Research On The Biological Function And Molecular Mechanism Of The Synonymous Mutation G235G At β₂-tubulin Of Fusarium Fujikuroi

Bakanae disease of rice,which was caused by G.fujikuroi species complex(GFC),is a kind of common disease of rice,and Fusarium fujikuroi was the main causative agent of"bakanae" in China.Benzimidazole fungicides has been used to control this disease for a long time in China,and has led to benzimidazole-resistance occurring.The early research in our laboratory demonstrated that the carbendazim-resistance of Fusarium fujikuroi was due to the 198th and 200th codon mutation at β2-tubulin gene.There were a total of three mutation sites:F200Y(nucleotide T900A)、E198V(nucleotide A894T)and synonymous mutation G235G(nucleotide C1006T).Synonymous mutation is the evolutionary substitution of one base for another in an exon of a gene coding for a protein,such that the produced amino acid sequence is not modified.Due to not inducing alteration in coding sequences,synonymous mutation was once considered neutral and inconsequential,but recent researches have demonstrated that some synonymous mutations can make great differenc by changing mRNA stability,mRNA secondary structure,mRNA spilicing,protein production,protein folding and so on.However,synonymous mutations were rarely investigated in filamentous fungus.In this study,five β2-tubulin site-directed mutants of Fusarium fujikuroi,which contained C1006T,T900A,A894T,C1006T+T900A and C1006T+A894T respectively,were constructed by using the true reversion technology.Comparing the biological phenotypes of wild type strain YC3 and the five β2-tubulin site-directed mutants,we found synonymous mutation C1006T could significantly enhance biological fitness of mutants,including sporulation,virulence,resistance to carbendazim(MBC),endurance to high temperature,tolerance against osmotic and heavy-metal ion stress,at the background of MBC resistant mutation T900A or A894T.However,isolated synonymous mutation C1006T did not have the same effect.These results suggested that the synonymous mutation C1006T has important biological function.To explore the molecular mechanism of how synonymous mutation C1006T increased biological fitness of MBC resistant mutants,the following reseaches were made:(1)The mRNA level,protein level and molecular weight of β2-TUBULIN and the mRNA level of β1-TUBULIN.Through real-time quantitative PCR experiment ofβ2-tubulin,we found this synonymous mutation decreased the mRNA level ofβ2-TUBULIN when combined with MBC resistant mutation T900A,but not decreased the mRNA level of β2-tubulin when combined with MBC resistant mutation A894T.Through western blotting experiment,we found this synonymous mutation could increase the protein level of β2-TUBULIN,when combined with MBC resistant mutation T900A.Through sequence analysis of the cDNA of β2-tubulin and western blotting experiment,we found this synonymous mutation did not affect mRNA spilicing and did not change the molecular weight of β2-TUBULIN.Through real-time quantitative PCR experiment β1-tubulin,we found this synonymous mutation did not affect the mRNA level of β1-TUBULIN.(2)The RNA secondary structure and codon usage bias.Using "The mfold Web Server",we obtained the secondary structure and corresponding Gibbs free energy ofβ2-tubulin mRNA,and excluded the probability that the mRNA stability was account for the observed differences in biological fitness.To test whether codon usage compromisesβ2-TUBULIN function,artificial synonymous mutations C1006A and C1006G was introduced into mutant M-T900A respectively to produce mutants M-T900A/C1006A and M-T900A/C1006G Relevant biological characteristics of M-T900A,M-T900A/C1006T,M-T900A/C1006A and M-T900A/C1006G were measured.Mutants M-T900A/C1006T,M-T900A/C1006A and M-T900A/C1006G all exhibited significantly higher biological fitness compared to M-T900A,including higher resistance to MBC,more spores in mung bean broth and higher pathogenicity on fresh tomato fruits.However,the enhance extent of biological fitness by the three synonymous mutations were different.Synonymous mutation C1006T made the largest enhancement on MBC resistance,and only this mutation increased mutant’s tolerance to high temperature.In conclusion,we report for the first time that the synonymous mutation G235G(nucleotide C1006T)at β2-tubulin of Fusarium fujikuroi has important biological function,and reveal how this synonymous mutation participates in the forming mechanism of carbendazim-resistance.