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Science 336 (6085): 1160-1164

Copyright © 2012 by the American Association for the Advancement of Science

Chitin-Induced Dimerization Activates a Plant Immune Receptor

Tingting Liu1,2,3,4,*, Zixu Liu4,5,*, Chuanjun Song6, Yunfei Hu7,8, Zhifu Han2,3, Ji She8, Fangfang Fan6, Jiawei Wang3, Changwen Jin7,8, Junbiao Chang6,{dagger}, Jian-Min Zhou4,9,{dagger}, and Jijie Chai2,3,{dagger}

1 Graduate Program in Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China.
2 Tsinghua-Peking Center for Life Sciences, Beijing 100084, China.
3 School of Life Sciences, Tsinghua University, Beijing 100084, China.
4 National Institute of Biological Sciences, No. 7 Science Park Road, Beijing 102206, China.
5 School of Life Science, Nanjing University, Nanjing 210093, China.
6 Department of Chemistry, Zhengzhou University, Zhengzhou 450001, China.
7 Beijing NMR Center, Peking University, Beijing 100871, China.
8 College of Life Sciences, Peking University, Beijing 100871, China.
9 State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China.


Figure 1
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  		Fig. 1. Tight packing of the three LysMs in AtCERK1-ECD. Overall structures of AtCERK1-ECD in two different orientations. The secondary-structure elements are labeled. Disulfide bonds (C29-C155, C25-C93, and C93-C153) are labeled and shown in yellow stick.

 

Figure 2
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  		Fig. 2. Specific recognition of a chitin oligomer by AtCERK1-ECD. (A) Chitin binds to a shallow surface groove on AtCERK1-ECD. AtCEKR1-ECD is shown in electrostatic surface (transparency) and cartoon. White, blue, and red indicate neutral, positive, and negative surfaces, respectively. Shown in magenta mesh is the omit electron density (Fo – Fc, 2.8{sigma}) around the bound chitin oligomer. Chitin residues are labeled (NAG1-4). (B) Detailed interactions between chitin and the AtCERK1-ECD. The side chains from AtCERK1 are shown in yellow. Red spheres represent oxygen atoms of water molecules. Four carbon atoms of the chitin oligomer are indicated (C1, C3, C4, and C6 in bold). The distance cut-off is 3.4 Å. (C) Sequence alignment of CERK1-LysM2 from different species. Red squares indicate the residues from LysM2 involved in interaction with chitin. At: Arabidopsis thaliana; Al: Arabidopsis lyrata; Vv: Vitis vinifera; Rc: Ricinus communis; Pt: Populus trichocarpa. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

 

Figure 3
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  		Fig. 3. Chitin-induced AtCERK1-ECD dimerization is critical for signaling. (A) Chitin octamer induces AtCERK1-ECD dimerization in vitro. An equal amount of His6- and HA-tagged AtCERK1-ECD proteins were mixed and supplemented with different chitin oligomers, as indicated, and PGN. After incubation, the mixture was loaded onto Ni-resin and then washed extensively. The eluted proteins were detected by Western blot with antibodies against His and HA. (B) (NAG)8 but not (NAG)4 induces AtCERK1-ECD oligomerization in solution. The translational diffusion coefficients D of free AtCERK1-ECD, and in the presence of excess (NAG)n, were determined by pulsed field gradient–NMR diffusion experiments. The molar ratio of (NAG)n: AtCERK1-ECD was 2:1. (C) Chitin induces AtCERK1-ECD dimerization in protoplasts. Top: HA- and FLAG- tagged AtCERK1-ECDs were coexpressed in wild-type (WT) Arabidopsis protoplasts. Coimmunoprecipitation assay was performed to detect chitin-induced dimerization after treatment with (+) or without (–) chitin (200 μg/ml). Bottom: A138H substitution attenuates chitin-induced AtCERK1-ECD dimerization. (D) Chitin induces dimerization of the full-length AtCERK1 protein in protoplasts. Top: HA- and FLAG-tagged AtCERK1 were coexpressed in WT Arabidopsis protoplasts under the control of native AtCERK1 promoter. Methods described in (C) were used to detect the proteins. Middle: A138H substitution attenuates chitin-induced dimerization of the full-length AtCERK1 protein. NP: native promoter. Bottom: A138H substitution attenuates (NAG)8-induced band shift of AtCERK1 protein in stable transgenic plants. Leaves of T1 transgenic plants were treated with H2O or 60 μM (NAG)8 for 15 min, and anti-FLAG immunoblot was used to detect band shift. (E) A138H attenuates chitin-induced FRK1::LUC expression. The cerk1 mutant Arabidopsis protoplasts were transfected with empty vector (EV), AtCERK1 (WT), or AtCERK1-A138H (A138H) along with 35S::R-LUC and FRK1::LUC. The FRK1::LUC activity was determined after protoplasts were treated with chitin (200 μg/ml) or H2O for 3 hours. Different letters above the bars indicate significant difference (mean + SD; n ≥ 3; P < 0.01).

 

Figure 4
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  		Fig. 4. Shorter chitin oligomers inhibit AtCERK1-mediated signaling. (A) (NAG)5 inhibits (NAG)8-induced AtCERK1-ECD dimerization in vitro. The experiments were conducted as described in Fig. 3A. (B) (NAG)5 inhibits (NAG)8-induced AtCERK1-ECD dimerization in protoplasts. HA- and FLAG-tagged AtCERK1-ECDs were coexpressed in WT Arabidopsis protoplasts, treated with H2O (–) and chitin oligomers, as indicated, for 10 min, and coimmunoprecipitation assay was conducted to detect the dimerization. (C) (NAG)5 and overexpression of AtCERK1-ECD (OXLysM) inhibit chitin-induced signaling. Top: Chitin induces heterodimerization between the overexpressed AtCERK1-ECD and the full-length AtCERK1. Bottom: OXLysM inhibits chitin-induced FRK1::LUC activity. WT Arabidopsis protopalsts were transfected with an empty vector (EV) or OXLysM plasmids along with FRK1::LUC and 35S::R-LUC. The activity of FRK1::LUC was analyzed after treatment with 60 μM (NAG)8 or a mixture of 60 μM (NAG)8 and 240 μM (NAG)5 [(NAG)8:5] for 3 hours. Different letters above the bars indicate significant difference (mean + SD; n ≥ 3 replicates; P < 0.01). (D) (NAG)5 inhibits (NAG)8-induced H2O2 production in planta. WT Arabidopsis leaves were treated with 30 μM (NAG)8 or a mixture of 30 μM (NAG)8 and (NAG)5 with different ratios [(NAG)8:5] as indicated (mean ± SD; n ≥ 4; P < 0.01). (E) A synthetic chitin derivative induces AtCERK1-ECD dimerization in vitro. The experiments were performed as described in Fig. 3A. (F) AtCERK1-ECD dimerization in protoplasts in response to synthetic chitin derivatives. The experiments were performed as described in Fig. 3C.

 

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