Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (9): 1894-1903.doi: 10.3864/j.issn.0578-1752.2021.09.007

• PLANT PROTECTION • Previous Articles     Next Articles

Functional Analysis of the Nucleoporin Gene FgNup42 in Fusarium graminearium

ZHANG ChengQi(),LIAO LuLu,QI YongXia,DING KeJian,CHEN Li()   

  1. School of Plant Protection, Anhui Agricultural University/Anhui Province Key Laboratory of Integrated Pest Management on Crops/Key Laboratory of Biology and Sustainable Management of Plant Diseases and Pests of Anhui Higher Education Institutes, Hefei 230036
  • Received:2020-09-14 Accepted:2020-09-24 Online:2021-05-01 Published:2021-05-10
  • Contact: Li CHEN E-mail:zhcq@ahau.edu.cn;chenlii@ahau.edu.cn

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Abstract:

【Objective】The nucleoporin Nup42 plays important roles in gene expression regulation, nuclear mRNA processing and export. The objective of this study is to analyze the function of the nucleoporin gene FgNup42 in growth and development, stress, pathogenicity and mycotoxins production of Fusarium graminearum. 【Method】Targeted gene deletion and mutant complementation constructs were generated using the double-joint PCR and yeast gap repair methods, respectively. Then the gene deletion mutant ΔFgNup42 and complemented strain ΔFgNup42-C were obtained by PEG-mediated protoplast transformation. The vegetative growth, asexual and sexual reproduction of the gene disrupted mutant ΔFgNup42 were examined. Moreover, the sensitivity of ΔFgNup42 to osmotic stress, fungicides and cell wall damaging agent were investigated. Pathogenicity of the mutant ΔFgNup42 was assayed by point-inoculated the flowering wheat heads and incubation on maize silks. Mycotoxins produced by ΔFgNup42 were quantified using the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The relative transcription levels of 7 TRIs involved in trichothecene mycotoxins biosynthesis were determined by qRT-PCR between PH-1 and ΔFgNup42. 【Result】Phenotype analysis showed that the growth rate of ΔFgNup42 was 50% of that of wild type PH-1, but more dense in hyphal branching. Microscopic examination revealed that the conidiation of ΔFgNup42 mutant decreased by 85.45% compared with the wild type PH-1, and percentage of conidia with septum range 0 to 2 was significantly increased. The ΔFgNup42 mutant produced more perithecia than the wild type strain in mating cultures suggesting improved sexual reproduction. The mutant ΔFgNup42 displayed increased tolerance to osmotic stress NaCl and KCl, fungicides tebuconazole and phenamacril, also cell wall damaging agent Congo red. Pathogenicity assays exhibited that disruption of gene FgNup42 caused a dramatic reduction in pathogenicity by inoculated the flowering wheat heads and maize silks. Furthermore, the amount of mycotoxins DON, 3ADON and 15ADON produced by ΔFgNup42 were significantly lower than that produced by wild type. 【Conclusion】The nucleoporin gene FgNup42 plays a key role in fungal growth and development, pathogenesis, mycotoxins production and responses to various environmental stresses in F. graminearum.

Key words: Fusarium graminearum, FgNup42, growth and development, environmental stress, pathogenicity

Table 1

Primers used in this study"

引物 Primer 序列 Sequence (5′-3′)
Nup42-up-F GCGCTCTCAAGAGAGTCACCG
Nup42-up-R CAAAATAGGCATTGATGTGTTGACCTCCCGCGGTAAGCTCTGGCTACCT
Nup42-down-F CTCGTCCGAGGGCAAAGGAATAGAGTAGCATAGGTAGGTAGGTATCTAC
Nup42-down-R TTGATTCCAGCCTCCAGCTG
HPH-F GGAGGTCAACACATCAATGCCTATT
HPH-R CTACTCTATTCCTTTGCCCT
Nup42-nest-F CGAGGGCCATGTCATTCCTG
Nup42-nest-R GAGTGAGATTCTAGGATCGT
Nup42-ID-F GTATCTACTATGGCGATATTGC
Nup42-ID-R GTATCCAACCAATCAGCGTGTC
Nup42-GFP-F ACTCACTATAGGGCGAATTGGGTACTCAAATTGGTTGCGCTCTCAAGAGAGTCACCG
Nup42-GFP-R CACCACCCCGGTGAACAGCTCCTCGCCCTTGCTCACAAAGTCCCACAATGTGCATTC
ID-Nup42-GFP-F CCGCAAGCTAACAATCCATTC
ID-GFP-R GTCAGCTTGCCGTAGGTGGCA
Tri1-RT-F GATGTTCCTTCTCGACAGCGT
Tri1-RT-R CACTGGTCGAAGATAGCTGG
Tri3-RT-F TGTTACGATCAATGGCTTGG
Tri3-RT-R TCCTCGTTGTAGTTTGCATCA
Tri4-RT-F ACGTGTGGCTACTCAGGAGAA
Tri4-RT-R TGGAATTGCCTTGGGGTA
Tri6-RT-F AAATGCCCATTCCCTAGTTG
Tri6-RT-R ATCTCGCATGTTATCCACCCT
Tri7-RT-F TACCGTCGTCTTCAAAACCA
Tri7-RT-R ACGCCAATGGTGTTCACAAA
Tri8-RT-F ATATAACGGTACCCCCAGATG
Tri8-RT-R TGTTTGTAGGACACTTCCGGT
Tri14-RT-F AACTCCCGTTGTGATCAAGCA
Tri14-RT-R AACAGTAATGTTGGCACCGT
actin-RT-F CCACGTCACCACTTTCAACT
actin-RT-R TGCTTGGAGATCCACATTTG

Fig. 1

Colony morphology and growth defects of the ΔFgNup42 mutant"

Fig. 2

Impacts of FgNup42 deletion on conidiogenesis of F. graminearum"

Fig. 3

The ΔFgNup42 mutant was defective in sexual development"

Fig. 4

Effects of FgNup42 disruption on the sensitivity to osmotic stresses, cell wall-damaging agent and fungicides"

Fig. 5

Involvement of FgNup42 in pathogenicity and trichothecene mycotoxins biosynthesis of F. graminearum"

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