尖孢镰刀菌FoPLC4参与调控孢子形成和致病性

doi:10.3864/j.issn.0578-1752.2014.12.008

摘要/Abstract

摘要： 【目的】黄瓜枯萎病是黄瓜生产中的重要病害，其病原菌为尖孢镰刀菌黄瓜专化型（Fusarium oxysporum f. sp. cucumerinum）。研究旨在明确尖孢镰刀菌黄瓜专化型编码磷脂酶C（phospholipase C，PLC）的FoPLC4在调控分生孢子形成和致病性等方面的功能。【方法】采用生物信息学方法，确定FoPLC4在染色体上的位置，分析FoPLC4结构。基于尖孢镰刀菌番茄专化型基因组数据库，采用特异性引物克隆黄瓜枯萎病菌FoPLC4。根据同源重组原理构建携带潮霉素磷酸转移酶（hygromycin phosphotransferase，HPH）基因的敲除载体，利用PEG（polyethylene glycol）介导的方法转化原生质体，获得黄瓜枯萎病菌的敲除突变体?FoPLC4和遗传回复突变体?FoPLC4/plc4。转化子经潮霉素B平板筛选后通过PCR和RT-PCR分子验证。在PDA（potato dextrose agar）培养基上测定?FoPLC4生物学性状。利用分生孢子接种黄瓜胚根测定?FoPLC4在黄瓜幼苗上的致病性。【结果】FoPLC4位于尖孢镰刀菌基因组的4号染色体，DNA全长3 282 bp，不含内含子，编码1 093个氨基酸；FoPLC4含有5个结构域，包括EF手形（EF-hand）区、PH（pleckstrin homology）区、C2区和2个催化区，属于PLCδ亚型。与野生型菌株相比，?FoPLC4菌落生长速率没有显著改变，但气生菌丝较疏松；?FoPLC4可以同时产生大型分生孢子和小型分生孢子，大型分生孢子只占孢子总数的12.1%，而野生型菌株在相同条件下只能产生小型分生孢子，?FoPLC4产孢量下降了82.2%。致病性测定表明?FoPLC4在黄瓜幼苗上的萎蔫症状明显减轻，接种10 d后病情指数下降了53.3%。回复突变体?FoPLC4/plc4在菌落生长、产孢和致病性等性状与野生型相比没有显著差别。【结论】尖孢镰刀菌黄瓜专化型FoPLC4全长3 282 bp，不含内含子，编码1 093个氨基酸。与野生型相比，?FoPLC4分生孢子的产孢类型明显改变、产孢数量和对黄瓜幼苗的致病性显著降低。FoPLC4在调控尖孢镰刀菌分生孢子形成和致病性中发挥重要作用。

关键词: 尖孢镰刀菌 , FoPLC4 , 孢子形成 , 致病性

Abstract: 【Objective】Cucumber Fusarium wilt, caused by Fusarium oxysporum f. sp. cucumerinum, is an important disease in cucumber (Cucumis sativus L.) and can result in serious economic loss. The objective of this study is to investigate the functions of phospholipase C4 (PLC4) in F. oxysporum.【Method】Chromosomal location of the FoPLC4 and structure of FoPLC4 were analyzed by bioinformatics methods. Based on the F. oxysporum f. sp. lycopersici genome database, the FoPLC4 was cloned using specific primers. The gene replacement vector carrying hygromycin phosphotransferase (HPH) gene was constructed based on the gene homologous recombination, and then transformed into protoplasts with PEG-mediated gene transformation. The FoPLC4 deletion mutant (?FoPLC4) was generated through hygromycin B selection and molecular assay. Biological characters of the mutant on PDA (potato dextrose agar) medium and its pathogenicity on cucumber seedlings were determined after inoculation of radicles with conidia suspensions.【Result】The FoPLC4 located on chromosome 4, contains 3 282 bp with no intron and encodes a long polypeptide of 1 093 amino acids (aa). The protein encoded by FoPLC4 resembled mammalian PLCδ and possessed five domains including an EF-hand domain, a pleckstrin homology domain, a C2 domain, and two catalytic domains. Transformants selected on hygromycin B plates were screened by PCR and RT-PCR. Compared with the wild type (WT) strain, the ?FoPLC4 mutant showed a similar colony growth rate, but exhibited sparse and fluffy aerial mycelium on PDA medium. The conidiation of the ?FoPLC4 mutant reduced by 82.2%, the ?FoPLC4 simultaneously produced micro- and macro-conidia while the WT strain did only produce micro-conidia, and macro-conidia accounted for 12.1% of all spores in the ?FoPLC4. Pathogenicity assay of the ?FoPLC4 showed that wilting symptoms of cucumber seedlings significantly reduced. After 10 days of inoculation, disease index decreased by 53.3% on inoculated seedlings. Furthermore, the functional complementation of ?FoPLC4 restored the characteristics of the WT strain.【Conclusion】FoPLC4 contains 3 282 bp with no intron and encodes a protein of 1 093 aa. Compared with WT, ?FoPLC4 significantly exhibited the change of spore types, decreased the number of spores and pathogenicity in cucumber seedlings. The results indicate that FoPLC4 plays multiple roles in the regulation of sporulation and pathogenicity in F. oxysporum.

Key words: Fusarium oxysporum , FoPLC4 , sporulation , pathogenicity

孙玲, 褚小静, 郝宇, 张洪滨, 梁元存. 尖孢镰刀菌FoPLC4参与调控孢子形成和致病性[J]. 中国农业科学, 2014, 47(12): 2357-2364.

SUN Ling, CHU Xiao-Jing, HAO Yu, ZHANG Hong-Bin, LIANG Yuan-Cun. FoPLC4, Encoding Phospholipase C4, Is Involved in Sporulation and Pathogenicity in Fusarium oxysporum[J]. Scientia Agricultura Sinica, 2014, 47(12): 2357-2364.

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