato, respectively, suggesting that P. syringae might be partly a necrotrophic pathogen (42, 43). As a necrotrophic pathogen, Pst DC3000 would benefit from nutrient release associated with host cell death. It is therefore possible that the high multiplication ability of WT CEL+ bacteria in plants results from the additive activities of CEL-encoded effectors in suppressing host defense and promoting nutrient leakage associated with host cell death. Such a dual virulence function could result from these effectors having multiple targets in the host or a single host target that is involved in both host cellular immunity and host cell survival.
To date, the most extensively characterized aspect of SA-mediated immunity is probably the expression of SA-responsive genes. However, yet to be elucidated are the entire SA-controlled defense network (including basal and inducible) and, more importantly, the specific host cellular modifications during SA-mediated immunity that ultimately inhibit bacterial pathogens. Study of HopPtoM/AvrE/DspA/E-mediated virulence functions may provide critical information about those particular SA-dependent host cellular changes that are actually responsible for the elusive bacterial resistance mechanism in plants. We show that CEL-mediated suppression of the SA-dependent basal immunity is not associated with a significant down-regulation of the expression of classical PR genes. Thus, HopPtoM and AvrE may act on SA-dependent basal immunity at a cellular process either downstream of or parallel to the expression of these known PR genes.
Results from our previous work on AvrPto (18) and the current work on AvrE and HopPtoM in the Arabidopsis-Pst DC3000 pathosystem illustrate that plant cell wall-based basal immunity is a key battlefield that has been subjected to repeated activation and inactivation by bacterial pathogens via SA-independent and SA-dependent plant-signaling pathways (Fig. 7). hrp mutants activate cell wall-based basal defense in an SA-independent manner, likely via the MAPK3/6 pathway, through perception of flagellin and other pathogen-associated molecular patterns (44). In the ΔCEL mutant, the AvrPto class of effectors inactivates the SA-independent basal defense. However, the host cell partially overcomes the AvrPto-mediated inactivation of SA-independent basal defense and activates an SA-dependent basal defense pathway. The bacterial factors involved in this SA-dependent activation are not known, but they may be type III effectors or the type III secretion process per se. In DC3000, HopPtoM and AvrE inactivate the SA-dependent basal defense and promote disease-associated host cell death.
Fig. 7.
A hypothetical model for the activation and inactivation of cell wall-based basal immunity during Pst DC3000 infection of susceptible Arabidopsis. MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; MAPKKK, MAPKK kinase.
In conclusion, we have identified a key group of conserved type III effectors in plant-pathogenic bacteria that target SA-dependent basal immunity and promote disease necrosis in plants. This finding provides an attractive explanation for the strict requirement of the HopPtoM and AvrE families of effectors for bacterial virulence in multiple pathosystems. Our results suggest that a synergistic combination of suppression of SA-dependent basal immunity and promotion of disease necrosis may be key to the aggressive colonization of plants by P. syringae, E. amylovora, and perhaps other necrosis-causing plant-pathogenic bacteria.

Supplementary Material

Supporting Information
Media (pnas_101_26_9927__.html) is missing or otherwise invalid.Media (pnas_101_26_9927__1.pdf) is missing or otherwise invalid.

Notes

This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: SA, salicylic acid; CEL, conserved effector locus; Pst, Pseudomonas syringae pv. tomato; HR, hypersensitive response; cfu, colony-forming units.

Acknowledgments

We thank Diane Cuppels, Marie-Anne Barny, and Alan Collmer for bacterial strains and John Ryals for nahG transgenic plants. Arabidopsis eds5 seeds were obtained from the Ohio State University Arabidopsis Stock Center. We thank the Michigan State University Genomic Technology Support Facility for assistance in microarray experiments, members of our laboratory for critical reading of the manuscript, and K. Bird for assistance in preparation of this paper. This study was supported by grants from the U.S. Department of Energy (to S.Y.H.), the U.S. National Science Foundation (to S.Y.H.), the U.S. Department of Agriculture (to R.T.), and a Korean government short-term overseas fellowship (to Y.-B.K.).

Supporting Information

Adobe PDF - 01601Fig8.pdf
Adobe PDF - 01601Fig8.pdf

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Published in

Go to Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences
Vol. 101 | No. 26
June 29, 2004
PubMed: 15210989

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Submission history

Received: March 6, 2004
Published online: June 21, 2004
Published in issue: June 29, 2004

Acknowledgments

We thank Diane Cuppels, Marie-Anne Barny, and Alan Collmer for bacterial strains and John Ryals for nahG transgenic plants. Arabidopsis eds5 seeds were obtained from the Ohio State University Arabidopsis Stock Center. We thank the Michigan State University Genomic Technology Support Facility for assistance in microarray experiments, members of our laboratory for critical reading of the manuscript, and K. Bird for assistance in preparation of this paper. This study was supported by grants from the U.S. Department of Energy (to S.Y.H.), the U.S. National Science Foundation (to S.Y.H.), the U.S. Department of Agriculture (to R.T.), and a Korean government short-term overseas fellowship (to Y.-B.K.).

Authors

Affiliations

Sruti DebRoy
Cell and Molecular Biology Program and Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI 48824
Roger Thilmony
Cell and Molecular Biology Program and Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI 48824
Yong-Bum Kwack
Cell and Molecular Biology Program and Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI 48824
Kinya Nomura
Cell and Molecular Biology Program and Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI 48824
Sheng Yang He
Cell and Molecular Biology Program and Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI 48824

Notes

To whom correspondence should be addressed. E-mail: [email protected].
Edited by Steven E. Lindow, University of California, Berkeley, CA, and approved May 12, 2004

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