Knockout and Functional Analysis of Transcription Factor SNT2 Coding Gene in Fusarium oxysporum f.sp.conglutinans

doi:10.7668/hbnxb.20195567

Abstract

Abstract:

Cabbage fusarium wilt is a serious soil-borne fungal disease caused by Fusarium oxysporum f.sp.conglutinans (FOC),which affects the yield and quality of cabbage.In order to clarify the biological function of the transcription factor SNT2 in this pathogen,the SNT2 gene knockout mutant ΔSNT2 in F.oxysporum was successfully obtained by homologous recombination and protoplast transformation,and its phenotype and pathogenicity were analyzed.The results showed that SNT2 in F.oxysporum encoded 1 529 amino acids,had a SANT domain that binds to DNA,and the protein belonged to a hydrophilic protein.Compared with the wild type strain,the mycelial growth rate of the ΔSNT2 mutant decreased and the septation increased significantly,and the conidial production decreased significantly.Based on the results of exogenous stress,ΔSNT2 was insensitive to osmotic stress of 1 mol/L sorbitol,but its tolerance to oxygen stress,salt stress and cell wall stress of 0.1% H₂O₂,2 mol/L NaCl and 0.05% Congo red was reduced.At the same time,the pathogenicity test showed that the disease index of ΔSNT2 mutant was significantly lower than that of wild type,and the deletion of SNT2 resulted in a significant decrease in the pathogenicity of F.oxysporum.In conclusion,the transcription factor SNT2 plays an important role in maintaining the integrity of the cell wall during the interaction between the pathogen and the host,and participates in the regulation of the growth and development of F.oxysporum and the expression of pathogenicity.

Key words: Fusarium oxysporum, SNT2, Phenotype, Pathogenicity, Functional analysis, Gene knockout

XU Xinrui, SONG Zelong, LI Erfeng. Knockout and Functional Analysis of Transcription Factor SNT2 Coding Gene in Fusarium oxysporum f.sp.conglutinans[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(2): 173-180. doi: 10.7668/hbnxb.20195567.

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Figures/Tables 8

Tab.1 Primer names and sequences

引物名称 Primer name	引物序列(5'—3') Primer sequences(5'—3')
up-F	AAATGCGGATGACTTGGCG
up-R	ACCTCCACTAGCTCCAGCCAAGAGTT GCGAAGGATGAAGACGG
down-F	GAATAGAGTAGATGCCGACCGGGTG AAAGTCTAAGAGGGCGAGC
down-R	AGTGCGAGAGAGCGGTATTT
hygR-F1	CTTGGCTGGAGCTAGTGGAGGT
hygR-R1	GGATGCCTCCGCTCGAAGTA
hygR-F2	CGTTGCAAGACCTGCCTGAA
hygR-R2	CCCGGTCGGCATCTACTCTATTC
upcheck-F	ATTGCTGTGTTGGTGCGTC
upcheck-R	ATTGCTGTGTTGGTGCGTC
downcheck-F	TCTGGACCGATGGCTGTGTAG
downcheck-R	CGTGTGTTGTTTCTCTCACCAT

Fig.1 Homologous recombination knockout strategy

Fig.2 SNT2 protein domain prediction

Fig.3 Amplification of homologous replacement arms M.DNA Marker;A.up fragment;B.down fragment;C.hygR1 fragment; D.hygR2 fragment;E.up-hygR1 fragment;F.hygR2-down fragment.

Fig.4 PCR and Southern Blot analysis of the deletion mutant A.PCR identification:M1.DNA Marker.B.Knockout mutant identification:M2.DNA Marker.

Fig.5 Comparison of mycelial growth and asexual reproduction between wild type and mutants A.Colony growth analysis between wild-type and mutants;B.The hyphal septum length of wild type and mutant;C.Mycelial morphology analysis between wild-type and mutants(m₁,m₂,m₃ and n₁,n₂,n₃ represent the length of hyphal septum);D.Sporulation between wild-type and mutants.Different lowercase letters represent significant differences(P<0.05).The same as Fig.6,7.

Fig.6 Comparison of growth stress between wild type and mutants A.Colonies growth analysis under different stresses;B.Relative inhibitory rate.

Fig.7 Comparison of pathogenicity between wild type and mutant A.The disease index of wild-type and mutant on cabbage from 6 to 15 days after inoculation;B.The growth status of cabbage inoculated with wild type and mutant on Day 11:the upper row is the leaf disease symptom feature map,and the lower row is the plant disease symptom map.

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