Pan-genome analysis of GT64 gene family and expression response to Verticillium wilt in cotton

Abstract

Background: The GT64 subfamily, belonging to the glycosyltransferase family, plays a critical function in plant adaptation to stress conditions and the modulation of plant growth, development, and organogenesis processes. However, a comprehensive identification and systematic analysis of GT64 in cotton are still lacking.

Results: This study used bioinformatics techniques to conduct a detailed investigation on the GT64 gene family members of eight cotton species for the first time. A total of 39 GT64 genes were detected, which could be classified into five subfamilies according to the phylogenetic tree. Among them, six genes were found in upland cotton. Furthermore, investigated the precise chromosomal positions of these genes and visually represented their gene structure details. Moreover, forecasted cis-regulatory elements in GhGT64s and ascertained the duplication type of the GT64 in the eight cotton species. Evaluation of the Ka/Ks ratio for similar gene pairs among the eight cotton species provided insights into the selective pressures acting on these homologous genes. Additionally, analyzed the expression profiles of the GT64 gene family. Overexpressing GhGT64_4 in tobacco improved its disease resistance. Subsequently, VIGS experiments conducted in cotton demonstrated reduced disease resistance upon silencing of the GhGT64_4, may indicate its involvement in affecting lignin and jasmonic acid biosynthesis pathways, thus impacting cotton resistance. Weighted Gene Co-expression Network Analysis (WGCNA) revealed an early immune response against Verticillium dahliae in G. barbadense compared to G. hirsutum. Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) analysis indicated that some GT64 genes might play a role under various biotic and abiotic stress conditions.

Conclusions: These discoveries enhance our knowledge of GT64 family members and lay the groundwork for future investigations into the disease resistance mechanisms of this gene in cotton.

Keywords: Expression pattern; GT64; Transgenic tobacco; Upland cotton; VIGS; WGCNA.

Fig. 1

A phylogenetic analysis of the GT64 family members in eight species of the cotton species

Fig. 2

The physical locations of the GT64 genes on the chromosomes, (A)-(D) represent G. arboreum, G. raimondii, G. hirsutum, G. barbadense

Fig. 3

The physical locations of the GT64 genes on the chromosomes, (A)-(D) represent G. herbaceum, G. darwinii, G. mustelinum, G. tomentosum

Fig. 4

The analysis of the GT64 genes involved a comprehensive investigation of gene structure, motifs, and cis-acting elements. The analysis was divided into five main categories: A:Phylogenetic tree of upland cotton, B:Motif composition and distribution, C:Conserved domains of the GT64 genes, D:Visualization of cis-acting components, E:Gene structure of GhGT64s

Fig. 5

Collinearity analysis of eight cotton genera

Fig. 6

Collinearity analysis was conducted for GT64s in different cotton specie. (A)-(E) represent G. barbadense, G. hirsutum, G. darwinii, G. mustelinum, and G. tomentosum, respectively. (F-H) Selection pressure analysis was carried out to examine the evolutionary dynamics of the GT64 gene family

Fig. 7

Generate a heatmap of the GT64 gene family members based on transcriptome data.(A)GhGT64s expression profiles in various cotton organs.(B)Heatmap showing expression of GhGT64_2 and GhGT64_4 in different cotton tissues.(C)Expression of GhGT64s in different tissues and at different periods of upland cotton.(D)GhGT64s expression profiles in ovule and fiber at different developmental stages.(E) GhGT64s expression levels in fuzz and fuzzless materials at various time points. (F)Expression levels of GhGT64s in ovule and fiber in materials with higher and lower lint percentages at different time points.(G)Expression patterns of GhGT64s in oil material.(H)Expression patterns of GhGT64s in CJ56 and CJ72 material

Fig. 8

Generate a heatmap of the GT64 gene family members based on transcriptome data.(A)Demonstration of GhGT64s activity in glanded and glandless variants.(B)Depiction of GhGT64s response in upland cotton to TDZ exposure.(C)Display of GhGT64s response under extreme temperature, salinity and drought at different time intervals.(D)Illustration of GhGT64s reaction in G. hirsutum when exposed to Verticillium dahliae at varying time points.(E)Portrayal of GbGT64s expression across different tissues and fiber development stages.(F)Exposition of GbGT64s expressions in G. barbadense varieties 5917 and PimaS7 at different fiber development stages.(G)Indication of GbGT64s reaction in G. barbadense under Fusarium oxysporum f. sp. vasinfectum stress conditions

Fig. 9

Characterization of tobacco resistance in transgenic GhGT64_4. (A) Assessment of disease resistance index in transgenic and wild type tobacco. (B)-(D) represent the phenotypes of wild-type tobacco, wild-type tobacco inoculated with vd592, and tobacco transformed with the GhGT64_4 and inoculated with vd592, respectively. (E)-(J) Analysis of gene expression levels. Statistical significance was observed in the experimental group compared to the control group at *P < 0.05,**P < 0.01, *** P < 0.001, **** P < 0.0001

Fig. 10

Validation of GhGT64_4 function.(A) Plant with albino phenotype (pTRV2-CLA, positive control; pTRV2, negative control). (B) The transgenic plants with silenced GhGT64_4. (C)The transgenic plants with silenced GhGT64_4 inoculated with vd592. (D) VIGS efficiency assessment of GhGT64_4 in upland cotton. (E) Disease resistance index of silenced and normal plants at 15 dpi.(F) Expression levels of resistance-related genes in pTRV2:00 and pTRV2: GhGT64_4 plants. (G) Expression levels of resistance-related genes in pTRV2:00 and pTRV2: GhGT64_4 plants after vd592 inoculation.(H) Fungal restoration experiment. (I) The lignin content of TRV: 00 and TRV: GhGT64_4 plants. Statistically significant differences from the control group indicated by *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001

Fig. 11

Transcriptome data were used to study GhGT64s in G. hirsutum and G. barbadense. (A) Expression analysis of GhGT64s in G. hirsutum. (B) Expression analysis of GhGT64s in G. barbadense. (C) Expression analysis of GhGT64_4 in G. hirsutum and G. barbadense. (D) Number of genes in the MEturquoise module of G. hirsutum and G. barbadense. (E) KEGG pathway enrichment analysis of the MEturquoise module in G. hirsutum. (F) KEGG pathway enrichment analysis of the MEturquoise module in G. barbadense

Fig. 12

WGCNA in G. hirsutum. (A) Gene clustering analysis outcomes from WGCNA on transcriptome data in G. hirsutum. (B) Heatmap illustrating correlations between modules and traits. (C) Development of the comprehensive network for GhGT64_4. (D) Perform KEGG pathway enrichment analysis on 150 genes that interact with GhGT64_4

Fig. 13

WGCNA in G. barbadense. (A) Gene clustering analysis outcomes from WGCNA on transcriptome data in G. barbadense. (B) Heatmap illustrating correlations between modules and traits. (C) Development of the comprehensive network for GhGT64_4. (D) Perform KEGG pathway enrichment analysis on 150 genes that interact with GhGT64_4

Fig. 14

qRT-PCR analysis of the GT64s in upland cotton and island cotton. (A) Expression analysis of GhGT64s in different oil materials. (B) Expression patterns of GhGT64s during fiber development. (C) Expression of GhGT64s after inoculation with vd592. (D) Expression patterns of GhGT64s under PEG stress. (E) Expression patterns of GhGT64s under salt stress. (F) Expression patterns of GbGT64s under FOV stress. The error bars indicate the means of three technical replicates ± standard errors. Statistically significant differences from the control group are denoted as *P < 0.05; **P < 0.01; ***P < 0.001