Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (15): 3219-3231.doi: 10.3864/j.issn.0578-1752.2021.15.007

• PLANT PROTECTION • Previous Articles     Next Articles

Identification of Xylosidase Genes from Verticillium dahliae and Functional Analysis Based on HIGS Technology

ZHANG XiaoXue(),SUN TianGe,ZHANG YingChun,CHEN LiHua,ZHANG XinYu,LI YanJun(),SUN Jie   

  1. College of Agriculture, Shihezi University, Shihezi 832003, Xinjiang
  • Received:2020-11-09 Accepted:2020-12-18 Online:2021-08-01 Published:2021-08-10
  • Contact: YanJun LI E-mail:943124507@qq.com;lyj20022002@sina.com

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Abstract:

【Objective】 The objective of this research is to identify xylosidase genes from Verticillium dahliae and study the relationship between xylosidase genes and pathogenicity of V. dahliae, which will provide a theoretical basis for exploring the molecular mechanism of pathogenicity of V. dahliae and a scientific basis for formulating better control strategies for verticillium wilt.【Method】 All xylosidase genes were identified from the genome database of V. dahliae by bioinformatics, and their protein domain, chromosomal location and phylogenetic relationship were analyzed. Quantitative real-time PCR (qRT-PCR) was used to detect the expression pattern of xylosidase genes in V. dahliae cultured with different root exudates from resistant and susceptible cotton varieties for 0, 6, 12, 24 and 48 h. The function of one of the xylosidase genes VdxyL3 in the infection of V. dahliae was analyzed by using host-induced gene silencing (HIGS) method. The target fragment of VdxyL3 was injected into cotton, and then V. dahliae Vd991 was inoculated to those plants injected with target fragment of VdxyL3 by using root-dip approach. The phenotype of transformed plants was observed and the disease index was counted, meanwhile, the biomass of fungi and the expression level of VdxyL3 in the plants were detected by qRT-PCR technique.【Result】 Bioinformatics analysis showed that there were 13 xylosidase genes (VdxyL1-VdxyL13) in V. dahliae, whose coding sequences ranged from 1 461 to 2 544 bp, molecular weight of the encoded proteins ranged from 38.78 to 90.97 kD, and theoretical isoelectric point ranged from 4.67 to 5.89. Protein domain phylogenetic relationship analysis showed that there were 9 glycoside hydrolase 43 family members, 1 glycoside hydrolase 3 family member and 3 glycoside hydrolase 31 family members included in xylosidase genes. The chromosomal location analysis showed that the 13 genes were distributed on 6 chromosomes and no gene clusters were formed. qRT-PCR analysis showed that the expression of 6 xylosidase genes was induced by cotton root exudates. After being cultured in one or more root exudates for 6 h or 12 h, expression levels of these genes were significantly increased and then decreased. Among 6 genes, the expression level of VdxyL3 increased significantly after sensing root exudates from sea island cotton. The results based on HIGS technology showed that after 14 and 21 days of inoculation, the disease symptom of cotton plants transformed with VdxyL3 interfering fragment was more serious, and the disease index (33.3 and 83.9) was significantly higher than that of empty vector control (21.7 and 66.1). qRT-PCR analysis showed that cotton plants transformed with VdxyL3 interfering fragment had higher fungal biomass but lower expression level of VdxyL3 compared to the empty vector control.【Conclusion】 The VdxyL3 gene silencing by using HIGS technology lead to a significant decrease of cotton resistance to V. dahliae, indicating that VdxyL3 may play a certain role in the pathogenesis of V. dahliae and host-pathogen interaction.

Key words: cotton, Verticillium dahliae, verticillium wilt, VdxyL3, xylosidase, host-induced gene silencing (HIGS)

Table 1

Primer sequences"

基因名称
Gene name		 引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 用途
Purpose
VdxyL3 (VDAG_01866) VdxyL3-F1 GGAATTCCTGGGCCTCTTCGCTCTATG PCR
VdxyL3-R1 GGGTACCACAGCACTGAGACCAGCATC PCR
VdxyL3-F CGGAAACATTCACCTCCCCA qRT-PCR
VdxyL3-R GGCGTAAGCCTCGAAAGCAT qRT-PCR
VdxyL4 (VDAG_04702) VdxyL4-F CAGCGCCATTGAAATCGAGG qRT-PCR
VdxyL4-R GGATTATTCCAGAGAAACCC qRT-PCR
VdxyL6 (VDAG_09393) VdxyL6-F CTCAACGCCAGCTTCTACGT qRT-PCR
VdxyL6-R CGTCGGGATCGTAGCGTAAA qRT-PCR
VdxyL8 (VDAG_05579) VdxyL8-F TAAGACCATTGAGAGCCGCG qRT-PCR
VdxyL8-R AGTTAACGTCGTGCCCTTGT qRT-PCR
VdxyL10 (VDAG_09302) VdxyL10-F CGCCAAACCCACTTCTCCTA qRT-PCR
VdxyL10-R TAGGACGACTCGGAGCTCAT qRT-PCR
VdxyL12 (VDAG_02226) VdxyL12-F CGATCTGCTGCTTGAGGAGT qRT-PCR
VdxyL12-R AGGTCGAGGAATGGCTGTTG qRT-PCR
Tubulin Tubulin-F TCCACCTTCGTCGGTAACTC qRT-PCR
Tubulin-R GCCTCCTCCTCGTACTCCTC qRT-PCR
Ve-ITS1 Ve-ITS1-F AAAGTTTTAATGGTTCGCTAAGA qRT-PCR
ST-VE1 ST-VE1-R CTTGGTCATTTAGAGGAAGTAA qRT-PCR
GhUBQ7 GhUBQ7-F GAAGGCATTCCACCTGACCAAC qRT-PCR
GhUBQ7-R CTTGACCTTCTTCTTCTTGTGCTTG qRT-PCR

Table 2

Identification of xylosidase genes in V. dahliae"

基因名称
Gene name		 序列号
Sequence number		 编码序列
Coding sequence (bp)		 编码蛋白
Coding protein (aa)		 分子量
Molecular weight (kD）		 等电点
pI		 基因的描述
Gene description		 亚细胞定位预测
Subcellular location prediction
VdxyL1 VDAG_01169 1786 558 62.33 5.16 β-木糖苷酶β-xylosidase 细胞膜外的蛋白质
Extracellular proteins
VdxyL2 VDAG_02166 1804 571 65.15 4.97 木糖苷酶/阿拉伯糖苷酶
Xylosidase/arabinosidase		 细胞膜外的蛋白质
Extracellular proteins
VdxyL3 VDAG_01866 2544 834 90.97 4.67 木糖苷酶/阿拉伯糖苷酶
Xylosidase/arabinosidase		 细胞膜外的蛋白质
Extracellular proteins
VdxyL4 VDAG_04702 1089 335 38.78 5.31 α-木糖苷酶α-xylosidase 细胞质Cytoplasm
VdxyL5 VDAG_07817 1611 512 56.13 5.04 β-木糖苷酶β-xylosidase 细胞膜外的蛋白质
Extracellular proteins
VdxyL6 VDAG_09393 1943 609 67.13 4.88 β-木糖苷酶β-xylosidase 细胞膜外的蛋白质
Extracellular proteins
VdxyL7 VDAG_03859 1881 572 62.66 5.89 β-葡糖苷酶/β-木糖苷酶
β-glucosidase/β-xylosidase		 溶酶体Lysosome
VdxyL8 VDAG_05579 2353 764 85.22 5.18 α-木糖苷酶α-xylosidase 溶酶体Lysosome
VdxyL9 VDAG_06173 2020 488 54.44 5.96 β-木糖苷酶β-xylosidase 高尔基体Golgi apparatus
VdxyL10 VDAG_09302 1614 537 60.70 5.68 β-木糖苷酶β-xylosidase 细胞质Cytoplasm
VdxyL11 VDAG_00716 1841 533 59.45 5.28 木糖苷酶/阿拉伯糖苷酶
Xylosidase/arabinosidase		 细胞核Cell nucleus
VdxyL12 VDAG_02226 1995 664 76.09 5.88 α-木糖苷酶α-xylosidase 细胞质Cytoplasm
VdxyL13 VDAG_03628 1461 400 44.70 5.78 木糖苷酶/阿拉伯糖苷酶
Xylosidase/arabinosidase		 细胞膜外的蛋白质
Extracellular proteins

Fig. 1

Domains analysis of xylosidase proteins in V. dahliae Black indicates the signal peptide; Blue indicates the Glyco_hydro 43 domain; Green indicates the Glyco_hydro 31 domain; Red indicates the Glyco_hydro 3 domain; Yellow indicates the Glyco_hydro 3_C domain; Orange indicates the Gal_mutarotas_2 domain"

Fig. 2

Phylogenetic tree analysis of xylosidase proteins in V. dahliae and other species Red indicates the Glyco_hydro 3 family; Purple indicates the Glyco_hydro 31 family, Blue indicates the Glyco_hydro 43 family"

Fig. 3

Structure analysis of xylosidase genes in V. dahliae"

Fig. 4

Chromosomal distribution of xylosidase genes in V. dahliae"

Fig. 5

The expression pattern of xylosidase genes in V. dahliae induced by root exudates from different cotton varieties"

Fig. 6

Detection of HIGS silencing effect A: Phenotypes of susceptible variety Xinluzao 8 after 10 days of injection with pTRV2-GhCHLI;B:qRT-PCR analysis of VdxyL3 expression in the pTRV2-00 and pTRV2-VdxyL3 treated plants. Total RNA was isolated from stems at 14 dpi. Tubulin was used as the control"

Fig. 7

Disease investigation on cotton plants treated with HIGS A: Disease symptom of the pTRV2-00 and pTRV2-VdxyL3 treated plants at 14 and 21 dpi;B:Plant height of the pTRV2-00 and pTRV2-VdxyL3 treated plants at 14 dpi;C:Disease index of the pTRV2-00 and pTRV2-VdxyL3 treated plants at 14 and 21 dpi;D: Disease symptom in stems of the pTRV2-00 and pTRV2-VdxyL3 treated plants at 14 dpi"

Fig. 8

Recovery experiment and relative content detection of V. dahliae in cotton plants treated with HIGS A:Quantification of the relative fungal biomass in stems of the pTRV2-00 and pTRV2-VdxyL3 treated plants at 14 dpi;B:Fungal isolation in the stem sections from pTRV2-00 and pTRV2-VdxyL3 treated plants at 14 dpi. Stems were plated on PDA medium. Photos were taken at 7 dpi of culture at 25℃"

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