The tubulin cofactor A is involved in hyphal growth, conidiation and cold sensitivity in Fusarium asiaticum

Tubulin cofactor A (TBCA), one of the members of tubulin cofactors, is of great importance in microtubule functions through participating in the folding of α/β-tubulin heterodimers in Saccharomyces cerevisiae. However, little is known about the roles of TBCA in filamentous fungi. In this study, we characterized a TBCA orthologue FaTBCA in Fusarium asiaticum. The deletion of FaTBCA caused dramatically reduced mycelial growth and abnormal conidiation. The FaTBCA deletion mutant (ΔFaTBCA-3) showed increased sensitivity to low temperatures and even lost the ability of growth at 4°C. Microscopic observation found that hyphae of ΔFaTBCA-3 exhibited blebbing phenotypes after shifting from 25 to 4°C for 1- or 3-day incubation and approximately 72% enlarged nodes contained several nuclei after 3-day incubation at 4°C. However, hyphae of the wild type incubated at 4°C were phenotypically indistinguishable from those incubated at 25°C. These results indicate that FaTBCA is involved in cell division under cold stress (4°C) in F. asiaticum. Unexpectedly, ΔFaTBCA-3 did not exhibit increased sensitivity to the anti-microtubule drug carbendazim although quantitative real-time assays showed that the expression of FaTBCA was up-regulated after treatment with carbendazim. In addition, pathogenicity assays showed that ΔFaTBCA-3 exhibited decreased virulence on wheat head and on non-host tomato. Taken together, results of this study indicate that FaTBCA plays crucial roles in vegetative growth, conidiation, temperature sensitivity and virulence in F. asiaticum.

TBCA, as a β-tubulin-interacting protein, was first purified from bovine testis [6]. To date, characterization of several TBCA in yeast, murine, Arabidopsis and human has demonstrated that TBCA regulates both the ratio between αand β-tubulin and the tubulin folding pathways for correct polymerization into microtubules [7][8][9][10][11]. Rbl2p, the TBCA yeast orthologue, is not an essential gene in S. cerevisiae but is required for normal meiosis [7]. In fission yeast Schizosaccharomyces pombe, TBCA orthologue Alp31 plays an important role in the maintenance of microtubule integrity and the determination of the cell polarity [11]. Mutations in KIS (TBCA orthologue) lead to defects similar to the phenotypes associated with impaired microtubule function in Arabidopsis [9]. TBCA knockdown by RNAi in human cell lines, results in decreased amounts of αand β-tubulin levels, subtle alterations in the microtubule cytoskeleton, G1 cell cycle arrest and cell death [10].
Fusarium asiaticum (teleomorph: Gibberella zeae) is one of major causal agents that are responsible for economically important Fusarium head blight (FHB) on various cereal crops [12][13][14][15]. In addition to yield reduction, this pathogen produces mycotoxins including deoxynivalenol (DON), acetyldeoxynivalenol (3-ADON or 15-ADON), nivalenol (NIV), and acetylnivalenol (4-ANIV) in infected plants, which pose a serious threat to human and animal health [16,17]. In China, F. asiaticum is more important than F. graminearum with respect to population quantity and mycotoxin production [15]. Because highly resistant wheat cultivars are not available [18], chemical control remains one of the major strategies for the management of FHB [19]. However, highly effective fungicides against FHB are limited [19]. Moreover, Fusarium spp. have developed resistance to several commercialized fungicides [20][21][22][23]. Therefore, the exploration of new compounds and potential targets is desperately needed for an effective management of FHB. In the therapies of human diseases, TBCA has been regarded as an attractive target for the treatment of clear cell renal cell carcinoma (ccRCC), since it plays crucial roles in the progression, invasion and metastasis of ccRCC [24]. Until now, little is known about the roles of TBCA in filamentous fungi. In this study, we were thus interested in investigating the functions of TBCA in F. asiaticum, which may help in its exploitation as a drug target for the design of new antifungal agents against FHB.
Sequence analyse of FaTBCA gene from F. asiaticum Based on the sequence of TBCA (FGSG_00510.3) gene in F. graminearum, one pair of primers A1 + A2 (Additional file 1) was designated to amplify FaTBCA from GJ33 DNA. PCR amplifications were purified, cloned, and sequenced. The sequence of FaTBCA was deposited in GenBank under accession number KM116518. On the basis of deduced amino acid sequences of FaTBCA and its orthologues, the phylogenetic tree was generated by the neighbor-joining method with 1000 bootstrap replicates using the Mega 4.1 software [25].

Yeast complementation assays
Full-length cDNA of FaTBCA was amplified using primer pair A3 + A4 listed in Additional file 1. PCR product was digested with appropriate enzymes and cloned into the pYES2 vector (Invitrogen Co., CA, USA), and transformed into the corresponding yeast mutant. Yeast transformants were then selected on synthetic medium lacking uracil. Additionally, the wild-type strain BY4741 and the mutant transformed with an empty pYES2 vector were used as controls. For complementation assays, the yeast transformants were grown at 30°C on YPRG medium (1% yeast extract, 2% bactopeptone, 2% galactose) supplied with 0.48% (v/v) HCl. The experiments were repeated three times independently. There are four replicates for each experiment.

Construction of gene deletion and complemented strains
FaTBCA deletion vector pBS-FaTBCA-Del was constructed by inserting two flanking sequences of FaTBCA into left and right sides of HPH (hygromycin resistance gene) in the pBS-HPH1 vector [26]. Briefly, by using primer pair A5 + A6 (Additional file 1), a 477 bp upstream flanking sequence of FaTBCA was amplified from GJ33 genomic DNA, and was inserted into XhoI-SalI sites of the pBS-HPH1 vector to generate a plasmid pBS-FaTBCA-Up. Subsequently, a 417 bp downstream flanking sequence of FaTBCA amplified from GJ33 genomic DNA using the primers A7 + A8 (Additional file 1) was inserted into HindIII-BamHI sites of the pBS-FaTBCA-Up vector to generate a plasmid pBS-FaTBCA-Del. Finally, the 2394 bp fragment containing FaTBCA-upstream-HPH-FaTBCA-downstream cassette was obtained by PCR amplification with primer pair A5 + A8 from the pBS-FaTBCA-Del. The resultant PCR product was purified and used for protoplast transformation. The PEG-mediated protoplast fungal transformation was performed as described previously [27]. For selective growth of transformants, PDA medium supplemented with hygromycin (100 mg l −1 ) were used.
To confirm that the phenotype of FaTBCA deletion mutant is due to disruption of the gene, genetic complementation was performed. The FaTBCA complement plasmid pCA-FaTBCA-Com was constructed using the backbone of pCAMBIA1300. First, a XhoI-KpnI neo cassette containing a trpC promoter was amplified from plasmid pBS-RP-Red-A8-NEO [28] with primers Neo-F + Neo-R (Additional file 1), and cloned into the XhoI-KpnI site of pCAMBIA1300 to create plasmid pCA-neo. Then, a 1862 bp of full length FaTBCA gene including 1437 bp promoter region was amplified using primer pair A11 + A12 (Additional file 1) from genomic DNA of the wild-type GJ33, and subsequently cloned into the KpnI-XbaI sites of pCAMBIA1300 to generate the complement plasmid pCA-FaTBCA-Com. Transformation of ΔFaTBCA-3 with the full-length FaTBCA was conducted as described above except that geneticin (100 mg l −1 ) was used as a selection agent. After single spore isolation, all of the mutants generated in this study were preserved in 15% glycerol at −80°C.

Microscopic examinations of hyphal and conidial morphology
Hyphal growth of each strain was tested on PDA, CM, MM and wheat-head media at 25°C for 3 days. The experiment was repeated three times, and each with two replicates. Hyphal morphology was examined under a Nikon ECLIPSE Ni-U microscope (Nikon Co., Tokyo, Japan) from mycelia that were incubated in potato dextrose broth (PDB; 200 g potato, 20 g dextrose and 1 l water) at 25°C for 1 day using a shaker then transferred to static incubation at 4, 10 and 25°C for 1 day or 3 days. Furthermore, nucleus of hyphae were examined under the same microscope after staining with 4',6-diamidino-2-phenylindole (DAPI), as described previously [29]. The experiments were repeated three times independently. A total of 150 hyphae were examined for each strain.
For conidiation assays, two methods were used. One method used was to count spores produced in CMC liquid media, using fresh mycelia (50 mg) of each strain taken from the edge of a 3-day-old colony to inoculate 100 ml of CMC liquid media. The flasks were incubated at 25°C for 5 days in a shaker (180 rpm). The other method used was to count spores produced on MBA, using 2 μl of a 1*10 6 ml −1 spore suspension to inoculate a MBA plate. After one week of incubation, two ml of water was added to the plate and spread over the mycelium to collect spores. The amount of conidia was counted with a hemacytometer. The experiments were repeated three times independently. There are three replicates for each experiment.
Additionally, conidia of each strain were re-suspended in 2% (w/v) sucrose solutions and incubated at 25°C for 4 hrs, then conidial germination was examined under a Nikon ECLIPSE Ni-U microscope. Conidial morphology was observed under the same microscope. Furthermore, septum and nucleus were examined after staining conidia from each strain with calcofluor white (CFW) and DAPI, respectively, as described previously [29]. The experiments were repeated three times independently. A total of 150 conidia were examined for each strain.

Sensitivity determination to different stress agents
To determine the sensitivity to different temperatures, 5-mm mycelial plugs of each strain taken from 3-dayold colony edge were inoculated on PDA and then incubated at 4, 10, 15, 20, and 25°C. Three replicate plates for each temperature were used for each strain. After incubation for 3.5 days, colony diameter in each plate was measured in two perpendicular directions with the original mycelial plug diameter (5 mm) subtracted from each measurement. For each plate, the average of the colony diameters was used for calculating the percentage of growth inhibition. The mycelium growth of each strain at 25°C was arbitrarily set as control. The experiment was repeated three times. There are two replicates for each experiment.
To determine the sensitivity to various stresses, 5-mm mycelial plugs of each strain taken from 3-day-old colony edge were inoculated on MM amended with antifungal compound carbendazim, tebuconazole, iprodione or fludioxonil, on fructose gelatin agar (FGA; 10 g fructose, 2 g gelatin, 1 g KH 2 PO 4 , 0.5 g MgSO 4°7 H 2 O, 2 g NaNO 3, 20 g agar and 1 l water, pH 7.0) amended with antifungal compound pyrimethanil, or on MM amended with cell wall stress agent congo red or SDS, osmotic agent NaCl or sorbitol, oxidative stress generator H 2 O 2 or paraquat, or metal ion CaCl 2 or MnCl 2 . The fungicides described above were kindly provided by the Institute of Zhejiang Chemical Industry or the Institute for the Control of Agrochemicals, Ministry of Agriculture (ICAMA), Beijing, China. The concentration of each compound is presented in Additional file 2. After incubation for 3.5 days, colony diameter in each plate was measured in two perpendicular directions with the original mycelial plug diameter (5 mm) subtracted from each measurement. For each plate, the average of the colony diameters was used for calculating the percentage of growth inhibition. The experiment was repeated three times. There are two replicates for each experiment.

Pathogenicity assays on flowering wheat heads and tomatoes
Pathogenicity test was performed with single floret injection method as previously described [30]. After incubation in CMC medium for 5 days, conidia of each strain were collected by filtration through three layers of gauze and subsequently resuspended in sterile distilled water to a concentration of 1 × 10 5 conidia ml −1 . A 10-μl aliquot of conidial suspension was injected into a floret in the central section spikelet of single flowering wheat heads of susceptible cultivar Jimai 22. There were ten replicates for each strain. After inoculation, the plants were kept at 22 ± 2°C under 95-100% humidity with 12 hrs of daylight. Fifteen days after inoculation, the infected spikelets in each inoculated wheat head were recorded. The experiment was repeated four times. To examine the ability to colonize tomato, a 10-μl aliquot of conidial suspension was injected into the wounded tomato after surface sterilization. There are five replicates for each strain. Inoculated tomatoes were incubated under the same conditions described above, and were photographed 3 days after inoculation. The experiment was repeated three times.

Determination of DON production
A 50-g aliquot of healthy wheat kernels was sterilized and inoculated with 50 mg fresh mycelia of each strain. After incubation at 25°C for 20 days, DON and fungal ergosterol were extracted using previously described protocols [31]. The DON extracts were purified with PuriToxSR DON column TC-T200 (Trilogy analytical laboratory), and amounts of DON and ergosterol in each sample were determined by using a HPLC system Waters 1525. The experiment was repeated three times, each with three replicates.

Analysis of gene expression
Total RNA of each strain was extracted using the TaKaRa RNAiso Reagent, and 10 μg of each RNA sample was used for reverse transcription with the oligo(dT) 18 primer using a RevertAid H Minus First Strand cDNA Synthesis kit. Expression of each gene was determined by quantitative reverse-transcriptase PCR (RT-PCR) with the corresponding primer pair (Additional file 1). The RT-PCR amplifications were performed in a DNA Engine Opticons 4 System (MJ Research) using the SYBR Green I fluorescent dye detection. Amplifications were conducted in 20-μl volumes containing 10 μl SYBR® Premix Ex Taq (TaKaRa Biotechnology Co., Ltd, Dalian, China), 2 μl template DNA and 1 μl each 4 μM primer (each primer pair per amplification). There were three replicates for each sample. The real-time PCR amplifications were performed with the following parameters: an initial preheat at 95°C for 2 min, followed by 35 cycles at 95°C for 15 s, 58°C for 20 s, 72°C for 20 s, and 80°C for 3 s in order to quantify the fluorescence at a temperature above the denaturation of primer-dimers. Once amplifications were completed, melting curves were obtained to identify PCR products. For each sample, PCR amplifications with primer pair Actin-F + Actin-R (Additional file 1) for the quantification of expression of ACTIN gene were performed as a reference. The relative expression levels of each gene in each strain or under the treatment were calculated using the 2 -ΔΔCt method [32]. The experiment was repeated three times.

Standard molecular methods
Fungal genomic DNA was extracted using a previous published protocol [33]. Plasmid DNA was isolated using a Plasmid Miniprep Purification Kit (BioDev Co., Beijing, China). Southern analysis of FaTBCA gene in F. asiaticum was performed using probe as indicated in Additional file 3. The probe was labeled with digoxigenin (DIG) using a High Prime DNA Labeling and Detection Starter kit II according to the manufacturer's instructions (Roche Diagnostics; Mannheim, Germany).

Statistical analysis
All data for gene expression, DON production, conidiation and conidial germination were subjected in an analysis of variance (ANOVA) and the means were separated using Fisher's protected least significant difference (P = 0.05). In the sensitivity to environmental stresses assays, the mycelial growth inhibition of ΔFaTBCA-3 under each stress was compared with that of the wild type using a t test.

Results
In silico analysis of FaTBCA in F. asiaticum One tubulin binding cofactor A (TBCA) (FGSG_00510.3) was retrieved by BLASTP searching the F. graminearum genome database (http://www.broadinstitute.org) with the S. cerevisiae TBCA orthologue Rbl2 as a query. Based on the DNA sequences of FGSG_00510.3, the corresponding orthologue FaTBCA was amplified and sequenced from F. asiaticum. Sequencing analysis showed that the nucleotide sequence of FaTBCA is 425 bp in length including one predicted intron, and is predicted to encode a 119 aminoacid protein. Phylogenic analyses showed that FaTBCA is homologous to their counterparts from other filamentous fungi and yeast (Additional file 4). In addition, protein domain analysis by Pfam (http://pfam.xfam.org/) revealed that FaTBCA contains a conserved TBCA domain (Additional file 4). However, the deduced amino acid sequence of TBCA from the wild-type GJ33 is not highly homologous to those from other fungi (Additional file 4). For example, the deduced amino acid sequence of TBCA in GJ33 shared 46.0, 37.7 and 30.3% identity to those from Magnaporthe oryzae (MagoCoA, XP_003709983.1), Aspergillus nidulans (AnCoA, CBF70033.1) and S. cerevisiae (SaCoA, EGA72990.1), respectively.

FaTBCA partially complement the yeast ΔRbl2 mutant
To characterize the functions of FaTBCA, we first tested whether this gene could complement an HCl-sensitive S. cerevisiae TBCA mutant (ΔRbl2) whose growth is dramatically inhibited on YPRG amended with 0.48% (v/v) HCl. An expression vector pYES2 containing the fulllength FaTBCA cDNA was transferred into ΔRbl2. As a negative control, the mutant was also transformed with an empty pYES2 vector. As shown in Figure 1, the reduced growth of yeast ΔRbl2 on YPRG +0.48% HCl was partially restored by F. asiaticum FaTBCA. These results indicate that FaTBCA could function as a TBCA in S. cerevisiae.
Deletion and complementation of FaTBCA in F. asiaticum To investigate the function of FaTBCA, we generated gene deletion mutants using a homologous recombination strategy (Additional file 3). Seven deletion mutants were identified from 13 hygromycin-resistant transformants by PCR analysis with the primer pair A9 + A10 (Additional file 1). The primer pair amplified 1710 and 460 bp fragments from FaTBCA deletion mutants and the wild-type progenitor GJ33, respectively. All seven deletion mutants showed identical growth defects on PDA plates. When probed with a 973 bp upstream DNA fragment of FaTBCA, the deletion mutant ΔFaTBCA-3 had an anticipated 5315 bp band, but lacked a 2205 bp band which was present in the wild-type GJ33 (Additional file 3). This Southern hybridization pattern confirmed that the transformant ΔFaTBCA-3 is a null mutant resulting from expected homologous recombination events at the FaTBCA locus. The complemented strain ΔFaTBCA-3C was a single copy of FaTBCA inserted into the genome of ΔFaTBCA-3 (Additional file 3).

Involvement of FaTBCA in vegetative growth and conidiation in F. asiaticum
The FaTBCA deletion mutant ΔFaTBCA-3 grew significantly slower than the wild-type GJ33 on PDA, CM, MM and wheat-head media (Figure 2). Compared to the wild-type GJ33, ΔFaTBCA-3 produced a similar number of conidia (Additional file 5), however, conidia of ΔFaTBCA-3 were shorter and with less septa ( Figure 3A and B). Microscopic examination showed that 81.7% of ΔFaTBCA-3 conidia had one to three septa with conidial lengths ranging from 23.6 to 57.7 μm ( Figure 3A, C and D). In contrast, 62.6% of the wild-type conidia had four or five septa, with lengths ranging from 62.7 to 80.4 μm ( Figure 3A, C and D). In addition, conidial germination assays found that conidia of ΔFaTBCA-3 did not show delayed germination (Additional file 5).

The FaTBCA deletion mutant showed increased sensitivity to low temperatures
Previous studies have reported that cold temperatures have disruptive effects on the stability of microtubules [34,35], we therefore determined the sensitivity of the FaTBCA deletion mutant to low temperatures. As shown in Figure 4A and B, the inhibition percentages of mycelial growth of the mutant ΔFaTBCA-3 at 10, 15 and 20°C were significantly higher than those of the wild type. Furthermore, after 7 days of incubation at 4°C, the wild type exhibited an obvious growth, however, ΔFaTBCA-3 did not show any sign of growth ( Figure 4C). After 21 days of incubation at 4°C, the colony diameter of the wild type was greater than 5.8 cm, whereas, the ΔFaTBCA-3 mutant was still unable to grow ( Figure 4C), indicating that the deletion of FaTBCA caused the stagnation of growth at 4°C. The ΔFaTBCA-3 mutant restored growth after shifting from 4 to 25°C (data not shown).
In order to explore why the mutant ΔFaTBCA-3 cannot grow at the cold temperature 4°C, we further observed the hyphal morphology and nuclear distribution of ΔFaTBCA-3 after 1-day or 3-day incubation at 4°C. After incubation in a shaker at 25°C for 1 day, the wild type and ΔFaTBCA-3 were immediately transferred to 4°C for 1 or 3 days. Microscopic observation showed that the hyphal tips of ΔFaTBCA-3 began to present blebbing phenotypes after 1-day incubation at 4°C ( Figure 5). Moreover, the enlarged nodes occurred not only in hyphal tips but also in hyphal middle sections after 3-day incubation ( Figure 5). By observing 150 hyphal tips, we found that 9 and 83% hyphal tips showed bulged nodes after 1 and 3 days of incubation at 4°C, respectively. Furthermore, 4',6-diamidino-2-phenylindole (DAPI) staining assays showed that approximately 72% enlarged nodes contained several nuclei by counting 100 enlarged nodes ( Figure 6). However, the hyphal morphology and nuclear distribution of the wild type after incubation at 4°C exhibited no obvious difference compared to those after incubation at 10 or 25°C ( Figure 5, 6and Additional file 6). Taken together, these results indicate that FaTBCA plays a critical role in the cell division under cold stress (4°C) in F. asiaticum.

The deletion of FaTBCA did not change the sensitivity to carbendazim
Since the fungicide carbendazim can cause the depolymerization of fungal microtubules, we tested the sensitivity of the mutant to this compound. Surprisingly, ΔFaTBCA-3 did not differ from the wild type in carbendazim sensitivity, as well as in other fungicides including tebuconazole, iprodione, fludioxonil and pyrimethanil (Additional file 2). In addition, the deletion of FaTBCA had no effect on the sensitivity of F. asiaticum to cell wall damaging agents, and to osmotic, oxidative and metal cation stresses (Additional file 2).
Given that the FaTBCA deletion mutant ΔFaTBCA-3 did not show increased sensitivity to carbendazim, we were interested in the expression of FaTBCA treated with carbendazim and the expression of two α-tubulin genes (encoded by FaTUA1 and FaTUA2) in ΔFaTBCA-3 mutant. As shown in Additional file 7, the expression of FaTBCA increased 3.1 times under the treatment of 3.5 μg ml −1 carbendazim. Quantitative realtime PCR analysis showed that the expression of FaTUA1 and FaTUA2 in ΔFaTBCA-3 showed no significant difference compared with the wild-type GJ33 (Additional file 7), indicating that there might be other unknown genes involved in regulating the balance of the α/β-tubulin monomers.
FaTBCA is required for full virulence and DON biosynthesis in F. asiaticum Pathogenicity assays indicated that disruption of FaTBCA caused a significant reduction in the virulence of F. asiaticum. As shown in Figure 7A, the wild-type strain GJ33 and ΔFaTBCA-3C caused the typical scab symptoms in the inoculated and nearby spikelets of flowering wheat heads 15 days after inoculation. Under the same conditions, however, scab symptoms caused by ΔFaTBCA-3 were only observed in the inoculated spikelet and in the adjacent two or three spikelets. Furthermore, three days after inoculation of non-host tomatoes, the water-soaked rot lesion caused by ΔFaTBCA-3 was significantly smaller than that caused by GJ33 or ΔFaTBCA-3C ( Figure 7B).
Previous studies have shown that DON is an important virulence factor and a prerequisite for colonization of wheat [36,37]. Therefore, we were interested in the effect of FaTBCA deletion on DON biosynthesis. After growth on sterilized wheat kernels for 20 days, the amount of DON produced by the wild type was 6.2 folds higher than that produced by ΔFaTBCA ( Figure 7C). To further confirm the results, we determined the expression levels of TRI6 and TRI10 by quantitative real-time PCR using RNA samples isolated from mycelia grown in minimal synthetic liquid medium (MS) [38] for 4 days at 25°C. The expression levels of TRI6 and TRI10 in ΔFaTBCA reduced by 94% and 76%, respectively, as compared to those in the wild type ( Figure 7D). These results indicate that FaTBCA plays a critical role in DON biosynthesis in F. asiaticum.

Discussion
In this study, we characterized a putative tubulin cofactor A FaTBCA, a homologue of S. cerevisiae Rbl2 in F. asiaticum. Although FaTBCA shares low homology with similar proteins found in other organisms, F. asiaticum FaTBCA can restore the sensitivity to HCl of the RBL2 deletion mutant of budding yeast. Similarly, expression of mouse TBCA not only suppresses the benomyl supersensitivity resulting from the deletion of RBL2 in S. cerevisiae [7], but also rescues all defects of a KIS (TBCA orthologue) mutant under the control of the 35S promoter in Arabidopsis [9]. The protein sequence of mouse TBCA is approximately 30% and 35% identical to Rbl2 and Kis, respectively. Therefore, the functions of TBCA homologues from yeast to higher eukaryotic organisms are evolutionally conserved.
Although TBCA in eukaryotic organisms are conserved, the detailed roles of TBCAs among different species are not fully consistent. In this present study, the deletion of FaTBCA leads to reduced vegetative growth and abnormal conidia with less septation in F. asiaticum ( Figures 2 and 3), which is not in agreement with the situations in yeast. In S. cerevisiae and S. pombe, TBCA homologues are dispensable for cell growth and asexual development, but played important roles in meiosis and growth polarity, respectively. Previous studies on F. asiaticum reported that the deletion of each β-tubulin results in reduced vegetative growth, abnormal conidial morphology or decreased conidiation [21,39,40]. Our FaTBCA deletion mutant ΔFaTBCA-3 shared similar phenotypes with β-tubulin disruption mutants of F. asiaticum, indicating that TBCA in F. asiaticum is involved in the maintenance of microtubule function as reported in other eukaryotic organisms.
Microtubules are dynamic by nature, with an equilibrium existing between soluble subunits and the polymerized filament that could influence normal cellular functions. Previous studies have found that low temperature generally shifts this equilibrium toward depolymerization and leads to intrinsic cold sensitivity of microtubule [34,35]. In this study, we found that the deletion of FaTBCA increased the sensitivity to low temperatures in F. asiaticum ( Figure 4A and B), indicating that deletion of FaTBCA might accelerate the depolymerization of microtubule at low temperatures. However, a precise monitoring of microtubule dynamics under cold stress would have to be addressed to confirm this hypothesis.
Under the cold temperature 4°C, the FaTBCA mutant exhibited growth stagnation accompanied with the phenotype of several nuclei in the enlarged hyphal nodes ( Figures 5 and 6), indicating that FaTBCA is involved in cell division. This might be a major contributor to the stagnation of growth at 4°C. In Arabidopsis, mesophyll cells in leaf sections of KIS mutant frequently were highly enlarged and the enlarged cells had one large nucleus or several nuclei [9]. In human HeLa cells transfected with TBCA siRNA also exhibits blebbing phenotypes, which are considered to be due to the changes of microtubule structures [10]. Taken together, our results showed that the TBCA in F. asiaticum might share some functions with Kis in Arabidopsis [9].
Carbendazim and other benzimidazole fungicides, which target β-tubulin and interrupt the polymerization of microtubules, have been extensively used to control various plant diseases caused by fungi [40]. The treatment of anti-microtubule drug carbendazim caused the up-regulation of FaTBCA in F. asiaticum (Additional file 7), which supported that TBCA serves as a reservoir of excess β-tubulin [1,8]. To our surprise, unlike the case in S. cerevisiae [7], the absence of FaTBCA did not render cells more sensitive to carbendazim (Additional file 2). In budding yeast, over-expression of either Rbl2 or α-tubulin suppresses β-tubulin lethality and causes Figure 4 The deletion of FaTBCA increased sensitivity to low temperatures. A. Mycelial plugs of the wild-type GJ33, ΔFaTBCA-3 and ΔFaTBCA-3C were inoculated on PDA and incubated at 4, 10, 15, 20 and 25°C for 3.5 days. B. Inhibition percentages of mycelial growth of GJ33, ΔFaTBCA-3 and ΔFaTBCA-3C at 4, 10, 15 and 20°C. After incubation for 3.5 days, colony diameter in each plate was measured in two perpendicular directions with the original mycelial plug diameter (5 mm) subtracted from each measurement. For each plate, the average of the colony diameters was used for calculating the percentage of growth inhibition. The mycelium growth of each strain at 25°C was arbitrarily set as control. Line bars in each column denote standard errors of three experiments. * = significant difference for each temperature at a 95% coincidence interval by performing a t test. C. The mutant ΔFaTBCA-3 was unable to grow at 4°C after incubation for 7 or 21 days. resistance to antimicrotubule drug benomyl [7]. However, our quantitative real-time PCR assays found that the deletion of FaTBCA did not result in significant change in the expression levels of two α-tubulin genes (Additional file 7), indicating that there might be other unknown genes involved in the control of the α/βtubulin monomer balance.
In addition to the involvement of FaTBCA in regulating mycelial growth, conidiation and low temperature sensitivity, this gene is also required for virulence of F. asiaticum. The reduced virulence in ΔFaTBCA may result from three defects of the mutant. First, ΔFaTBCA showed drastic mycelium growth defect, which might be a major contributor. As observed on PDA, the mutant also grew significantly slower than the wild-type strain on the wheat-head medium ( Figure 2). Second, the ability of ΔFaTBCA to produce trichothecene mycotoxins in infected wheat kernels was greatly decreased. DON, the end product of the trichothecene biosynthetic pathway, plays an important role in the spread of FHB within a spike [36,37], thus this may also contribute to the reduced virulence of the mutant. Third, TBCA has been found to play a crucial role in correct polymerization of microtubules [7,9]. Moreover, previous studies have reported that extracellular secretion of virulence factors is dependent on microtubulemediated vesicle transport in pathogenic fungi [41][42][43]. Thus, the reduced virulence in ΔFaTBCA-3 might be associated with the disturbance of microtubule-dependent vesicle transport.

Conclusion
Our FaTBCA studies in F. asiaticum found that FaTBCA plays critical roles in vegetative growth, conidial morphology, sensitivity to low temperatures and virulence. To our knowledge, this is the first report about the functions of TBCA in filamentous fungi. Our results indicate that

Additional files
Additional file 1: Oligonucleotide primers used in this study.
Additional file 2: Sensitivity of GJ33, ΔFaTBCA-3 and ΔFaTBCA-3C to environmental stresses. A. Comparisons were made on MM plates amended with 10 μg ml −1 iprodione, 0.25 μg ml −1 tebuconazole, 0.5 μg ml −1 carbendazim, 0.05 μg ml −1 rapamycin, 0.25 M CaCl 2 , 10 μg ml −1 paraquat, 0.05% H 2 O 2 , 0.7 M NaCl, 0.7 M sorbitol, 0.01% SDS, and 0.2 g l −1 congo red. B. Inhibition percentages of mycelial growth of GJ33, ΔFaTBCA-3 and ΔFaTBCA-3C under each stress. After incubation for 3.5 days, colony diameter in each plate was measured in two perpendicular directions with the original mycelial plug diameter (5 mm) subtracted from each measurement. For each plate, the average of the colony diameters was used for calculating the percentage of growth inhibition. Line bars in each column denote standard errors of three experiments. A t test was performed to determine significant differences, * = significant difference for each stress at a 95% coincidence interval. The phylogenetic tree was generated by the neighbor-joining method with 1000 bootstrap replicates using the Mega 4.1 software. B. Each TBCA orthologue contains one functional domain, tubulin binding cofactor A, which were identified by Pfam (http://pfam.xfam.org/). C. Alignment of the predicted amino acid sequences of TBCA domains from F. asiaticum, M. oryzae, N. crassa, A. nidulans, S. cerevisiae, S. pombe, and C. albicans. The Boxshade program was used to highlight identical (black shading) or similar (grey shading) amino acids.
Additional file 5: Effect of FaTBCA deletion on conidiation and conidium germination of F. asiaticum. A. Conidia were counted after incubation of the wild-type GJ33, the mutant ΔFaTBCA-3 and the complemented strain ΔFaTBCA-3C in carboxymethyl cellulose liquid medium (CMC) for 5 days in a shaker at 25°C. B. Conidia were counted after incubation of each strain on mung bean agar (MBA) for one week Figure 7 FaTBCA is required for full virulence and DON biosynthesis in F. asiaticum. A. Flowering wheat heads were point inoculated with a conidial suspension at 10 5 conidia ml −1 of the wild-type GJ33, ΔFaTBCA-3 and ΔFaTBCA-3C, and infected wheat heads were photographed 15 days after inoculation. B. Tomatoes were inoculated with a conidial suspension at 10 5 conidia ml −1 of each strain and infected fruits were photographed 3 days after inoculation. C. The amount of DON (per mg fungal ergosterol) produced by each strain in infected wheat kernels was determined after 20 days of inoculation. Line bars in each column denote standard errors of three experiments. Values on the black bars followed by the same letter are not significantly different according to a Fisher's least significant difference (LSD) test at P = 0.05. D. Relative expression of DON biosynthetic TRI genes in each strain and bars denote standard errors from three experiments. Values on the black bars followed by the same letter for each gene are not significantly different according to a Fisher's least significant difference (LSD) test at P = 0.05.