Effects of smy1 Gene Knockout on Physiological Function of Fusarium oxysporum f. sp. cubense race 4

doi:10.3969/j.issn.1000-2561.2019.12.019

Abstract

Abstract:

Smy1 is a kinesin involved in the regulation of fungal growth. In the study, the smy1 kinesin gene of Fusarium oxysporum f. sp. cubense race 4 (Foc 4) was cloned. The smy1 knockout mutant of Foc 4 was obtained by homologous recombination technique. Besides observing the mycelial morphological characteristic, growth rate and sporulation of the mutants, pathogenicity to banana seedlings and sensitivity to fungicide were studied in order to reveal the role of smy1 played in the growth and development of F. oxysporum and resistance to fungicide. The results showed that, compared with the Foc 4 wild-type strain, the knockout mutants grew slowly, the mycelial morphology changed, the sporulation increased, and the pathogenicity to banana plantlets seedlings decreased, but the resistance level to fungicide changed little. This indicates that the smy1 gene may play an important role in the growth, sporulation and pathogenicity in Foc 4.

Key words: Fusarium oxysporum, gene knockout, pathogenicity, smy1 gene

CLC Number:

S436.681

HE Zhuang,QI Yanxiang,ZENG Fanyun,DING Zhaojian,LIANG Junwei,ZHANG Xin,PENG Jun,XIE Peilan,XIE Yixian. Effects of smy1 Gene Knockout on Physiological Function of Fusarium oxysporum f. sp. cubense race 4[J]. Chinese Journal of Tropical Crops, 2019, 40(12): 2447-2455.

Figures/Tables 9

Tab.1 Primer sequences used in the assay

引物名称 Primer name	序列（5′-3′） Sequence (5′-3′)
HYG-F	CTTGGCTGGAGCTAGTGGAGGT
HYG-R	CCCGGTCGGCATCTACTCTATTC
HYG-R1	GGATGCCTCCGCTCGAAGTA
HYG-F1	CGTTGCAAGACCTGCCTGAA
HPT-LBCK	GACAGACGTCGCGGTGAGTT
HPT-RBCK	TCTGGACCGATGGCTGTGTAG
Foc 4-smy1-LBCK	ACTCTTTCCACCGCTGCCTAT
Foc 4-smy1-HPH-LB-R	ACCTCCACTAGCTCCAGCCAAGGGTTGTTGGTCTTGTTTCACTCG
Foc 4-smy1-LB-F	CACCTAACTCCTCCGCTGTGG
Foc 4-smy1-HPH-RB-F	GAATAGAGTAGATGCCGACCGGGGGCGAAGATGACACAGAGGG
Foc 4-smy1-RBCK	TGGTGAGGTTGGATAGAATAGAGGG
Foc 4-smy1-RB-R	GCCAATCTGCCTTGTAAGCG
Foc 4-smy1-CK-F	CAGATGTGGCGTGCTGGTG
Foc 4-smy1-CK-R	ACTCGCTTGAGGAAGTGCTGT

Fig. 1 Inhibition of wild-type strain Foc 4 by different concentrations of phenamacril

Fig. 2 Phylogenetic analysis of smy1

Fig. 3 Schematic diagram of smy1 gene knockdown recombinant fragment and amplification electrophoresis A: schematic diagram of homologous recombination knockout of smy1 gene; B: upstream and downstream fragments amplified by two rounds of PCR (1-2: first round PCR amplification of upstream and downstream fragments; 3: amplification of HYG gene; 4-5: second round of PCR amplification of the upstream and downstream fragments; M: DNA Marker DL 5000).

Fig. 4 Identification of smy1 knockout mutants by PCR A: 1-2: PCR amplification of the upstream and downstream recombinant fragments of the knockout mutant was verified, the band sizes were 2393 bp, 2170 bp, H2O and Foc4 were controls, M: DNA Marker DL 5000; B: 1-4: Knockout mutant in vivo gene amplification by PCR, 5: H2O control, 6: Foc4 control, band size 697 bp, M: DNA Marker DL 5000; C: 1-4: knockout mutation PCR amplification of the hygromycin gene, 5: H2O control, 6: Foc4 control, band size 1376 bp, M: DNA Marker DL 5000.

Fig. 5 Colony growth of wild-type strains Foc 4 and smy1 knockout mutants on PDA plates with different concentrations of phenamacril

Fig. 6 Effects of different stress factors on the growth of wild-type strains and smy1 knockout mutant colonies

Fig. 7 Biological characteristics of smy1 knockout mutants A: The colonies morphology of Foc 4 and smy1 (2, 4, 6 and 7 d); B: The diameter of colonies; C: The conidia of the smy1 mutants and Foc 4; D: The mycelial growth form of Foc 4; E: The mycelial growth form of smy1 mutants. The error bar represents the standard error of three biological repetitions, and different lowercase letters represent significant difference (P<0.05).

Fig. 8 Pathogenicity test of smy1 mutant strain A: Symptoms of the Brazilian banana bulb infected by Foc 4 wild-type strain and smy1 strain; B: Brazilian banana seedling disease index map. The error bar represents the standard error of three biological repetitions, and different lowercase letters represent significant difference (P<0.05).

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