SWR1 Chromatin-Remodeling Complex Subunits and H2A.Z Have Non-overlapping Functions in Immunity and Gene Regulation in Arabidopsis

Abstract

Incorporation of the histone variant H2A.Z into nucleosomes by the SWR1 chromatin remodeling complex is a critical step in eukaryotic gene regulation. In Arabidopsis, SWR1c and H2A.Z have been shown to control gene expression underlying development and environmental responses. Although they have been implicated in defense, the specific roles of the complex subunits and H2A.Z in immunity are not well understood. In this study, we analyzed the roles of the SWR1c subunits, PHOTOPERIOD-INDEPENDENT EARLY FLOWERING1 (PIE1), ACTIN-RELATED PROTEIN6 (ARP6), and SWR1 COMPLEX 6 (SWC6), as well as H2A.Z, in defense and gene regulation. We found that SWR1c components play different roles in resistance to different pathogens. Loss of PIE1 and SWC6 function as well as depletion of H2A.Z led to reduced basal resistance, while loss of ARP6 fucntion resulted in enhanced resistance. We found that mutations in PIE1 and SWC6 resulted in impaired effector-triggered immunity. Mutation in SWR1c components and H2A.Z also resulted in compromised jasmonic acid/ethylene-mediated immunity. Genome-wide expression analyses similarly reveal distinct roles for H2A.Z and SWR1c components in gene regulation, and suggest a potential role for PIE1 in the regulation of the cross talk between defense signaling pathways. Our data show that although they are part of the same complex, Arabidopsis SWR1c components could have non-redundant functions in plant immunity and gene regulation.

Keywords: H2A.Z; SWR1; arabidopsis; chromatin remodeling; gene regulation; immunity.

Figure 1

Developmental and Basal Resistance Phenotypes in SWR1c Component and H2A.Z Mutants. (A) Current understanding of the SWR1c: the complex subunits function in a single complex to catalyze H2A.Z incorporation. The subunits for which viable mutants exist in Arabidopsis are shown in color. (B) Phenotype of mutants grown in LD conditions. arp6, swc6-1, and hta9-1 hta11-1 showing early flowering phenotype. The pie1-2 mutant shows a very stunted growth phenotype. In SD conditions, the SWR1c mutants (pie1-2, arp6-1, and swc6-1) and hta9-1 hta11-1 show more similar phenotypes: serrated leaves with elongated petioles. The pie1-2 phenotype is less severe in these conditions. (C) Trypan blue staining of leaves from SWR1c and H2A.Z mutant plants grown under SD photoperiods showing no cell death, similar to wild-type plants. (D) Disease symptoms 3 dpi with virulent Pst DC3000 in the indicated genotypes. (E) Resistance phenotype after spray inoculation with Pst DC3000 bacterial suspension in the indicated genotypes. Bacterial titers were determined 2 h (day 0, white bars) and 3 days (day 3, black bars) post infection. Bars are average of data from four plants (n = 4), and error bars show SD. Asterisks indicate statistically significant differences compared with Col-0, with *P <0.05 and **P < 0.01 analyzed with Student's t-test. Similar results were obtained in at least three independent experiments. See also Supplemental Figures 1–3.

Figure 2

ETI Responses in the SWR1c Component and H2A.Z Mutants. (A and B) Resistance phenotype after spray inoculation with avirulent Pst DC3000 AvrRps4 (A) and Pst DC3000 AvrRpt2 (B) bacterial suspension in the indicated genotypes. Bacterial titers were determined a 2 h (day 0, white bars) and 3 days (day 3, black bars). Bars are average from four plants (n = 4), and error bars show SD. Asterisks indicate statistically significant differences compared with Col-0, with **P < 0.01 analyzed with Student's t-test. Similar results were obtained in at least three independent experiments. (C) Quantification of free SA content before (T0, light gray bars) and 24 h after spray inoculation with Pst DC3000 AvrRps4 (black bars) or after mock treatment with 10 mM MgCl₂ (dark gray bars). Bars represent the average of three independent biological replicates and error bars show SD. **P < 0.01 analyzed with Student's t-test.

Figure 3

SWR1c Components Have Different Roles in Resistance to Necrotrophic Pathogen. (A) Resistance to Botrytis cineria: average lesion diameter for all inoculation sites at 3 dpi. Asterisks indicate statistically significant differences compared with Col-0, with *P < 0.05 analyzed with Student's t-test. Similar results were obtained in at least two independent experiments (n = 30). (B) Frequency of outgrowing lesions (a size bigger than the inoculation site, 2 mm) in the different mutants background at 3 dpi with Botrytis cinerea. Type II, outgrowing lesion between 2 and 4 mm in diameter; type III, 4 and 6 mm in diameter; and type IV, >6 mm in diameter. See also Supplemental Figure 5.

Figure 4

SWR1c Subunits Have Distinct Functions in Gene Regulation. (A and B) Venn diagrams of differentially expressed genes showing overlap in genes whose expression levels are uniquely or concordantly upregulated (≥2-fold) (A) or downregulated (≥2-fold) (B) in the mutants. Total number of ≥2-fold up- or downregulated genes in each mutant is shown in parentheses. (C) Principal component analysis of the RNA-seq data. The results are depicted three-dimensionally with PC1 (44.4%), PC2 (30.5%), and PC3 (17.1%) as the X, Y, and Z axes, respectively. (D–G) Scatter plot comparing differential expression of all genes in respective mutant backgrounds. Each point represents the log 2 change in expression of individual genes in the respective mutant. The solid black line shows linear regression representing the correlation coefficient (r²). See also Supplemental Figures 6 and 7.

Figure 5

Defense Gene Expression Is Altered in SWR1c Mutants. (A–D) Gene expression of SAR genes in SWR1c mutants: PR1(A) is strongly upregulated in the pie1 mutant. PR5(B) shows upregulation in all the mutants, with the strongest effect in pie1. EDS1(C) is specifically upregulated in pie1, while NIMIN1 (D) is upregulated in arp6 and pie1 only. (E) Dot density plot showing misregulation of the WRKY gene family in the SWR1c component mutants. The pie1 mutant displays a general upregulation of the WRKY genes. (F)WRKY38 and (G)WRKY62 show strong upregulation in the pie1 mutant. Data represented are expression values from RNA-seq data of three independent biological replicates, normalized to EF1α. Error bars show SD from three biological replicates.

Figure 6

PIE1 Is Required to Maintain the Fine Balance between SA and JA Signaling Pathways. (A–I) Genes involved in SA/JA signaling trade-off are upregulated in the pie1 mutant: (A) SA-inducible GRX480, known to antagonize the JA-responsive transcription of PDF1.2, is strongly upregulated in the pie1 mutant. JA biosynthesis genes AOC2(B), OPR3(C), and LOX2 (D) show strong upregulation in pie1. Genes involved in JA signaling JAZ1(E) and JAZ10(F) are specifically upregulated in pie1, but downregulated in swc6 and hta9 hta11. JA-responsive genes VSP2(G), PDF1.2 (H), and JR2/TAT1(I) are upregulated in pie1. (J) JA-induced genes show increased expression in pie1 suggesting that PIE1 activity is essential for SA-mediated repression of JA-responsive gene expression.

Figure 7

SWC6 Is Epistatic to ARP6 in Disease Resistance to P. syringae. Analysis of the arp6 swc6 double mutant for resistance to Pst DC3000. Bacterial titers were determined 3 days post spray inoculation with Pst DC3000 bacterial suspension in the indicated genotypes. Bars are average of six plants (n = 6), and error bars show SD. Asterisks indicate statistically significant differences compared with Col-0, with **P < 0.01 analyzed with Student's t-test. Similar results were obtained in at least two independent experiments.

Figure 8

SWR1c Function Is Required for Pathogen-Responsive Gene Expression. Analysis of PR1(A) and PR5(B) gene expression in SWR1c mutants in response to bacterial infection by Pst DC3000. Four-week-old Col-0, arp6, pie1, swc6, and hta9 hta11 were inoculated with Pst DC3000 or mock treated with MgCl₂. RNA was isolated 6 h post treatment, and gene expression was analyzed by qRT–PCR (three biological replicates and three technical replicates). Insets show log fold induction upon Pst DC3000 treatment. Asterisks indicate significant difference from respective Col-0 treatment as analyzed by Student's t-test (*P <0.05, **P < 0.01, and ***P < 0.001).