Folpcs1 Is Responsible for Asexual Reproduction and Vegetative Growth in Fusarium oxysporum f. sp. Lini.

doi:10.13523/j.cb.1908059

China Biotechnology

2020, Vol. 40

Issue (3): 48-64 DOI: 10.13523/j.cb.1908059

Orginal Article

Folpcs1 Is Responsible for Asexual Reproduction and Vegetative Growth in Fusarium oxysporum f. sp. Lini.

GUO Jing,HOU Zhan-ming(

)

College of Life Sciences and Technology, Inner Mongolia Normal University, Hohhot 010022, China

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Abstract

Fusarium oxysporum f.sp.Lini, a forma specialis of Fusarium oxysporum on flax, is soil-borne fungus which causes Fusarium wilt of flax, a serious disease of flax resulting in great loss of yield and quality of the crop. It has been identified that C2H2 zinc finger transcription factor Pcs1 in Fusarium graminearum is responsible for the generation of conidia produced from intercalary phialides on hyphae. Objective:The gene disruption of the Folpcs1 is performed according the principle of homologous recombination for identifying the function of the Folpcs1, the homologous gene of the pcs1, in Fusarium oxysporum f. sp. Lini. Methods:The disruption of the gene was made by the Split-Marker strategy after the genomic and complementary DNA was sequenced. The deletion cassettes containing a hygromycin resistance gene (hph) was constructed and transformed into protoplast of the wild type of the fungus mediated by polyethylene glycol. The deletion mutants, ΔFolpcs1s, were obtained and confirmed by PCR using positive and negative primer. To make complementation for the deletion mutuant, the coding frame sequence of the Folpcs1 gene, together with its upstream and downstream flanking sequence, was cloned into the pZWH1 containing neomycin resistant gene and transformed into the deletion mutant. Results:The sequencing revealed that the gene contained one intron of 654bp and the whole cDNA sequence consisted of 2 846bp. The morphological and microscopic observation found that growth rate of deletion mutant significantly reduced and the conidium was hardly observed in culture mixture of the mutant. The revertant recovered in normal growth and conidiogenesis of the wild type. Conclusion:The results suggest that Folpcs1 is responsible for asexual reproduction and vegetative growth in Fusarium oxysporum f. sp. Lini.

Key words： Fusarium oxysporum Schl.f.sp.Lini Folpcs1 Split-marker Gene disruption

Received: 29 August 2019 Published: 18 April 2020

ZTFLH:

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Corresponding Authors: Zhan-ming HOU E-mail: houzhm@imnu.edu.cn

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Cite this article:

GUO Jing,HOU Zhan-ming. Folpcs1 Is Responsible for Asexual Reproduction and Vegetative Growth in Fusarium oxysporum f. sp. Lini.. China Biotechnology, 2020, 40(3): 48-64.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.1908059 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I3/48

Table 1 pMD19-T vector cloning system

Table 2 This experiment involves important primers

Table 3 ClonExpress?Ⅱone step cloning system

Fig.1 Fusarium oxysporum Schl.f.sp.Lini total genomic DNA and RNA DL1000 DNA maker (a) Fusarium oxysporum Schl.f.sp.Lini total genomic DNA (b) Fusarium oxysporum Schl.f.sp.Lini total genomic total RNA(1-6)

Fig.2 Folpcs1 gene cDNA terminal cloning M: DL1000 DNA maker (a) PCR products of 3'RACE 1:Outer PCR products of 3'RACE; 2: Inner PCR products of 3'RACE (b) PCR products of 5'RACE 1:Inner PCR products of 5'RACE (c) PCR products of 3'RACE verification(1-5) (d) PCR products of 5'RACE verification(1-5)

Fig.3 DNA and cDNA products of Folpcs1 M: 1000LD DNA marker (a) PCR products of DNA 1: PCR products with Folpcs1G1F/G2R as primer using genomic DNA as template at different temperatures; 2: PCR products with Folpcs1G3F/G4R as primer using genomic DNA as template at different temperatures (b) PCR products of DNA 1: PCR products with Folpcs1G5F/G6R as primer using genomic DNA as template at different temperatures; 2: PCR products with Folpcs1G7F/G8R as primer using genomic DNA as template at different temperatures (c) PCR products of DNA 1: PCR products with Folpcs1 G11F(3)/G12R(3) as primer using genomic DNA as template (d) PCR products of DNA 1: PCR products with Folpcs1G9F(2)/G10R as primer using genomic DNA as template (e) PCR products of cDNA,PCR products with Folpcs1G7F/G8R as primer 1: Using cDNA as template at different temperatures;2: Using DNA as template (f) PCR products of cDNA,PCR products with Folpcs1 G3F/4R as primer 1:Using cDNA as template at different temperatures;2: Using DNA as template

Fig.4 Amino acid sequence alignment of four fungus

Fig.5 Phylogenetic tree constructed with Folpcs1 and Pcs1 proteins from the other fungus

Fig.6 Construction for deletion cassette of the Folpcs1 M: LD1000 DNA maker (a) PCR products of amplification of upstream and downstream sequences of Folpcs1 gene 1: PCR products of amplification of upstream sequences of Folpcs1 gene; 2: PCR products of amplification of downstream sequences of Folpcs1 gene (b) PCR products of amplification of upstream and downstream sequences of hph gene 1: PCR products of amplification of upstream sequences of hph gene; 2:PCR products of amplification of downstream sequences of hph gene (c) Split-Marker overlapping PCR products of Folpcs1 gene 1: pcs1 ko1F/HY-R overlapping PCR products of Folpcs1 gene; 2: YG-F/Folpcs1 ko4R overlapping PCR products of Folpcs1 gene

Fig.7 Screening for knock-out mutant of Folpcs1 M:LD1000 DNA marker (a) Genomic DNA of Folpcs1 transformants (b) Negative screening for knock-out mutant of Folpcs1 (c) Positive Screening 1 for knock-out mutant of Folpcs1 (d) Positive screening 2 for knock-out mutant of Folpcs1 (b)-(d) 1-5:Genomic DNA of Folpcs1-2, Folpcs1-3, Folpcs1-9, Folpcs1-11, Folpcs1-12 transformants as template; 6: Genomic DNA of the wild type as template;7: ddH₂O as template for a negative control

Fig.8 Phenotypic analysis experiment of different strains (a)Colony morphology of wild type hm, deletion mutant ΔFolpcs1-3 and ΔFolpcs1-12 cultured on TCC (b) The mycelium growth rate of hm, ΔFolpcs1-3 and ΔFolpcs1-12 (c) Conidia production of hm, ΔFolpcs1-3, ΔFolpcs1-12 and ec Folpcs1-2 in CMC medium; The black triangle points to the intercalary phialides (d) Hyphal morphology of hm, ΔFolpcs1-3, ΔFolpcs1-12 and ec Folpcs1-2 in YEPD medium (e) Infection of flax seedlings by conidium suspension

Table 4 Statistical analyses on conidia number of different strains

Fig.9 pZWH1 vector linearized M: LD1000 DNA marker (a) 1-2:pZWH1 plasmid DNA (b) Linearized vector pZWH1

Fig.10 Folpcs1 together with its upstream and downstream flanking sequence of PCR product

Fig. 11 Screening of recombinant plasmid pZGH1 M:LD1000 DNA marker (a) 1-3: PCR product of Folpcs1 N1F/ N2R as primer and colony as template (b) 1-3: Colony 4,10,14 plasmid DNA (c) 1-3: PCR detection of Folpcs1 CEⅡF/ CEⅡR as primer and plasmid DNA as template (d) 1-3: PCR detection of NeoF/NeoR as primer and plasmid DNA as template (e)1-3: EcoRⅠenzyme-digested product (f) BamHⅠenzyme-digested product

Fig.12 Screening of Folpcs1-C PCR M:LD1000 DNA marker (a) 1-5: Transformant 1-5 DNA (b) 1-5: Transformants 1-5 HYG-F/HYG-R PCR screening; 6: ΔFolpcs1-3 DNA HYG-F/HYG-R PCR control; 7:hm DNA HYG-F/HYG-R PCR control (c) 1-5: Transformants 1-5 Folpcs1 N1F/N2R PCR screening; 6: ΔFolpcs1-3 DNA Folpcs1 N1F/N2R PCR control; 7:hm DNA Folpcs1 N1F/N2R PCR control (d) 1-5: Transformants 1-5 NeoF/NeoR PCR screening; 6: ΔFolpcs1-3 DNA NeoF/NeoR PCR control; 7:hm DNA NeoF/NeoR PCR control

Fig.13 Phenotypic analysis experiment of different strains (a) Colony morphology of wild type hm, ΔFolpcs1 and Folpcs1-C1 cultured on TCC (b) The mycelium growth rate of hm,Folpcs1-C1 and ΔFolpcs1 (c) Conidia production of hm, ΔFolpcs1, Folpcs1-C1 in CMC medium; The black triangle points to the intercalary phialides


[1]	Placinta C M , D'Mello J P F, Macdonald A M C.Areview of worldwide contamination of cereal grains and animal feed with Fusarium mycotoxins. Animal Feed Science Technongy, 1999,78(1-2):21-37.

[2]	Mirocha C J, Schauerhamer B, Christensen C M , et al. Incidence of zearalenol(Fusarium mycotoxin)in animal feed. Applied and Environmental Microbiology, 1979,38(4):749-750.

[3]	Ma L J , Van Der Does H C, Borkovich K A, et al. Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium. Nature, 2010,464(7287):367-373.

[4]	Cuomo C A, Güldener U , Xu J R, al et. The Fusarium graminearum genome reveals a link between localized polymorphism and pathogen specialization. Science, 2007,317(5843):1400-1402.

[5]	Ospina-Giraldo M D, Mu Llins E, Kang S . Loss of function of the Fusarium oxysporum SNF1 gene reduces viru Lence on cabbage and Arabidopsis. Current genetics, 2003,44(1):49-57.

[6]	Calero-Nieto F, Di Pietro A , Roncero M I G, et al. Role of the transcriptional activator xln R of Fusarium oxysporum in regu Lation of xylanase genes and virulence. Molecu Lar Plant-microbe Interactions, 2007,20(8):977-985.

[7]	Di Pietro A , García-Maceira F I, Meglecz E, et al. A MAP kinase of the vascu Lar wilt fungus Fusarium oxysporum is essential for root penetration and pathogenesis. Molecu Lar microbiology, 2001,39(5):1140-1152.

[8]	张艳君 , 尖孢镰刀菌亚麻专化型FolMKK2基因在MGV1 MAPK 信号级联途径中的功能解析. 呼和浩特:内蒙古师范大学, 2019.

[8]	Zhang Y J , Functional analysis of FolMKK2 gene in MGV1MAPK signaling cascade pathway of Fusarium oxysporum Schl. f.sp.Lini. Hohhot: Inner Mongolia Normal University, 2019.

[9]	李丹 , 尖孢镶刀菌亚麻专化型 FoIBCK1基因敲除及功能研究. 呼和浩特:内蒙古师范大学, 2019.

[9]	Li D , A Research on the gene knockout and its function of FolBCK1 gene in Fusarium oxysporum Schl.f.sp.Lini(Bolley) Snyder & Hansen. Hohhot: Inner Mongolia Normal University, 2019.

[10]	Chantal O, Sophie T , Marie-noelle B, et al. Colonization of flax roots and early physiological responses of flax cell inoculated with pathogenic and nonpathogenic strains of Fusarium oxysporum. Applied and Environmental Microbiology, 2003,69(9):5453-5460

[11]	杨学, 王玉富, 关凤芝 , 等. 亚麻枯萎病发生规律及其综合防治措施. 中国麻业, 2002,24(1):23-26.

[11]	Yang X, Wang Y F, Guan F Z , et al. The measurement of comprehensive prevention and the law of occurrence of flax blight, Plant Fiber and Products, 2002,24(1):23-26.

[12]	王政逸, 李德葆 . 尖孢镰刀菌的遗传多态性. 植物病理学报, 2000,30(30):193-200.

[12]	Wang Z Y, Li D B . Genetic diversity in Fusarium oxysporum. Acta Phytopathologica Sinica, 2000,30(30):193-200.

[13]	杨万荣, 薄天岳 . 高抗萎蔫病胡麻品种资源的筛选利用及抗病性遗传浅析. 华北农学报, 1994,9(S1):100-104.

[13]	Yang W R, Bo T Y . Utilization and screening of linseed cultivars for high-resistance to Fusarium Wilt and genetic analysis of the disease resistanee,Acta Agriculturace Boreali-sinica, 1994,9(S1):100-104.

[14]	程琳 . 尖孢镰刀菌Qip基因功能初步研究,硕士学位论文. 临汾: 山西师范大学, 2015.

[14]	Cheng L . Primary function research on Qip gene in Fusarium oxysporum. Linfen: Shanxi Normal University, 2015.

[15]	Booth C. 镰刀菌属. 陈其译.北京: 农业出版社, 1984: 321-322.

[15]	Booth C. Fusarium. Cheng Q. BeiJing: Agricultural Press, 1984: 321-322.

[16]	Dahlberg K R , Van Etten J L. Physiology and biochemistry of fungal sporulation. Annu Rev Phytopathol, 1982,20:218-301.

[17]	Toshiaki O, Takashi T . FoSTUA, encoding a basic helix-loop-helix protein, differentially regulates development of three kinds of asexual spores,macroconidia, microconidia, and chlamydospores, in the fungal plant pathogen Fusarium oxysporum. American Society for Microbiology, 2004,3(6):1412-1422.

[18]	Son H, Seo Y S, Min K , et al. A phenome-based functional analysis of transcription factors in the cereal head blight fungus, Fusarium graminearum. PLoS Pathogens, 2011,7(10):e1002310.

[19]	Boknam J, Jungwook P, Son H . A Putative transcription factor pcs1 positively regulates both conidiation and sexual reproduction in the cereal pathogen Fusarium graminearum. The Plant Pathology Journal, 2014,30(3):236-244.

[20]	杨学 . 亚麻苗期病害发生特点及防治技术研究. 中国麻叶, 2003,25(5):223-227.

[20]	Yang X . The occurance chacteristics and control of diseases at sendling stage of flax. Plant Fiber and Products, 2003,25(5):223-227.

[21]	吴彬 . 小麦赤霉菌Fg AC1基因敲除及功能研究. 呼和浩特:内蒙古师范大学, 2011.

[21]	Wu B , Characterization of Fg AC1 gene of Fusarium graminearum. Hohhot: Inner Mongolia Normal University, 2011.

[22]	Catlett N L, Lee B, Yoder O C , et al. Split-marker recombination for efficient targeted deletion of fungal genes. Fungal Genetics Newsletter, 2002,50(49):9-11.

[23]	郭雪芳, 侯占铭 , 小麦赤霉菌FGSG_08948基因敲除及功能研究. 分子植物育种, 2018,16(8):2409-2414.

[23]	Guo X-F, Hou Z-M , Knockout and function research of FGSG_08948 gene in Fusarium graminearum. Molecular Plant Breeding, 2018,16(8):2409-2414.

[24]	彭海丽, 侯占铭 . 小麦赤霉菌FGSG_04871基因敲除及功能研究. 内蒙古师范大学学报, 2017,46(3):394-400.

[24]	Peng H L, Hou Z M . Research on knockout and function of FGSG_04871 gene in Fusarium graminearum. Journal of Inner Mongolia Normal University, 2017,46(3):394-400.

[25]	Tommy C. Sewall, Charles W , et al. abaA controls phialide differentiation in Aspergillus nidulans. The Plant Cell, 1990,2:731-739.

[26]	Son H, Kim M G, Min K , et al. AbaA regulates conidiogenesis in the ascomycete fungus Fusarium graminearum. Public Library of Science, 2013,8(9):e72915.

[27]	Shengli D, Rahim M, Cornelia K , Transducin beta-like gene FTL1 is essential for pathogenesis in Fusarium graminearum. Eukaryotic Cell, 2009,8:867-876.

[28]	Lysoe E, Pasquali M, Breakspear A , et al. The transcription factor FgStuAp influences spore development, pathogenicity, and secondary metabo-lism in Fusarium graminearum. Mol Plant -Microbe Interact, 2001,24:54-67.

[29]	Zheng W, Zhao X, Xie Q , et al. A conserved homeobox transcription factor Htf1 is required for phialide development and conidiogenesis in Fusarium species. PLoS One, 2012,7:e45432.

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