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, 113 (36), 10204-9

Cytosolic Activation of Cell Death and Stem Rust Resistance by Cereal MLA-family CC-NLR Proteins

Affiliations

Cytosolic Activation of Cell Death and Stem Rust Resistance by Cereal MLA-family CC-NLR Proteins

Stella Cesari et al. Proc Natl Acad Sci U S A.

Abstract

Plants possess intracellular immune receptors designated "nucleotide-binding domain and leucine-rich repeat" (NLR) proteins that translate pathogen-specific recognition into disease-resistance signaling. The wheat immune receptors Sr33 and Sr50 belong to the class of coiled-coil (CC) NLRs. They confer resistance against a broad spectrum of field isolates of Puccinia graminis f. sp. tritici, including the Ug99 lineage, and are homologs of the barley powdery mildew-resistance protein MLA10. Here, we show that, similarly to MLA10, the Sr33 and Sr50 CC domains are sufficient to induce cell death in Nicotiana benthamiana Autoactive CC domains and full-length Sr33 and Sr50 proteins self-associate in planta In contrast, truncated CC domains equivalent in size to an MLA10 fragment for which a crystal structure was previously determined fail to induce cell death and do not self-associate. Mutations in the truncated region also abolish self-association and cell-death signaling. Analysis of Sr33 and Sr50 CC domains fused to YFP and either nuclear localization or nuclear export signals in N benthamiana showed that cell-death induction occurs in the cytosol. In stable transgenic wheat plants, full-length Sr33 proteins targeted to the cytosol provided rust resistance, whereas nuclear-targeted Sr33 was not functional. These data are consistent with CC-mediated induction of both cell-death signaling and stem rust resistance in the cytosolic compartment, whereas previous research had suggested that MLA10-mediated cell-death and disease resistance signaling occur independently, in the cytosol and nucleus, respectively.

Keywords: cell death; plant immunity; resistance protein; signaling; wheat stem rust.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The full CC domains of MLA10, Sr33, and Sr50 are sufficient for autoactivity in planta. Indicated fragments of the MLA10, Sr33, and Sr50 proteins fused to HA or CFP were transiently expressed in N. benthamiana leaves by Agrobacterium tumefaciens infiltration along with the autoactive RGA4:HA or RGA4:CFP positive controls and the inactive rga4TYG/MHD:HA or rga4TYG/MHD:CFP or mock inoculation as negative controls. Pictures were taken 3 d (A) or 5 d (B and C) after infiltration. Equivalent results were obtained in three independent experiments.
Fig. 2.
Fig. 2.
The full CC domains of Sr33, Sr50, and MLA10 can self-associate in planta. (A) The full CC domains of MLA10, Sr33, and Sr50 fused to CFP or HA tags were transiently expressed in N. benthamiana leaves in the indicated combinations (+, agro-infiltrated construct; −, non–agro-infiltrated construct), and proteins were extracted after 24 h. Tagged proteins were detected in the extract (input) and after immunoprecipitation with anti-GFP beads (IP-GFP) by immunoblotting with anti-HA (α-HA) and anti-GFP (α-GFP) antibodies. The RGA4 CC fused to CFP was used as a control for specificity. Protein loading in the input is shown by Ponceau staining of the large RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) subunit. The experiment was carried out three times with identical results. (B) The truncated CC domains of MLA10, Sr33, and Sr50 fused to CFP or HA were transiently expressed in N. benthamiana leaves in the indicated combinations. Samples were processed as described in A. The experiment was carried out twice with identical results. (C) The procedure described in A was applied to the full-length Sr33 and Sr50 proteins.
Fig. S1.
Fig. S1.
Short 1–120 CC domains of MLA10, Sr33, and Sr50 are truncated. (A) Protein sequences were aligned using ClustalX (59) and visualized with GeneDoc (www.nrbsc.org/gfx/genedoc/ebinet.htm). Domains equivalent to MLA10 1–120, 1–160, and 1–225 are indicated by the red numbers 1, 2, and 3, respectively. (B) The probability that a residue in a protein is part of a CC structure was assessed by comparing its flanking sequences with sequences of known CC proteins using the CC prediction tool (15). Predictions were made using 14-, 21-, and 28-residue windows.
Fig. S2.
Fig. S2.
Cell-death assays in N. benthamiana. (A) The MLA10 CC1–120, Sr33 CC1–120, and Sr50 CC1–123 domains fused to HA were transiently expressed in N. benthamiana leaves. The autoactive RGA4:HA construct was used as a positive control, and the inactive rga4TYG/MHD:HA and a mock inoculation were used as negative controls. Cell death was visualized 4 d after infiltration. Equivalent results were obtained in three independent experiments. (B) The indicated Sr33 and Sr50 genomic constructs (including their native promoter sequences) or cDNA sequences (under the control of the 35S promoter) were transiently expressed in N. benthamiana leaves by Agrobacterium-mediated transformation. Cell death was visualized 4 d after infiltration. Equivalent results were obtained in three independent experiments.
Fig. S3.
Fig. S3.
(AF) Immunoblotting showing expression of HA-, CFP-, or YFP-fused proteins. The indicated proteins were extracted from transiently transformed N. benthamiana leaves 24 h after infiltration and were analyzed by immunoblotting with anti-GFP or anti-HA antibodies. Ponceau staining of the large RuBisCO subunit was used to verify equal protein loading. An asterisk marks unspecific background.
Fig. S4.
Fig. S4.
The CC domains of MLA10 and Sr50 do not self-associate in yeast. (A) The yeast strain HF7c was cotransformed with the specified constructs, and self-interaction of MLA10 and Sr50 CC domain fragments (BD:MLA101–160/AD:MLA101–160, BD:MLA101–225/AD:MLA101–225, BD:Sr501–163/AD:Sr501–163, BD:Sr501–228/Ad:Sr501–228, and BD:Sr501–128/AD:Sr501–128) was assayed by yeast two-hybrid assay. AD:RGA51–228, BD:RGA51–228, AD:L6-TIR, and BD:L6-TIR constructs were used as controls. Cultures of cotransformed yeast clones were adjusted to an OD of 0.2, and three dilutions (1/1, 1/10, 1/100) were spotted on synthetic −LTH medium (−Trp/−Leu/−His) to assay for interactions and on synthetic double-dropout −LT medium (−Trp/−Leu) to monitor proper growth. Photographs were taken after 4 d of growth. (B) Myc- or HA-tagged proteins were extracted from yeasts and detected by immunoblotting using anti-Myc or anti-HA antibodies, respectively.
Fig. S5.
Fig. S5.
Cell-death and in planta coimmunoprecipitation assays. (A) The indicated combinations of constructs fused to either the CFP or the HA tag were transiently expressed in N. benthamiana leaves. Samples were collected for coimmunoprecipitation assays 24 h after infiltration, and pictures were taken 3 d after infiltration. (B) The truncated CC domains of MLA10, Sr33, and Sr50 do not self-associate in planta. Coimmunoprecipitation was performed as described in Fig. 2. The Sr50 CC1160 positive control was included in the experiment. (C) The full-length Sr33 protein self-associates in planta. Coimmunoprecipitation was performed as described in Fig. 2. An additional control was included to test Sr33-unspecific binding to the GFP-trap_M beads. (D) Full-length Sr33 and Sr50 homo- and hetero-associate in planta. Coimmunoprecipitation was performed as described in Fig. 2. A control was included to test Sr33- and Sr50-unspecific binding to the GFP-trap_M beads.
Fig. 3.
Fig. 3.
Residues within the 120–144 CC region of Sr33 are crucial for self-association and autoactivity. (A) Alignment showing the 120–160 fragment of MLA10, Sr33, and Sr50. Six mutations (numbered 1–6) were generated in which conserved hydrophobic residues (red) were replaced by glutamate, or a hydrophilic stretch (blue) was replaced by alanines. (B) WT and mutated constructs of the Sr33 CC1–160 domain or full-length protein fused to HA or CFP were transiently expressed in N. benthamiana, and cell death was visualized after 4 d. Equivalent results were obtained in three independent experiments. (C) The indicated mutants of the Sr33 CC1–160 domain fused to CFP or HA tags were coexpressed in N. benthamiana and analyzed by coimmunoprecipitation as described in Fig. 2A. RGA41–171:CFP was used as a control for specificity, and WT Sr331–160:HA and Sr331–160:CFP were used as positive controls. Three independent experiments gave equivalent results.
Fig. 4.
Fig. 4.
The CC domains of Sr33, Sr50, and MLA10 induce cell-death signaling in the cytosol of N. benthamiana cells. (A) Fluorescence microscopy images (20 h postinfiltration) of the localization of the Sr33 CC1–160 domain fused to YFP:NLS, YFP:nls, YFP:NES, or YFP:nes in transiently transformed N. benthamiana leaf epidermal cells. Fluorescence channels for YFP and chloroplasts are merged on the overlay picture, and all channels are merged on the overlay + bright-field picture. White arrows indicate nuclei. (Scale bars, 20 µm.) (B) MLA10 CC1–160, Sr33 CC1–160, and Sr50 CC1–163 domains fused to YFP:NLS, YFP:nls, YFP:NES, or YFP:nes were transiently expressed in N. benthamiana leaves by A. tumefaciens infiltration. Pictures were taken 40 h after infiltration. Equivalent results were obtained in three independent experiments.
Fig. S6.
Fig. S6.
Confocal imaging in N. benthamiana and wheat epidermal cells. (A) CC domains fused to YFP:NLS, YFP:nls, YFP:NES, or YFP:nes were transiently expressed in N. benthamiana. Confocal images were taken 20 h after infiltration. Arrows indicate nuclei. (Scale bars, 20 μm.) (B) Confocal images showing localization of Sr33 fused to the YFPv, YFPv:NLS, YFPv:nls, YFPv:NES, or YFPv:nes in transgenic wheat leaf epidermal cells were taken on 10- to14-d-old T1 plants. YFPv fluorescence appears in green; stomatal and chloroplastic autofluorescence emissions are shown in blue and red, respectively. White arrowheads mark nuclei. (Scale bars, 20 μm.)
Fig. 5.
Fig. 5.
Sr33 induces disease-resistance signaling from the cytosol of wheat cells. (A) Untransformed Fielder and two independent T1 transgenic lines per construct expressing Sr33 fused to YFPv, YFPv:NLS, YFPv:nls, YFPv:NES, or YFPv:nes plants were inoculated with Pgt strain 98-1,2,3,5,6 at the two-leaf stage. Pictures were taken 13 d after inoculation. The presence of the transgenes was determined by PCR with Sr33-specific primers. (B) Immunoblot of transgenic wheat seedlings expressing YFP-tagged proteins detected with anti-GFP.
Fig. 6.
Fig. 6.
Localization of Sr33 fusion proteins in wheat mesophyll protoplasts. (A) Linear unmixing confocal images showing the localization of Sr33 fused to the YFPv, YFPv:NLS, YFPv:nls, YFPv:NES, or YFPv:nes in transgenic wheat mesophyll protoplasts. Protoplasts were isolated from 10- to 14-d-old T1 transgenic plants and were observed within 8 h following isolation. YFP-specific fluorescence appears in green; specific vacuolar and chloroplastic autofluorescence emissions are shown in blue and red, respectively. White circles mark nuclei locations on bright-field pictures. (Scale bars, 5 µm.)

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