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. 2010 Apr 1;6(4):e1000894.
doi: 10.1371/journal.pgen.1000894.

Allele-specific virulence attenuation of the Pseudomonas syringae HopZ1a type III effector via the Arabidopsis ZAR1 resistance protein

Jennifer D Lewis  1 , Ronald Wu, David S Guttman, Darrell Desveaux
Affiliations

Allele-specific virulence attenuation of the Pseudomonas syringae HopZ1a type III effector via the Arabidopsis ZAR1 resistance protein

Jennifer D Lewis et al. PLoS Genet. .

Abstract

Plant resistance (R) proteins provide a robust surveillance system to defend against potential pathogens. Despite their importance in plant innate immunity, relatively few of the approximately 170 R proteins in Arabidopsis have well-characterized resistance specificity. In order to identify the R protein responsible for recognition of the Pseudomonas syringae type III secreted effector (T3SE) HopZ1a, we assembled an Arabidopsis R gene T-DNA Insertion Collection (ARTIC) from publicly available Arabidopsis thaliana insertion lines and screened it for plants lacking HopZ1a-induced immunity. This reverse genetic screen revealed that the Arabidopsis R protein HOPZ-activated resistance 1 (ZAR1; At3g50950) is required for recognition of HopZ1a in Arabidopsis. ZAR1 belongs to the coiled-coil (CC) class of nucleotide binding site and leucine-rich repeat (NBS-LRR) containing R proteins; however, the ZAR1 CC domain phylogenetically clusters in a clade distinct from other related Arabidopsis R proteins. ZAR1-mediated immunity is independent of several genes required by other R protein signaling pathways, including NDR1 and RAR1, suggesting that ZAR1 possesses distinct signaling requirements. The closely-related T3SE protein, HopZ1b, is still recognized by zar1 Arabidopsis plants indicating that Arabidopsis has evolved at least two independent R proteins to recognize the HopZ T3SE family. Also, in Arabidopsis zar1 plants HopZ1a promotes P. syringae growth indicative of an ancestral virulence function for this T3SE prior to the evolution of recognition by the host resistance protein ZAR1. Our results demonstrate that the Arabidopsis resistance protein ZAR1 confers allele-specific recognition and virulence attenuation of the Pseudomonas syringae T3SE protein HopZ1a.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. HopZ1a recognition is independent of known signaling components of R gene- mediated immunity.
(A) Half-leaves of Arabidopsis Col-0, Ws-0 or mutant plants were infiltrated with 10 mM MgCl2 or with PtoDC3000 expressing the empty vector (Ev), AvrRpt2, or HopZ1a or HopZ1aC216A (C/A) with a C-terminal HA tag under its endogenous promoter. C216 of HopZ1a is part of the predicted catalytic triad and the mutant protein is expressed at a similar level to HopZ1a . The bacteria were syringe infiltrated into the leaves at 5×107 cfu/mL. Photos were taken 22 hours post-infiltration. The number of leaves showing an HR is indicated below the appropriate construct. HRs are marked with an asterisk. Patchy HRs are marked with a double asterisk. Scale bar is 1 cm. (B–I) PtoDC3000 expressing the indicated construct was syringe infiltrated at 1×105 cfu/mL into Arabidopsis Col-0 or mutant leaves and bacterial counts were determined one hour post-infection (Day 0) and 3 days post-infection (Day 3). Two-tailed homoschedastic t-tests were performed to test for significant differences. Within a plant genotype, treatments were compared to empty vector and significant differences are indicated by an asterisk (* P<0.01). To compare between plant genotypes, growth of PtoDC3000 carrying HopZ1a, AvrRpt2 or AvrRps4 was normalized to the average growth of PtoDC3000(Ev). Significant differences in growth of a P. syringae strain between a mutant genotype and wild type Col-0 or Ws are indicated by a triangle (▴ P<0.01). Error bars indicate the standard deviation from the mean of 10 samples. Growth assays were performed at least 3 times. Arabidopsis genotypes are: (B) sgt1a (C) sgt1b (D) ndr1rar1 (E) eds1 (F) pad4 (G) rbohd/f (H) nahG (I) eds16.
Figure 2
Figure 2. ZAR1 recognizes HopZ1a in Arabidopsis.
(A) Half-leaves of Arabidopsis Col-0 or zar1-1 plants were infiltrated with 10 mM MgCl2 or with PtoDC3000 expressing the empty vector (Ev), AvrRpt2, or HopZ1a or HopZ1aC216A (C/A) with a C-terminal HA tag under its endogenous promoter. C216 of HopZ1a is part of the predicted catalytic triad and the mutant protein is expressed at a similar level to HopZ1a . The bacteria were syringe infiltrated into the leaves at 5×107 cfu/mL. Photos were taken 22 hours post-infiltration. The number of leaves showing an HR is indicated below the appropriate construct. HRs are marked with an asterisk. Scale bar is 1 cm. (B) At3g50950 is ZAR1. The promoter is shown by grey boxes and the exon by a large black box. There is an intron in the promoter, shown by a black line. The position of the T-DNA insertion lines is shown below the locus. (C) Half-leaves of Arabidopsis Col-0, zar1-2, zar1-3, zar1-4, or zar1-5 plants were infiltrated with 10 mM MgCl2 or with PtoDC3000 expressing the empty vector (Ev), AvrRpt2, or HopZ1a or HopZ1aC216A (C/A) with a C-terminal HA tag under its endogenous promoter.
Figure 3
Figure 3. zar1-1 Arabidopsis plants do not display immunity against HopZ1a.
(A) Trypan blue staining of PtoDC3000-infiltrated Arabidopsis Col-0 or zar1-1 leaves. The bacteria were syringe infiltrated into the leaves at 5×107 cfu/mL. Scale bar is 1 cm. C/A indicates the C216A mutation of HopZ1a in the predicted catalytic triad. The mutant protein is expressed at a similar level to HopZ1a . (B) Electrolyte leakage of Arabidopsis Col-0 or zar1-1 leaf discs after infiltration with PtoDC3000 expressing the indicated constructs. The bacteria were syringe infiltrated into the leaves at 2×107 cfu/mL. Error bars indicate the standard deviation from the mean of 6 samples. C/A indicates the C216A mutation. Two-tailed homoschedastic t-tests were performed to test for significant differences. Within a plant genotype, treatments were compared to empty vector and significant differences are indicated by an asterisk (* P<0.01). To compare between plant genotypes, ion leakage from PtoDC3000 carrying HopZ1a or AvrRpt2 was normalized to the average ion leakage of PtoDC3000(Ev) in the same genotype. Significant growth differences between zar1-1 and wild-type Col-0 are indicated by a triangle (▴ P<0.01). (C) PtoDC3000 expressing the indicated construct was syringe infiltrated at 1×105 cfu/mL into Arabidopsis Col-0 or zar1-1 leaves and bacterial counts were determined one hour post-infection (Day 0) and 3 days post-infection (Day 3). Two-tailed homoschedastic t-tests were performed to test for significant differences. Within a plant genotype, treatments were compared to empty vector and significant differences are indicated by an asterisk (* P<0.01). To compare between plant genotypes, growth of PtoDC3000 carrying HopZ1a or AvrRpt2 was normalized to the average growth of PtoDC3000(Ev). Significant growth differences between zar1-1 and wild-type Col-0 are indicated by a triangle (▴ P<0.01). Error bars indicate the standard deviation from the mean of 10 samples. Growth assays were performed at least 3 times.
Figure 4
Figure 4. HopZ1a has a virulence function in zar1-1 Arabidopsis plants.
Pci0788-9 expressing the indicated construct was syringe infiltrated at 1×105 cfu/mL into Arabidopsis Col-0 or zar1-1 leaves and bacterial counts were determined one hour post-infection (Day 0) and 3 days post-infection (Day 3). C/A indicates the C216A mutation of HopZ1a in the predicted catalytic triad and the mutant protein is expressed at a similar level to HopZ1a . Two-tailed homoschedastic t-tests were performed to test for significant differences. Within a plant genotype, treatments were compared to empty vector and significant differences are indicated by an asterisk (* P<0.01). To compare between plant genotypes, growth of Pci0788-9 carrying HopZ1a, or HopZ1aC216A (HopZ1aC/A) was normalized to the average growth of Pci0788-9(Ev). Significant differences between zar1-1 and Col-0 are indicated by a triangle (▴ P<0.01). Error bars indicate the standard deviation from the mean of 10 samples. Growth assays were performed at least 3 times.
Figure 5
Figure 5. Evolutionary relationships of 95 ZAR1 coiled-coil domain homologs.
The evolutionary relationships of the homologous amino acid sequences were inferred using Neighbor-Joining, with the robustness of the tree assessed via bootstrapping (500 replicates, with bootstrap values greater than 60% shown above the appropriate nodes). The tree is drawn to scale, with branch lengths scaled to evolutionary distances (scale shown at the bottom of the tree). All Arabidopsis ZAR1 coiled-coil domain homologs are shown in reverse type, while the ZAR1 sequence is found at the top of the tree. The data were parsed to remove redundant sequences as described in the Materials and Methods. “put” indicates a putative R protein while “hyp” is hypothetical. The major structure of this tree (e.g. clustering of ZAR1 and other Arabidopsis homologs) is identical to that observed in trees produced by maximum likelihood and maximum parsimony analysis (data not shown).
Figure 6
Figure 6. ZAR1 does not recognize HopZ1b.
(A) Transgenic homozygous HopZ1b or HopZ1bC/A plants were sprayed with 30 µM dexamethasone or water. C/A indicates the C212A mutation of HopZ1b in the predicted catalytic triad. Photos were taken 24–72 hours post-spraying. The number of plants showing a macroscopic HR is indicated in each box. Scale bar is 1 cm. (B) Immunoblot analysis of HopZ1b or HopZ1bC/A protein expressed in transgenic lines after treatment with 30 µM dexamethasone or water. C/A indicates the C212A mutation of HopZ1b in the predicted catalytic triad. The Ponceau Red stained blot serves as the loading control. The predicted size of HopZ1b-HA is 42.4 kDa. (C) Half-leaves of Arabidopsis Col-0 or zar1-1 plants were infiltrated with 10 mM MgCl2 or with PtoDC3000 expressing the empty vector (Ev), HopZ1a or HopZ1b with a C-terminal HA tag under its endogenous promoter. The bacteria were syringe infiltrated into the leaves at 5×107 cfu/mL. Photos were taken 24 hours post-infiltration. The number of leaves showing an HR is indicated below the appropriate construct. HRs are marked with an asterisk. Scale bar is 1 cm.
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