尖孢镰刀菌黏团专化型β-葡萄糖苷酶Foglu1的功能

doi:10.3969/j.issn.1004-1524.2023.02.15

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (2): 373-382.DOI: 10.3969/j.issn.1004-1524.2023.02.15

尖孢镰刀菌黏团专化型β-葡萄糖苷酶Foglu1的功能

王腾(), 王碧香, 李诗瑶, 尉婧, 李二峰()

天津农学院园艺园林学院,天津 300392

收稿日期:2022-04-08 出版日期:2023-02-25 发布日期:2023-03-14
通讯作者: 李二峰
作者简介:*李二峰,E-mail:lef143@126.com
王腾(1996—),男,湖北黄冈人,硕士研究生,主要从事植物病理学研究。E-mail:811151928@qq.com
基金资助:
国家自然科学基金(31801682);国家重点研发计划(2018YFD0201204)

Functional study of a β-glucosidase Foglu1 in Fusarium oxysporum f. sp. conglutinans

WANG Teng(), WANG Bixiang, LI Shiyao, WEI Jing, LI Erfeng()

College of Horticulture and Landscape Architecture, Tianjin Agricultural University, Tianjin 300392, China

Received:2022-04-08 Online:2023-02-25 Published:2023-03-14
Contact: LI Erfeng

摘要/Abstract

摘要：

甘蓝枯萎病是由尖孢镰刀菌黏团专化型(Fusarium oxysporum f. sp. conglutinans, FOC)引起的。β-葡萄糖苷酶(β-glucosidase)是纤维素降解过程中的限速酶。本研究中,为明确FOC中一种β-葡萄糖苷酶在其生长发育和致病中的生物学功能,通过split-marker同源重组技术对该病原菌中一种β-葡萄糖苷酶基因(Foglu1)进行敲除与回补,分析野生型菌株与ΔFoglu1敲除突变体菌株、ΔFoglu1-C回补菌株间的表型差异。结果表明,在细胞膜胁迫试验(0.05% SDS)中,ΔFoglu1突变体生长5 d后的菌落直径与野生型相比降低19.6%,推测Foglu1基因对病原菌细胞膜功能具有一定的作用;氧胁迫试验(0.1% H₂O₂)中菌落直径比野生型减小14%,表明ΔFoglu1突变体比野生型对氧胁迫更加敏感;ΔFoglu1突变体产孢量较野生型降低约22.3%,说明Foglu1基因影响FOC的产孢能力;在接种寄主甘蓝时,发现接种ΔFoglu1菌株的甘蓝植株初期发病缓慢,接种后第8天的病情指数比接种野生菌株的低39.5%,接种后第11、15和18天,野生菌株和ΔFoglu1突变体致病力无显著差异,说明Foglu1基因影响病原菌与寄主间的早期互作,延缓发病但对其致病力无影响。上述结果显示,β-葡萄糖苷酶在FOC细胞膜完整性、氧化胁迫、产孢和致病过程中可能起重要作用。

关键词: 甘蓝枯萎病, 基因敲除, β-葡萄糖苷酶, 致病力

Abstract:

Fusarium oxysporum f. sp. conglutinans(FOC) is a causal agent of cabbage Fusarium wilt. The β-glucosidase is a rate-limiting enzyme for cellulose degradation. In this study, to determine biological roles of a β-glucosidase (Foglu1) in fungal development and virulence of FOC, the Foglu1 gene was knocked out by split-marker homologous recombination and complementation strains were generated by reintroducing Foglu1 gene into ΔFoglu1 mutant. Then, phenotypes of the wild type strain, deletion mutant ΔFoglu1 and complemented strain ΔFoglu1-C were analyzed. The result showed that the colony diameter of ΔFoglu1 mutant was significantly reduced by 19.6% compared to that of the wild type in cell membrane stress experiment (0.05% SDS), indicating that Foglu1 was required for cell integrity in FOC. Also, in oxygen stress experiment (0.1% H₂O₂), colony diameter of ΔFoglu1 mutant significantly decreased by 14% compared with the wild type, indicating that deletion of Foglu1 resulted in the increase of sensitivity to oxygen stress. Meanwhile, conidial production of ΔFoglu1 mutant was reduced by 22.3% compared to the wild type, indicating that Foglu1 gene was important for sporulation in FOC. Interestingly, at 8 days after inoculation, disease index of ΔFoglu1 mutant was significantly reduced and 39.5% lower than that of the wild type. The disease indexes of seedlings inoculated with the wild type, ΔFoglu1 and ΔFoglu1-C were no differences at 11, 15 and 18 days after inoculation. It showed that Foglu1 played roles in the early interaction between the pathogen and the host. It could delay the disease development but had no effect on pathogenicity. In conclusion, these findings suggested that β-glucosidase (Foglu1) played an important role in FOC cell membrane integrity, oxidative stress, sporulation and pathogenesis.

Key words: cabbage Fusarium wilt, gene knockout, β-glucosidase, pathogenicity

中图分类号:

S432.1

王腾, 王碧香, 李诗瑶, 尉婧, 李二峰. 尖孢镰刀菌黏团专化型β-葡萄糖苷酶Foglu1的功能[J]. 浙江农业学报, 2023, 35(2): 373-382.

WANG Teng, WANG Bixiang, LI Shiyao, WEI Jing, LI Erfeng. Functional study of a β-glucosidase Foglu1 in Fusarium oxysporum f. sp. conglutinans[J]. Acta Agriculturae Zhejiangensis, 2023, 35(2): 373-382.

图/表 8

表1 引物名称与序列

Table 1 Names and sequences of primers

引物名称 Primer name	引物序列 Primer sequence (5'→3')
UP-F	GCCACCTGCTTCACTTCTGC
UP-R	CATTGTTGACCTCCACTAGCTCCAGCCAAGGGAGGGGAGAGAAGCCTCAG
DOWN-F	GGCAAAGGAATAGAGTAGATGCCGACCGGGCAGCTGTATGTCAGCCTCGG
DOWN-R	GACATCCAAGGAACAGCGCC
Foglu1-CHECK-F	GCCAGAACTCCAAGCTCCTC
Foglu1-CHECK-R	GCTCATCTCCCTGTTGCCAG
H-F	GGACTAGTCCCGAGCAAGGACTCATGCGTG
H-R	AACTGCAGAACCAATGCATTGGCCACGACACTGTTCCTTCCG
NEO-CHECK-F	CCAGGGTTTTCCCAGTCACG
NEO-CHECK-R	GATCGACAAGACCGGCTTCC
HPT-F	CTTGGCTGGAGCTAGTGGAGGT
HPT-R	CCCGGTCGGCATCTACTCTATTC
HPT-R1	GGATGCCTCCGCTCGAAGTA
HPT-F2	CGTTGCAAGACCTGCCTGAA
UPCHECK-F	CCTGCATCTGCATACCACCC
UPCHECK-R	GGATGCCTCCGCTCGAAGTA
DOWNCHECK-F	CGTTGCAAGACCTGCCTGAA
DOWNCHECK-R	CTGCTTCCAGCCTCATACGC
EF1α-F	CATCGGCCACGTCGACTCT
EF1α-R	AGAACCCAGGCGTACTTGAA
Foglu1-F	CCAACAACGAATGGGCCCAG
Foglu1-R	GCTCATCTCCCTGTTGCCAG
hpt-F	GTCACGTTGCAAGACCTGCC
hpt-R	CGCGCATATGAAATCACGCC

图1 尖孢镰刀菌β-葡萄糖苷酶Foglu1蛋白结构域分析和不同真菌中GH3家族β-葡萄糖苷酶同源蛋白系统发育分析 A,尖孢镰刀菌β-葡萄糖苷酶Foglu1蛋白结构域分析。红色区域代表糖苷水解酶3N末端结构域,绿色区域代表糖苷水解酶3C末端结构域,紫色区域代表fibronectin type Ⅲ-like domain. B,β-葡萄糖苷酶同源蛋白系统发育分析。尖孢镰刀菌黏团专化型Foglu1用蓝色三角标注。

Fig.1 Structural domain and phylogenetic relationship analysis of the β-glucosidase homologs from different fungal species A, Conserved domains of β-glucosidase in F. oxysporum f. sp. conglutinans based on Pfam analysis. The red represented the glycosyl hydrolase family 3 N-terminal domain. The green represented the glycosyl hydrolase family 3 C-terminal domain. The purple represented the fibronectin type Ⅲ-like domain. B, Phylogenetic relationship of the β-glucosidase homologs from different fungal species. F. oxysporum f. sp. conglutinans was marked with blue triangle.

图2 Foglu1基因敲除 A,同源置换臂的扩增。M,Trans 2K Plus Maker;1,UP-HPT1片段;2,HPT2-DOWN片段。B,基因敲除策略。

Fig.2 Knockout of Foglu1 gene A, Amplification of homologous replacement arms. M, Trans 2K Plus Maker; 1, Fragment of UP-HPT1; 2, Fragment of HPT2-DOWN. B, Strategy of gene knockout.

图3 ΔFoglu1突变体的验证 A,缺失突变体的PCR验证。M,Trans 2K Plus Maker;1,ΔFoglu1-1突变体;2,ΔFoglu1-2突变体;WT,野生型。B,qRT-PCR验证ΔFoglu1突变体基因拷贝数。

Fig.3 Verification of ΔFoglu1 mutants A, PCR verification of ΔFoglu1 mutants. M, Trans 2K Plus Maker; 1, ΔFoglu1-1 mutant; 2, ΔFoglu1-2 mutant; WT, Wild type. B, qRT-PCR verification of gene copy number in ΔFoglu1 mutants.

图4 PKN-Foglu1质粒的酶切验证与ΔFoglu1-C验证 A,PKN-Foglu1的酶切验证。M,Trans 15K maker;1,PKN质粒单酶切;2,PKN-Foglu1质粒单酶切;3,PKN-Foglu1质粒双酶切;4,Foglu1回补片段双酶切。B,回补菌株ΔFoglu1-C的验证。M,Trans 2K Plus Maker;1,ΔFoglu1-C的NEO-CHECK片段;2,ΔFoglu1突变体的NEO-CHECK片段;3,ΔFoglu1-C的Foglu1-CHECK片段;4,ΔFoglu1突变体的Foglu1-CHECK片段。

Fig.4 PKN-Foglu1 enzyme digestion and ΔFoglu1-C validation A, Enzymatic digestion validation of PKN-Foglu1. M, Trans 15K maker; 1, Single cleavage of PKN plasmid; 2, Single cleavage of PKN-Foglu1 plasmid; 3, Double digestion of PKN-Foglu1 plasmid; 4, Double digestion of Foglu1 complement fragment. B, Validation of complement strain ΔFoglu1-C. M, Trans 2K Plus Maker; 1, NEO-CHECK fragment of ΔFoglu1-C; 2, NEO-CHECK fragment of ΔFoglu1; 3, Foglu1-CHECK fragment of ΔFoglu1-C; 4, Foglu1-CHECK fragment of ΔFoglu1.

图5 ΔFoglu1突变体生长胁迫测定 A,不同胁迫条件下生长5 d的菌落;B,菌落直径的方差分析。柱形上方相同字母表示数据在单因素方差分析中差异不显著(P>0.05),不同字母表示差异显著(P<0.05)。下同。

Fig.5 Stress measurement of ΔFoglu1 mutant growth A, Colonies under different stresses for 5 days; B, Variance analysis of colony diameter. The same letters above the column indicated that the data were not significant (P>0.05) in one-way ANOVA, different letters indicated significant (P<0.05). The same as below.

图6 野生型和突变体菌株的产孢能力

Fig.6 Conidia production of wild type and mutant stains

图7 缺失Foglu1基因导致FOC侵染初期的致病力减弱 A,接种尖孢镰刀菌的甘蓝在第8、11、15天的生长情况;B,野生菌株、缺失突变菌株ΔFoglu1、回补菌株ΔFoglu1-C接种甘蓝4~18 d的病情指数。

Fig.7 Deletion of the Foglu1 gene leads to reduced virulence in the initial stage of FOC infected cabbage A, The growth of cabbage inoculated with FOC at 8 days, 11 days and 15 days after inoculation; B, The disease indexes of wild type, ΔFoglu1 and ΔFoglu1-C from 4 to 18 days after inoculation.

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尖孢镰刀菌黏团专化型β-葡萄糖苷酶Foglu1的功能

Functional study of a β-glucosidase Foglu1 in Fusarium oxysporum f. sp. conglutinans

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