Elongator subunit 3 positively regulates plant immunity through its histone acetyltransferase and radical S-adenosylmethionine domains

Abstract

Background: Pathogen infection triggers a large-scale transcriptional reprogramming in plants, and the speed of this reprogramming affects the outcome of the infection. Our understanding of this process has significantly benefited from mutants that display either delayed or accelerated defense gene induction. In our previous work we demonstrated that the Arabidopsis Elongator complex subunit 2 (AtELP2) plays an important role in both basal immunity and effector-triggered immunity (ETI), and more recently showed that AtELP2 is involved in dynamic changes in histone acetylation and DNA methylation at several defense genes. However, the function of other Elongator subunits in plant immunity has not been characterized.

Results: In the same genetic screen used to identify Atelp2, we found another Elongator mutant, Atelp3-10, which mimics Atelp2 in that it exhibits a delay in defense gene induction following salicylic acid treatment or pathogen infection. Similarly to AtELP2, AtELP3 is required for basal immunity and ETI, but not for systemic acquired resistance (SAR). Furthermore, we demonstrate that both the histone acetyltransferase and radical S-adenosylmethionine domains of AtELP3 are essential for its function in plant immunity.

Conclusion: Our results indicate that the entire Elongator complex is involved in basal immunity and ETI, but not in SAR, and support that Elongator may play a role in facilitating the transcriptional induction of defense genes through alterations to their chromatin.

Figure 1

Characterization of the gns2 Mutant. (A) Seeds of wild type (WT), npr1, and gns2 npr1 were placed on half-strength MS agar medium containing 0.26 mM SA. After three days of stratification, the plate was transferred to a growth chamber and photographed ten days later. (B) Four-week-old soil-grown WT, npr1, and gns2 npr1 plants were inoculated with the virulent bacterial pathogen Psm ES4326 (OD₆₀₀ = 0.001). Twenty-four hours later, the inoculated leaves were collected for SA measurement. Data represent the mean of three independent samples with standard deviation (SD). Different letters above the bars indicate significant differences (p < 0.05, Student’s t-test). (C) Total SA levels in Psm ES4326-infected WT, npr1, and gns2 npr1 plants. The experiment was carried out as in (B). SAG, 2-O-β-D-glucosylsalicylic acid. (D) Leaves of four-week-old soil-grown WT, npr1, and gns2 npr1 plants were inoculated with Psm ES4326 (OD₆₀₀ = 0.0001). The in planta bacterial titers were determined immediately and three days postinoculation. Cfu, colony-forming units. Data represent the mean of eight independent samples with SD. Different letters above the bars indicate significant differences (p < 0.05, Student’s t-test). (E) Morphology of npr1 and gns2 npr1 plants. The plants were grown on soil for four weeks before the photos were taken. The gns2 npr1 plant is a lighter shade of green than the npr1 plant.

Figure 2

Map-Based Cloning of gns2. (A) Schematic representation of the mapping strategy for identifying the gns2 mutation. New molecular markers used in this study are presented in Additional file 1: Table S1. (B) A dCAPS marker distinguishing gns2 and WT. A deletion of a guanine (39 bp from ATG) was detected in the first exon of AtELP3, which allowed the development of a dCAPS marker to genotypically distinguish the mutant from the wild type (Additional file 1: Table S1). (C) Structure of the GNS2/ELO3/AtELP3 gene and the positions of the gns2 and elo3-1 mutations [39]. Boxes denote the translated regions and lines between boxes denote introns. (D) Ten-day-old seedlings of WT, npr1, Atelp3-10 npr1, and two complementation lines Com(n)^#1 and Com(n)^#2 grown on half-strength MS agar medium containing 0.26 mM SA. Com(n), 35S::AtELP3 Atelp3-10 npr1 transgenic plants. (E) Four-week-old soil-grown WT, npr1, Atelp3-10 npr1, and Com(n) plants. (F) Free SA levels in Psm ES4326-infected WT, npr1, Atelp3-10 npr1, and Com(n) plants. Plants were inoculated with Psm ES4326 (OD₆₀₀ = 0.001). Twenty-four hours later, the inoculated leaves were collected for SA measurement. Data represent the mean of four independent samples with SD. Different letters above the bars indicate significant differences (p < 0.05, Student’s t-test). (G) Total SA levels in Psm ES4326-infected plants. The experiment was carried out as in (F). (H) Growth of Psm ES4326 in WT, npr1, Atelp3-10 npr1, and Com(n) plants. Cfu, colony-forming units. Leaves of four-week-old plants were inoculated with Psm ES4326 (OD₆₀₀ = 0.0001). The in planta bacterial titers were determined immediately and three days postinoculation. Cfu, colony-forming units. Data represent the mean of eight independent samples with SD. Different letters above the bars indicate significant differences (p < 0.05, Student’s t-test).

Figure 3

Pathogen-Induced PR Gene Expression in Atelp3-10 npr1 and Atelp3-10 Plants. (A) Expression of PR genes in Psm ES4326-infected WT, npr1, Atelp3-10 npr1, Com(n), Atelp3-10, and Com(C) plants. Com(n), 35S::AtELP3 Atelp3-10 npr1 transgenic plants; Com(C), 35S::AtELP3 Atelp3-10 transgenic plants. HPI, hours postinoculation. Plants were inoculated with Psm ES4326 (OD₆₀₀ = 0.001). Leaf tissues were collected at the indicated time points after the inoculation. Total RNA was extracted and subjected to RNA gel blot analysis. The RNA samples were run on the same agarose gel and transferred onto the same blotting membrane. The rRNA bands in the ethidium bromide-stained gel were photographed as a loading control prior to blotting. (B) Growth of Psm ES4326 in WT, Atelp3-10, and Com(C) plants. Cfu, colony-forming units. Leaves of four-week-old plants were inoculated with Psm ES4326 (OD₆₀₀ = 0.0001). The in planta bacterial titers were determined immediately and three days postinoculation. Data represent the mean of eight independent samples with SD. Different letters above the bars indicate significant differences (p < 0.05, Student’s t-test).

Figure 4

SA-Induced PR Gene Expression and Resistance in Atelp3-10. (A) Expression of PR genes in SA-treated WT, Atelp3-10, and Com(C) plants. Com(C), 35S::AtELP3 Atelp3-10 transgenic plants. Plants were treated with soil drenches of 1 mM SA water solution or water. Leaf tissue was collected at the indicated time points after the treatment. Total RNA was extracted and subjected to RNA gel blot analysis. The rRNA bands in the ethidium bromide-stained gel were photographed as a loading control prior to blotting. (B) Growth of Psm ES4326 in SA-treated WT, Atelp3-10, and Com(C) plants. Plants were treated with soil drenches of 1 mM SA solution or water. Twenty-four hours later, the plants were inoculated with Psm ES4326 (OD₆₀₀ = 0.001). The in planta bacterial titers were determined immediately and three days postinoculation. Cfu, colony-forming units. Data represent the mean of eight independent samples with SD. Different letters above the bars indicate significant differences (p < 0.05, Student’s t-test).

Figure 5

Characterization of ETI in Atelp3-10 Plants. (A) Expression of nine ETI-inducible genes in Pst DC3000/avrRpt2-infected WT and Atelp3-10 plants. Plants were inoculated with Pst DC3000/avrRpt2 (OD₆₀₀ = 0.001). Leaf tissues were collected at the indicated time points. Total RNA was extracted and analyzed for the expression of indicated genes using real-time qPCR. The y-axes indicate relative expression levels monitored by qPCR. Expression was normalized against constitutively expressed UBQ5. The x-axes indicate hours after Pst DC3000/avrRpt2 infection. Data represent the mean of three independent samples with SD. An asterisk (*) indicates that the expression level of the gene in the WT was significantly different from that in Atelp3-10 (p < 0.05, Student’s t-test). (B) Growth of Pst DC3000/avrRpt2 in WT, npr1, Atelp3-10, Atelp3-10 npr1, and rps2 plants. Plants were inoculated with Pst DC3000/avrRpt2 (OD₆₀₀ = 0.0001). The in planta bacterial titers were determined immediately and three days postinoculation. Cfu, colony-forming units. Data represent the mean of eight independent samples with SD. Different letters above the bars indicate significant differences (p < 0.05, Student’s t-test).

Figure 6

SAR Induction in Atelp3-10 Plants. (A) Expression of six SAR-associated genes in systemic leaves of WT, npr1, Atelp3-10, and Com(C) plants. Com(C), 35S::AtELP3 Atelp3-10 transgenic plants. Three lower leaves on each plant were inoculated with Psm ES4326 (OD₆₀₀ = 0.002) (+SAR) or mock-treated with 10 mM MgCl₂ (−SAR). Two days later, total RNA was extracted from the upper uninfected/untreated leaves and analyzed for the expression of indicated genes by real-time qPCR. Expression was normalized against constitutively expressed UBQ5. Data represent the mean of three independent samples with SD. Different letters above the bars indicate significant differences (p < 0.05, Student’s t-test). Note that the comparison was made among WT, npr1, Atelp3-10, and Com(C) for each gene/treatment. (B) SAR induction in WT, npr1, Atelp3-10, and Com(C) plants. Three lower leaves on each plant were inoculated with Psm ES4326 (OD₆₀₀ = 0.002) (+SAR) or mock-treated with 10 mM MgCl₂ (−SAR). Two days later, two upper uninfected/untreated leaves were challenge-inoculated with Psm ES4326 (OD₆₀₀ = 0.001). The in planta bacterial titers were determined immediately and three days after challenge inoculation. Cfu, colony-forming units. Data represent the mean of ten independent samples with SD. Psm ES4326 grew significantly less in the SAR-induced WT, Atelp3-10, and Com(C) plants than in the mock-treated plants (*All p < 0.05, Student’s t-test).

Figure 7

Characterization of the HAT and Radical SAM Domain Mutants of AtELP3. (A) Schematic representation of the HAT and radical SAM domain mutations. The radical SAM and HAT domains of AtELP3 are aligned with those of the yeast (ScELP3) and mouse ELP3 (MmELP3). Only sequences that are part of the alignment are shown. The conserved amino acid residues are labeled in red. Arrows indicate the mutations created in the SAM and HAT mutants. (B) Ten-day-old seedlings of WT, npr1, Atelp3-10 npr1, HAT(n), and SAM(n) grown on half-strength MS agar medium containing 0.26 mM SA. HAT(n) and SAM(n), transgenic lines in the Atelp3-10 npr1 genetic background expressing the HAT and the SAM mutant, respectively. (C) Free SA levels in Psm ES4326-infected WT, npr1, Atelp3-10 npr1, HAT(n), and SAM(n) plants. Plants were inoculated with Psm ES4326 (OD₆₀₀ = 0.001). Twenty-four hours later, the inoculated leaves were collected for SA measurement. Data represent the mean of three independent samples with SD. Different letters above the bars indicate significant differences (p < 0.05, Student’s t-test). (D) Total SA levels in Psm ES4326-infected WT, npr1, Atelp3-10 npr1, HAT(n), and SAM(n) plants. Experiment was performed as in (C). (E) Expression of eight defense genes in Pst DC3000/avrRpt2-infected WT, Atelp3-10, HAT(C), and SAM(C) plants. HAT(C) and SAM(C), transgenic lines in the Atelp3-10 genetic background expressing the HAT and the SAM mutant, respectively. Data represent the mean of three independent samples with SD. An asterisk (*) indicates that the expression level of the gene in the WT was significantly higher than that in Atelp3-10, HAT(C), and SAM(C) (p < 0.05, Student’s t-test). (F) Growth of Psm ES4326 in WT, Atelp3-10, HAT(C), and SAM(C) plants. Cfu, colony-forming units. Data represent the mean of eight independent samples with standard deviation. Different letters above the bars indicate significant differences (p < 0.05, Student’s t-test).