The Analysis of the Glycosyltransferase Activity Gene Family in Gossypium hirsutum and Functional Verification of GTs Conferring Resistance to Verticillium Wilt

Abstract

Glycosyltransferases (GTs) play an important role in plant growth and development, as well as responses to biotic and abiotic stresses. However, the function of the GT family in cotton resistance to Verticillium wilt is limited. In the present study, transcriptome analysis revealed eight GTs upregulated in susceptible cotton varieties and downregulated in resistant cotton varieties during early Verticillium dahliae inoculation, indicating they were involved in regulating the infection of V. dahliae in cotton. Promoter analysis revealed a high prevalence of MeJA (methyl jasmonate) and ABA (abscisic acid)-related cis-acting elements among these GTs. Genome-wide and location analysis of the homologous genes showed that these GTs were relatively conserved in evolution. Furthermore, a Virus-Induced Gene Silencing (VIGS) experimental results demonstrated a reduction in disease resistance after GhGT61 silencing. These insights not only deepen our understanding of the GT family's role in cotton, but also provide a foundation for future research on the disease resistance mechanisms of these genes.

Keywords: GhGT61; Glycosyltransferases; Gossypium hirsutum; Verticillium wilt.

Figure 1

GO analysis of DEGs in response to V. dahliae infection at 12 hpi in resistant and susceptible upland cotton. (A) GO analysis of up-regulating DEGs in response to V. dahliae infection at 12 hpi in susceptible upland cotton Z8. (B) GO analysis of down-regulating DEGs in response to V. dahliae infection at 12 hpi in resistant upland cotton JK1775. The transferase activity and transferring glycosyl group pathway were marked with red boxes.

Figure 2

Expression analysis of eight GTs in response to V. dahliae infection in resistant and susceptible upland cotton at 12 hpi. (A) Heatmap of eight GTs in resistant upland cotton JK1775 in response to V. dahliae infection at 12 hpi. (B) Heatmap of eight GTs in susceptible upland cotton Z8 in response to V. dahliae infection at 12 hpi. (C) qRT-PCR verification of GTs transcriptome data. The data are mean ± SD (n = 3). Significance is determined using Duncan’s multiple range test, indicated by * p ≤ 0.05 and ** p ≤ 0.01.

Figure 3

Phylogenetic tree, gene structure, and conservative motif analysis of eight GTs. (A) Neighbor-joining phylogenetic tree of eight GTs. (B) Prediction of conserved motifs in the GTs, with different colors representing distinct motifs. (C) Analysis of the intron-exon structure of the GTs, where exons and introns are depicted by yellow boxes and gray lines, respectively.

Figure 4

Phylogenetic analysis of GTs proteins from G. hirsutum, G. barbadense, G. arboreum, G. raimondii, and A. thaliana. The GTs proteins from the five species are indicated by different colors. The phylogenetic tree is constructed using MEGA X with the maximum likelihood method and 1000 bootstrap replications. Eight GTs were marked with red stars.

Figure 5

Cis-acting elements of GTs in upland cotton. (A) Predicted cis-elements in the promoter regions of GTs. (B) Proportion of different types of cis-elements. Ten groups of cis-acting elements are identified and represented by color-coded boxes.

Figure 6

Chromosome distribution of GTs in G. hirsutum. The heatmap visualizes the chromosome density, with a blue-to-red color scale representing low to high-density values. The chromosome number is annotated along the left side in orange, while eight GTs (red) and their homologous genes (black) are labeled on the right flanking axis, respectively.

Figure 7

Intraspecific and interspecific collinearity analysis of eight GTs. (A) Synteny analysis between At and Dt subgenomes in G. hirsutum. Duplicated gene pairs are denoted by red connecting lines, whereas gray lines indicate identical gene pairs genome-wide. Eight GTs are labeled in red, with homologous genes distinguished by black labeling. (B) Synteny of repetitive gene pairs in three cotton species (G. hirsutum, G. arboretum, and G. raimondii).

Figure 8

Function of GhGT61 on the resistance of cotton to V. dahliae. (A) VIGS-mediated silencing of GhCLA1 results in an albino phenotype in cotton. (B) Phenotype of silenced and normal plants inoculated with V. dahliae at 13 dpi. (C) Vascular bundle browning observed in longitudinal stem sections of silenced and control plants. (D) Fungal biomass recovery experiment in stems of silenced and control plants. (E) The expression level of GhGT61 in silenced and normal plants. (F) Disease index of silenced and normal plants at 13 dpi. (G). qRT-PCR quantification of fungal biomass in stems in silenced and control plants. The data are presented as mean ± SD (n = 3). Significance is determined using Duncan’s multiple range test, indicated by ** p ≤ 0.01.