OsCUL3a Negatively Regulates Cell Death and Immunity by Degrading OsNPR1 in Rice

Abstract

Cullin3-based RING E3 ubiquitin ligases (CRL3), composed of Cullin3 (CUL3), RBX1, and BTB proteins, are involved in plant immunity, but the function of CUL3 in the process is largely unknown. Here, we show that rice (Oryza sativa) OsCUL3a is important for the regulation of cell death and immunity. The rice lesion mimic mutant oscul3a displays a significant increase in the accumulation of flg22- and chitin-induced reactive oxygen species, and in pathogenesis-related gene expression as well as resistance to Magnaporthe oryzae and Xanthomonas oryzae pv oryzae. We cloned the OsCUL3a gene via a map-based strategy and found that the lesion mimic phenotype of oscul3a is associated with the early termination of OsCUL3a protein. Interaction assays showed that OsCUL3a interacts with both OsRBX1a and OsRBX1b to form a multisubunit CRL in rice. Strikingly, OsCUL3a interacts with and degrades OsNPR1, which acts as a positive regulator of cell death in rice. Accumulation of OsNPR1 protein is greater in the oscul3a mutant than in the wild type. Furthermore, the oscul3a osnpr1 double mutant does not exhibit the lesion mimic phenotype of the oscul3a mutant. Our data demonstrate that OsCUL3a negatively regulates cell death and immunity by degrading OsNPR1 in rice.

Figure 1.

Identification of the Lesion Mimic Mutant oscul3a. (A) oscul3a mutant and ZH11 plants were grown in the field and photographed at 60 dps. (B) Leaves of oscul3a mutant and ZH11 plants at 60 dps. (C) DAB staining of the oscul3a mutant and ZH11 leaves shown in (B). (D) ROS accumulation dynamics in oscul3a mutant and ZH11 plants after flg22 and water (mock) treatments. Error bars represent the se; n = 3. (E) ROS accumulation dynamics in oscul3a mutant and ZH11 plants after chitin and water (mock) treatments. Error bars represent the se; n = 3.

Figure 2.

The oscul3a Mutant Displays Enhanced Resistance to Both M. oryzae and Xoo. (A) and (B) Punch inoculation of oscul3a mutant and ZH11 plants with the compatible M. Oryzae isolate RB22. (A) Leaves were photographed at 12 dpi. (B) Lesion area (left) and fungal biomass (right) of the inoculated leaves of oscul3a mutant and ZH11 plants as shown in (A) at 12 dpi. Error bars represent the se; n = 3 (t test between mutant and ZH11 values; *P < 0.05). (C) and (D) Leaves of oscul3a mutant and ZH11 plants were inoculated with three compatible Xoo isolates. (C) Leaves at 15 dpi. (D) Lesion lengths on the leaves at 15 dpi. Error bars represent the se; n = 3 (t test between mutant and ZH11 values; ***P < 0.001). (E) Total RNA was extracted from the leaves (second from the top) of oscul3a mutant and ZH11 plants at 60 dps. qRT-PCR was used to analyze the expression of OsPR1a, OsPR1b, OsPR10, OsPAL1, OsWRKY45, and OsAOS2. Data were normalized to the expression level of the constitutively expressed OsACTIN gene. Error bars represent the se; n = 3 (t test between mutant and ZH11 values; *P < 0.05, **P < 0.01, and ***P < 0.001).

Figure 3.

Map-Based Cloning of oscul3a. (A) Delimitation of the candidate genomic region of oscul3a. (a) Preliminary mapping of the oscul3a gene using 94 recessive F2 plants with simple sequence repeat markers. The numbers under the linkage map represent the number of recombinants. (b) Fine mapping of the oscul3a locus with the InDel markers. The numbers under the linkage map represent the number of recombinants. (c) The arrows denote the candidates (ORFs) within the genomic region between the ZN36 and ZN9 markers. (d) Structure of the OsCUL3a gene and the mutation site. The line represents the intron; black boxes represent the exons. (B) Protein alignment between wild-type OsCUL3a and the deduced 69 amino acid product in oscul3a. The red arrow indicates the frameshift mutation site. (C) Phenotype of 2-month-old complemented T1 plants grown in the field. pOsCUL3a complementation plants were developed by transforming the whole genomic fragment of OsCUL3a into the oscul3a mutant background. pOsCUL3a plants were photographed at 60 dps. (D) Leaves (second from the top) of oscul3a mutant and pOsCUL3a plants at 60 dps. (E) DAB staining of the oscul3a mutant and pOsCUL3a leaves shown in (D). (F) to (H) Punch inoculation of oscul3a mutant and pOsCUL3a plants with the compatible M. oryzae isolate RB22. (F) Leaves were photographed at 12 dpi. (G) Lesion area of the inoculated leaves at 12 dpi. Error bar represent the se; n = 3 (t test; *P < 0.05). (H) Fungal biomass of the inoculated leaves at 12 dpi. Error bars represent the se; n = 3 (t test; *P < 0.05).

Figure 4.

OsCUL3a Assembles with OsRBX1a or OsRBX1b to Form a CULLIN-RING-Like E3 Ligase Complex. (A) OsCUL3a interacts with OsRBX1a as indicated by LCI assay. OsCUL3a-NLuc and CLuc-OsRBX1a were transiently expressed in N. benthamiana by coinfiltration; NLuc and CLuc were the negative controls. Luminescence was monitored with a low-light, cooled, CCD imaging apparatus at 3 dai. (B) Quantification of LUC activity in the leaves shown in (A). Error bars represent the se; n = 3 (t test between OsCUL3a-NLuc+CLuc-OsRBX1a and control group value; **P < 0.01). (C) OsCUL3a interacts with OsRBX1b as indicated by LCI assay. OsCUL3a-NLuc and CLuc-OsRBX1b were transiently expressed in N. benthamiana by coinfiltration; NLuc and CLuc were the negative controls. Luminescence was monitored with a low-light, cooled, CCD imaging apparatus at 3 dai. (D) Quantification of LUC activity in the leaves shown in (C). Error bars represent the se; n = 3 (t test between OsCUL3a-NLuc+CLuc-OsRBX1b and control group value; **P < 0.01). (E) BiFC assays showing the interaction between OsCUL3a and OsRBX1a in N. benthamiana. The OsCUL3a and OsRBX1a proteins were fused to the N- or C-terminal fragment of eYFP and transiently expressed in N. benthamiana. The YFP signal was evaluated via confocal microscopy at 3 dai. The N terminus of OsCUL3a was used as the negative control. Bar = 50 µm. (F) BiFC assays for the interaction between OsCUL3a and OsRBX1b in N. benthamiana. The OsCUL3a and OsRBX1b proteins were fused to the N- or C-terminal fragment of eYFP and transiently expressed in N. benthamiana. The YFP signal was evaluated via confocal microscopy at 3 dai. The N terminus of OsCUL3a was used as the negative control. Bar = 50 µm.

Figure 5.

OsCUL3a Interacts with OsNPR1 and Promotes Its Degradation in Vivo. (A) OsCUL3a interacts with OsNPR1 as indicated by LCI assay. OsCUL3a-NLuc and CLuc-OsNPR1 were transiently expressed in N. benthamiana by coinfiltration; NLuc and CLuc were the negative controls. Luminescence was monitored with a low-light, cooled, CCD imaging apparatus at 3 dai. (B) Quantification of the LUC activity in the leaves shown in (A). Error bars represent the se; n = 3 (t test between OsCUL3a-NLuc+CLuc-OsNPR1 and control group value; **P < 0.01). (C) OsCUL3a interacts with OsNPR1 in a co-IP assay. Myc-tagged OsNPR1 or LUC was transiently expressed with HA-tagged OsCUL3a in rice protoplasts by cotransfection. Following total protein extraction, samples were immunoprecipitated with anti-HA antibody. Crude and immunoprecipitated proteins were analyzed with anti-HA or anti-Myc antibodies. (D) OsCUL3a is required for OsNPR1 degradation in rice protoplasts. OsNPR1 was fused with the Myc tag and coexpressed with GFP in rice protoplasts isolated from ZH11, oscul3a, and pOsCUL3a plants. The transfected protoplasts were treated with or without MG132 for 20 h. Total protein was detected with anti-Myc and anti-GFP antibodies. The expression of OsNPR1 and OsACTIN was analyzed by RT-PCR. The relative abundance of OsNPR1 was calculated by comparing to GFP using ImageJ software. The OsNPR1/GFP relative quantity in ZH11 without MG132 was defined as 1.0 (lane 1). (E) OsCUL3a-mediated degradation of OsNPR1 via the 26S proteasome. Myc-OsNPR1 was transiently expressed in rice protoplasts isolated from ZH11 and oscul3a plants with or without cycloheximide and MG132 treatments. Total protein was extracted at 48 h after transfection for immunoblotting analysis with anti-cMyc and anti-HSP antibodies.

Figure 6.

Characterization of the oscul3a osnpr1 Double Mutant. (A) Domain structures of wild-type OsNPR1 and OsNPR1 from each of the two types of CRISPR/Cas9-derived (osnpr1) mutants. (B) ZH11, oscul3a, and oscul3a osnpr1 double mutants were grown in a greenhouse, and leaves (second from the top) were collected and photographed at 60 dps. The cell death phenotype of the oscul3a mutant was abolished in the oscul3a osnpr1-2 double mutants. (C) The expression levels of OsPR1a, OsPR1b, and OsPR10 in leaves (as in [B]) were analyzed using qRT-PCR and were normalized against OsACTIN. Error bars represent the se; n = 3 (t test between ZH11 and oscul3a value or ZH11 and oscul3a osnpr1-1 value; ***P < 0.001).

Figure 7.

A Proposed Model of OsCUL3a Action. OsCUL3a assembles with OsRBX1 and an unknown BTB domain-containing protein to form an E3 ligase complex that promotes the degradation of OsNPR1 via the 26S proteasome system. OsNPR1 highly accumulates in oscul3a mutant plants, resulting in cell death and immunity.