Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (10): 1915-1929.doi: 10.3864/j.issn.0578-1752.2024.10.006

• PLANT PROTECTION • Previous Articles     Next Articles

Identification and Functional Analysis of the 5-Oxoprolinase Genes in Fusarium oxysporum

LOU Hui1(), ZHU JinCheng2, HAN ZeGang3(), ZHANG Wei1()   

  1. 1 School of Agriculture, Shihezi University/Key Laboratory of Oasis Ecological Agriculture of Xinjiang Production and Construction Corps, Shihezi 832000, Xinjiang
    2 Institute of Biotechnology, Xinjiang Academy of Reclamation Sciences/Key Laboratory of Corps of Crop Germplasm Innovation and Gene Resource Utilization, Shihezi 832000, Xinjiang
    3 College of Agriculture and Biotechnology/The Advanced Seed Institute, Zhejiang University, Hangzhou 310058
  • Received:2023-12-18 Accepted:2024-01-13 Online:2024-05-16 Published:2024-05-23
  • Contact: HAN ZeGang, ZHANG Wei

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Abstract:

【Objective】 5-Oxoprolinase (OXP) is one of the six core enzymes in the γ-glutamine cycle. The objective of this study is to characterize the OXP gene of Fusarium oxysporum, clarify its relationship with the pathogenicity of F. oxysporum, and to provide a theoretical basis for the analysis of the molecular mechanism of F. oxysporum pathogenicity and the prevention and control of cotton wilt.【Method】 Bioinformatics methods were used to identify the FoOXPs from the genome of F. oxysporum and analyze their gene structure, domains of the encoded proteins, chromosomal localization and evolutionary relationships. The phenotypes of F. oxysporum after FoOXP2 mutation were analyzed by using knockout mutant and complementary strain and the pathogenicity differences between the mutant and wild-type strains on cotton seedlings were detected. Furthermore, the disease grade rate and disease index on cotton were investigated by using host-induced gene silencing (HIGS) technology. The fungal biomass in cotton and expression of FoOXP2 in cotton stems transformed with interfering fragments of the FoOXP2 were examined by real-time quantitative PCR (qRT-PCR).【Result】 A total of two FoOXPs (FoOXP1 and FoOXP2) were identified in the F. oxysporum genome with coding sequence lengths of 4 080 and 3 921 bp, encoding 1 359 and 1 306 amino acids, respectively. Their protein molecular weights are 14.90 and 14.07 kDa, and theoretical isoelectric points are 5.73 and 5.30, respectively. The FoOXP1 protein is located in the mitochondria and the FoOXP2 protein is located in the cytoskeleton. FoOXP1 and FoOXP2 locate on chromosome JH657921 and chromosome JH657938, do not form gene clusters, with a sequence similarity of 52.00%. Phylogenetic analysis revealed that FoOXP1 and FoOXP2 belonged to two subgroups. Compared with the wild-type strain, the FoOXP2 knockout mutant strain showed significantly lower spore production and spore germination, loss of penetration ability, and increased tolerance to CR and sorbitol, but was more sensitive to cell wall stress (SDS and CFW), oxidative stress (H2O2), and osmotic stress (NaCl and KCl). Moreover, the pathogenicity on cotton was significantly reduced. The results of the HIGS assay illuminated that after 14 and 21 d of inoculation, the incidence of FoOXP2 silenced cotton plants was significantly reduced, whose disease indexes (17.3 and 40.2) were significantly lower than those of the control (28.2 and 77.1). The expression of FoOXP2 and fungal biomass were significantly lower than those of the control.【Conclusion】 The FoOXP2 positively regulates the pathogenicity of F. oxysporum and may play an important role in host-pathogen interactions.

Key words: cotton, Fusarium oxysporum, wilt, 5-oxoprolinase (OXP), host-induced gene silencing (HIGS)

Table 1

Primers used in this study"

引物Primer 引物序列Primer sequence (5′-3′)
TT0003-F GTCACCAGCCCCTGGGTTGAATTCGTTATCACCGACTCCC
TT0003-R AATGCTCCTTCAATATCAGAATTCGGTACATATCGCCCAC
TT0004-F CGACCTGCAGGCATGCAAGCTTTCATTGCCACTGGTTG
TT0004-R AACGACGGCCAGTGCCAAGCTTCATCGCAGGACTCACG
Z0074-F GCCCTTCCTCCCTTTATTTC
Z0074-R GATGTTGGCGACCTCGTATT
ECFoOXP2-F CCACTGTGAGTACTCTCCTCG
ECFoOXP2-R CCCAGGCGTACTTGAAGGAAC
FoOXP2-qPCR-F CCACCATCACGCCTGTGT
FoOXP2-qPCR-R ATACCGCCGATGTCTGCG
FoOXP2-F GTGCCAGTGCTGTTCATACGC
FoOXP2-R TCTGACCCTCTTCTCCTCCCTC
EF-F CACCTTAACGTCGTCGTCATC
EF-R GGAAGTACCAGTGATCATGTT
β-tubulin-F CGGAACCGACGACCAACA
β-tubulin-R GGCGGAAAAGCTGGCCTA

Fig. 1

Bioinformatics analysis of FoOXPs"

Fig. 2

Multiple sequence comparison of FoOXP1, FoOXP2 and ScOXP1"

Fig. 3

Transmembrane structure prediction and structural analysis of FoOXP proteins"

Fig. 4

FoOXP2 knockout, complementary and relative expression level determination"

Fig. 5

Growth phenotype of FoOXP2 knockout mutant and complementary strain"

Fig. 6

Spore and mycelial morphology of FoOXP2 knockout mutant and complementary strain (Bar=20 μm)"

Fig. 7

Cellophane penetration experiment"

Fig. 8

Sensitivity of each F. oxysporum strain under different stresses"

Fig. 9

Pathogenicity phenotypes of FoOXP2 knockout mutant and complementary strain"

Fig. 10

Pathogenicity determination of FoOXP2 knockout mutant and complementary strain"

Fig. 11

Silence of FoOXP2 by HIGS"

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