Folcdc15 Gene Involved in the Conidiogenesis and Hyphal Growth in Fusarium oxysporum f.sp.lini

doi:10.7668/hbnxb.20193898

Abstract

Abstract:

To identify the Folcdc15 gene and its function in Fusarium oxysporum f.sp.lini through disruption of the gene and complementation test.After DNA sequencing of Folcdc15 gene,Split-Marker strategy was used to construct a deletion cassette containing hygromycin resistance gene(hph),which was transformed into protoplast of wild-type hm by PEG mediated method.Transformants were collected in the medium containing hygromycin and Folcdc15 deleted mutants were screened through PCR using positive and negative primers.The complementary vector pZLY1 containing Folcdc15 was constructed by using pZWH1 vector and transformed into the knockout mutant for complementation test.Compared with wild-type hm,the growth rate of deletion mutant ko1 decreased by 73%,and the mycelia morphology changed significantly and became shorter and more bifurcated.The number of conidia decreased significantly,and the septum and length of conidia increased.The infection experiment in flax showed that the virulence of deletion mutant ko1 was significantly reduced.The complementation test revealed that the revertant ca1 recovered phenotype of wild-type hm from the defects in conidiation,morphology of conidium,growth rate of colony,morphology of colony and pathogenicity caused by the Folcdc15 gene deletion.Folcdc15 is involved in the regulation of conidiogenesis and hyphal growth in F.oxysporum f.sp.lini.

Key words: Fusarium oxysporum f.sp.lini, Flax blight, Split-Marker, Folcdc15, Gene function, Pathogenicity

LI Chunyang, HOU Zhanming. Folcdc15 Gene Involved in the Conidiogenesis and Hyphal Growth in Fusarium oxysporum f.sp.lini[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(6): 175-183. doi: 10.7668/hbnxb.20193898.

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Figures/Tables 9

Tab.1 Primers

引物 Primer	引物序列(5'—3') Primer sequences(5'—3')
FGSG-1Fα	GCGCATGATCGAGGTGGGTTG
FGSG-2Rα	CCACTGCTTGACTTCCTCCACG
FGSG-3R	TCGAGCTTCTTCAAGGCAAG
FGSG-4R	CCGCTATTGATGATTCAGGTC
FGSG-5Fα	CTCACGACAGAGCGATGCG	N1F
FGSG-6Rα	GCTGAGTGCAGCGACTC	N2R
FGSG-7Fβ	GGTCAACGGTATCTGGAACG	N3F
FGSG-8Rα	GGACTGTTGCCGTTGGC	N4R
FGSG-9F	GCGCATGATTGATTGGAGATC
FGSG-10R	CCGACAACCTGTGCCTCC
FGSG-11Fα	CCGTGCACGATATAGATCGTC
FGSG-12R	CGACTATTCGAGCCTCTCGC
FGSG-13F	CGTCATCCGGAAGACGAGC
FGSG-20R	CGCCTCATGTCTATCRTG
HYG-F	GGCTTGGCTGGAGCTAGTGGAGGTCAA
HY-R	GTATTGACCGATTCCTTGCGGTCCGAA
YG-F	GTATTGACCGATTCCTTGCGGTCCGAA
HYG-R	GAACCCGCGGTCGGCATCTACTCTAT
FGSG-KO1F	CTGTGAATGGATGATGCCACACG
FGSG-KO2R	TTGACCTCCACTAGCTCCAGCCAAGC
	CGACCGATTGTGTCGTCGTCG
FGSG-KO3F	ATAGAGTAGATGCCGACCGCGGGTTC
	GGGTCGCAGATACTTATGGCAG
FGSG-KO4R	CGACGTGGACTGGCTGCTAC
Com-1F	GCTATGACCATGATTACGCCAAGCTT
	GCCACACGAGAGTGAGCTAGTGG
Com-2R	GTCGACCTGCAGGCATGC AAGCTTC
	TTCATGTCGTGCATTCCACTCG
Neo-F	GAGAGGCTATTCGGCTATGACT
Neo-R	GGCCACAGTCGATGAATCCAGA

Fig.1 Split-Marker and pZWH1 plasmid A.Split-Marker diagram;B.pZWH1 plasmid.H. Hind Ⅲ;E.EcoR Ⅰ.

Fig.2 Single gene evolutionary tree

Fig.3 Construction of knockout voctor using Split-Marker and knockout transformant screening A.Upstream and downstream sequences of hph:1.Upstream sequences of hph,2.Downstream sequences of hph;B.Upstream and downstream homologous arm sequences of Folcdc15:1.Upstream homologous arm sequences of Folcdc15,2.Downstream homologous arm sequences of Folcdc15;C.Overlapping PCR sequences:1,3.Overlap the upstream PCR sequences;2,4.Overlap the downstream PCR sequences;D.Positive screening of Folcdc15 knockout mutants using KO1F/HY-R,YG-F/KO4R,HYG-F/HYG-R as primers:1—3.ko1,4—6.ko1-4,7—9.ko2-1;E.Negative screening of Folcdc15 knockout mutants using N1F/N2R as primers:1.ko1,2.ko1-4,3.ko2-1,4.hm,M.1 kb plus DNA Ladder.

Fig.4 Construction of complementary vector and screening of transformants A:1.pZWH1 plasmid DNA,2.pZWH1 plasmid DNA digested by Hind Ⅲ,3.Folcdc15 gene DNA;B:2,3,5.White colony PCR positive;C:1.pZLY1 plasmid DNA,2.pZLY1 plasmid DNA digested by EcoR Ⅰ;D.pZLY1 plasmid DNA digested by Hind Ⅲ;E.Selection of transformants using HYG-F/HYG-R as primer;F:1—2.Selection of transformants using Neo-F/Neo-R as primer,3—4.Selection of transformants using N1F/N2R as primer;G.Selection of transformants using Com-1F/Com-2R as primer,M.1 kb plus DNA Ladder.

Fig.5 Conidial morphology A.The spore morphology of hm;B.The spore morphology of ko1;C.The spore morphology of ca1.

Tab.2 Conidial count,colony diameter and spore diaphragm of different strains

菌株 Strain	孢子产量(×10⁶) Conidia number	菌落直径/mm Colony diameter	隔膜数量 Septal number
hm	3.12±1.10Aa	74.33±0.57Aa	2.00~3.00±1.00Aa
ko1	0.82±0.20Bb	22.17±0.76Bb	7.00~8.00±1.00Bb
ca1	3.14±1.20Aa	73.33±0.57Aa	2.00~3.00±1.00Aa

Fig.6 Colony mycelial morphology and growth curve A.Growth curve;B.Colony morphology of different media;C.Mycelial morphology of different strains.

Fig.7 Infection of flax seedlings by different strains A.Positive view of infected flax seedlings;B.Top view of infected flax seedlings.

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