Aspartyl Protease-Mediated Cleavage of BAG6 Is Necessary for Autophagy and Fungal Resistance in Plants

Abstract

The Bcl-2-associated athanogene (BAG) family is an evolutionarily conserved group of cochaperones that modulate numerous cellular processes. Previously we found that Arabidopsis thaliana BAG6 is required for basal immunity against the fungal phytopathogen Botrytis cinerea. However, the mechanisms by which BAG6 controls immunity are obscure. Here, we address this important question by determining the molecular mechanisms responsible for BAG6-mediated basal resistance. We show that Arabidopsis BAG6 is cleaved in vivo in a caspase-1-like-dependent manner and via a combination of pull-downs, mass spectrometry, yeast two-hybrid assays, and chemical genomics, we demonstrate that BAG6 interacts with a C2 GRAM domain protein (BAGP1) and an aspartyl protease (APCB1), both of which are required for BAG6 processing. Furthermore, fluorescence and transmission electron microscopy established that BAG6 cleavage triggers autophagy in the host that coincides with disease resistance. Targeted inactivation of BAGP1 or APCB1 results in the blocking of BAG6 processing and loss of resistance. Mutation of the cleavage site blocks cleavage and inhibits autophagy in plants; disease resistance is also compromised. Taken together, these results identify a mechanism that couples an aspartyl protease with a molecular cochaperone to trigger autophagy and plant defense, providing a key link between fungal recognition and the induction of cell death and resistance.

Figure 1.

Overexpression of BAG6 in Arabidopsis Induces Lesion Mimic Development. Arabidopsis bag6-1 T-DNA insertion mutant was transformed with N-terminal HA- or YFP-tagged BAG6 under either 35S promoter or native promoter (NP) by the floral dip method. Stable transgenic plants harboring BAG6 driven by the 35S promoter (35S:HA-BAG6 and 35S:YFP-BAG6) exhibited apparent lesion mimics on leaves ([D] and [F]), while plants expressing BAG6 driven by the native promoter (NP:HA-BAG6 and NP:YFP-BAG6) ([C] and [H]) were similar to wild-type Col-0 and bag6-1 mutant ([A] and [B]). Overexpression of a cleavage-deficient form of BAG6, BAG6^D760A, driven by 35S promoter (35S:HA-BAG6^D760A and 35S:YFP-BAG6 ^D760A), abolished the lesion mimic phenotype in the bag6 mutant background ([E] and [G]). Representative plants were photographed. White circles show the typical lesion mimic phenotype.

Figure 2.

BAG6 Is Cleaved by Human Caspase-1 in Vitro and by Pant Caspase-1-Like Protease Activity in Vivo. (A) Structure of BAG6, showing the conserved BAG domain, nuclear localization signal (NLS) and calmodulin binding motif (CaM). The arrow indicates the amino acid position (amino acid 760) cleaved by the caspase-1 protease. aa, amino acid. (B) Arabidopsis BAG6 is cleaved by human caspase-1 in vitro. HA-tagged full-length BAG6 and BAG6^D760A under 35S promoter were transiently expressed in N. benthamiana for 3 d. Equal amounts of purified HA-BAG6 (10 μg) were treated with recombinant human caspase-1 (2 units) at 37°C for 2 h and analyzed by immunoblotting with anti-HA antibody. CBS, Coomassie blue staining (C) BAG6 is cleaved by plant caspase-1-like protease activity in vivo. Proteins were extracted from Arabidopsis plants expressing HA-BAG6 and HA-BAG6^D760A under the 35S promoter or native promoter in response to B. cinerea 24 h after inoculation and subjected to immunoblotting using anti-HA antibody (left panel). Proteins from leaves with or without lesion mimics in Arabidopsis plants expressing HA-BAG6 under 35S promoter were monitored for BAG6 cleavage using anti-HA antibody (right panel). Equal loading was confirmed by SDS-PAGE and Coomassie blue staining.

Figure 3.

The Processing of BAG6 Is Required for Resistance to B. cinerea. (A) Agar plugs containing actively growing cultures of B. cinerea were inoculated onto leaves of Arabidopsis plants. Representative images were taken 48 h after inoculation. (B) Lesion diameters were measured at 24 and 48 h after inoculation. Data represent means ± sd from three replicates.

Figure 4.

The Cleavage of BAG6 by Caspase-1-Like Activity Is Abolished by Aspartyl Protease Activity. N. benthamiana leaves transiently expressing HA-fused Arabidopsis BAG6 under the 35S promoter were infiltrated with 200 μg/mL chitin (to induce BAG6 cleavage), in combination with individual protease inhibitor (1 μM pepstatin, 1 mM aprotitin, 0.5 mM bestatin, 1 mM leupeptin, 10 μM E-64, and 5 mM N-ethylmaleimide) 30 min before harvesting. BAG6 cleavage was detected by anti-HA immunoblotting. Equal loading was confirmed by SDS-PAGE and Coomassie blue staining (CBS).

Figure 5.

BAG6 Interacts with a GRAM Domain-Containing Protein (BAGP1) and an Aspartyl Protease (APCB1) in Yeast and in Planta. (A) Yeast two-hybrid assay was used to determine the interactions of BAG6 with BAGP1 and APCB1. pGADT7-T and pGBKT7-53, and pGADT7-T and pGBKT-Lam were used as positive CK (+) and negative CK (–), respectively. (B) BAG6, BAGP1, and APCB1 interact in planta. 35S promoter-driven-HA-BAG6 was coexpressed with FLAG-BAGP1 and Myc-APCB1 in Arabidopsis Col-0 protoplasts. Co-IP was performed with an anti-HA antibody (IP) and proteins analyzed using immunoblotting with either anti-FLAG or anti-Myc antibody (IB).

Figure 6.

BAGP1 and APCB1 Are Required for Disease Resistance and BAG6 Cleavage. (A) Detached leaves of wild-type Col-0, bag6-1, bagp1-1, and apcb1-1 mutants were agar plug inoculated with B. cinerea. Lesion development was monitored and representative photographs were taken 48 h after inoculation. (B) Lesion diameters were measured at 24 and 48 h after inoculation. Data represent means ± sd from three replicates. (C) Arabidopsis Col-0, bagp1, and apcb1 protoplasts were transfected with either HA-AtBAG6 or HA-AtBAG6^D760A under 35S promoter in the presence or absence of chitin (lanes 1 to 4 and 6). BAG6 cleavage was monitored using immunoblotting. BAG6 cleavage was restored when the bagp1 and apcb1 mutants were complemented (lanes 5 and 7). A catalytically inactive form of APCB1 (Myc-APCB1^D223A/D431A) was unable to restore BAG6 cleavage (lane 8). Equal protein samples were separated by 10% SDS-PAGE and stained with Coomassie blue (CBS); the presence of HA-BAG6 was detected by an anti-HA antibody.

Figure 7.

BAG6 Induces Autophagic Structures in Plants. (A) Wild-type Col-0, bag6 mutant, and the autophagy-deficient mutant atg18 (SALK_081770C) were inoculated with B. cinerea using colonized agar plugs, and lesions monitored over time. Photographs of representative leaves were taken 48 h after inoculation. (B) Lesion diameters were measured 24 and 48 h after inoculation with B. cinerea. Data represent means ± sd from three replicates. (C) Autophagic activity was detected using the fluorescent stain MDC. Leaves were vacuum-infiltrated with a 100 μM final concentration of MDC for 30 min at 37°C, 24 h after B. cinerea inoculation. Fluorescence was visualized using an Olympus IX81 inverted fluorescence confocal microscope with excitation and emission wavelengths of 335 and 508 nm, respectively. All images were collected using Olympus DP controller and processed using Olympus Fluoview software. Bar = 20 μm. (D) Representative TEM images of tobacco tissues expressing the full-length and cleaved form of AtBAG6. Tissues were processed 48 h after infiltration. Arrows indicate autophagosomal structures. Bar = 2 μm.

Figure 8.

BAG6-Induced Autophagy Is Specific to B. cinerea. (A) Autophagic activity was detected using MDC staining. Col-0 and bag6 Arabidopsis plants were treated with B. cinerea (24 h), tunicamycin (0.5 μg/mL, 8 h), or heat stress (50°C, 30 min). MDC-stained structures were detected in Col-0 plants in response to all three treatments, but only in response to tunicamycin and heat stress in bag6 plants. Fluorescence was visualized using an Olympus IX81 inverted fluorescence confocal microscope with excitation and emission wavelengths of 335 and 508 nm, respectively. All images were collected using Olympus DP controller and processed using Olympus Fluoview software. Bar = 20 μm. (B) Similarly, the lipidation pattern of ATG8 was analyzed in Col-0 and bag6 plants following all three treatments. Protein extracts were subjected to SDS-PAGE followed by immunoblotting with anti-ATG8 antibody. The positions of ATG8 and ATG8-PE are indicated. Equal loading was confirmed by SDS-PAGE and Coomassie blue staining (CBS).