Genome-wide identification and characterization of LRR-RLKs reveal functional conservation of the SIF subfamily in cotton (Gossypium hirsutum)

Abstract

Background: As one of the largest subfamilies of the receptor-like protein kinases (RLKs) in plants, Leucine Rich Repeats-RLKs (LRR-RLKs) are involved in many critical biological processes including growth, development and stress responses in addition to various physiological roles. Arabidopsis contains 234 LRR-RLKs, and four members of Stress Induced Factor (SIF) subfamily (AtSIF1-AtSIF4) which are involved in abiotic and biotic stress responses. Herein, we aimed at identification and functional characterization of SIF subfamily in cultivated tetraploid cotton Gossypium hirsutum.

Results: Genome-wide analysis of cotton LRR-RLK gene family identified 543 members and phylogenetic analysis led to the identification of 6 cotton LRR-RLKs with high homology to Arabidopsis SIFs. Of the six SIF homologs, GhSIF1 is highly conserved exhibiting 46-47% of homology with AtSIF subfamily in amino acid sequence. The GhSIF1 was transiently silenced using Virus-Induced Gene Silencing system specifically targeting the 3' Untranslated Region. The transiently silenced cotton seedlings showed enhanced salt tolerance compared to the control plants. Further, the transiently silenced plants showed better growth, lower electrolyte leakage, and higher chlorophyll and biomass contents.

Conclusions: Overall, 543 LRR-RLK genes were identified using genome-wide analysis in cultivated tetraploid cotton G. hirsutum. The present investigation also demonstrated the conserved salt tolerance function of SIF family member in cotton. The GhSIF1 gene can be knocked out using genome editing technologies to improve salt tolerance in cotton.

Keywords: Genome-wide analysis; Gossypium hirsutum; LRR-RLKs; Salt tolerance.

Fig. 1

Phylogenetic analysis of Gossypium hirsutum LRR-RLK protein sequences. The evolutionary history was inferred using the Neighbor-Joining method with 1000 bootstrap replication. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid substitutions per site. The analysis involved 543 G. hirsutum LRR-RLK protein sequences and 234 Arabidopsis thaliana LRR-RLK protein sequences. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA6

Fig. 2

Phylogenetic tree of Arabidopsis thaliana SIF family and G. hirsutum LRR-RLK subclade I protein kinases. a The phylogenetic tree is constructed using the Maximum Likelihood method based on the JTT matrix-based model with MEGA 6. The analysis involved 13 G. hirsutum LRR-RLK subclade I protein sequences with 4 of Arabidopsis thaliana SIF family protein sequences. All positions containing gaps and missing data were eliminated. b Alignment of Malectin-like domain and (c) LRR domain of AtSIFs and GhLRR-RLKs protein sequences. Protein alignment analysis was conducted with Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). In the alignment, amino acid residues are depicted with different colors for distinguishing. Ellipses represent amino acid gaps. The numbers indicate the positions of amino acid residues. Malectin-like domain in (b) and LRR domains in (c) are highlighted with red boxes. In (c), ‘L--L--L--L-L--N-L--G-IP-’ indicates the conserved amino acid sequence of LRR domain, and the predicted β-strand/β-turn structure is underlined as --L-L--, where the ‘-’ stands for non-conserved amino acid residues, the ‘L’ represents Leu or Ile, and the ‘I’ represents Val or Ile

Fig. 3

Chromosomal localization and distribution of G. hirsutum LRR-RLKs. Chromosomal coordinates of GhLRR-RLKs were plotted on the G. hirsutum A-subgenome and D-subgenome specific chromosomes. Genes in red color and green indicate the tandem duplication. Genes located on unanchored scaffolds are not included in this figure. All the chromosomes are drawn using the scale (in Mb) shown in the figure

Fig. 4

Expression analysis of G. hirsutum LRR-RLKs. Hierarchically clustered heatmap for individual subclades of G. hirsutum LRR-RLK genes in − 3 dpa ovule, − 1 dpa ovule, − 0 dpa ovule, 1 dpa ovule, 3 dpa ovule, 5 dpa fiber, 10 dpa fiber, 20 dpa fiber, 25 dpa fiber, and leaves. Scales used to prepare heatmap is included with individual subclade specific heatmaps

Fig. 5

Real-time RT-PCR analysis of G. hirsutum LRR-RLKs expression. Ovule, fiber, and leaf tissue samples were collected at 5 dpa from cotton plants grown in the green house for real-time RT-PCR analysis. The expressions of 26 G. hirsutum LRR-RLKs in various subclades were analyzed. GhActin2 was used as the internal reference gene. Data shown are an average of three technical replicates for three independent biological replicates. Error bars represent S.D. (n = 9). The statistically significant difference between fiber and other tissues was determined by t-test. P < 0.05 was marked as *. P < 0.01 was marked as **

Fig. 6

Expression and phenotypic analyses of GhSIF1 under salt treatment and in VIGS treat cotton plants. (a) Cotton (G. hirsutum) seeds germinated on ½ MS medium were transferred to ½ MS with or without 300 mM NaCl medium. Ten days later, leaves and roots were collected for real-time PCR analysis. GhActin2 was used as the reference gene. (b) 10 days old cotton plants (G. hirsutum) with two cotyledon leaves were infiltrated with TRV1 and empty TRV2 (as control) or TRV2-GhSIF1 (targeting GhSIF1 mRNA). Ten days later, leaf samples were collected for real-time PCR analysis. GhActin2 was used as the reference gene. Data shown are an average of three technical replicates for two independent biological replicates. Error bars represent S.D. (n = 6). The statistically significant difference was determined by t-test. P < 0.05 was marked as *. P < 0.01 was marked as **. Pictures were taken (c) before salt treatment and (d) 18 days after salt treatment

Fig. 7

Down-regulation of GhSIF1 leads to enhanced salt tolerance in VIGS treated cotton plants. Ten days old cotton plants (G. hirsutum) with two cotyledon leaves were infiltrated with TRV1 and empty TRV2 (as control) or TRV2-GhSIF1 (targeting GhSIF1 mRNA). Ten days later, plants were treated with 300 mM NaCl for 2 weeks. a Pictures were taken 18 days after salt treatment. b Shoot length and Root length, c fresh weight, d dry weight, e chlorophyll content, and f electrolyte leakage of control plants and GhSIF1 targeting plants were measured. For (B-D) data shown are an average of eight independent biological replicates. Error bars represent S.D. (n = 8). P < 0.05 was marked as *. P < 0.01 was marked as **. For (E-F) data shown are an average of three technical replicates for five independent biological replicates. Error bars represent S.D. (n = 15). P < 0.05 was marked as *. P < 0.01 was marked as **. VIGS(empty): control plant. VIGS(GhSIF1): GhSIF1 targeting plant