death control - Wiley Online Library

The Plant Journal (2016) 85, 520–531

doi: 10.1111/tpj.13125

Nucleocytoplasmic trafficking is essential for BAK1- and BKK1-mediated cell-death control Junbo Du1,2, Yang Gao1, Yanyan Zhan1, Shasha Zhang1, Yujun Wu1, Yao Xiao1, Bo Zou3, Kai He1, Xiaoping Gou1, Guojing Li3, Honghui Lin4 and Jia Li1,* 1 Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730000, China, 2 College of Agronomy, Sichuan Agricultural University, Chengdu 611130, China, 3 College of Life Sciences, Inner Mongolia Agricultural University, Hohhot 010018, China, and 4 Ministry of Education Key Laboratory of Bio-Resource and Eco-Environment, College of Life Sciences, Sichuan University, Chengdu 610064, China Received 28 August 2015; revised 30 December 2015; accepted 11 January 2016; published online 18 January 2016. *For correspondence (email [email protected]).

SUMMARY BRI1-ASSOCIATED KINASE 1 (BAK1) was initially identified as a co-receptor of the brassinosteroid (BR) receptor BRI1. Genetic analyses also revealed that BAK1 and its closest homolog BAK1-LIKE 1 (BKK1) regulate a BR-independent cell-death control pathway. The double null mutant bak1 bkk1 displays a salicylic acid- and light-dependent cell-death phenotype even without pathogen invasion. Molecular mechanisms of the spontaneous cell death mediated by BAK1 and BKK1 remain unknown. Here we report our identification of a suppressor of bak1 bkk1 (sbb1–1). Genetic analyses indicated that cell-death symptoms in a weak double mutant, bak1–3 bkk1–1, were completely suppressed by the loss-of-function mutation in SBB1, which encodes a nucleoporin (NUP) 85-like protein. Genetic analyses also demonstrated that individually knocking out three other nucleoporin genes from the SBB1-located sub-complex was also able to rescue the celldeath phenotype of bak1–3 bkk1–1. In addition, a DEAD-box RNA helicase, DRH1, was identified in the same protein complex as SBB1 via a proteomic approach. The drh1 mutation also rescues the cell-death symptoms of bak1–3 bkk1–1. Further analyses indicated that export of poly(A)+ RNA was greatly blocked in the nup and drh1 mutants, resulting in accumulation of significant levels of mRNAs in the nuclei. Over-expression of a bacterial NahG gene to inactivate salicylic acid also rescues the cell-death phenotype of bak1–3 bkk1–1. Mutants suppressing cell-death symptoms always showed greatly reduced salicylic acid contents. These results suggest that nucleocytoplasmic trafficking, especially of molecules directly or indirectly involved in endogenous salicylic acid accumulation, is critical in BAK1- and BKK1-mediated cell-death control. Keywords: receptor-like kinase, BAK1, BKK1, nucleoporin, cell death, Arabidopsis thaliana.

INTRODUCTION As sessile organisms, plants have developed sophisticated adaptation mechanisms to meet the challenges of adverse environmental conditions such as intensive UV light, unfavorable temperatures, drought, nutrient deficiencies and pathogen attacks. Plant cells often use cell-surface receptor-like protein kinases (RLKs) to sense various signals from adjacent cells or surrounding environments and relay the signals into the cell to initiate cellular signaling processes for adequate responses. RLKs are single-pass transmembrane proteins that typically contain an extracellular domain, a single-pass transmembrane domain, and a cyto520

plasmic kinase domain (Li, 2010). The extracellular domain of an RLK is responsible for perceiving extracellular signals such as phytohormones and small peptides. The transmembrane domain anchors the RLK to the plasma membrane. The cytoplasmic kinase domain possesses either serine/threonine kinase activity or serine/threonine/tyrosine dual kinase activity (Horn and Walker, 1994; Oh et al., 2009; Li, 2010). Usually an external signaling molecule (ligand) binds to the extracellular domain of a ligand-binding RLK, which triggers homodimerization or heterodimerization with a second RLK molecule. Although not yet proven, it is © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd

Nucleoporins and BAK1-associated cell-death control 521 possible that signal perception by the extracellular domain of an RLK may also be achieved via a chemical reaction triggered by signals such as reactive oxygen species. The dimerization process allows the kinase domain to be activated via intermolecular phosphorylation. The activated RLK then transduces the signal to downstream components via a reversible phosphorylation and dephosphorylation cascade, altering the gene expression profile in response to the extracellular signal. More than 610 RLK and receptor-like cytoplasmic kinase genes have been found in the Arabidopsis genome (Shiu and Bleecker, 2001). Some of the RLKs were found to be involved in many different developmental and stress/defense related signaling pathways. For example, somatic embryogenesis receptor-like kinases (SERKs) are a small group of leucine-rich repeat RLKs that play critical roles in the early events of brassinosteroid (BR) signal transduction (Gou et al., 2012). SERKs belong to the type II leucine-rich repeat RLK family (Li et al., 2002; Nam and Li, 2002; He et al., 2007; Li, 2010). DcSERK was originally identified from carrot (Daucus carota) as a molecular marker that is indicative of the transition of cells from somatic to competent and embryogenic cells (Schmidt et al., 1997). There are five homologs of DcSERK in Arabidopsis: SERK1–SERK5 (Hecht et al., 2001). SERK3 and its closest homolog SERK4 are also named BRI1-ASSOCIATED KINASE 1 (BAK1) and BAK1LIKE 1 (BKK1), respectively (He et al., 2007). Previous studies have revealed that SERKs regulate multiple distinctive signaling pathways, including the BR pathway (Li et al., 2002; Nam and Li, 2002; He et al., 2007; Gou et al., 2012), root development (Du et al., 2012), anther development (Albrecht et al., 2005; Colcombet et al., 2005), and pathogenesis signaling pathways mediated by FLAGELLIN SENSING 2 (FLS2) and EF–TU RECEPTOR (EFR) (Zipfel et al., 2006; Chinchilla et al., 2007). Several lines of evidence from genetic, biochemical and protein structural studies demonstrated that BAK1 and its homologs are authentic co-receptors of BRI1, FLS2 and EFR (Li et al., 2002; Nam and Li, 2002; Zipfel et al., 2006; Chinchilla et al., 2007; He et al., 2007; Hothorn et al., 2011; Roux et al., 2011; She et al., 2011; Gou et al., 2012; Sun et al., 2013). BAK1 and BKK1 were also found to regulate a BR-independent pathway and an undefined cell-death control pathway (He et al., 2007; Kemmerling et al., 2007). This pathway may interact with but is clearly distinctive from known BAK1-mediated pathogen-triggered innate immunity (Chinchilla et al., 2007), and is therefore worth further investigating. The double null mutant bak1–4 bkk1–1 shows a spontaneous cell-death phenotype when grown on pathogen-free medium under light conditions (He et al., 2007). As a consequence, the plant is completely dead at the seedling stage. Further analyses showed that the cell death of bak1 bkk1 is dependent on salicylic acid (SA), and PR genes are drastically up-regulated in the bak1–4 bkk1–1

(He et al., 2007). The lethality of bak1–4 bkk1–1 makes this double mutant less valuable for genetic suppressor screens. However, a different double mutant, bak1–3 bkk1– 1, with leaky expression of BAK1, exhibits a semi-dwarfed but fertile phenotype, with lesions present from the cotyledon stage (He et al., 2007; Gou et al., 2012), and is therefore an excellent material for genetic modifier screens. Here we report our identification of a genetic suppressor of bak1 bkk1 that fully rescues the cell death phenotype of bak1–3 bkk1–1. Genetic data showed that suppression of the cell-death phenotype of bak1–3 bkk1–1 is caused by loss of function of a putative nucleoporin-encoding gene: SBB1. Sequence alignment showed that SBB1 displays high similarity with an NUP85-like protein, a member of the NUP107– 160 sub-complex of the nuclear pore complex. Further genetic investigation showed that individual knockout mutants of several other members of the NUP107–160 subcomplex, including NUP160, NUP96 and SEC13 homolog 1, also rescue the cell-death phenotype of bak1–3 bkk1–1. Proteomic analysis revealed that many proteins are present in the SBB1-associated complex. Of those, a DEAD-box RNA helicase (DRH1) was further characterized and shown to be involved in the cell-death control of bak1 bkk1. Furthermore, knocking out NUP genes and DRH1 resulted in mRNA accumulation in the nuclei. Our data suggest that these mutations may result in accumulation of SA biosynthesis-related transcripts in the nucleus. As a consequence, SA content is controlled at a lower level and cell death is inhibited. This study offers new insights into nucleocytoplasmic trafficking in BAK1- and BKK1-mediated cell-death control. RESULTS sbb1 is a genetic suppressor of the bak1–3 bkk1–1 double mutant To understand the molecular mechanisms of BAK1- and BKK1-mediated cell-death control, we transformed an activation-tagging vector (pBASTA-AT2) into bak1–3 bkk1–1 to screen for genetic modifiers of the double mutant (Yuan et al., 2007). Within T2 progeny generated from approximately 16 000 self-pollinated T1 lines, one of the suppressors showed a fully rescued cell-death phenotype, which was subsequently named sbb1–1 (suppressor of bak1 bkk1) (Figure 1a). After back-crossing bak1–3 bkk1–1 sbb1–1 with bak1–3 bkk1–1, all the F1 progeny exhibited a phenotype similar to bak1–3 bkk1–1. However, in the F2 progeny, approximately a quarter of the plants showed a wild-type (WT)-like phenotype and three-quarters exhibited a typical cell death phenotype (Table S1), suggesting that a single recessive locus is responsible for the suppression phenotype. Molecular cloning of the genomic sequences flanking the T–DNA insertion site revealed that a T–DNA was inserted in the sixth exon of At4g32910, which encodes a putative NUP85-like protein (Figure 1b and Figure S1).

© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), 85, 520–531

522 Junbo Du et al.

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

(k)

Figure 1. Identification of a genetic suppressor of bak1–3 bkk1–1 as a T– DNA inactivation mutant of nucleoporin-encoding gene SBB1. (a) Phenotypes of 4-week-old Col–0, bak1–3 bkk1–1 and bak1–3 bkk1–1 sbb1–1 seedlings grown in soil. (b) Positions of T–DNA insertions in two sbb1 mutants. (c) Confocal analyses of pSBB1::SBB1-GFP transgenic seedling roots, showing that SBB1 localizes to the nuclear envelope in Arabidopsis. Left panel: pSBB1::SBB1-GFP transgenic plant root cells. Middle panel, root cells stained with propidium iodide (PI). Right panel, merged images from the left and middle panels. Scale bars = 1 cm (a) and 10 lm (c).

Confocal microscopic analysis indicated that the SBB1–GFP fusion protein is indeed localized on the nuclear envelope (Figure 1c). An sbb1–1 single mutant was then generated by back-crossing bak1–3 bkk1–1 sbb1–1 with Col–0. The second T–DNA insertion allele of SBB1, sbb1–2/ nup85–1 (SALK_086258C), was obtained from the Arabidopsis Biological Resource Center (Figures 1b and 2a). RT–PCR analyses showed that transcripts of full-length SBB1 were not present in either sbb1–1 or sbb1–2 mutants (Figure S2), confirming that both alleles are null alleles. Neither allele alone displays any visible phenotypes compared to Col–0 (Figure 2a). To examine whether cell death in bak1–3 bkk1–1 is truly suppressed by removal of SBB1, trypan blue staining was used to detect dead cells in the cotyledons of bak1 bkk1 sbb1 triple mutant plants and various other mutants. The

Figure 2. Both sbb1–1 and sbb1–2 suppress the cell-death phenotype, elevated expression of PR genes and SA accumulation in bak1–3 bkk1–1. (a) Knocking out SBB1 rescues the cell-death phenotype of 3-week-old bak1–3 bkk1–1 seedlings. Scale bar = 1 cm. (b–g) Trypan blue-stained cotyledons showing that the cell-death phenotype of bak1–3 bkk1–1 is suppressed by sbb1: (b) Col–0, (c) bak1–3 bkk1–1, (d) sbb1–1, (e) sbb1–2, (f) bak1–3 bkk1–1 sbb1–1, and (g) bak1–3 bkk1–1 sbb1–2. Scale bars = 100 lm. (h–j) Expression of PR genes is decreased in the bak1 bkk1 sbb1 triple mutants. (k) SA levels in various genotypes. Values in (h–k) are means and SD.

results showed that cell death only occurs extensively in the bak1–3 bkk1–1 double mutant, but not in WT, sbb1 single mutants, or bak1–3 bkk1–1 sbb1–1 and bak1–3 bkk1–1 sbb1–2 triple mutants (Figure 2b–g). In our previous studies, we found that the SA-responsive genes PR1, PR2 and PR5 are significantly up-regulated in the bak1–4 bkk1–1


Nucleoporins and BAK1-associated cell-death control 523 double mutant compared to Col–0 (He et al., 2007). Expression of these three genes is drastically elevated in the bak1–3 bkk1–1 double mutant compared to Col–0, but greatly suppressed in the bak1–3 bkk1–1 sbb1–1 and bak1– 3 bkk1–1 sbb1–2 triple mutants (Figure 2h–j). Consistent with this, the endogenous level of total SA is increased in the bak1–3 bkk1–1 double mutant and is almost restored to a WT-like level in the bak1–3 bkk1–1 sbb1–1 and bak1–3 bkk1–1 sbb1–2 triple mutants (Figure 2k). To further confirm that sbb1 is responsible for suppression of cell death in bak1–3 bkk1–1, a vector expressing SBB1 genomic DNA, driven by a native promoter (pSBB1::SBB1g), was transformed into the bak1–3 bkk1–1 sbb1–1 triple mutant, and the results showed that pSBB1::SBB1 g recapitulates the cell-death phenotype (Figure 3a–f). The genetic backgrounds were verified by RT–PCR analyses (Figure S3). Relative expression of PR genes and the endogenous SA level in bak1–3 bkk1–1 were partially restored by pSBB1::SBB1g (Figure 3g–j). These results demonstrate that the putative nucleoporin SBB1 negatively regulates BAK1- and BKK1controlled cell death.

(a)

(b)

(c)

(d)

(e)

(g)

(h)

(i)

(j)

(f)

SA is a central player in BAK1- and BKK1-mediated celldeath control To verify that the cell death seen in both the bak1–4 bkk1–1 and bak1–3 bkk1–1 double mutants is SA-dependent, we over-expressed NahG, a bacterial gene that encodes a hydroxylase that inactivates SA (Gaffney et al., 1993), in the double mutants. Over-expression of NahG partially rescued the cell-death phenotype of the null double mutant bak1–4 bkk1–1 (He et al., 2007), and almost fully restored the dwarf and cell-death phenotypes of the weak double mutant bak1–3 bkk1–1 (Figure 4a–d). The relative expression of PR genes and the endogenous SA content in bak1–3 bkk1–1 were restored to WT-like levels by 35S::NahG (Figure 4e–h). These results suggest that the cell death in bak1 bkk1 is SAdependent. Although the bak1–3 bkk1–1, sbb1–1, sbb1–2, bak1–3 bkk1–1 sbb1–1 and bak1–3 bkk1–1 sbb1–2 plants did not display distinctive phenotypes when grown on SA-free medium for 2 weeks, bak1–3 bkk1–1 sbb1–1 and bak1–3 bkk1–1 sbb1–2 were less sensitive to exogenous SA treatment than the bak1–3 bkk1–1 double mutant (Figures 4i and S4). Our results indicate that, compared to bak1–3 bkk1–1, SA accumulation is restored in the bak1 bkk1 sbb1 triple mutants. These data suggest that SBB1 modulates cell death by altering the endogenous level of SA in bak1–3 bkk1–1. It is likely that SA is one of the main regulators of the cell death seen in the bak1 bkk1 double mutants. Some but not all the members of the NUP107–160 subcomplex are essential for BAK1- and BKK1-mediated celldeath control The NUP107–160 sub-complex, located on the outer ring of the nuclear pore complex, is the largest sub-complex

Figure 3. The cell-death phenotype, elevated expression of PR genes and SA accumulation observed in bak1–3 bkk1–1 are recapitulated by transforming pSBB1::SBB1 g into bak1–3 bkk1–1 sbb1–2. (a) Restoration of the cell-death phenotype of bak1–3 bkk1–1 sbb1–2 by expression of SBB1 genomic DNA driven by its native promoter. Scale bar = 1 cm. (b–f) Trypan blue-stained cotyledons showing that the cell-death phenotype of bak1–3 bkk1–1 sbb1–2 is restored by the transformation with pSBB1:: SBB1 g: (b) Col–0, (c) bak1–3 bkk1–1, (d) bak1–3 bkk1–1 sbb1–2, (e) line 1 of bak1–3 bkk1–1 pSBB1::SBB1 g, (f) and line 2 of bak1–3 bkk1–1 pSBB1:: SBB1 g. Scale bar = 100 lm. (g–j) Relative expression of PR1 (g), PR2 (h) and PR5 (i), and endogenous SA content (j) are restored in the pSBB1::SBB1 g bak1–3 bkk1–1 sbb1–2 transgenic plants. Values in (g–j) are means SD.

conserved in vertebrates and plants. In vertebrates, NUP75, a homolog of SBB1, belongs to the NUP107–160 sub-complex (Alber et al., 2007; Zuccolo et al., 2007). In plants, The NUP107–160 sub-complex contains eight putative NUPs, NUP160/SAR1, NUP133, NUP107, NUP96/ MOS3/SAR3, SBB1/NUP85, NUP43, SEH1 and SEC13, for


524 Junbo Du et al.

(a)

(b)

(d)

(c)

(e)

(f)

(g)

(h)

Figure 4. BAK1- and BKK1- regulated cell death is SA-dependent. (a) Over-expression of NahG rescues the cell-death phenotype of bak1–3 bkk1–1. Scale bar = 1 cm. (b–d) Trypan blue-stained cotyledons showing that cell-death phenotypes are suppressed by over-expression of NahG: (b) Col–0, (c) bak1–3 bkk1–1, and (d) bak1–3 bkk1–1 35S::NahG. Scale bars = 100 lm. (e–g) Relative expression of PR1, PR2 and PR5 in bak1–3 bkk1–1 is suppressed by NahG over-expression. (h) The SA level of bak1–3 bkk1–1 is decreased by over-expression of NahG. (i) Fresh weights of individual plants from various genotypes treated with two SA concentrations. Values are means SD. Student’s t test was used to assess the significance of differences between Col–0 and the mutants under each treatment. Asterisks indicate statistically significant differences compared with Col–0 (P < 0.0001).

overlapping functions of putative members of the NUP107–160 sub-complex prompted us to test whether additional NUPs other than SBB1 in this sub-complex contribute to BAK1- and BKK1-mediated cell-death control. Seven available T–DNA insertion mutants of the NUPs in the NUP107–160 sub-complex were crossed with bak1–3 bkk1–1 to generate triple mutants (Figures 5a–c and S5a– d). RT–PCR analyses verified the genetic backgrounds of these mutants (Figure S6a–g). Trypan blue staining showed that individual knockout of SEH1, NUP160 or NUP96, but not NUP107, NUP133, NUP43 or SEC13, is able to almost fully rescue the cell-death phenotype of bak1–3 bkk1–1 in cotyledons (Figures 5e–p and S5e–t). The expression of PR genes and endogenous SA levels were drastically down-regulated in the bak1–3 bkk1–1 seh1–1, bak1–3 bkk1–1 nup160–3 and bak1–3 bkk1–1 nup96–1 triple mutants compared to bak1–3 bkk1–1 (Figure 5u–w). Although the expressions of PR genes and endogenous SA contents were partially suppressed in bak1–3 bkk1–1 nup133–3, bak1–3 bkk1–1 nup107–1, bak1–3 bkk1–1 nup43– 1 and bak1–3 bkk1–1 sec13b–1 compared to bak1–3 bkk1–1 (Figure S5u–x), the SA levels remain relatively high in the triple mutants, and the suppression of SA levels was not enough to suppress the cell death of bak1–3 bkk1–1. These results indicated that four members of the NUP107–160 sub-complex play more significant roles in BAK1- and BKK1-mediated cell-death control.

(i)

DRH1 is associated with the NUP complex and required for mRNA export

which only limited functions have been determined in plants (Zhang and Li, 2005; Parry et al., 2006; Xu and Meier, 2008; Tamura et al., 2010; Wiermer et al., 2012; Tamura and Hara-Nishimura, 2013). The specificity and

Nucleocytoplasmic trafficking is an essential means of subcellular communication between cytoplasm and nucleus (Heese-Peck and Raikhel, 1998), in which the nuclear pore complex plays a central role. Studies of plant NUPs are still in their infancy compared to those in vertebrates, humans and yeasts. Identification of novel components associated with the plant nuclear pore complex largely relies on proteomic approaches. Recently, Tamura et al. (2010) discovered 24 NUPs using interactive proteomics and linear ion trap mass spectrometer analyses. Using SBB1-GFP transgenic plants, we used a proteomic method to discover


Nucleoporins and BAK1-associated cell-death control 525

(a)

(b)

(c)

(d)

(e)

(f)

(i)

(j)

(m)

(n)

(q)

(r)

(g)

(h)

(k)

(l)

(o)

(p)

(s)

(t)

(u)

(v)

(w)

(x)

Figure 5. Loss-of-function of SEH1, NUP160, NUP96 or DRH1 individually is able to rescue the cell-death phenotype of bak1–3 bkk1–1. (a–d) The cell-death phenotype of bak1–3 bkk1–1 is suppressed by knocking out SEH1, NUP160, NUP96 or DRH1 individually. Scale bar = 1 cm. (e–t) Trypan blue-stained cotyledons showing that the cell-death phenotype is rescued by seh1, nup160, nup96 or drh1: (e) Col–0; (f) bak1–3 bkk1–1; (g) seh1–1; (h) bak1–3 bkk1–1 seh1–1; (i) Col–0; (j) bak1–3 bkk1–1; (k) nup160–3; (l) bak1–3 bkk1–1 nup160–3; (m) Col–0; (n) bak1–3 bkk1–1; (o) nup96–1; (p) bak1–3 bkk1–1 nup96–1. (q) Col–0; (r) bak1–3 bkk1–1; (s) drh1–1; (t) bak1–3 bkk1–1 drh1–1. Scale bars = 200 lm. (u–x) The relative expression of PR1, PR2 and PR5, and the SA level in bak1–3 bkk1–1 are restored by (u) seh1–1, (v) nup160–3, (w) nup96–1 and (x) drh1–1.

novel components associated with SBB1. After immunoprecipitation followed by an LC-MS/MS assay, a number of SBB1-interacting proteins, including DEAD-box RNA

helicases, were obtained (Appendix S1). It was previously reported that some DEAD-box RNA helicases and RNAbinding proteins contribute to plant responses to various


526 Junbo Du et al. biotic and abiotic stresses (Gong et al., 2005; Li et al., 2008; Germain et al., 2010; Guan et al., 2013). Transient expression of 35S-DRH1-YFP showed that DRH1 localizes in the nucleus (Figure S7). We therefore selected DEAD-BOX RNA HELICASE 1 (DRH1, At3g01540) (Okanami et al., 1998), which was co-immunoprecipitated with SBB1, for further analyses. A bak1–3 bkk1–1 drh1–1 triple mutant was subsequently generated by genetic crossing (Figure 5d). The genetic backgrounds were verified by RT–PCR analyses (Figure S6h). The dwarfism, cell-death phenotype, expression of PR genes and endogenous SA content of bak1–3 bkk1–1 were all suppressed by the loss-of-function mutation of DRH1 (Figure 5q–t,x). To further investigate whether DRH1 is associated with the NUP107–160 sub-complex, we performed yeast two-hybrid analyses to examine interactions of DRH1 with members of the NUP107–160 sub-complex. The results showed that DRH1 is able to physically interact with NUP107, suggesting that DRH1 is indeed

(a)

(c)

(e)

(b)

(d)

(f)

(g)

(i)

(k)

(h)

(j)

(l)

associated with the NUP107–160 sub-complex (Figure S8). These results indicate that DRH1 is indeed required for BAK1- and BKK1-mediated cell-death control, possibly by coordinating with NUPs. The fact that sbb1 and drh1 suppress the cell-death phenotype of bak1–3 bkk1–1 suggests that RNA regulation such as mRNA export is critical for cell-death control. Thus, whole-mount in situ hybridization of mRNA was performed in various sbb1 and drh1 mutants. Our data showed extensive mRNA accumulation in the nuclei of the sbb1–1 and drh1–1 single mutants, as well as the bak1–3 bkk1–1 sbb1–1, bak1–3 bkk1–1 seh1–1, bak1–3 bkk1–1 nup160–3, bak1–3 bkk1–1 nup96–1 and bak1–3 bkk1–1 drh1–1 triple mutants (Figure 6a–l). Translocation of mRNAs is also more or less blocked in the nuclei of nup133–3, nup107–1 and nup43–1 single mutants and bak1–3 bkk1–1 nup133–3, bak1–3 bkk1–1 nup107–1 and bak1–3 bkk1–1 nup43–1 triple mutants, but not in sec13b–1 or bak1–3 bkk1– 1 sec13b–1 mutants (Figure S9). These results clearly indicate Figure 6. Loss-of-function mutations of SBB1, SEH1, NUP160 and DRH1 all show blocked mRNA export. Whole-mount in situ hybridization of mRNA showing that poly(A)+ RNAs are almost evenly distributed in the cells of (a) Col–0 and (b) bak1–3 bkk1–1, but accumulate in the nucleus of (c) sbb1–1, (d) bak1–3 bkk1–1 sbb1–1, (e) seh1–1, (f) bak1–3 bkk1–1 seh1–1, (g) nup160–3, (h) bak1–3 bkk1–1 nup160–3, (i) nup96–1, (j) bak1–3 bkk1–1 nup96–1, (k) drh1–1 and (l) bak1–3 bkk1–1 drh1–1. Scale bars = 10 lm.


Nucleoporins and BAK1-associated cell-death control 527 that, in addition to SEH1, NUP160 and NUP96, whose functions in RNA export have been reported previously in Arabidopsis (Parry et al., 2006; Wiermer et al., 2012), SBB1, DRH1, NUP133, NUP107 and NUP43 also contribute significantly to mRNA export. To examine whether the SA biosynthetic mRNAs remain in the nuclei, we chose sbb1–1 and drh1–1 mutants, which are able to fully restore the cell-death phenotype of bak1–3 bkk1–1, for further investigation. We analyzed the relative expression of EDS1, EDS5, PAD4 and SID2, key genes that regulate SA biosynthesis, in the cytoplasmic and nuclear fractions of Col–0, sbb1–1 and drh1–1 mutants. However, expression of these genes was too low to be detectable in separated cytoplasmic or nuclear fractions, even when using 400 ng of cDNA as the template for a 45cycle amplification, especially for SID2. In addition, expression of these SA-related genes was not obviously increased in the nuclei of sbb1 and drh1 mutants compared with Col–0 (Figure S10), suggesting that mutations of SBB1 or DRH1 affect export of certain unknown mRNAs that may indirectly regulate SA production. DISCUSSION Our genetic evidence clearly demonstrated that the function of members of the NUP107–160 sub-complex, including SBB1, SEH1, NUP160 and NUP96, and nucleoporin-associated RNA-binding protein DRH1 is required for the cell-death phenotype of the bak1–3 bkk1–1 mutant, indicating the essential roles of nucleocytoplasmic trafficking in BAK1- and BKK1-mediated cell-death control. Two independent alleles of the SBB1 mutants, sbb1–1 and sbb1–2, completely restore the early senescence phenotype of the bak1–3 bkk1–1 double mutant. The high accumulation of SA in bak1–3 bkk1–1 is also suppressed to almost WT level by introducing the sbb1 mutant alleles. Transgenic plants expressing SBB1 in the bak1–3 bkk1–1 sbb1–1 or bak1–3 bkk1–1 sbb1–2 triple mutant background under the control of its own promoter recapitulate the early senescence and cell-death symptoms of the double mutant. Interestingly, our results indicated that mRNA processing and nuclear accumulation are apparently critical for BAK1- and BKK1-mediated cell-death control. SA is probably one of the key mediators of BAK1- and BKK1-controlled cell-death. Removal of SA by over-expressing bacterial NahG in bak1–3 bkk1–1 restores the double mutant to an almost WT-like phenotype. In addition, bak1–3 bkk1–1 is significantly more sensitive to the exogenous SA treatment than WT. Taken together, our results indicate that BAK1and BKK1-mediated cell death is regulated by nucleocytoplasmic trafficking, which may modulate an SA burst by affecting the export of SA-related mRNAs. Previous reports suggested that members of the NUP107–160 sub-complex contribute to a variety of physiological processes. SAR1/NUP160 and SAR3/NUP96 were shown to play a role in the import of AXR3/IAA17 and the

export of mRNAs (Parry et al., 2006). NUP160 is involved in regulating flowering time and cold stress tolerance (Dong et al., 2006). NUP133 and NUP85 are responsible for Ca2+ spiking and symbiotic signaling pathways mediated by rhizobial bacteria and mycorrhizal fungi in Lotus japonicus (Kanamori et al., 2006; Saito et al., 2007). Intriguingly, previous studies indicated that SEH1, NUP160 and NUP96 are involved in an R protein-mediated pathogen defense response. Knockout of any one of the SEH1, NUP160 and NUP96 genes is capable of suppressing the phenotype of snc1, a gain-of-function R gene mutant that exhibits constitutive autoimmunity (Zhang and Li, 2005; Wiermer et al., 2012), suggesting that R proteins may act as regulatory components in BAK1- and BKK1-mediated cell-death control pathway. SEH1, NUP160 and NUP96 were previously identified as important regulators in defense responses in which SBB1 was found not to participate (Zhang and Li, 2005; Wiermer et al., 2012), suggesting overlapping and distinctive functions of NUPs in basal defense and BAK1and BKK1-mediated cell-death control. We speculate that SEH1, NUP160 and NUP96 and SBB1 form a small subcomplex in the outer ring of the nuclear pore complex that may possess certain specificity in regulating BAK1- and BKK1-mediated cell death. Disruption of any single member in this small sub-complex may interrupt the function of the four-member sub-complex. In eukaryotes, mRNA is transcribed and processed in the nucleus and exported through the nuclear pores to the cytoplasm for protein translation. In yeast and humans, some RNA transport factors (heterodimers Mex67–Mtr2 in yeast and Tap–P15 in humans), DEAD-box proteins and NUPs play important roles in mRNA export (Chinnusamy et al., 2008; Meier, 2012). However, the molecular mechanisms of mRNA export in plants are largely unknown. No homologs of Mex67/Mtr2 and Tap/P15 have been reported in plants (Meier, 2012). Recent reports have revealed that NUPs such as NUP160/SAR1, NUP96/SAR3/MOS3, NUA/ TPR, NUP136, RAE1 and SEH1 (Zhang and Li, 2005; Dong et al., 2006; Parry et al., 2006; Jacob et al., 2007; Xu et al., 2007; Lee et al., 2009; Lu et al., 2010; Tamura et al., 2010; Wiermer et al., 2012), and DEAD-box proteins such as Arabidopsis LOW EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 4 (LOS4) and rice OsBIRH1 control mRNA export in plants (Gong et al., 2005; Li et al., 2008), as indicated by the accumulation of mRNA in the nuclei of the corresponding mutant plants. Arabidopsis LOS4, a homolog of human DEAD-box protein 5 (DBP5), is required for abiotic stress responses (Tseng et al., 1998; Gong et al., 2005). Over-expression of OsBIRH1, a rice gene encoding a DEAD-box RNA helicase with high identity to Arabidopsis AtRH50 (At3g06980), enhanced disease resistance and oxidative stress responses (Li et al., 2008). Knockout of an Arabidopsis RNA binding protein, MOS11, a homolog of human RNA binding protein CIP29, partially suppressed


528 Junbo Du et al. the enhanced disease resistance phenotypes of snc1 (Germain et al., 2010). Mutation of a cold-inducible DEAD-box RNA helicase, REGULATOR OF CBF GENE EXPRESSION 1 (RCF1), is hypersensitive to cold stress and results in misspliced cDNAs for several cold-responsive genes (Guan et al., 2013). A recent study demonstrated that loss of function of the polyadenylation factor CPSF30 was able to suppress the cell-death symptoms of lsd1, cpr5, cat2 and mpk4 in an SA-dependent manner (Hajouj et al., 2000). The participation of nuclear RNA processing in cell-death control is further supported by our results showing that SBB1 and DRH1 play central roles in the BAK1- and BKK1mediated cell death pathway, most likely by affecting mRNA export. However, the mRNAs of selective genes that regulate SA accumulation do not significantly accumulate in the nuclear fractions of sbb1–1 and drh1–1 relative to WT plants. Because SA biosynthetic routes have not yet been completely elucidated (Dempsey et al., 2011), it remains to be discovered which genes with interrupted mRNA export in the bak1–3 bkk1–1 sbb1–1 or bak1–3 bkk1–1 drh1–1 triple mutants are associated with BAK1/SBB1/DRH1 regulation, and whether these genes act as specific regulators in the BAK1/SBB1/DRH1 pathway. Recent studies showed that the Arabidopsis receptor-like kinase BIR1 and the Arabidopsis copine protein BON1 physically interact with BAK1 to modulate cell death and defense responses (Gao et al., 2009; Wang et al., 2011). Our study shows that sbb1 is not able to suppress the dwarfism and cell-death phenotype of bir1 (Figure S11a–e) or bon1 (Figure S11f–j). These results indicate that BIR1 and BON1 may function independently of SBB1, suggesting that SBB1 may have some specificity in regulating celldeath pathways.

BAK1 serves as a co-receptor in BRI1-mediated BR signaling and FLS2-mediated innate immunity signaling pathways (Li et al., 2002; Chinchilla et al., 2007; Roux et al., 2011). We therefore hypothesize that BAK1 and BKK1 may function as co-receptors of an unknown RLK to regulate intracellular SA homeostasis, preventing accumulation of an excessive amount of SA under normal growth conditions (Figure 7). In the bak1–3 bkk1–1 double mutant, SArelated genes are extensively up-regulated, causing an endogenous SA burst that eventually triggers cell death. Loss-of-function mutations of SBB1 and several other members of the NUP107–160 sub-complex, and RNA-binding protein DRH1, may result in the disruption of nucleocytoplasmic trafficking, and as a consequence, SA-related transcripts accumulate in the nucleus and are not translocated to the cytoplasm for translation (Figure 7). In bak1–3 bkk1–1 sbb1–1 or other relevant triple mutants, the SA burst is effectively blocked and the cell-death process cannot be initiated (Figure 7). Future studies will focus on revealing detailed molecular mechanisms, including the specificity of NUPs and DRH1 in coordinating BAK1- and BKK1-mediated cell-death control. EXPERIMENTAL PROCEDURES Plant materials, growth conditions, genetic modifier screen and generation of double and triple mutants All of the Arabidopsis seeds used in these studies are in the Col–0 background. Plants were grown at 22°C under long-day growth conditions (16 h light/8 h dark) in a greenhouse except for those specified treatments. bak1–3 bkk1–1 was generated by genetic crossing as previously described (He et al., 2007). A pBASTA-AT2 activation-tagging binary vector was transformed into bak1–3 bkk1–1 using the flo-

Figure 7. A hypothetical model of the role of SBB1 and DRH1 in regulating BAK1- and BKK1-mediated cell-death control. In WT plants, BAK1 and BKK1 may interact with another unknown RLK to regulate normal expression of SA-related genes and maintain basal levels of endogenous SA. In the bak1 bkk1 double mutant, highly expressed SA-related genes may trigger an SA burst, resulting in cell death. However, if mRNA export is blocked, as in sbb1 and drh1 mutants, SA biosynthesis-related transcripts cannot be exported and translated. Therefore, cell death cannot be initiated.


Nucleoporins and BAK1-associated cell-death control 529 ral-dip method (Clough and Bent, 1998; Yuan et al., 2007). sbb1–1 was identified in T2 progeny that are resistant to BASTA herbicide. bak1–3 bkk1–1 sbb1–1 was crossed with Col–0 for several generations to segregate bak1–3 bkk1–1, and back-crossed with bak1–3 bkk1–1 to segregate other possible T–DNA insertions in the genome. Mutants obtained from the Arabidopsis Biological Resource Center were crossed to generate double or triple mutants. PCR and RT–PCR were performed to verify genotypes. The T–DNA lines used in these studies are sbb1–2/nup85–1 (SALK_086258C), seh1–1 (SALK_ 022717C), nup160–3 (SAIL_877_B01), nup96–1 (SALK_117966), drh1– 1 (SALK_063362C), nup133–3 (WiscdsLox354H11), nup107–1 (SALK_ 057072C), nup43–1 (SALK_000711) and sec13b–1 (SALK_045825C). The primers used for nup mutant background verification by RT–PCR are listed in Table S2.

Identification of T–DNA insertions in sbb1–1, and construction of SBB1 and NahG expression vectors Adapter ligation-mediated PCR was used to amplify the flanking genome sequences of the T–DNA from pBASTA-AT2-transformed plants (O’Malley et al., 2007). Sequencing and RT–PCR were performed in the sbb1 mutant. The full-length coding sequence or genomic DNA with the 1520 bp promoter of SBB1 were amplified by regular PCR and cloned using a Gatewayâ cloning approach (Invitrogen, www.thermofisher.com/cn/zh/home/brands/invitrogen. html). A pBASTA-35S-NahG plasmid was constructed by PCR and Gatewayâ cloning from an NahG transgenic plant.

Trypan blue staining Leaves of soil-grown seedlings were used for trypan blue staining as described previously (He et al., 2007).

Cytoplasmic/nuclear fractionation, RNA/protein isolation and detection Seven-day-old seedlings were used for cytoplasmic and nuclear fractionation and RNA and protein analyses as described previously (Wiermer et al., 2012).

Quantitative PCR analyses Two micrograms of total RNA were used for reverse transcription with M–MLV (Invitrogen, www.thermofisher.com/cn/zh/home/ brands/invitrogen.html). Real-time PCR was performed using SYBRâ Premix Ex TaqTM II (TaKaRa, www.takara.com.cn), and relative expression of genes compared to ACT2 was calculated using the DDCT method (Livak and Schmittgen, 2006). The primers used were PR1–F (50 -CATACACTCTGGTGGGCCTTA-30 ), PR1–R (50 PR2–F (50 -CGGGA CGCTAACCCACATGTTCACG-30 ), CGAGTGTGGAAAAC-30 ), PR2–R (50 -ATAGCTTTCCCTGGCCTTCT30 ), PR5–F (50 -TCACCCACAGCACAGAGACA-30 ), PR5–R (50 -CAATG CCGCTTGTGATGAAC-30 ), EDS1–F (50 -GCTTACCTAACCGAGCGCT AT-30 ), EDS1–R (50 -TGTCCGGATCGAAGAAATCT-30 ), EDS5–F (50 -G TGGCCGTTTATCCTTGTTG-30 ), EDS5–R (50 -AATGATCGTTGCTGC AGCTA-30 ), PAD4–F (50 -TCTCCACCTCAATTTCACGAT-30 ), PAD4–R (50 -GGCCAGAATTGTTCATTGCT-30 ), SID2–F (50 -GCCGCCACTGAAA GGCTAAT-30 ), SID2–R (50 -AGAAGATCGGGACGACCAAC-30 ), ACT2– F (50 -TGTGCCAATCTACGAGGGTTT-30 ) and ACT2–R (50 -TTTCCCG CTCTGCTGTTGT-30 ).

SA treatment assay Seeds were surface-sterilized and grown on half-strength Murashige and Skoog (MS) medium (pH 5.7) supplemented with 1% sucrose, 0.6% agar and various concentrations of SA, and

then placed in a growth chamber at 22°C under 16 h light/8 h dark. Two-week-old seedlings were photographed and weighed. The treatments were repeated four times, and at least ten seedlings were measured for each genotype under each treatment. Student’s t test was used to examine the significance of difference in fresh weights between WT plants and the mutants.

SA content measurements SA extraction from plants was performed as described previously with some modifications (Marek et al., 2010). Briefly, 0.1 g of 3week-old soil-grown plants were ground into fine powder in liquid nitrogen. Then 1 ml of 90% methanol was added to the ground tissues and vortexed. The mixture was centrifuged at 14 000 g for 10 min, followed by extraction with another 0.5 ml of 100% methanol. The supernatants of the extractions were combined and vacuum-dried. The residue was re-suspended using 250 ll hydrolysis buffer (0.1 M NaAC, pH 5.5). Ten units of b–glucosidase (Sigma, www.sigmaaldrich.com/) were added, and the solution was incubated at 37°C for 90 min followed by addition of 250 ll of 10% trichloroacetic acid to stop the reaction. Then 1 ml of extraction solvent (100:99:1 ethyl acetate/cyclopentane/isopropanol) was added, followed by vortexing. The organic phase was vacuumdried for HPLC analyses.

Immunoprecipitation and LC-MS/MS Total proteins were extracted from 35S-SBB1-GFP transgenic plants using extraction buffer containing 50 mM HEPES/KOH, pH 7.5, 0.15 M NaCl, 0.5% v/v Triton X–100, 0.1% v/v Tween–20 and 1% protein inhibitor cocktail (Sangon, www.sangon.com/). Immunoprecipitation was performed using a–GFP antibodies (Invitrogen). The immunoprecipitated proteins was separated by SDS–PAGE, and analyzed by (LC-MS/MS were performed by BGI, http://www.genomics.cn/), as described previously (Tamura et al., 2010).

Whole-mount in situ hybridization of mRNA Leaves of 7-day-old seedlings grown on half-strength MS medium were fixed in formaldehyde – acetic acid – ethanol solution, and poly(A)+ RNAs were hybridized with a 50 -Alexa Fluor 488-labeled oligo(dT)45 probe as described previously (Gong et al., 2005; Wiermer et al., 2012). The probed RNA signals were observed under a FluoView FV1000 confocal microscope (Olympus, www.olympusglobal.com).

DRH1 cloning and transient expression in protoplast of Arabidopsis leaves The methods for DRH1 cloning and transient expression in protoplast of Arabidopsis leaves are described in Methods S1.

Yeast two-hybrid assay The method for the yeast two-hybrid assay is described in Methods S2.

ACKNOWLEDGMENTS These studies were supported by National Basic Research Program of China grant 2011CB915401 (to J.L.), and National Natural Science Foundation of China grants 90917019, 91117008 and 91317311 (to J.L.). We are grateful to Marcel Wiermer (Michael Smith Laboratories, University of British Columbia, Vancouver, Canada) for sharing the protocol for nuclear/cytoplasmic fractiona-


530 Junbo Du et al. tion, to the Arabidopsis Biological Resource Center for providing Arabidopsis mutant seeds, and to Ben H. Holt III (Department of Botany and microbiology, University of Oklahoma, Norman) for the NahG transgenic plants.

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article. Figure S1. Sequence alignment showing that Arabidopsis SBB1 is similar to NUP85 homologs from rice, human and yeast. Figure S2. RT–PCR analyses showing the genetic backgrounds of two T–DNA insertion alleles of sbb1. Figure S3. The genetic backgrounds of pSBB1::SBB1g transgenic plants and the corresponding mutants were verified by RT–PCR analyses. Figure S4. bak1–3 bkk1–1 sbb1 is less sensitive to SA than bak1–3 bkk1–1. Figure S5. Loss-of-function mutations of NUP133, NUP107, NUP43 and SEC13B cannot fully suppress the cell-death phenotype of bak1–3 bkk1–1. Figure S6. RT–PCR analyses showing the genetic backgrounds of various mutants. Figure S7. DRH1 localizes in the nucleus. Figure S8. DRH1 physically interacts with NUP107 by in a yeast two-hybrid assay. Figure S9. mRNA export is affected in nup133, nup107 and nup43 mutants but not in sec13b. Figure S10. Nuclear accumulation of the mRNAs of several genes that regulate SA biosynthesis is not obviously affected in the sbb1–1 and drh1–1 mutants compared with Col–0. Figure S11. Cell death of bir1 and bon1 is not be suppressed by the SBB1 mutation. Table S1. Segregation ratios in F2 progeny of bak1–3 bkk1–1 sbb1– 1 9 bak1–3 bkk1–1. Table S2. Primers used for RT–PCR analyses of NUP genes. Methods S1. DRH1 cloning and transient expression in protoplast of Arabidopsis leaves. Methods S2. Yeast two-hybrid assay. Appendix S1. A proteomic approach was used to discover new components involved in SBB1-controlled cell-death signaling pathways.

REFERENCES Alber, F., Dokudovskaya, S., Veenhoff, L.M. et al. (2007) The molecular architecture of the nuclear pore complex. Nature, 450, 695–701. Albrecht, C., Russinova, E., Hecht, V., Baaijens, E. and de Vries, S. (2005) The Arabidopsis thaliana SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASES1 and 2 control male sporogenesis. Plant Cell, 17, 3337–3349. Chinchilla, D., Zipfel, C., Robatzek, S., Kemmerling, B., Nurnberger, T., Jones, J.D.G., Felix, G. and Boller, T. (2007) A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence. Nature, 448, 497–500. Chinnusamy, V., Gong, Z. and Zhu, J.K. (2008) Nuclear RNA export and its importance in abiotic stress responses of plants. In Nuclear pre-MRNA Processing in Plants (Reddy, A.N. and Golovkin, M., eds). Berlin/Heidelberg: Springer, pp. 235–255. Clough, S.J. and Bent, A.F. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743. Colcombet, J., Boisson-Dernier, A., Ros-Palau, R., Vera, C.E. and Schroeder, J.I. (2005) Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASES1 and 2 are essential for tapetum development and microspore maturation. Plant Cell, 17, 3350–3361.

Dempsey, D.M.A., Vlot, A.C., Wildermuth, M.C. and Klessig, D.F. (2011) Salicylic acid biosynthesis and metabolism. Arabidopsis Book, 9, e0156. Dong, C., Hu, X., Tang, W., Zheng, X., Kim, Y.S., Lee, B. and Zhu, J. (2006) A putative Arabidopsis nucleoporin, AtNUP160, is critical for RNA export and required for plant tolerance to cold stress. Mol. Cell. Biol. 26, 9533– 9543. Du, J., Yin, H., Zhang, S., Wei, Z., Zhao, B., Zhang, J., Gou, X., Lin, H. and Li, J. (2012) Somatic embryogenesis receptor kinases control root development mainly via brassinosteroid-independent actions in Arabidopsis thaliana. J. Integr. Plant Biol. 54, 388–399. Gaffney, T., Friedrich, L., Vernooij, B., Negrotto, D., Nye, G., Uknes, S., Ward, E., Kessmann, H. and Ryals, J. (1993) Requirement of salicylic acid for the induction of systemic acquired resistance. Science, 261, 754–756. Gao, M., Wang, X., Wang, D. et al. (2009) Regulation of cell death and innate immunity by two receptor-like kinases in Arabidopsis. Cell Host Microbe, 6, 34–44. Germain, H., Qu, N., Cheng, Y.T. et al. (2010) MOS11: a new component in the mRNA export pathway. PLoS Genet. 6, e1001250. Gong, Z., Dong, C., Lee, H., Zhu, J., Xiong, L., Gong, D., Stevenson, B. and Zhu, J. (2005) A DEAD box RNA helicase is essential for mRNA export and important for development and stress responses in Arabidopsis. Plant Cell, 17, 256–267. Gou, X., Yin, H., He, K., Du, J., Yi, J., Xu, S., Lin, H., Clouse, S.D. and Li, J. (2012) Genetic evidence for an indispensable role of somatic embryogenesis receptor kinases in brassinosteroid signaling. PLoS Genet. 8, e1002452. Guan, Q., Wu, J., Zhang, Y., Jiang, C., Liu, R., Chai, C. and Zhu, J. (2013) A DEAD box RNA helicase is critical for pre-mRNA splicing, cold-responsive gene regulation, and cold tolerance in Arabidopsis. Plant Cell, 25, 342–356. Hajouj, T., Michelis, R. and Gepstein, S. (2000) Cloning and characterization of a receptor-like protein kinase gene associated with senescence. Plant Physiol. 124, 1305–1314. He, K., Gou, X., Yuan, T., Lin, H., Asami, T., Yoshida, S., Russell, S.D. and Li, J. (2007) BAK1 and BKK1 regulate brassinosteroid-dependent growth and brassinosteroid-independent cell-death pathways. Curr. Biol. 17, 1109–1115. Hecht, V., Vielle-Calzada, J.-P., Hartog, M.V., Schmidt, E.D.L., Boutilier, K., Grossniklaus, U. and de Vries, S.C. (2001) The Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1 gene is expressed in developing ovules and embryos and enhances embryogenic competence in culture. Plant Physiol. 127, 803–816. Heese-Peck, A. and Raikhel, N. (1998) The nuclear pore complex. Plant Mol. Biol. 38, 145–162. Horn, M.A. and Walker, J.C. (1994) Biochemical properties of the autophosphorylation of RLK5, a receptor-like protein kinase from Arabidopsis thaliana. Biochim. Biophys. Acta, 1208, 65–74. Hothorn, M., Belkhadir, Y., Dreux, M., Dabi, T., Noel, J.P., Wilson, I.A. and Chory, J. (2011) Structural basis of steroid hormone perception by the receptor kinase BRI1. Nature, 474, 467–471. Jacob, Y., Mongkolsiriwatana, C., Veley, K.M., Kim, S.Y. and Michaels, S.D. (2007) The nuclear pore protein AtTPR is required for RNA homeostasis, flowering time, and auxin signaling. Plant Physiol. 144, 1383–1390. Kanamori, N., Madsen, L.H., Radutoiu, S. et al. (2006) A nucleoporin is required for induction of Ca2+ spiking in legume nodule development and essential for rhizobial and fungal symbiosis. Proc. Natl Acad. Sci. USA, 103, 359–364. Kemmerling, B., Schwedt, A., Rodriguez, P. et al. (2007) The BRI1-associated kinase 1, BAK1, has a brassinolide-independent role in plant celldeath control. Curr. Biol. 17, 1116–1122. Lee, J.-Y., Lee, H.-S., Wi, S.-J., Park, K.Y., Schmit, A.-C. and Pai, H.-S. (2009) Dual functions of Nicotiana benthamiana Rae1 in interphase and mitosis. Plant J. 59, 278–291. Li, J. (2010) Multi-tasking of somatic embryogenesis receptor-like protein kinases. Curr. Opin. Plant Biol. 13, 509–514. Li, J., Wen, J., Lease, K.A., Doke, J.T., Tax, F.E. and Walker, J.C. (2002) BAK1, an Arabidopsis LRR receptor-like protein kinase, interacts with BRI1 and modulates brassinosteroid signaling. Cell, 110, 213–222. Li, D., Liu, H., Zhang, H., Wang, X. and Song, F. (2008) OsBIRH1, a DEADbox RNA helicase with functions in modulating defence responses


Nucleoporins and BAK1-associated cell-death control 531 against pathogen infection and oxidative stress. J. Exp. Bot. 59, 2133– 2146. Livak, K.J. and Schmittgen, T.D. (2006) Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2CT Method. Methods, 25, 402–408. Lu, Q., Tang, X., Tian, G. et al. (2010) Arabidopsis homolog of the yeast TREX–2 mRNA export complex: components and anchoring nucleoporin. Plant J. 61, 259–270. Marek, G., Carver, R., Ding, Y., Sathyanarayan, D., Zhang, X. and Mou, Z. (2010) A high-throughput method for isolation of salicylic acid metabolic mutants. Plant Methods, 6, 21. Meier, I. (2012) mRNA export and sumoylation – lessons from plants. Biochim. Biophys. Acta, 1819, 531–537. Nam, K.H. and Li, J. (2002) BRI1/BAK1, a receptor kinase pair mediating brassinosteroid signaling. Cell, 110, 203–212. Oh, M.-H., Wang, X., Kota, U., Goshe, M.B., Clouse, S.D. and Huber, S.C. (2009) Tyrosine phosphorylation of the BRI1 receptor kinase emerges as a component of brassinosteroid signaling in Arabidopsis. Proc. Natl Acad. Sci. USA, 106, 658–663. Okanami, M., Meshi, T. and Iwabuchi, M. (1998) Characterization of a DEAD box ATPase/RNA helicase protein of Arabidopsis thaliana. Nucleic Acids Res. 26, 2638–2643. O’Malley, R.C., Alonso, J.M., Kim, C.J., Leisse, T.J. and Ecker, J.R. (2007) An adapter ligation-mediated PCR method for high-throughput mapping of T-DNA inserts in the Arabidopsis genome. Nat. Protoc. 2, 2910–2917. Parry, G., Ward, S., Cernac, A., Dharmasiri, S. and Estelle, M. (2006) The Arabidopsis SUPPRESSOR OF AUXIN RESISTANCE proteins are nucleoporins with an important role in hormone signaling and development. Plant Cell, 18, 1590–1603. Roux, M., Schwessinger, B., Albrecht, C., Chinchilla, D., Jones, A., Holton, € r, M., de Vries, S. and Zipfel, C. (2011) The AraN., Malinovsky, F.G., To bidopsis leucine-rich repeat receptor-like kinases BAK1/SERK3 and BKK1/ SERK4 are required for innate immunity to hemibiotrophic and biotrophic pathogens. Plant Cell, 23, 2440–2455. Saito, K., Yoshikawa, M., Yano, K. et al. (2007) NUCLEOPORIN85 is required for calcium spiking, fungal and bacterial symbioses, and seed production in Lotus japonicus. Plant Cell, 19, 610–624. Schmidt, E.D., Guzzo, F., Toonen, M.A. and de Vries, S.C. (1997) A leucinerich repeat containing receptor-like kinase marks somatic plant cells competent to form embryos. Development, 124, 2049–2062. She, J., Han, Z., Kim, T. et al. (2011) Structural insight into brassinosteroid perception by BRI1. Nature, 474, 472–476.

Shiu, S.H. and Bleecker, A.B. (2001) Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases. Proc. Natl Acad. Sci. USA, 98, 10763–10768. Sun, Y., Han, Z., Tang, J., Hu, Z., Chai, C., Zhou, B. and Chai, J. (2013) Structure reveals that BAK1 as a co-receptor recognizes the BRI1-bound brassinolide. Cell Res. 23, 1326–1329. Tamura, K. and Hara-Nishimura, I. (2013) The molecular architecture of the plant nuclear pore complex. J. Exp. Bot. 64, 823–832. Tamura, K., Fukao, Y., Iwamoto, M., Haraguchi, T. and Hara-Nishimura, I. (2010) Identification and characterization of nuclear pore complex components in Arabidopsis thaliana. Plant Cell, 22, 4084–4097. Tseng, S.S.I., Weaver, P.L., Liu, Y., Hitomi, M., Tartakoff, A.M. and Chang, T.-H. (1998) Dbp5p, a cytosolic RNA helicase, is required for poly(A)+ RNA export. EMBO J. 17, 2651–2662. Wang, Z., Meng, P., Zhang, X., Ren, D. and Yang, S. (2011) BON1 interacts with the protein kinases BIR1 and BAK1 in modulation of temperaturedependent plant growth and cell death in Arabidopsis. Plant J. 67, 1081– 1093. Wiermer, M., Cheng, Y.T., Imkampe, J., Li, M., Wang, D., Lipka, V. and Li, X. (2012) Putative members of the Arabidopsis Nup107–160 nuclear pore sub-complex contribute to pathogen defense. Plant J. 70, 796–808. Xu, X.M. and Meier, I. (2008) The nuclear pore comes to the fore. Trends Plant Sci. 13, 20–27. Xu, X.M., Rose, A., Muthuswamy, S., Jeong, S.Y., Venkatakrishnan, S., Zhao, Q. and Meier, I. (2007) NUCLEAR PORE ANCHOR, the Arabidopsis homolog of Tpr/Mlp1/Mlp2/Megator, is involved in mRNA export and SUMO homeostasis and affects diverse aspects of plant development. Plant Cell, 19, 1537–1548. Yuan, T., Fujioka, S., Takatsuto, S., Matsumoto, S., Gou, X., He, K., Russell, S.D. and Li, J. (2007) BEN1, a gene encoding a dihydroflavonol 4–reductase (DFR)-like protein, regulates the levels of brassinosteroids in Arabidopsis thaliana. Plant J. 51, 220–233. Zhang, Y. and Li, X. (2005) A putative nucleoporin 96 is required for both basal defense and constitutive resistance responses mediated by suppressor of npr1–1, constitutive 1. Plant Cell, 17, 1306–1316. Zipfel, C., Kunze, G., Chinchilla, D., Caniard, A., Jones, J.D.G., Boller, T. and Felix, G. (2006) Perception of the bacterial PAMP EF–Tu by the receptor EFR restricts Agrobacterium-mediated transformation. Cell, 125, 749–760. Zuccolo, M., Alves, A., Galy, V. et al. (2007) The human Nup107–160 nuclear pore subcomplex contributes to proper kinetochore functions. EMBO J. 26, 1853–1864.
