Role of C2H2 Zinc Finger Transcription Factor FpCzf7 in the Growth and Pathogenicity of Fusarium pseudograminearum

doi:10.13560/j.cnki.biotech.bull.1985.2021-1457

Abstract

Abstract:

Fusarium crown rot caused by Fusarium pseudograminearum has become one of the most destructive diseases of wheat in China. However,its molecular mechanism of pathogenicity is known little. C2H2 zinc finger transcription factors have been characterized from a wide variety of organisms,including animals,plants and fungi,and they are involved in the physiological processes such as growth,development and stress responses. In this study,a gene FpCzf7 encoding C2H2 zinc finger transcription factor was identified,and its deletion mutants（Δfpczf7）was obtained by PEG-mediated genetic transformation. Further analysis revealed that Δfpczf7 mutants grew significantly slowly on PDA plates and less aerial hyphae compared with that of wild type（WT）and the complement strains（Δfpczf7-cp）. Conidiation of the FpCzf7 deletion mutant in CMC liquid reduced as well,but their conidia morphology and germination were normal. Also,Compared to WT and Δfpczf7-cp,the pathogenicity caused by the FpCzf7 deletion mutant decreased on wheat coleoptile and spike and barley leaves,and DON toxin production reduced in the Δfpczf7 strain. Collectively,these results indicate that FpCzf7 is involved in growth,conidiation and pathogenicity and toxin production in F. pseudograminearum.

Key words: Fusarium crown rot, Fusarium pseudograminearum, C2H2 zinc finger protein, pathogenicity

ZHAO Jing-ya, PENG Meng-ya, ZHANG Shi-yu, SHAN Yi-xuan, XING Xiao-ping, SHI Yan, LI Hai-yang, YANG Xue, LI Hong-lian, CHEN Lin-lin. Role of C2H2 Zinc Finger Transcription Factor FpCzf7 in the Growth and Pathogenicity of Fusarium pseudograminearum[J]. Biotechnology Bulletin, 2022, 38(8): 216-224.

Figures/Tables 8

Table 1 Primers used in this study

Primer	Sequence（5'-3'）
F1	CATCAAATGCAGGAACGGAGC
R1	CAATATCATCTTCTGTCGACGGTTTCATGGGCTGC- TAGAAATC
F2	ATAGAGTAGATGCCGACCGCGGGTTCCATCGTCT- TGCCGTGGTGAATTG
R2	CAATTGAACGAGGAAACACG
HYG/F^[16]	GTCGACAGAAGATGATATTG
HYG/R^[16]	GAACCCGCGGTCGGCATCTACTCTAT
YG/F^[16]	GATGTAGGAGGGCGTGGATATGTCCT
HY/R^[16]	GTATTGACCGATTCCTTGCGGTCCGAA
F3	GACCTACCCATGTTTACTTG
R3	CTTCGAAATCTGCATCGCATG
G1	CAACATCATATCGAACCCAC
G2	GAGAGTGAGAAGACATGG
H850F^[16]	TTCCTCCCTTTATTTCAGATTCAA
H852R^[16]	ATGTTGGCGACCTCGTATTGG
H855R^[16]	GCTGATCTGACCAGTTGC
H856F^[16]	GTCGATGCGACGCAATCGT
PKNTG-F	CTATAGGGCGAATTGGGTACCGACCTACCCATGTT- TACTTG
PKNTG-R	GCAGGCATGCAAGCTTATCGATGGAGCTTTTGCT- GCTCTTG
nei-F	GACCACGGTTTCCACTACTC
GFP-R	GATGCCCTTCAGCTCGATGCGGTTCA
RTF	TTCAACACCACCAGCATCA
RTR	TGTGGTGCAAGTTCTCGTT
TEF1a-RTF	TCACCACTGAAGTCAAGTCC
TEF1a-RTR	ACCAGCGACGTTACCACGTC

Fig. 1 Domains and expression levels of FpCzf7 in F. pseudograminearum A：The C2H2 zinc finger motifs in FpCzf7. B：The relative expression levels of FpCzf7 during infection stages. MY and IF18h,IF30h,IF48h,IF3d,IF5d,IF7d represent the wild-type mycelia and the F. pseudograminearum infected the wheat coleoptile at 18 h,30 h,48 h,3 d,5 d,and 7 d,respectively

Fig. 2 FpCzf7 gene knockout in F. pseudograminearum A：Schematic representation of the FpCzf7 deletion strategy and PCR amplification（M：DNA size marker;A1：a 964 bp fragment of FpCzf7 upstream;A2：a 997 -bp fragment of FpCzf7 downstream;HPH：a 1 350 bp fragment of the hygromycin phosphotransferase gene;A1H1：a 1 762 bp fragment of FpCzf7 and HPH upstream cassette;H2A2：a 1 928 bp fragment of FpCzf7 and HPH downstream cassette）. B：PCR assay of the FpCzf7 deletion mutants（M：DNA size marker;H：a 750 bp fragment of the HPH;G：a 751 bp fragment of the FpCzf7 gene;F：a 1 673 bp fragment of FpCzf7 and HPH upstream cassette;R：a 1 568 bp fragment of FpCzf7 and HPH downstream cassette）

Fig. 3 Mycelia growth and morphology assays of WT,Δfpczf7 and Δfpczf7-cp on PDA plates A：Colonies morphology for 3 d. B：Colony diameters for 3 d（**P < 0.01,t-test）. C：Hyphal morphology for 24 h（Bar=50 µm）

Fig. 4 Conidia production and germination assays A：Conidia and germinated tubes of WT,Δfpczf7 and Δfpczf7-cp. B：Production of conidia of WT,Δfpczf7 and Δfpczf7-cp（**P < 0.01,t-test）. C：Conidia germination rates of WT,Δfpczf7 and Δfpczf7-cp

Fig. 5 Inoculation on barley leaves and wheat coleoptiles A：Barley leaves inoculated with mycelial blocks of WT,Δfpczf7 and Δfpczf7-cp at 3 d. B：The lesion diameters on barley leaves（**P < 0.01,t-test）. C：Wheat coleoptiles were inoculated with mycelial blocks of WT,Δfpczf7 and Δfpczf7-cp at 3 d. D：Lesion lengths on wheat coleoptiles（**P < 0.01,t-test）

Fig. 6 Experiments of pot-culture and wheat heads inoculation A：Wheat seedlings inoculated by WT,Δfpczf7 and Δfpczf7-cp for 10 d. B：The disease severity index of Fusarium crown rot.（**P < 0.01,t-test）. C：Wheat heads were inoculated with conidial suspension of WT,Δfpczf7 and Δfpczf7-cp strains for 20 d

Fig. 7 DON production **P < 0.01,t-test

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