Gene knockout and phenotype analysis of the β2-tubulin in Fusarium oxysporum f. sp. cubense race 4

Abstract: 【Objective】The objective of this study was to clone and identify the β2-tubulin (β2-tub) gene of Fusarium oxysporum f. sp. cubense race 4 (Foc4) , and reveal the function of β2-tub in resistance of Foc4 to carbendazim and its role in the pathogenic process of Foc4.【Methods】Based on the sequences of β2-tub gene (XM＿011327927.1) of Fusarium graminearum and the complete genome sequence of the Foc4 54006 strain (JH658279.1) , specific primers were designed. A full-length sequence of β2-tub gene from Foc4 was cloned by PCR amplification, and sequence characterization, phylogenetic clustering and protein domains of β2-tub gene were also analyzed, respectively. The signature domains were analyzed by SMART software and the phylogenetic tree was built by MEGA 6.0. β2-tub knockout mutants of Foc4 were obtained by split-marker homologous recombination technology. Sensitivity of Foc4 and its knockout mutants to carbendazim was tested by inoculating their mycelium onto PDA plates with different concentrations of carbendazim, respectively. The biological phenotypes in-cluding the growth rate of colonies, the amount of sporulation, the form of mycelium, selective pressure of cell wall, oxidation pressure and osmotic pressure of Foc4 and its knockout mutants were tested. In addition, the pathogenicity of Foc4 and its knockout mutants was tested by pot culture root drenching method. Foc4 and its knockout mutants were treated with 15 Brazil banana seedlings (Cavendish AAA) .Each seedling was irrigated with 20 mL spores suspension, and aseptic water was used as a control. The corm of Brazil banana was cut longitudinally after 30 d, and the browning degree of the bulb and the condition of the chlorosis of the outer leaves were observed.【Results】Bioinformatics analysis revealed that the complete DNA and cDNA sequences of β2-tub from Foc4 were 1 694 bp and 1 347 bp, respectively. The cDNA of β2-tub included 5 exons and 4 introns and encoded a protein of 448 amino-acids.Moreover, the β2-tub was highly conserved. Compared with other plant pathogenic fungi, the gene was highly homologous. In Foc4, the β1-tub protein encoded 446 amino-acids. The homology of DNA sequence of β1-tub and β2-tub was 64.67%, the homology of amino acid sequence was 76.34%, and they had the same domain. The phylogenetic tree showed that β2-tub was closely related to F. oxysporum f.sp. lycopersici and F. graminearum. The β2-tub gene is highly conserved in biological evolution with other common plant pathogenic fungi. After two rounds of PCR amplification, the upstream and downstream recombination fragments required for homologous recombination for protoplast transformation were successfully amplified by split-marker. The sensitivity test of Foc4 and its knockout mutants to carbendazim showed that the β2-tub gene was related to the resistance of Carbendazim. The EC50 value of Foc4 to carbendazim was 0.51 μg · mL-1, and the EC50 values of β2-tub gene knockout mutant to carbendazim was 0.36 μg · mL-1. Both were sensitive to carbendazim, however, the sensitivity to carbendazim of β2-tub gene knockout mutant increased significantly. Compared with Foc4, the growth rate, the sporulation quantity and the mycelial morphology of the β2-tub gene knockout mutant were not changed, and the difference was not significant. Sensitivity analysis of stress factors showed that β2-tub gene knockout mutants had no influence on cell wall selective pressure, oxidative stress and osmotic pressure. The results of pathogenicity test showed that the leaves of the plants inoculated with wild strains were yellow and wilted, most of which were yellow in the lower leaves, and the browning of the bulbs was serious by the incision of the bulbs. However, a few leaves of the plants inoculated with the β2-tub gene knockout mutant strain were yellow, and the bulbs appeared slightly browning. There was no leaf yellowing in the control plants inoculated with H2 O, and no browning in the corms. According to the disease index statistics, the results showed that the average disease index (42.24) of the inoculated strain of β2-tub gene knockout mutant was significantly lower than the average disease index (59.17) of Foc4 wild fungi.【Conclusion】Foc4 wild strains and β2-tub gene knockout mutant strains all maintained high sensitivity to carbendazim. Furthermore, the knockout mutants showed a significant increase in sensitivity to carbendazim, indicating that the change of β2-tub gene caused the resistance of wild strain to carbendazim. The gene of β2-tub was highly conserved and had a typical microtubule protein domain. The biological phenotypes of β2-tub knockout mutants, including growth rate, sporulation, hyphae morphology, selective pressure on cell wall, oxidative pressure and osmotic pressure, were not significantly different, but their pathogenicity to Brazil banana seedlings decreased significantly.