Functions Of Two B-Tubulin Genes And Carbendazim-Resistance Mechanisms In Fusarium Graminearum Schwabe

Microtubules, as steady-state dynamic polymers composed ofα-andβ-tubulin, are ubiquitous eukaryotic cell structures that are involved in a variety of intracellular processes including morphogenesis, cell growth and division. Beta-tubulin is the target of benzimidazoles, and the resistance of these fungicides is associated with amino acid substitutions at special codons ofβ-tubulin. Fusarium graminearum is the primary pathogen causing Fusarium head blight of wheat in China, and carbendazim (methyl benzimidazol-2-yl carbendazim, MBC) is one of the key fungicides used to control this disease, but the action and resistance phenotypes of this agent are different for F. graminearum than for most other filamentous pathogenic fungi.The purposes of this dissertation are to (1) understand the action mechanism of carbendazim on F. graminearum by compare the conidial germination and mitosis in germlings of carbendazim-sensitive and-resistant strains of F. graminearum and B. cinerea; (2) determine the target of carbendazim in F. graminearum by deletion theβ1-orβ2-tubulin gene and microtubules labeled by expressing the GFP taggedβ1-orβ2-tubulin; (3) study the physiological functions of the two P-tubulin genes in F. graminearum by compare the characteristics of mutants deletedβ1-orβ2-tubulin gene; (4) analysis the expression of the two P-tubulin genes in F. graminearum by taggingβ1-andβ2-tubulin with GFP and RFP; (5) confirm the carbendazim resistance mechanism by construction the putative carbendazim-resistantβ2-tubulin gene-deletion and-complementation mutants of F. graminearum. These results provide a theoretical foundation to control Fusarium head blight of wheat and manage carbendazim-resistance in F. graminearum.1 Mitotic Division in Germ Tube of Fusarium graminearumThe nuclear phase and mitosis in germ tube of Fusarium graminearum were studied with staining by Giemsa techniques. There was single nucleus in conidial cell. After division in conidial cell, the nuclei entered into germ tube and divided repeatedly, and therefore the number of nuclei in germ tube variably multiplied. The mitosis of F. graminearum has four phases. The chromosomes gradually shortened and thickened during prophase, and at metaphase the chromosomes were distinctly visible. The chromatids separated at anaphase and moved to opposite poles, and then, daughter nuclei were shaped at telophase. Synchronous and asynchronous chromosome separation was seen, and it was more common that the lagging chromosomes formed anaphase bridges.2 Effects of Carbendazim on Conidial Germination and Mitosis in Germlings of Fusarium graminearumPrevious data indicated that the action mechanism of benzimidazoles on most filamentous pathogenic fungi, e.g. Botrytis cinerea, is inhibition mitotic division, and resistance to benzimidazoles has been associated with mutations in theβ-tubulin gene. The effects and the different resistance mechanism of carbendazim in Fusarium graminearum was found, by comparison of the effects of carbendazim on conidial germination and mitosis in germlings of F. graminearum and B. cinerea. Conidia and germlings of F. graminearum wild type strains, as B. cinerea, germinated or grew in the presence of carbendazim to produce distorted, more branched, and elongation-restrained germlings and more branched foreparts. And in carbendazim-treated germlings there were irregularly distribution of chromosomes masses and no normal nuclei division was able to be observed. The CMIs (chromosome mitosis index) rapidly increased within 60 min and after that rapidly dropped. Compairing the carbendazim-resistants, however, F. graminearum was different from B. cinerea in growth and mitosis when they were treated with carbendazim. F. graminearum carbendazim-resistant strains, as ones of their wild-type, they produced significantly more branches and the CMIs have significant variation although abnormal mitosis was not observed under treatment of carbendazim. While B. cinerea carbendazim-resistant strains expressed normally in morphology of growth and mitosis after carbendazim treatment. The results show that carbendazim inhibits mitosis in F. graminearum and there should be a novel mechanism for carbendazim resistance in F. graminearum.3 Target of Carbendazim on Fusarium graminearumIn this study, we deleted theβ1-andβ2-tubulin gene of F. graminearum, respectively, and measure the sensitivity of carbendazim on deletion mutants. The results indicated that theβ1-orβ2-tubulin gene-deletion mutants are carbendazim-sensitive, but there different sensitivity in these mutants to carbendazim. The same results have been obtained by acquirement the mutants which microtubules labeled by expression the GFP taggedβ1-or P2-tubulin, and determination the carbendazim-binding affinity onβ1-orβ2-tubulin of F. graminearum by observation the characteristics of microtubules in these mutants under carbendazim treatment. The results show that the affinities are different forβ1-tubulin than forβ2-tubulin.The present results indicate that bothβ1-andβ2-tubulin of F. graminearum are the targets of carbendazim, and there is different carbendazim-binding affinity of the twoβ-tubulins. This difference may be associated with amino acid diversity at codon 240 ofβ-tubulin. The present work also reveals clearly that bothβ1-and P2-tubulin of F. graminearum, tagged with GFP and replaced one or two endogenous P-tubulins with a tagged version of the same gene, is apparently biologically active, and expression does not impact cell physiology.4 Functions of Twoβ-Tubulin Genes in Fusarium graminearumThe biological characteristics of theβ1-andβ2-tubulin gene deletion mutants of F. graminearum were assayed, including vegetative growth, asexual and sexual reproduction, mitosis and pathogenicity, to detect the physiological functions of the two P-tubulin genes. The results indicate that there are no significant different biological phenotypes inβ1-tubulin gene deletion mutants from wild type strains. Although they are active and can complete their life cycles, P2-tubulin gene deletion mutants have slow growth, more branches, decreased asexual and sexual reproduction ability, and poor pathogenicity. All mutants exhibit normal mitosis. These data indicate thatβ2-tubulin gene is more essential for activity thanβ1-tubulin gene, and there may be some functional differences between them, but the two P-tubulins are functionally interchangeable.The same results have been obtained by acquirement the mutants which microtubules labeled by expression the GFP taggedβ1-orβ2-tubulin, and determination the carbendazim-binding affinity onβ1- orβ2-tubulin of F. graminearum by observation the characteristics of microtubules in these mutants under carbendazim treatment.We constructed gene fusion mutants whichβ1-or/andβ2-tubulin was/were replaced by a GFP/RFP tagged version of the same gene, and determined temporal and spatial expression of the two P-tubulin genes. These data indicate that the two highly divergent P-tubulin genes in F. graminearum are both constitutively expressed during all lifecycle stages in all cells and individual microtubules can be composed of combinations between these different isotypes.All these data demonstrate that the twoβ-tubulins are substantially functionally interchangeable, but suggest that there may be subtle functional differences between them. 5 Molecular Mechanisms of Carbendazim Resistance in Fusarium graminearumBenzimidazole fungicides are thought to act by bindingβ-tubulin and preventing its polymerisation to microtubules. Resistance is believed to be associated with mutations in theβ-tubulin gene resulting in altered binding affinity in most filamentous pathogenic fungi. Previous studies have indicated that two P-tubulin structural genes in F. graminearum, and carbendazim-resistance is not due toβ1-tubulin but due toβ2-tubulin mutation included at codon 167,198 and 200. More evidence is required to confirm this carbendazim-resistance mechanism in F. graminearum.In this study, we deleted theβ1-andβ2-tubulin gene of field carbendazim-resistant strain of F. graminearum, respectively, and measure the sensitivity of carbendazim on deletion mutants. The results indicated that theβ2-tubulin gene-deletion mutants are super-sensitive to carbendazim, same as the mutant deleted theP2-tubulin gene of wild type strain. But theβ1-tubulin gene-deletion mutants are very high resistant to carbendazim, namely, more resistant to carbendazim than the field carbendazim-resistant strain whichβ1-tubulin gene was deleted. These results are confirmed by observation the effects of carbendazim on mitosis of these mutants through Giemsa-stain method. We obtained the mutants which microtubules labeled by expression the GFP taggedβ1-orβ2-tubulin of the field carbendazim-resistant strain, and determination the carbendazim-binding affinity onβ1-orβ2-tubulin by observation the characteristics of microtubules in these mutants under carbendazim treatment. The results further directly verify that the carbendazim-binding affinity onβ2-tubulin of this field resistant strain is significantly lower than wild type strain. Four kinds of amino acid substitutions inβ2-tubulin gene of carbendazim field resistant strains were used to replace the P2-tubulin gene of wild type strain. All mutants are resistant to carbendazim, and the resistance level of these mutants is consistent with these field resistant strains.Altogether these observations confirm that carbendazim-resistance in F. graminearum is due toβ2-tubulin gene mutation, different amino acid substitutions inβ2-tubulin are correlated with different sensitivity to crabendazim andβ2-tubulin gene of carbendazim-resistant strains can be used as a dominant selectable marker in F. graminearum transformation.