Genome-wide identification of mitogen-activated protein kinase gene family in Gossypium raimondii and the function of their corresponding orthologs in tetraploid cultivated cotton

Abstract

Background: Mitogen-activated protein kinase (MAPK) cascades play a crucial role in plant growth and development as well as biotic and abiotic stress responses. Knowledge about the MAPK gene family in cotton is limited, and systematic investigation of MAPK family proteins has not been reported.

Results: By performing a bioinformatics homology search, we identified 28 putative MAPK genes in the Gossypium raimondii genome. These MAPK members were anchored onto 11 chromosomes in G. raimondii, with uneven distribution. Phylogenetic analysis showed that the MAPK candidates could be classified into the four known A, B, C and D groups, with more MAPKs containing the TEY phosphorylation site (18 members) than the TDY motif (10 members). Furthermore, 21 cDNA sequences of MAPKs with complete open reading frames (ORFs) were identified in G. hirsutum via PCR-based approaches, including 13 novel MAPKs and eight with homologs reported previously in tetraploid cotton. The expression patterns of 23 MAPK genes reveal their important roles in diverse functions in cotton, in both various developmental stages of vegetative and reproductive growth and in the stress response. Using a reverse genetics approach based on tobacco rattle virus-induced gene silencing (TRV-VIGS), we further verified that MPK9, MPK13 and MPK25 confer resistance to defoliating isolates of Verticillium dahliae in cotton. Silencing of MPK9, MPK13 and MPK25 can significantly enhance cotton susceptibility to this pathogen.

Conclusions: This study presents a comprehensive identification of 28 mitogen-activated protein kinase genes in G. raimondii. Their phylogenetic relationships, transcript expression patterns and responses to various stressors were verified. This study provides the first systematic analysis of MAPKs in cotton, improving our understanding of defense responses in general and laying the foundation for future crop improvement using MAPKs.

Figure 1

Chromosomal distribution of MAPK genes in G. raimondii. The chromosome numbers are indicated at the top of each bar. The chromosome numbers from D1 to D5, and D7 to D12 were consistent with our newly-updated interspecific genetic map in allotetraploid cultivated cotton species reported recently (Zhao et al. [32]), and the scaffolds name from G. raimindii genome was showed in the bracket. Lines were drawn to connect duplicated genes. The nomaclature of MAPKs were based on the order of the chromosomes in G. raimondii.

Figure 2

Comparison of the amino acid sequences of GrMAPKs. Roman numerals indicate regions containing the 11 domains (I–XI) found in the cotton PK subdomains. The A-Loop, CD-domain and phosphorylation-activation motif (TEY and TDY) are indicated with red boxes.

Figure 3

Intron and exon organization of G. raimondii MAPK genes ( GrMPKs ). Introns and exons are represented by black lines and colored boxes, respectively. GrMPKs were grouped according to phylogenetic classification. Phylogenetic analysis was done using the ML method with 1,000 resampling replicates. Bootstrap values (%) based on 1000 replicates are indicated beside the nodes.

Figure 4

Phylogenetic relationships of MAPK family genes from G. raimondii , A. thaliana , O. sativa , and G. max. Amino acid sequences were aligned using ClustalX software and subjected to phylogenetic analysis using the ML method with 1,000 resampling replicates. Bootstrap values (%) based on 1000 replicates are indicated beside the nodes. GrMAPKs are highlighted in red and the other MAPKs from A. thaliana, O. sativa and G. max are shown in different colors.

Figure 5

Real-time qRT-PCR analysis of MAPK genes in different tissues and organs in G. hirsutum acc. TM-1. A total of eight cotton tissues (root; stem; leaf; petal; anther; ovule at 0 day post anthesis (DPA); fiber at 10 DPA; and fiber at 21 DPA) were sampled to analyze. Differences in gene expression intensities are shown in colors indicated in the scale. Phylogenetic analysis was done using the ML method with 1,000 resampling replicates. Bootstrap values (%) based on 1000 replicates are indicated beside the nodes.

Figure 6

Relative expression of G. hirsutum MAPK genes under stress-related signal treatments. The data are presented in clusters using the fold-change (E/C) of relative expression for all MAPK genes in response to stress-siganl treatments (Experiment), in comparison to their respective controls (Control). Red and blue colors represent increased or decreased expression levels, respectively, in comparison to controls. The stress-related signals included JA, H₂O₂, ABA and SA, respectively.

Figure 7

Relative expression of G. hirsutum MAPK genes under different stress treatments. The data are presented in clusters using the fold-change (E/C) of relative expression for all MAPK genes in response to different treatments (Experiment), in comparison to their respective controls (Control). Red and blue colors represent increased or decreased expression levels, respectively, in comparison to controls. The stressors included NaCl, PEG, 4°C, 37°C, and wounding treatment, respectively.

Figure 8

Expression patterns of three MAPK genes induced by V. dahliae and VIGS analysis. (a) Q-PCR analysis of the expression of three MAPK genes after inoculation by V. dahliae. The error bars were calculated based on three biological replicates using standard deviation. “*”: significant difference (P < 0.05); “**”: significant difference (p < 0.01). (b) Phenotypes after TRV-VIGS silencing of three MAPK genes and GhCLA1. After two weeks post-treatment with TRV1 and TRV2/TRV2-MPKs, the treated plants exhibited normal growth. TRV1- and TRV-GhCLA1-treated plants exhibited a photobleaching phenotype. (c) Gene expression of MPK9, MPK13 and MPK25 in silenced and control plant leaves by Q-PCR analysis; The error bars were calculated based on three biological replicates using standard deviation. “*”: significant difference (P < 0.05); “**”: significant difference (p < 0.01). The cotton histone 3 (AF026714) was used as the reference gene.

Figure 9

Silencing of MPK9 , MPK13 and MPK25 enhances plant susceptibility to Verticillium dahlia infection. Individual genes of the cotton cultivar Hai7124 were first silenced by VIGS, and the plants were then inoculated with V. dahliae suspension spores at a concentration of 1 × 10⁷/mL. (a) Plant phenotypes at 28 days after V. dahlia inoculation. (b) Percentage of diseased leaves after V. dahlia inoculation. The percentage of diseased leaves was scored. The experiments were repeated using 15 plants per treatment. The error bars were calculated based on three biological replicates using standard deviation.