GhMPK9‐GhRAF39_1‐GhWRKY40a Regulates the GhERF1b‐ and GhABF2‐Mediated Pathways to Increase Cotton Disease Resistance

Abstract Mitogen‐activated protein kinase (MAPK) cascade is the center of plant signal transduction system that amplify immune signals into cellular responses by phosphorylating diverse substrates. The MAPK cascade consisting of MAPK kinase kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs is well characterized in plants, in which Raf‐like kinases are generally regarded as MAPKKKs. However, it is rarely reported that Raf‐like MAPKKKs function as middle regulators to link MAPK and its downstream transcription factors in plant immunity. Verticillium wilt, caused by the soil‐borne vascular fungus Verticillium dahliae, is a serious disease in many plants, including cotton. The previous studies showed that GhMPK9 (a MAPK) is involved in the response to Verticillium wilt. Here, the Raf‐like kinase GhRAF39_1 is reported as helper regulates the phosphorylation of WRKY transcription factor GhWRKY40a by GhMPK9. The phosphorylated GhWRKY40a can further activate the transcription of GhERF1b to up‐regulate defense‐related genes while inhibit the transcription of GhABF2 to regulate the stomatal opening, thus improving the resistance to Verticillium wilt in cotton. This study reveals a new signaling module of GhMPK9‐GhRAF39_1‐GhWRKY40a to regulate GhERF1b‐ and GhABF2‐mediated defense responses, which triggers plant defense against Verticillium wilt.


Supplemental Figures
Table S2.The primers used in this study.

Figure S1 .
Figure S1.The roles of GhMPK9 in the cotton defense response to Verticillium wilt.(A) Phenotypes of TRV:GhCLA seedlings on 14 th day post VIGS (dpv).(B-C) The relative expression level (REL) of GhMPK9 and GhMPK13 in TRV:00 and TRV:GhMPK9 seedlings on 14 dpv.The values are normalized to those of Histone3.(D) Phenotypes of TRV:00, TRV:GhMPK9 and Junmian1 (susceptible control) seedlings from 15 th day post V991 inoculation (dpi) to 35 dpi.(E) The disease index of TRV:00, TRV:GhMPK9 and Junmian1 during 15 to 35 days after V991 inoculation.Data are presented as means ± SD in (B, C and E), n = 9 (B and C), n = 30 (E), statistical analyses were performed using Student's t test: *, P < 0.05 and **, P < 0.01.

Figure S2 .
Figure S2.The phenotype observation of GhMPK9-RNAi and GhMPK9-OE transgenic plants.(A) DNA detection of GhMPK9-RNAi lines by PCR analysis.PC: positive control; WT: wild-type; RNAi: GhMPK9 suppression transgenic lines.(B) The relative expression level (REL) of GhMPK9 in wild-type and transgenic plants.Expression levels were normalized to those of Histone3.(C) The phenotype of stigma, stamen and pistil in wild-type and transgenic cotton plants (RNAi-1 and RNAi-2).(D) The phenotype of boll after pollination in GhMPK9-RNAi transgenic cotton.At 7day post anthesis after pollination, the ovules fall off.(E) The phenotype of leaf in GhMPK9-OE transgenic cotton at 30-day post V991 inoculation.OE: GhMPK9 overexpression transgenic lines.Each OE line contains 30 plants.Data are presented as means ± SD in (B), n = 9, statistical analyses were performed using Student's t test: **, P < 0.01.

Figure S3 .
Figure S3.Weighted correlation network analysis (WGCNA) for the overlapped 1458 DEGs.(A-B) The identification of soft threshold (power) in WGCNA.The horizontal axis represents weight parameters β. (A) The vertical axis on the left represents the square of the correlation coefficient between log(k) and log(p(k)) in the corresponding network.(B) The vertical axis on the right represents the mean of all gene adjacency functions in the corresponding gene module.(C) Gene clustering dendrogram obtained by hierarchical clustering of adjacency-based dissimilarity.The color row below the dendrogram indicates module membership.Gray color denotes the genes unclassified into any module.(D) The upper row shows the heatmap of the MEblue module genes across the transcriptome.The lower row shows the corresponding module eigengene (ME) expression values (y-axis) versus the same samples.Note that the ME takes on low values in samples where a lot of module genes are under-expressed (green color in the heatmap).Conversely, the ME takes on high values where module genes are overexpressed (red in the heatmap).

Figure S4 .
Figure S4.Structural and expression characteristics of GhWRKY40s.(A) Alignments of protein sequences among AtWRKY40, AtWRKY18, AtWRKY60 and their homologous genes in cotton.There are two copies in tetraploid cotton genome for each pair, with each one in A and D subgenome, respectively.(B) The phylogenetic tree of AtWRKY40, AtWRKY18, AtWRKY60 and five pairs of homologs of GhWRKY40 (a-e).(C) The expression patterns in different tissues and organs for five pairs of homologs of GhWRKY40 (a-e).Colored squares indicate expression levels of the selected genes from 0 (blue) to 9 (red) normalized by Log2 (TPM+1).

Figure S5 .
Figure S5.Structural and expression characteristics of GhRAF39s.(A) Alignments of protein sequences among AtRAF39 and its homologous genes in cotton.(B) The phylogenetic tree of AtRAF39 and six pairs of homologs of GhRAF39 (1-6).GhRAF39-3 and GhRAF39-4 have only one copy in D subgenome, others have two copies with each one in A and D subgenome, respectively.(C) The expression patterns in different tissues and organs for six pairs of homologs of GhRAF39 (1-6).Colored squares indicate expression levels of the selected genes from 0 (blue) to 7 (red) normalized by Log2 (TPM+1).(D)The prediction analysis of phosphorylation sites in GhRAF39 phospho-peptides.

Figure S6 .
Figure S6.Sequence alignments of MPK9 (A), RAF39_1 (B) and WRKY40a (C) between G. hirsutum and G. barbadense with their corresponding silenced fragment in VIGS experiment.The identical sequences are shaded in black.

Figure S7 .
Figure S7.The predicted binding sites of GhWRKY40a in the promoter region of GhERF1b.(A) Schematic diagram of W-box binding sites contained in GhERF1b-promoter region.The promoter was divided into eight segments (P1-P8).W-box motifs are labeled in red.(B) The predicted W-box motifs in the GhERF1b-promoter.

Figure S8 .
Figure S8.The predicted binding sites of GhWRKY40a in the promoter region of GhABF2.(A) Schematic diagram of W-box binding sites in GhABF2-promoter region.The promoter was divided into two segments (P1, P2).W-box motifs are labeled in red.(B) The predicted W-box motif in the GhABF2-promoter.