The Arabidopsis BAP1 and BAP2 genes are general inhibitors of programmed cell death

Abstract

Here we identify the BAP1 and BAP2 genes of Arabidopsis (Arabidopsis thaliana) as general inhibitors of programmed cell death (PCD) across the kingdoms. These two homologous genes encode small proteins containing a calcium-dependent phospholipid-binding C2 domain. BAP1 and its functional partner BON1 have been shown to negatively regulate defense responses and a disease resistance gene SNC1. Genetic studies here reveal an overlapping function of the BAP1 and BAP2 genes in cell death control. The loss of BAP2 function induces accelerated hypersensitive responses but does not compromise plant growth or confer enhanced resistance to virulent bacterial or oomycete pathogens. The loss of both BAP1 and BAP2 confers seedling lethality mediated by PAD4 and EDS1, two regulators of cell death and defense responses. Overexpression of BAP1 or BAP2 with their partner BON1 inhibits PCD induced by pathogens, the proapoptotic gene BAX, and superoxide-generating paraquat in Arabidopsis or Nicotiana benthamiana. Moreover, expressing BAP1 or BAP2 in yeast (Saccharomyces cerevisiae) alleviates cell death induced by hydrogen peroxide. Thus, the BAP genes function as general negative regulators of PCD induced by biotic and abiotic stimuli including reactive oxygen species. The dual roles of BAP and BON genes in repressing defense responses mediated by disease resistance genes and in inhibiting general PCD has implications in understanding the evolution of plant innate immunity.

Figure 1.

BAP2 has an overlapping function with BAP1. A, Alignment of the amino acid sequences of BAP1 and BAP2. Identical residues are shaded in black and similar residues are shaded in gray. The C2 domains are underlined. B, Expression patterns of the BAP1 and BAP2 genes. Shown are representative GUS stainings of transgenic plants containing pBAP1∷GUS and pBAP2∷GUS at the seedling and flowering stages. Note expression in roots, young leaves, stems, and floral buds. C, BAP2 overexpression largely rescues the bap1-1 defect. bap1-1 has a dwarf phenotype compared to the wild-type Col. Shown on the right are three independent transgenic lines carrying p35S∷BAP2 in bap1-1. D, BAP1 and BAP2 interact with BON1, BON2, and BON3 in the yeast two-hybrid system. GBD:BAP1 and GBD:BAP2 are fusions of BAP1 and BAP2 with the GAL4 DNA-binding domain, respectively. GAD:BON1A, GAD:BON2A, and GAD:BON3A are fusions of the A domains of BON1, BON2, and BON3 with the GAL4 activation domain, respectively. Yeast cells containing both the GAD and GBD constructs were patched on SC medium selecting for protein-protein interaction 3 d after streaking. Note the combinations of the BAP proteins with the BON proteins, but not with the GAD vector controls, grow on this medium. E, bap2-4 has no obvious growth defects. Shown are 3-week-old seedlings of the wild-type Col, bap1-1, and bap2-4. F and G, bap2 has an altered HR in response to Pst DC3000 avrRpt2. At 8 hpi, most of the bap2 leaves but not the Col leaves exhibited HR indicated by white arrows (F). The percentage of leaves exhibiting HR is shown during the course of 30 h after inoculation (G). Replicated experiments yielded a similar alteration. H, The bap1bap2 double mutant is seedling lethal. Shown are seedlings several days after germination. The two on the left are the bap1 single mutants and the two on the right are the bap1bap2 double mutants. I, bap1 and bap2 have dominant interactions. Shown are plants after bolting. bap1bap2/+ and bap1/+bap2 have more severe phenotype than the bap1 and the bap2 single mutants, respectively. Insert shows a bended and yellow inflorescence stem in bap1/+bap2.

Figure 2.

Cell death occurs in bap1 and bap2 mutant combinations. A, Trypan blue staining of representative leaves of Col, bon1, and bap1bap2/+. In contrast to wild-type Col and the bap1 and bap2 single mutants (data not shown), bap1bap2/+ has a strong trypan blue staining similar to bon1. B, Autofluorescence of leaves from Col, bap1, bap2, bon1, bap1bap2/+, bap1/+bap2, bap1bap2pad4, and bap1bap2eds1. bap1bap2/+ has the strongest autofluorescence while the bap1 and bap2 single mutants have no significant amount. Autofluorescence is absent in bap1bap2pad4 and bap1bap2eds1. C, Accumulation of H₂O₂ in various mutants. Top section shows DAB staining of 2-week-old plants grown under constant lights and the bottom section shows DAB staining of individual leaves from plants grown under 12 h light and 12 h of darkness. Note the weak staining in bap1, a strong staining in bap1bap2/+, bap1/+bap2, but no staining in wild-type Col, bap2, bap1pad4, or bap1bap2pad4. D, Both pad4 and eds1 rescued the lethal phenotype of bap1bap2. Shown are 3-week-old seedlings of the wild-type Col, bap1bap2pad4, and bap1bap2eds1 grown at 22°C. bap1bap2 was dead at this stage. E, High temperature partially rescued the bap1bap2 mutant phenotype. Shown are 3-week-old seedlings of the wild-type Col, bap1/+bap2, bap1bap2/+, and bap1bap grown at 28°C. Note bap1bap2/+ and bap1/+bap2 are wild-type looking and bap1bap2 is surviving but yellowing at this stage.

Figure 3.

The bap2 mutant does not have an enhanced disease resistance. A, bap2 is susceptible to Pseudomonas syringe pv tomato DC3000. Plants were infected with Pst DC3000 and the amount of bacterial growth in the leaves was determined at 0 and 4 d post inoculation (dpi). Bacterial growth was inhibited in bap1 but not in bap2 compared to the wild-type Col. bap1/+bap2 and bap1bap2/+ had approximately the same amount of growth as bap2 and bap1, respectively. bap1bap2pad4 supports the same amount of bacterial growth as the pad4 single mutant. B, bap2 is susceptible to virulent growth of Hyalopernonspora parasitica. H. parasitica Noco2 strain was used to infect Col, bap1, and bap2. Shown is the distribution of the number of sporangiophores per leaf formed a week later for each genotype. In contrast to bap1, bap2 had the same amount of sporangiophore formation as the wild-type Col.

Figure 4.

Overexpression of BAP1 and BAP2 suppresses PCD. A, BAP1 and BON1 together suppress HR triggered by Pst DC3000 harboring avrRpt2 and avrRpm1. DC3000 strains were inoculated on Arabidopsis leaves to induce HR indicated by the collapse of cells (marked by white arrows). p35S∷BAP1, p35S∷BON1, or an empty vector were agroinfiltrated together with DC3000. Shown are leaves 8 hpi for avrRpm1 and 17 hpi for avrRpt2. Combination of p35S∷BAP1 and p35S∷BON1 significantly inhibits HR induced by both AvrRpt2 and AvrRpm1. p35S∷BAP1 appears to have a weak suppression of HR induced by AvrRpt2, but it was not consistently observed. B, Both BON1 and BAP1 inhibit HR induced by the R protein Rx in N. benthamiana. Rx and its effector CP were agroinfiltrated on leaves (marked by red circles) to induce HR. Except for the control area, all other areas were coagroinfiltrated with BON1, BAP1, or the empty vector singly or combined with the same total amount of Agrobacterium cells in each infiltrated area. Shown is a representative leaf at 60 hpi. Note the cell collapse in the vector and the control areas (indicated by white arrows). C, BAP1 and BAP2 suppress Bax-induced cell death in N. benthamiana. Leaf areas marked by black circles were agroinfiltrated with pDEX∷Bax and Bax expression was induced by spraying the whole leaf with DEX. These areas were also agroinfiltrated with BAP1, BAP2, BON1, and the empty vector either singly or combined with the same total amount of agrobacteria for each area. Shown is a representative leaf at 72 hpi. Cell death occurred in area coinfiltrated with the vector control, BON1, and BAP1 singly (indicated by white arrows). Cell death is slightly suppressed by BAP2 and is greatly suppressed by coexpression of BAP1 or BAP2 together with BON1. D, BAP1 overexpression confers paraquat resistance. Leaf discs from the wild-type Col and 35S∷BAP1 transgenic plants were floated on paraquat solution (4 μm) for 2 d before pictures were taken. E, Yeast strains transformed with BAP1, BAP2, or the empty vector pAD4M were treated with 10 mm of H₂O₂ or water (mock). Shown is the amount of live cells 12 h after treatment in two BAP1, two BAP2, and one vector transformants from three replicates. BAP1 and especially BAP2 greatly increased the survival rates of yeast cells treated with H₂O₂.