Effect Of Site-Directed Mutagenesis And Gene Deletion Of Gibberella Zeaeβ-Tubulin On Its Sensitivity To Carbendazim And Gene Expression Proflie

Microtubules, stiff, cylindrical heterodimers of a-and P-tubulins, are ubiquitous eukaryotic cell structures that are involved in a variety of intracellular processes including mitosis, cell growth and cell differentiation. Beta-tubulin is the target of benzimidazoles, and the resistance to these fungicides is associated with amino acid substitutions at special codons of β-tubulin. Fusarium head blight (FHB), caused by Fusarium graminearum (teleomorph, Gibberella zeae), is a destructive fungal disease of wheat and other small grain cereals worldwide. In addition to substantial yield losses, the scabby grain is often contaminated with trichothecene mycotoxins, such as deoxynivalenol (DON), making it unsuitable for human food or animal feed. Various tactics have been developed to control Fusarium head blight and to reduce DON contamination of cereals, with chemical control, especially carbendazim having an important role in an integrated FHB control strategy. However, G. zeae has developed serious resistance to MBC with a site-specific mode of action, leading to inefficacy of this fungicide. To better understand the relationship between carbendazim resistance mechanism and β-tubulin genes to provide the theoretical foundation to control Fusarium head blight of wheat and manage carbendazim-resistance in G. zeae, we did the following research.Mutated fragments of G. zeae P2-tubulin gene (β2-tub) at codons50,167,198and200were constructed by overlap PCR. The mutated fragments were transformed into β2-tub deletion mutant to replace selective markers and site-directed mutants were obtained. The mutants were tested for their sensitivity to benzimidazole fungicides. Mutation at codon50(Tyr→Cys)in β2-tub conferred MBC low resistance; Mutation at codon167(Phe→Tyr)in β2-tub conferred MBC medium resistance; Mutation at codon198(Glu→Lys)in β2-tub conferred MBC low resistance; Mutation at codonl98(Glu→Gln)in β2-tub conferred MBC high resistance; Mutation at codon200(Phe→Tyr)in β2-tub conferred MBC medium resistance. Resistance level depended on mutation site and mutation mode. The wild type strain and site-directed mutants did not differ in mycelial growth rate, pathogenicity, sporulation or production of perithecia. Homology modelling was performed on the SWISS MODEL web server in the first approach mode (http://www.expasy.ch/swissmod/); a three-dimensional model of the G zeae β2-tub was built. The resulting structure was viewed with SwissPdb Viewer V.3.7. These mutation sites were marked on the model; it is interesting that mutations at amino acids167,198and200are within the SDETFC motif of β2-tub in G. zeae, which is similar to the’benzimidazole-resistant box’motif in other filamentous fungi.There is the other P-tubulin gene (β1-tub) in G. zeae. Deletion vector containing hygromycin selective marker was constructed in vitro. It was transformed into wild type strain and site-directed mutants to replace β1-tub locus. The biological characteristics of the β1-tub deletion mutants of G. zeae were assayed, including vegetative growth, asexual reproduction, pathogenicity and sensitivity to carbendazim. β1-tub deletion mutants had slow growth rate and poor pathogenicity, while increased asexual reproduction ability. All β1-tub deletion mutants were more resistant to carbendazim with the enhanced expression level of β2-tub. In order to verify this correlation, β2-tub overexpression mutant was constructed by connecting strong promoter of Aspergillus nidulans with β2-tub. The results indicated that there were no significantly different biological phenotypes in β2-tub overexpression mutants from wild type strains, except the elevated production of spores. β2-tub overexpression mutants were more resistant to carbendazim than wild type strain. Though β2-tub is related to carbendazim resistance, it plays only a minor role, as the minimum inhibitory concentration of wile type β2-tub overexpression mutant is still less than1.4μg-mL-1. Homology modelling of G. zeae β1-tub was performed on the SWISS MODEL web server in the first approach mode (http://www.expasy.ch/swissmod/). The resulting structure was viewed with SwissPdb Viewer V.3.7. Above mutation sites were marked on the model; it is interesting that mutations at amino acids167,198and200are within the SDETFC motif of β2-tub in G. zeae, which is similar to the ’benzimidazole-resistant box’motif in other filamentous fungi. The deduced amino acid sequences of β1-tub and β2-tub in G. zeae are only76%identical. Except for codon240, the codons involved in benzimidazole resistance are same on β1-tub and β2-tub. Amino acid diversity at codon240of P-tubulin may be associated with recognition of benzimidazole molecule and the difference of carbendazim binding with the β-tubulin genes. The availability of the genome sequences of the fungal pathogen have not only accelerated the discovery of genes involved in pathogenicity but also provided a new platform to understand molecular pathogenesis at the genome level in recent years. Here, we analysed the transcriptome of G. zeae β2-tub deletion mutant (DN83), site-directed mutant (F167Y) and wide type (2021) by high-throughput tag-sequencing (Tag-seq) technique and we ultimately sequenced about6million raw tags for each sample. After the removal of low quality tags, we obtained a total of5678673,6013236and5893596clean sequences corresponding to94393,84202, and103338distinct tags for2021, DN83and F167Y, respectively. All clean tags were aligned to the Fusarium databases. About15%of the distinct tags were uniquely mapped to the reference genes. Analyses of differentially expressed genes revealed the substantial changes in gene type and abundance between the wild type and mutant libraries. Comparing with2021, DN83was detected an increased expression of246genes and decreased expression of1883genes. Among the total,14putative multifunctional transcription factors were significantly down regulated. Meanwhile,17genes found in G. zeae kinome important for vegetative growth, conidiation and pathogenicity also displayed decreased expression in the deletion mutant. Several pathogenicity-related genes were also severely down-regulated in the mutant.14ABC transporters involved in fungicide or toxin efflux presented decreased expression. These results showed β2-tub could participate in life cycle of G. zeae via correlating with the above genes through some pathways.1442genes were differently expressed between2021and F167Y. Of these,1036were up-regulated and401down-regulated. Quantitative real time polymerase chain reaction (qRT-PCR) results proved that mutation at codon167in β2-tub could increase DON production by activating genes involved in isoprenoid biosynthetic pathway and trichothecene biosynthetic pathway.