bZIP10-LSD1 antagonism modulates basal defense and cell death in Arabidopsis following infection

Abstract

Plants use sophisticated strategies to balance responses to oxidative stress. Programmed cell death, including the hypersensitive response (HR) associated with successful pathogen recognition, is one cellular response regulated by reactive oxygen in various cellular contexts. The Arabidopsis basic leucine zipper (bZIP) transcription factor AtbZIP10 shuttles between the nucleus and the cytoplasm and binds consensus G- and C-box DNA sequences. Surprisingly, AtbZIP10 can be retained outside the nucleus by LSD1, a protein that protects Arabidopsis cells from death in the face of oxidative stress signals. We demonstrate that AtbZIP10 is a positive mediator of the uncontrolled cell death observed in lsd1 mutants. AtbZIP10 and LSD1 act antagonistically in both pathogen-induced HR and basal defense responses. LSD1 likely functions as a cellular hub, where its interaction with AtbZIP10 and additional, as yet unidentified, proteins contributes significantly to plant oxidative stress responses.

Figure 1

AtbZIP10 shuttles between the nucleus and the cytoplasm in plant protoplasts. (A) Bright-field (I), epifluorescence (II) and confocal laser scanning images (III) of parsley protoplasts expressing AtbZIP10-GFP or AtbZIP63-GFP. Images in I and II show identical protoplasts, and confocal images (III) are from independent cells. c, cytoplasm; n, nucleus; v, vacuole. Scale bars, 20 μm. Confocal images are false colored red. (B) Confocal laser scanning images of transiently transformed tobacco BY-2 protoplasts expressing GFP-NLS-CHS-NES (I), AtbZIP10-GFP (II) or GFP (III). Protoplasts, 16 h after transformation, were treated for 4 h with 2 μM of the nuclear export inhibitor LMB (+LMB) or mock treated (−LMB). c, cytoplasm; n, nucleus; nl, nucleolus; v, vacuole. Scale bar, 30 μm.

Figure 2

LSD1 expression prevents AtbZIP10 nuclear function in yeast. (A) AtbZIP10 and AtbZIP63 are transported into the nucleus in yeast. Schematic representation of the yeast NTT system (left panel). AtbZIP10 and AtbZIP63 were expressed in yeast (strain EGY48) as NES-BD_LEXA-AD_GAL4 (LexAD) fusions. Nuclear accumulation of LexAD-AtbZIP10 and LexAD-AtbZIP63, caused by the bZIP factor's NLS, induces the Leu reporter gene activity, enabling the growth of yeast cells on media without Leu (leu^–, right panel). (B) Genetic screening for proteins that retain AtbZIP10 in the yeast cytosol. One of 11 colonies showed reproducibly reduced growth on CSM-H,U,L and contained a cDNA encoding full-length LSD1 (right panel). AtbZIP10/control: yeast EGY48 coexpressing LexAD-AtbZIP10 and a control protein. (C) Cytoplasmic retention activity of LSD1 in yeast is specific for AtbZIP10. Constructs encoding the indicated proteins were cotransformed into yeast strain EGY48. Nuclear localization was determined by comparative growth assay on plasmid-selective CSM-H,U media or on CSM-H,U,L media selective for nuclear accumulation (see Materials and methods). (D) LSD1 localizes to the yeast cytoplasm. The constructs encoding the indicated LexAD fusions were transformed in yeast strain EGY48. Nuclear localization was determined by comparative growth assay on CSM-H and CSM-H,L as described in (C).

Figure 3

LSD1 interacts specifically with AtbZIP10, and inhibits the in vitro DNA binding capacity of AtbZIP10. (A) Specific interaction of LSD1 with AtbZIP10 tested in the yeast two-hybrid system. The indicated Gal4 DNA binding (BD) and Gal4 transactivation (AD) fusion constructs were cotransformed into PJ69-4A. Activity of reporter genes was determined either by growth on interaction selective CSM-L,W,A media or by quantitative β-galactosidase activity assay (LacZ). Error bars are s.d. (n>3). (B) Identification of the LSD1 domain required for interaction with AtbZIP10. The BD constructs encoding the indicated LSD1 polypeptides and the AD construct of full-length AtbZIP10 were cotransformed into EGY48[p2op-lacZ]. Interaction of the fusion proteins was tested using a β-galactosidase assay. 1, 2, 3 indicate the three zinc fingers, and PPP a proline-rich motif, in LSD1. (C) Identification of the AtbZIP10 domain required for interaction with LSD1. The AD constructs encoding the indicated AtbZIP10 polypeptides and the BD construct of full-length LSD1 were cotransformed and examined for protein–protein interaction as described in (B). NLS/basic indicates the NLS-containing DNA-binding domain, ZIP the leucine zipper and PPP the proline-rich transactivation domain of AtbZIP10. (D) Coomassie brilliant blue staining of SDS–PAGE-separated (His)₆-tagged AtbZIP10, AtbZIP63 and LSD1 used in this study. Recombinant proteins were expressed in E. coli and purified by Ni-NTA affinity chromatography. (E) The DNA binding capacity of AtbZIP10 is inhibited by LSD1. EMSA of AtbZIP10 following coincubation with increasing amounts of LSD1 (black triangle) either before (LSD1 → DNA) or after (DNA → LSD1) the addition of radioactively labeled C-box DNA (left panels). As a control, the same experiment (LSD1 → DNA) was performed with AtbZIP63 (right panel). AtbZIP/DNA complexes are indicated.

Figure 4

LSD1 can alter AtbZIP10 distribution and the two proteins can interact in planta. (A) F1 plants from (lsd1-2 [Est-AtbZIP10-HA] × Col-0) (left) or (lsd1-2 [Est-AtbZIP10-HA] × lsd1-2) (right) were sprayed with 20 μM estradiol. Tissue was harvested at 0, 60, 90 and 180 min after application of estradiol. Protein was extracted in sucrose buffer and fractionated. Data from one of two independent experiments are shown. (B) F1 plants from (35S-AtbZIP10-HA × Est-LSD1-myc) were sprayed with 20 μM estradiol. Tissue was harvested at 0, 24 and 48 h after application of estradiol. Protein was extracted in sucrose buffer and fractionated. Relative amounts of AtbZIP10 protein in the soluble fraction or the microsomal and nucleus-enriched fractions were determined using Image J and are presented as percent of total AtbZIP10 protein at each time point. One of three independent experiments is shown. (A, B) T, total extract; S, soluble fraction; M, microsomal and nucleus-enriched fraction; α-HA, detects HA-epitope-tagged AtbZIP10 protein; α-myc, myc-epitope-tagged LSD1 protein; α-APX, soluble ascorbate peroxidase; α-RD28, membrane-specific RD28 marker; α-H3, nuclear histone H3 protein; min, minutes after application; hpa, hours post estradiol application. (C) Arabidopsis protoplasts were transiently transformed with P_35S:AtbZIP10-GFP or P_35S-AtbZIP63-GFP either alone (upper row) or in combination with P_LSD1:LSD1 (lower row). One day after transfection, protoplasts were analyzed by epifluorescence microscopy (see text). One of two independent experiments is shown. Representative images are shown. n, nucleus; c, cytoplasm; v, vacuole. Scale bars, 20 μm. (D) BiFC using AtbZIP10 and LSD1 in N. benthamiana epidermal leaf cells. Leaves were infiltrated with mixtures of Agrobacterium suspensions carrying plasmids encoding the indicated C-terminal (YFP^C) or N-terminal (YFP^N) YFP fusions of AtbZIP10, LSD1 and, as a negative control, AtbZIP63. One to two days after infiltration, the epidermal cells were analyzed by epifluorescence microscopy. An infiltrated epidermal area of 1–2 cm² was scanned for cells showing a BiFC signal (see text). The experiments were repeated three times with similar outcomes. Representative images are shown. n, nucleus; c, cytoplasm; v, vacuole. Scale bars, 60 μm.

Figure 5

AtbZIP10 is a positive regulator of runaway cell death in lsd1. Five-week-old plants of the genotypes denoted at right were sprayed with 150 μM BTH and tissues were harvested at either 64 hpa (A) or 18 hpa (B) and processed as described in Materials and methods. Error bars represent 2 × standard error. The experiment was repeated two times. hpa, hours post application.

Figure 6

AtbZIP10 and LSD1 antagonistically control pathogen-induced cell death and disease resistance responses. (A, B) Twelve-day-old seedlings were inoculated with 5 × 10⁴ spores/ml of the avirulent Hp isolate Cala2. Cotyledons were stained with Trypan blue at 5 dpi. (A) Presentation of HR (hypersensitive response; left panel), scale bar=100 μm; TN (trailing necrosis; middle panel), scale bar=250 μm; and FH (free hyphae; right panel), scale bar=100 μm. h denotes an Hp haustorium. (B) One randomly chosen interaction site per cotyledon from 87 to 104 interaction sites per genotype was microscopically evaluated and classified as HR, TN or FH. Control genotypes are indicated in black; (+Est) indicates treatment with 20 μM estradiol 24 h before inoculation with Hp. Three separate experiments were performed. (C, D) Twelve-day-old seedlings were inoculated with 5 × 10⁴ spores/ml of the virulent Hp isolate Emco5. Sporangiophores per cotyledon were determined at 5 dpi (C) or 4 dpi (D). Cotyledons were classified as supporting no sporulation (0 sporangiophores), light sporulation (classes 1–5 and 6–10 sporangiophores/cotyledon), medium sporulation (class 11–20 sporangiophores/cotyledon) or heavy sporulation (>20 sporangiophores/cotyledon). Sporangiophores were counted on 100 cotyledons per genotype. Controls are indicated in black, and mutant and transgenic genotypes in red. The experiment was performed three (C) or two (D) times. For ^* in (C), see Materials and methods. For (B–D), we used standard contingency table analyses to make sequential pairwise comparisons between relevant genotypes designated alphabetically at top (Materials and methods; Supplementary Tables 1, 2 and 3). Significant comparisons are listed at right.