Nup82 functions redundantly with Nup136 in a salicylic acid-dependent defense response of Arabidopsis thaliana

Abstract

The nuclear pore complex (NPC) comprises more than 30 nucleoporins (Nups). NPC mediates macromolecular trafficking between the nucleoplasm and the cytoplasm, but specific roles of individual Nups are poorly understood in higher plants. Here, we show that the novel nucleoporin unique to angiosperm plants (designated as Nup82) functions in a salicylic acid-dependent defense in a redundant manner with Nup136, which is a component of the nuclear basket in the NPC. Arabidopsis thaliana Nup82 had a similar amino acid sequence to the N-terminal half of Nup136 and a Nup82-GFP fusion was localized on the nuclear envelope. Immunoprecipitation and bimolecular fluorescence complementation analyses revealed that Nup82 interacts with the NPC components Nup136 and RAE1. The double knockout mutant nup82 nup136 showed severe growth defects, while the single knockout mutant nup82 did not, suggesting that Nup82 functions redundantly with Nup136. nup82 nup136 impaired benzothiadiazole (an analog of salicylic acid)-induced resistance to the virulent bacteria Pseudomonas syringae pv. tomato DC3000. Furthermore, transcriptome analysis of nup82 nup136 indicates that deficiency of Nup82 and Nup136 causes noticeable downregulation of immune-related genes. These results suggest that Nup82 and Nup136 are redundantly involved in transcriptional regulation of salicylic acid-responsive genes through nuclear transport of signaling molecules.

Keywords: Arabidopsis thaliana; Nup136/Nup1; Nup82; nuclear envelope; nucleoporin; plant immunity; proteome; transcriptome.

Figure 1.

Nup82 is a nucleoporin that is unique to angiosperms and is localized on the nuclear envelope. (A) The phylogenetic tree of Nup82 (yellow box) and Nup136 (purple box) proteins from representative angiosperms (Amborella trichopoda), monocots (Oryza sativa and Zea mays), eudicots (Nelumbo nucifera), and dicots (Glycine max, Populus trichocarpa and Arabidopsis thaliana). The aligned amino acid sequences were assembled into a phylogenetic tree using the boot-strapped neighbor-joining algorithm in MEGA 6.06 with 1000 trials (http://www.megasoftware.net/). Bootstrap values are indicated as percentages of 1000 trials at their respective nodes. Labels at the branch tips indicate NCBI Reference Sequence Numbers or gene names. (B) Confocal fluorescence images of root tip cells from transgenic Arabidopsis stably expressing Nup82-GFP. (C) Confocal fluorescence images of protoplasts of Arabidopsis cultured cells transiently expressing Nup82-GFP. (D) Bimolecular fluorescence complementation (BiFC) assays in tobacco leaf epidermal cells. Histone-RFP was coexpressed as a marker for visualizing nuclei in the transformed cells. nYFP, N-terminal half of YFP. cYFP, C-terminal half of YFP.

Figure 2.

Isolation of nup82 nup136 double mutants. (A) A schematic representation of the NUP82 gene, which contains 8 exons. The positions of T-DNA insertions in nup82–1 and nup82–2 are shown. Closed boxes and solid lines indicate exons and introns, respectively. Black arrows indicate the orientation of the left border sequence. Red arrows indicate the primers used for RT-PCR in (B). (B) RT-PCR analysis of NUP82 and ACTIN2 (ACT2) transcripts in the wild-type (WT), nup82–1, and nup82–2. Amplification of NUP82 and ACT2 required 40 and 27 PCR cycles, respectively. These data are representative of 2 biologic replicates and 2 technical replicates. (C) Thirteen-day-old seedlings of wild-type (WT), nup136–2, nup136–2 nup82–1, and nup136–2 nup82–2 plants. (D) Five-week-old wild-type (WT), nup136–2, nup136–2 nup82–1, and nup136–2 nup82–2 plants. (E) Siliques of wild-type (WT), nup82–1, nup136–2, and nup136–2 nup82–1 plants.

Figure 3.

Defense-related genes are downregulated in the nup82 nup136 double mutant. (A) An enrichment analysis of Gene Ontology (GO) terms in the biologic processes category. REViGO amalgamated GO terms associated with the downregulated genes in the nup136 nup82 double mutant. (B) The top 10 genes in the group of defense response, which were downregulated in the nup82 nup136 double mutant. Bacterial response indicates whether gene expression is induced by bacterial infection, according to a public microarray analysis (AtGeneExpress). fold, fold change in gene expression between Control P.s. at 24 h vs Nonhost P.s. at 24 h. (C) Quantitative RT-PCR of defense-related genes in 14-day-old seedlings of the wild-type (WT), nup82–1, nup136–2, and nup82–1 nup136–2 plants. Data represent mean values of 3 independent experiments with standard deviations.

Figure 4.

Nup136 and Nup82 are involved in benzothiadiazole (BTH)-induced resistance against Pseudomonas syringae pv tomato virulent strain DC3000 (Pto DC3000). (A and B) Bacterial growth at 2 d after inoculation with Pto DC3000 (OD600 = 0.0001) in the leaves of nup136–2, nup82–1, and nup136–2 nup82–1 mutants that were pretreated without (A) or with (B) 150 µM BTH for 1 day. *, p < 0.05; **, p < 0.01 compared with the wild-type plants. Data represent mean values of 2 independent experiments (n = 16). Error bars indicate standard errors (Student's t test, *p < 0.05, **p < 0.01). (C) Quantitative RT-PCR of defense-related genes in 14-day-old seedling of WT and nup82–1 nup136–2 with (+BTH) and without benzothiazole (-BTH) treatment. Data represent mean values of 3 independent experiments with standard deviations.