Dual regulation of gene expression mediated by extended MAPK activation and salicylic acid contributes to robust innate immunity in Arabidopsis thaliana

Abstract

Network robustness is a crucial property of the plant immune signaling network because pathogens are under a strong selection pressure to perturb plant network components to dampen plant immune responses. Nevertheless, modulation of network robustness is an area of network biology that has rarely been explored. While two modes of plant immunity, Effector-Triggered Immunity (ETI) and Pattern-Triggered Immunity (PTI), extensively share signaling machinery, the network output is much more robust against perturbations during ETI than PTI, suggesting modulation of network robustness. Here, we report a molecular mechanism underlying the modulation of the network robustness in Arabidopsis thaliana. The salicylic acid (SA) signaling sector regulates a major portion of the plant immune response and is important in immunity against biotrophic and hemibiotrophic pathogens. In Arabidopsis, SA signaling was required for the proper regulation of the vast majority of SA-responsive genes during PTI. However, during ETI, regulation of most SA-responsive genes, including the canonical SA marker gene PR1, could be controlled by SA-independent mechanisms as well as by SA. The activation of the two immune-related MAPKs, MPK3 and MPK6, persisted for several hours during ETI but less than one hour during PTI. Sustained MAPK activation was sufficient to confer SA-independent regulation of most SA-responsive genes. Furthermore, the MPK3 and SA signaling sectors were compensatory to each other for inhibition of bacterial growth as well as for PR1 expression during ETI. These results indicate that the duration of the MAPK activation is a critical determinant for modulation of robustness of the immune signaling network. Our findings with the plant immune signaling network imply that the robustness level of a biological network can be modulated by the activities of network components.

Figure 1. SA-independent regulation of PR1 during ETI.

(A) The PR1 expression level in leaves at 6 or 24 hpi with Pto strains (OD₆₀₀ = 0.001) or mock was determined by qRT-PCR. Bars represent means and standard errors of two biological replicates calculated using a mixed linear model. The vertical axis shows the log₂ expression level relative to Actin2 (At2g18780). (B) The free SA levels in leaf samples corresponding to those in (A) were determined. Bars represent means and standard errors of two biological replicates calculated using a mixed linear model. The SA level is shown on a log₁₀ scale. Asterisks indicate significant differences from mock (P<0.01, two-tailed t-tests).

Figure 2. Sustained MAPK activation supports transcriptional regulation of a majority of SA-responsive genes without SA.

(A). A heatmap of the SA-responsive genes. Leaves were collected at 24 hpi with the indicated Pto strains (OD₆₀₀ = 0.001) or mock. Independently, leaves of DEX-MKK4DD plants were collected at 24 hpi with 2 µM DEX or mock and subjected to mRNA profiling analysis using a whole genome DNA microarray. SA-responsive genes were selected for reproducible SID2-dependent responsiveness to the Pto strains as described in Experimental Procedures. The log₂ ratios compared to mock for 187 SA-responsive genes were subjected to agglomerative hierarchical clustering analysis. The log₂ ratio of DEX/mock for the DEX-MKK4DD sid2 plant (MKK4DD/sid2) samples was weighted by a factor of 0.5 to reduce its effects on the clustering pattern. The log₂ ratios used were averaged from three independent experiments. Green indicates negative values, red indicates positive values and black indicates zero: see the color scale. The arrow indicates the position of PR1. Means and standard errors of (B) Cluster I, 85 genes; (C) Cluster II, 20 genes; (D) Cluster III, 25 genes are shown.

Figure 3. MAPK activation is sustained in ETI but transient in PTI.

(A) MAPK activation during flg22-PTI. Seedlings were treated with 10 nM flg22 for the indicated times in a liquid medium. For the 10 min* sample, fresh seedlings were treated with the flg22-containing liquid medium used for the 3 h sample, revealing that flg22 was not degraded in the 3 h sample. (B,C) MAPK activation during AvrRpt2-ETI. Seedlings of the transgenic lines that carry the estradiol-inducible AvrRpt2 transgene in wild-type (B) or rps2 (C) background (XVE-AvrRpt2/WT or rps2) were treated with 20 nM estradiol for the indicated times in a liquid medium. Activated MAPKs, MPK3 and MPK6 were detected by immunoblot using anti-p44/42 MAPK, anti-AtMPK3 and anti-AtMPK6 antibody, respectively. Ponceau S stained blots are shown for loading controls. Experiments were conducted three times with similar results. (D) The AvrRpt2 mRNA levels in seedlings treated with 20 nM estradiol for the indicated times were determined by qRT-PCR. Bars represent means and standard errors of two biological replicates calculated using a mixed linear model. The vertical axis shows the log₂ expression level relative to Actin2 (At2g18780). Asterisks indicate significant differences from the untreated controls (P<0.01, two-tailed t-tests).

Figure 4. MAPK activation is sustained in ETI but transient in non-ETI conditions.

Leaves of Col (A, B) and rpm1 rps2 (C) plants were infiltrated with Pto hrcC, Pto EV, Pto AvrRpt2 (OD₆₀₀ = 0.01) or water (mock) and samples were collected at the indicated time points. Activated MAPKs were detected by immunoblot using anti-p44/42 MAPK antibody. Ponceau S stained blots are shown for loading controls. Experiments were conducted three times, yielding similar results.

Figure 5. Sustained MAPK activation is sufficient for PR1 induction.

The PR1 (A) or FRK1 (B) expression levels in DEX-MKK4DD (MKK4DD) or -MKK5DD (MKK5DD) at the indicated times after treatment with 2 µM DEX were determined by qRT-PCR. Bars represent means and standard errors of three biological replicates calculated using a mixed linear model. The vertical axis shows the log₂ expression level relative to Actin2 (At2g18780). Asterisks indicate significant differences from untreated samples (0 h) (P<0.01, two-tailed t-tests).

Figure 6. SA signaling is not involved in PR1 induction by sustained MAPK activation.

(A) The free SA levels in leaves of wild-type (Col) or DEX-GUS (GUS), -MKK4DD (MKK4DD) or -MKK5DD (MKK5DD) plants 9 hours after treatment with 2 µM DEX (DEX) or mock. For the Col (flg22) sample, leaves of Col plants were infiltrated with 1 µM flg22 or mock, and the result is shown as a positive control for induced SA accumulation. Bars represent means and standard errors of four biological replicates calculated using a mixed linear model. The SA level is shown on a log₁₀ scale. (B) The PR1 expression levels in leaves of the plant lines carrying the DEX-GUS (GUS), -MKK4DD (MKK4DD) or -MKK5DD (MKK5DD) transgenes in wild-type (Col), sid2 or npr1 backgrounds 24 hours after treatment with 2 µM DEX (DEX) or mock were determined by qRT-PCR. Bars represent means and standard errors of two biological replicates calculated using a mixed linear model. The vertical axis shows the log₂ expression level relative to Actin2 (At2g18780). Asterisks indicate significant differences from mock (P<0.01, two-tailed t-tests).

Figure 7. Compensation between MPK3 and SA contributes to the robust ETI levels.

(A) The PR1 expression level in leaves of the indicated genotypes at 24 hpi with Pto AvrRpt2 (blue bars) or AvrRpm1 (red bars) (OD₆₀₀ = 0.001) was determined by qRT-PCR. Bars represent means and standard errors of three biological replicates calculated using a mixed linear model. The vertical axis shows the log₂ expression level relative to Actin2 (At2g18780). Statistically significant differences are indicated by different letters (P<0.01, two-tailed t-tests). (B) The signaling allocations for the PR1 expression level shown in (A) were estimated for MPK3 and SID2 (upper panel) or MPK6 and SID2 (lower panel). (C) The bacterial counts of Pto EV (left panel) or AvrRpt2 (right panel) (inoculation dose, OD₆₀₀ = 0.0001) at 0 or 2 dpi in leaves of the indicated genotypes were measured. Bars represent means and standard errors of three independent experiments with at least 4 or 12 biological replicates for 0 dpi or 2 dpi, respectively. Statistically significant differences are indicated by different letters per strain per dpi (P<0.01, two-tailed t-tests). (D) The signaling allocations for AvrRpt2-ETI shown in (C, 2 dpi) were estimated for MPK3 and SID2 (left panel) or MPK6 and SID2 (right panel). (B,D) Bars represent means and standard errors determined using a mixed linear model. Asterisks indicate significant effects or interaction (P<0.01).

Figure 8. A model of signaling activated by sustained MAPK activation or SA signaling that regulates the common genes during AvrRpt2-ETI, resulting in robust immunity.

During non-ETI, such as PTI, MAPK activation is transient. Transient MAPK activation is not sufficient for regulating the SA-responsive genes. However, during AvrRpt2-ETI, sustained MAPK activation can regulate the SA-responsive genes independently of SA. The differential duration of the MAPK activation can modulate the network property of robustness.