Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (16): 3428-3439.doi: 10.3864/j.issn.0578-1752.2021.16.006

• PLANT PROTECTION • Previous Articles     Next Articles

Identification and Functional Analysis of Transcription Factors FpAPSES in Fusarium pseudograminearum

ZHAO JingYa(),XIA HuiQing,PENG MengYa,FAN Zhuo,YIN Yue,XU SaiBo,ZHANG Nan,CHEN WenBo,CHEN LinLin()   

  1. College of Plant Protection, Henan Agricultural University/National Key Laboratory of Wheat and Maize Crop Science, Zhengzhou 450002
  • Received:2020-11-26 Accepted:2021-01-08 Online:2021-08-16 Published:2021-08-24
  • Contact: LinLin CHEN E-mail:zhaojy0108@163.com;llchensky@163.com

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Abstract:

【Objective】Fusarium crown rot (FCR) caused by Fusarium pseudograminearum seriously affects wheat production in China. The objective of this study is to find and analyze the functions of APSES transcription factors in F. pseudograminearum, and to provide theoretical basis for revealing the pathogenic mechanism of F. pseudograminearum and prevention and treatment of FCR.【Method】The known APSES proteins were obtained from GenBank, and APSES candidates in F. pseudograminearum were found by BLASTP. Domains of FpAPSES proteins were determined using Pfam. The phylogenetic tree of APSES proteins was constructed using MEGA 5.05. The relative expression levels of FpAPSES1 and FpAPSES4 during infection were examined by quantitative RT-PCR (qRT-PCR). FpAPSES1 and FpAPSES4 deletion mutants were generated by polyethylene glycol (PEG)-mediated protoplast fungal transformation, and screened by PCR. The wild type, Δfpapses1 and Δfpapses4 strains were activated on PDA medium, and cultured on PDA for mycelial growth and morphology assays; mycelial blocks were introduced into liquid CMC medium to assess conidiation and conidia morphology; conidia were cultured in sterile distilled water to explore conidia germination; mycelial blocks or conidia suspension were prepared and inoculated wheat coleoptiles and barely leaves to explore pathogenicity. The pot-culture experiment was also used for virulence assay. ELISA was used to detect deoxynivalenol (DON) in inoculation millet. 【Result】 Four APSES candidates were found in F. pseudograminearum, which all contained the conserved DNA binding domain HTH. The phylogenetic tree analysis showed that FpAPSES1, FpAPSES2 and FpAPSES4 were divided into A group, while FpAPSES3 belonged to the C group. The qRT-PCR analysis revealed that FpAPSES1 and FpAPSES4 were up-regulated at infection stages, which suggested that FpAPSES1 and FpAPSES4 might play important roles in infection. Two FpAPSES1 deletion mutants (Δfpapses1-T10 and Δfpapses1-T27) and two FpAPSES4 deletion mutants (Δfpapses4-T1 and Δfpapses4-T2) were obtained. Compared with the wild type strain, both FpAPSES1- and FpAPSES4-deleted mutants exhibited significantly reduced colony growth rates. FpAPSES1-deleted mutants had normal hyphal branches, while FpAPSES4-deleted mutants exhibited curved and more branched hypha. Both FpAPSES1- and FpAPSES4-deleted mutants exhibited significantly reduced conidiation in CMC, and the conidia numbers were respectively reduced by 99.5% and 97.4% comparing with that of the wild type. Furthermore, conidia of FpAPSES1- and FpAPSES4-deleted mutants were shorter, less septa and lower germination rates. The wheat coleoptiles were point-inoculated with mycelial blocks or conidia, and the virulence of FpAPSES1- and FpAPSES4-deleted mutants was significantly reduced comparing to the wild type. Reduced pathogenicity was further observed by barely leaves inoculation and pot culture experiment. The DON levels in millet upon infection with FpAPSES1- and FpAPSES4-deleted mutants were reduced by 78% and 44% comparing to the wild type, respectively.【Conclusion】Both A group APSES homologs FpAPSES1 and FpAPSES4 play important roles in growth, conidiation and pathogenicity of F. pseudograminearum.

Key words: Fusarium crown rot, Fusarium pseudograminearum, APSES transcription factor, pathogenicity

Table 1

Primers used in the study"

引物Primer 引物序列Primer sequence (5′-3′)
FpAPSES1-F1 GTGGGTCTCATGATGATTC
FpAPSES4-F1 GAGTCTTCAACCCCTCTC
FpAPSES1-R1 CAATATCATCTTCTGTCGACGTTGAAAGATTGTTGAGATG
FpAPSES4-R1 CAATATCATCTTCTGTCGACGTTGGCGACTAAGATGAAGC
FpAPSES1-F2 ATAGAGTAGATGCCGACCGCGGGTTCTACCGGCACTCACCTCTC
FpAPSES4-F2 ATAGAGTAGATGCCGACCGCGGGTTCTTAACATCATTGGCCTATC
FpAPSES1-R2 CATACAAACATGCTGCCTCTC
FpAPSES4-R2 CAATTGTCCGGTGCTGTC
YG-F GATGTAGGAGGGCGTGGATATGTCCT
HY-R GTATTGACCGATTCCTTGCGGTCCGAA
HYG-F GTCGACAGAAGATGATATTG
HYG-R GAACCCGCGGTCGGCATCTACTCTAT
FpAPSES1-F3 CATTAGGCTCTTCGGGTGTG
FpAPSES4-F3 CACACCAACCCAATCCCTC
FpAPSES1-R3 CATACGAGACTCTACAACAG
FpAPSES4-R3 GTTCTGGTTGCTGGTCCTG
FpAPSES1-G1 CAACGACTCATGGCTCAATG
FpAPSES4-G1 CATGCATATTCAGAACCCAG
FpAPSES1-G2 GTAAGGGAGAGGTTCAAGAG
FpAPSES4-G2 GTGCTGTGTGATCCTCTG
H1R GCTGATCTGACCAGTTGC
H1F GTCGATGCGACGCAATCGT
H2F TTCCTCCCTTTATTTCAGATTCAA
H2R ATGTTGGCGACCTCGTATTGG
TEF1a-RTF TCACCACTGAAGTCAAGTCC
TEF1a-RTR ACCAGCGACGTTACCACGTC
FpAPSES1-RTF AACGTGGATATGTATGCGGC
FpAPSES4-RTF GCTATGGGTGATTTGGATGTCG
FpAPSES1-RTR TCGACATCTG GAGACATCTC
FpAPSES4-RTR GTTCTTTCATGACCATAGG

Table 2

Basic properties of FpAPSES transcription factors"

蛋白名称
Protein name		 基因ID
Gene ID		 转录号
Transcription number		 蛋白大小
Size (aa)		 蛋白分子量Molecular weight (kD) 蛋白等电点
Isoelectric point (PI)
FpAPSES1 XP_009261816.1 FPSE_10424 794 86.28 6.02
FpAPSES2 XP_009258602.1 FPSE_07209 420 46.08 5.78
FpAPSES3 XP_009254016.1 FPSE_02622 702 77.12 6.34
FpAPSES4 XP_009258789.1 FPSE_07396 675 75.55 5.30

Fig. 1

Domains and phylogenetic tree of FpAPSES proteins"

Fig. 2

Expression profiles of FpAPSES1 and FpAPSES4 at F. pseudograminearum infection stages"

Fig. 3

Gene knockout for FpAPSES1 and FpAPSES4"

Fig. 4

Mycelial growth assays of F. pseudograminearum"

Fig. 5

Conidia production and germination assays of F. pseudograminearum"

Fig. 6

Pathogenicity assays of F. pseudograminearum WT, Δfpapses1 and Δfpapses4 on wheat coleoptiles"

Fig. 7

Pathogenicity assays of F. pseudograminearum WT, Δfpapses1 and Δfpapses4 on barley leaves and wheat stem bases"

Fig. 8

DON production assays of F. pseudograminearum WT, Δfpapses1 and Δfpapses4 strains"

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