Figure 1 Interaction between CERK1-BD and HSP1-AD in yeast cells (A) The HSP1 protein was fused with AD at the N-terminal, and CERK1-CD was fused with BD at the C-terminal, then these two recombinants were transformed into Y187 and AH109 yeast strains, respectively. After hybridization, it was plated on SD-Trp-Leu/SD-Ade-Trp-Leu/SD-His-Ade-Trp-Leu plates, and cultivated at 28°C, P53 and Lam represent positive and negative controls, respectively. (B) C-terminal of CERK1 was fused with cYFP, HSP1 and CPK5 were fused with nYFP, and then co-injected into Nicotiana benthamiana mesophyll cells, CPK5 and HSP1 served as negative control. After 72 hours of injection, the fluorescence intensity of YFP fluorescent protein was detected under fluorescence microscope; (C) Interaction between HSP1 and CERK1 was detected in N. benthamiana by Western blot (WB). C-terminal of CERK1D441V was fused with Flag tag, and C-terminal of HSP1 was fused with GFP tag, then were co-injected into N. benthamiana mesophyll cell. The CERK1 protein was precipitated using a Flag antibody, and the HSP1-GFP was detected by CoIP. The experiment was repeated for three times with similar results.

Figure 2 Identification of hsp1 v63 deletion mutants in Arabidopsis thaliana (A) The hsp1 genomic DNA was extracted, then the HSP1 gene was amplified by PCR, and the PCR product was used for sequencing analysis (by comparing the sequencing results, it was found that the 63 rd valine of HSP1 was knocked out, the blue sequence represents the target site of gRNA, and the red sequence represents the NGG sequence; (B) Analysis of the conservation of the amino acid sequence of HSP1 in Arabidopsis thaliana, A. lyrata, Camelina sativa, Arabis and Brassica using Clustal W alignment (* represent the conserved amino acid sites); (C) The hsp1 v63 deletion mutant (left) and Col-0 (right) grown for 4 weeks, and their growth conditions were similar.

Figure 3 Determination of defense responses in the hsp1 v63 deletion mutant of Arabidopsis thaliana (A), (B) Chitin-induced gene expression analysis of WRKY53 and WRKY33 in Arabidopsis hsp1 v63 deletion mutant (the tran-scriptional level of WRKY53 and WRKY33 genes in hsp1 v63 deletion mutant and Col-0 seedlings was measured by qRT-PCR before and after chitin induction. All expression levels were normalized using the internal reference gene ACTIN2, data are pre-sented as means±SD, one-way ANOVA, Tukey post-test, *** P<0.001, n=3); (C) Chitin-induced MAPK activation experiments in Arabidopsis cerk1, hsp1 v63 deletion mutant, wild-type Col-0 (chitin-induced MAPK phosphorylation was examined in cerk1, hsp1 v63 deletion mutant and wild-type Col-0. The A. thaliana seedlings were treated with chitin for the time indicated in the figure. After the samples were collected, the phosphorylation of MAPK3/6 was detected by immunoblot. The antibody used in this experiment was Anti-P44/P42. The experiment was repeated three times with similar results).

Figure 4 Detection of chitin-induced CERK1 protein levels in cerk1, hsp1 v63 deletion mutants and wild-type Col-0 of Arabidop-sis (A) Arabidopsis thaliana seedlings were soaked in MS plate for 7 days, and the seedlings were treated with 25 μg·mL-1 chitin for the indicated times as shown in the figure. After samples were collected, CERK1 protein level was detected by Western blot (WB) by CERK1 antibody; (B) CERK1 protein level were measured after treated with 50 nmol·L-1 Kif for 0, 5, 15, and 30 minutes under hsp1 v63 deletion mutant; (C) Comparison the phosphorylation of MAPK3/6 after treated by 25 μg·mL-1 Chitin or 25 μg·mL-1 Chitin and 50 nmol·L-1 Kif mixture for indicated time. The experiment was repeated three times with similar results.