Overexpression of the endoplasmic reticulum chaperone BiP3 regulates XA21-mediated innate immunity in rice

Abstract

Recognition of pathogen-associated molecular patterns by pattern recognition receptors (PRRs) activates the innate immune response. Although PRR-mediated signaling events are critical to the survival of plants and animals, secretion and localization of PRRs have not yet been clearly elucidated. Here we report the in vivo interaction of the endoplasmic reticulum (ER) chaperone BiP3 with the rice XA21 PRR, which confers resistance to the Gram negative bacterium, Xanthomonas oryzae pv. oryzae (Xoo). We show that XA21 is glycosylated and is primarily localized to the ER and also to the plasma membrane (PM). In BiP3-overexpressing rice plants, XA21-mediated immunity is compromised, XA21 stability is significantly decreased, and XA21 proteolytic cleavage is inhibited. BiP3 overexpression does not affect the general rice defense response, cell death or brassinolide-induced responses. These results indicate that BiP3 regulates XA21 protein stability and processing and that this regulation is critical for resistance to Xoo.

Figure 1. Rice BiP3 Interacts with XA21 In Vivo.

(A) An XA21 complex was isolated from Ubi Myc-XA21 transgenic rice after Xoo strain PXO99Az inoculation. Five grams of leaves from Ubi Myc-XA21 or Kit were treated with Xoo or water for 12 h. After separation by SDS-PAGE, co-immunoprecipitated proteins were detected by silver staining. A 75 kDa protein co-immunoprecipitated with the XA21 protein. (B) XA21 was detected after co-immunoprecipitation. Myc-XA21 and Myc-XA21^cp displayed bands at about 140 and 100 kDa, respectively, as reported previously , . (C) BiP3 co-immunoprecipitated with XA21 before (Mock) and after Xoo strain PXO99Az inoculation (Xoo) in transgenic rice carrying Myc-Xa21 under the control of its native promoter. The precipitates were used for protein gel blot analysis using anti-Myc antibody (left) or anti-BiP antibody (right). Myc-XA21 and Myc-XA21^cp displayed bands at about 140 and 100 kDa, respectively, and BiP3 was detected as a 75 kDa band.

Figure 2. Overexpression of BiP3 Compromises XA21-Mediated Resistance.

(A) Rice lines 14 days after inoculation with Xoo strain PXO99Az. From left to right: Kitaake (Kit), transgenic line (Nat XA21) carrying Xa21 driven from its native promoter, and transgenic lines carrying BiP3 ox (BiP3 ox/Nat XA21). (B) Lesion length measurements of Xoo strain PXO99Az inoculated plants, BiP3 ox/Nat XA21 (4A-10, 5A-6, 7B-10, and 8A-12), Kitaake (Kit), and Nat XA21 control lines over 12 days. Each data point represents the average and standard deviation of at least four samples. (C) Xoo strain PXO99Az populations were monitored over 12 days in BiP3 ox/Nat XA21 (4A-10, 5A-6, 7B-10, and 8A-12), Kit, and Nat XA21 rice lines. For each time point, bacterial populations were determined in three separate leaves for each genotype. Capped vertical bars represent standard deviation values (cfu/leaf) obtained from the three samples.

Figure 3. XA21 Is Mainly Localized to the Endoplasmic Reticulum in Rice Leaf Sheath Tissue.

In planta subcellular localization of Nat XA21-YFP (top) and non-transgenic Kitaake (bottom) were determined using confocal microscopy. Intact adaxial sheath epidermal cells were imaged with an Olympus FV1000 confocal microscope equipped with a 60×oil immersion lens [numerical aperture, 1.42]. YFP signal was excited at 515 nm and emission was collected between 530-560 nm. Scale bar, 5 µm.

Figure 4. XA21 Is a Glycosylated Protein.

Myc-XA21 protein was immunoprecipitated from Ubi Myc-XA21 rice using anti-Myc antibody. After washing the Myc-XA21 protein bound to anti-Myc antibody-conjugated agarose beads, Myc-XA21 protein was digested with PNGase F for 2 h at 37°C. Immunoprecipitated Myc-XA21 (IPed) was loaded in the first lane. IPed Myc-XA21 was denatured (second lane) and then treated with PNGase F (third lane). The samples were then subjected to SDS-PAGE for western blot analysis with anti-Myc antibody.

Figure 5. Transgenic Lines Overexpressing BiP3 Fail to Accumulate the XA21 and Its Cleavage Product.

(A) Lesion length measurements of F₂ population segregating for Myc-XA21 and overexpressed BiP3 (BiP3 ox). The F₂ segregants (Xa21/BiP3ox; +/−, +/+, −/+, −/−) were inoculated with Xoo strain PXO99Az and lesion lengths were measured 12 days post-inoculation. Nat Myc-XA21: Xa21 driven by the native promoter. Ubi Myc-XA21: Xa21 driven by the maize ubiquitin promoter. (B) RNA accumulation of the Myc-Xa21 and BiP3 transcripts in F₂ segregants 3A 1–5 and 3A 2–7 before and after Xoo strain PXO99Az inoculation. Lines are as described in (A). Total RNA was extracted and RT-PCR was performed using Myc-Xa21 and BiP3-specific primers. Control RT-PCR reactions were carried out with 18S rRNA-specific primers. (C) Protein accumulation of the Myc-XA21 and BiP3 in F₂ segregants 1–5 and 1–7 before and after Xoo strain PXO99Az inoculation. Equal amounts (100 µg) of total protein form Ubi Myc-XA21, Ubi Myc-XA21×BiP3ox3A 1-5, Nat Myc-XA21, and Nat Myc-XA21×BiP3ox3A 1–7 were extracted after Xoo strain PXO99Az inoculation, analyzed by SDS-PAGE, and immunoblotted with anti-Myc and anti-BiP antibodies. (D) Total protein was extracted from Kit, Nat Myc-XA21/BiP3 ox, and Nat Myc-XA21 plants after Xoo strain PXO99Az inoculation. Equal amounts (300 µg) of total protein were analyzed by SDS-PAGE and immunobloted with anti-Myc antibody. Equal total protein loading was confirmed with anti-actin antibody. A nonspecific band (n.s.) of 95 kD was detected. (E) After immunoprecipitation with anti-Myc antibody, western blot analysis was performed to visualize XA21 and its cleavage product. Immunoprecipitates from Nat Myc-XA21 were diluted ten-fold, resulting in similar amounts of XA21 in Nat Myc-XA21/BiP3 ox and in Nat Myc-XA21.

Figure 6. BiP3 Does Not Interfere with Brassinolid-Induced Responses.

(A) Seeds from Kitaake (Kit) and the BiP3 ox 3A line (BiP3ox) were germinated on MS agar in the presence (+) or absence (−) of 0.1 µM BL. Seedlings were examined 3 days after germination. (B) Effect of BL on coleoptile and root elongation in Kit and BiP3 ox seedlings. The plants were germinated in MS agar plates containing the indicated concentration of BL. Data presented are the means of results from four plants. Bars indicate SD.

Figure 7. A Model for Regulation of XA21-Mediated Innate Immunity by BiP3.

The XA21 LRR domain is responsible for recognition of Xoo strains carrying Ax21 , , . XA21/Ax21 binding is hypothesized to activate the non-RD kinase domain leading to XA21 autophosphorylation and/or transphosphorylation of downstream target proteins , , . XA21 transphosphorylates the RING finger ubiquitin ligase XB3, which is required for effective XA21-mediated resistance . XB10 (OsWRKY62) and other WRKY transcription factors either activate or repress PR genes , . In plants, pathogen infection upregulates expression of PR genes , , , , resulting in increased translations of the corresponding proteins in the ER. The transiently accumulated unfolded and/or misfolded proteins cause ER stress, which, in Arabidopsis, activates BiPs . During systemic acquired resistance, BiP helps to secrete accumulated PRs . Our results suggest that excessive loading of unfolded and/or misfolded proteins during prolonged ER stress attenuates the signal transduction pathway causing the ER stress. In support of this model, BiP3 overexpression drives XA21 proteolysis and down-regulates the XA21-mediated immune response.