Functional Verification of GhDIR1 Gene Against Verticillium Wilt in Cotton

doi:10.11983/CBB24135

Abstract

Abstract: INTRODUCTION: Verticillium wilt (VW), caused by Verticillium dahliae, severely reduces cotton yield and fiber quality. Previous transcriptomic analysis in V. dahliae-inoculated Arabidopsis thaliana identified the pathogen-induced DIR1-like gene AT3G53980.2. In cotton, we discovered a homologous gene, GhDIR1 (Gh_A09G180700.1), encoding a lipid transfer protein. This study investigates its role in cotton resistance to V. dahliae. RATIONALE: We characterized GhDIR1’s molecular features, expression patterns under pathogen stress, and functional impact using bioinformatics, subcellular localization, qRT-PCR, and virus-induced gene silencing (VIGS) analyses. Transcriptomic analysis of wild-type and GhDIR1-silenced plants were conducted to unravel downstream regulatory networks, focusing on metabolic pathways linked to plant immunity. RESULTS: The results showed that GhDIR1 contains a 351 bp ORF encoding 116 amino acids. Subcellular localization confirmed its presence on the cell membrane. qRT-PCR showed rapid induction of GhDIR1 by V. dahliae. Silencing GhDIR1 increased cotton susceptibility to the pathogen. Transcriptomic data revealed that differentially expressed genes in silenced plants were enriched in flavonoid biosynthesis, sesquiterpene/triterpene biosynthesis, and α-linolenic acid metabolism. Key genes (GhCHS, GhDFR, GhCAD, GhSEQ, GhLOX, and GhAOC) in these pathways were downregulated, suggesting impaired synthesis of protective metabolites. CONCLUSION: It is speculated that GhDIR1 positively regulates cotton resistance to VW by modulating flavonoid and terpenoid biosynthesis and jasmonic acid-related signaling. Its silencing disrupts critical defense pathways, highlighting its role in coordinating immune responses. These findings propose GhDIR1 as a potential target for enhancing disease resistance in cotton.

The induced expression pattern of GhDIR1 and related genes after inoculation with Verticillium dahliae.

Key words: Gossypium hirsutum, cotton Verticillium wilt, GhDIR1, virus-induced gene silencing, transcriptome analysis

Huang Yuxin, Xie Tao, Wang Xingfen, Guo Huiming, Cheng Hongmei, Ma Bojun, Chen Xifeng, Su Xiaofeng. Functional Verification of GhDIR1 Gene Against Verticillium Wilt in Cotton[J]. Chinese Bulletin of Botany, 2025, 60(5): 816-830.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB24135

https://www.chinbullbotany.com/EN/Y2025/V60/I5/816

Figures/Tables 8

Table 1 Primer sequences used in this study

Primer names	Primer sequences (5′-3′)
GhDIR1-F	ATGGCAGCTGCAATGAAACTC
GhDIR1-R	TCAAGGAAGTGTATATGCTCCAC
qGhDIR1-F	CCTGGGAAAGGTGGTTGATG
qGhDIR1-R	TCTCAGGCTTGATTCCAGATGC
qGhCHS-F	GCTGCTGTTATAGTAGGTGCGGATC
qGhCHS-R	CTCAGCTAGGCTCTTTTCAATGTTC
qGhDFR-F	GCTCTGGGTTCATTGGTTCATGG
qGhDFR-R	CCTCTTCAGCTAAATCTGCTTTCC
qGhCAD-F	CCTGGCATTTGGGGAGATATCTTC
qGhCAD-R	GACACCCAACCTTTGGACTGC
qGhSEQ-F	GCAAAGGAGAATGCTCGCCTG
qGhSEQ-R	CCTGGCTGCAACAGTTCTCCAAC
qGhLOX-F	GATCGTGCTTGATGGTCTCACTG
qGhLOX-R	CGTTCCCTCTCAATGCCTGC
qGhAOC-F	CGGATAGGAATAACAGCAGGGATG
qGhAOC-R	GCCAGATCCACCAGTAATAGCG
Vd-ITS-F	TCCGTAGGTGAACCTGCGG
Vd-ITS-R	TCCTCCGCTTATTGATATGC
GhDIR1-VIGS-F	TCTGTGAGTAAGGTTACC GAATTCG- TTCTGGGATTGATTGTGCTTATT
GhDIR1-VIGS-R	ACGCGTGAGCTCGGTACC GGAT C C- ATGCTCCACATCTGTAACCGACT
p1132-GhDIR1-F	TCTGTGAGTAAGGTTACC GAATTCC- CTGGGAAAGGTGGTTGATG
p1132-GhDIR1-R	ACGCGTGAGCTCGGTACC GGATCC- TCTCAGGCTTGATTCCAGATGC
UBQ-F	AGCTCGGATACGATTGATAACG
UBQ-R	GAAGACGAAGAACAAGGGGAAG

Figure 1 Physicochemical properties of GhDIR1 and analysis of cis-acting element of the GhDIR1 gene promoter (A) Hydrophilicity analysis of GhDIR1 protein; (B) GhDIR1 structural domain analysis; (C) Tertiary structure prediction of GhDIR1 protein; (D) Transmembrane structural domain prediction of GhDIR1 protein; (E) Analysis of cis-acting element of the GhDIR1 gene promoter

Figure 2 Genome collinearity analysis of GhDIR1 Dark blue regions represent the chromosomes of Gossypium hirsutum. The gray lines depict all collinear pairs across the 26 chromosomes. The red lines highlight collinear gene pairs associated with segmental duplications of the target gene. The innermost and outermost light blue rings illustrate two distinct representations of gene density.

Figure 3 Expression of GhDIR1 in cotton inoculated with Verticillium dahliae Heat map analysis of GhDIR1 gene expressions in different organs (roots, stems) and different inoculation times (0, 0.5, 1, 2, 4, 8, and 12 h) of Gossypium hirsutum with V. dahliae. The color from blue to yellow indicates low to high level of expression.

Figure 4 Subcellular localization of GhDIR1 The fusion proteins GFP and GhDIR1-GFP were co-localized with the PM-mCherry membrane localization marker in tobacco. The GFP and GhDIR1-GFP fusion proteins exhibited green fluorescence in the green fluorescence channel, while the PM-mCherry membrane localization marker displayed red fluorescence in the mCherry channel only. (A) GFP subcellular localization map (bars=20 μm); (B) GhDIR1-GFP subcellular localization map (bars=10 μm)

Figure 5 Detection of GhDIR1 gene silencing efficiency and the resistant analysis of GhDIR1 silenced seedlings against cotton Verticillium wilt (A) TRV2::CLA1 plants (bar=5 cm); (B) Gene expression of GhDIR1 in silenced cotton plants (three biological replicates (n=3); ANOVA was used for the statistical analysis; *** P<0.001); (C) Phenotype of TRV2::00 and TRV2::GhDIR1 plants at 14 days post inoculation (dpi) with Verticillium dahliae (bar=5 cm); (D) Stem phenotype of TRV2::00 and TRV2::GhDIR1 plants at 14 dpi with V. dahliae (bar=1 cm); (E) Statistical analysis of disease index in cotton plants of TRV2::00 and TRV2::GhDIR1 at 14 dpi (grades 1-4 represent disease severity classifications. 1: Cotyledons turn yellow, no symptoms on true leaves; 2: All cotyledons show symptoms, 1-3 true leaves show necrosis or chlorosis; 3: More than 5 cotton leaves, including cotyledons, show symptoms; 4: All leaves show symptoms, and leaves fall off/plant dies); (F) Fungal biomass of TRV2::00 and TRV2::GhDIR1 plants at 14 dpi with V. dahliae (three biological replicates (n=3); ANOVA was used for the statistical analysis; *** P<0.001)

Figure 6 RNA-seq comparison between TRV2::00 and TRV2::GhDIR1 cottons (A) Heat map of differentially expressed genes (DEGs) in each sample; (B) Volcano map of DEGs; (C) GO terms; (D) KEGG terms. WT: Wild type; VIGS: Virus-induced gene silencing plant; BP: Biological process; CC: Cellular component; MF: Molecular function

Figure 7 qRT-PCR verification of the expression levels of six key differentially expressed genes (DEGs) The blue bars represent the relative expression levels of genes in TRV2::00, normalized to 1, serving as the control group. The purple bars indicate the relative expression levels of genes in TRV2::GhDIR1. Three biological replicates (n= 3). ANOVA was used for the statistical analysis. ** P<0.01; *** P<0.001

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