JXB Advance Access originally published online on April 10, 2007
Journal of Experimental Botany 2007 58(7):1617-1626; doi:10.1093/jxb/erl298
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RESEARCH PAPER |
Phytotoxic effects of trichothecenes on the growth and morphology of Arabidopsis thaliana
1Division of Life Science, Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa 920-1192, Japan
2Division of Functional Genomics, Advanced Science Research Center, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-0934, Japan
3Plant and Microbial Metabolic Engineering Research Unit and Laboratory for Remediation Research, Discovery Research Institute (DRI), Plant Science Center (PSC1), RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
To whom correspondence should be addressed. E-mail: tnish9{at}kenroku.kanazawa-u.ac.jp
Received 5 October 2006; Revised 8 December 2006 Accepted 11 December 2006
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionReferences |
|---|
Non-volatile sesquiterpenoids, a trichothecene family of phytotoxins such as deoxynivalenol (DON) and T-2 toxin, contain numerous molecular species and are synthesized by phytopathogenic Fusarium species. Although trichothecene chemotypes might play a role in the virulence of individual Fusarium strains, the phytotoxic action of individual trichothecenes has not been systematically studied. To perform a comparative analysis of the phytotoxic action of representative trichothecenes, the growth and morphology of Arabidopsis thaliana growing on media containing these compounds was investigated. Both DON and diacetoxyscirpenol (DAS) preferentially inhibited root elongation. DON-treated roots were less organized compared with control roots. Moreover, preferential inhibition of root growth by DON was also observed in wheat plants. In addition, T-2 toxin-treated seedlings exhibited dwarfism with aberrant morphological changes (e.g. petiole shortening, curled dark-green leaves, and reduced cell size). These results imply that the phytotoxic action of trichothecenes differed among their molecular species. Cycloheximide (CHX)-treated seedlings displayed neither feature, although it is known that trichothecenes inhibit translation in eukaryotic ribosomes. Microarray analyses suggested that T-2 toxin caused a defence response, the inactivation of brassinosteroid (BR), and the generation of reactive oxygen species in Arabidopsis. This observation is in agreement with our previous reports in which trichothecenes such as T-2 toxin have an elicitor-like activity when infiltrated into the leaves of Arabidopsis. Since it has been reported that BR plays an important role in a broad range of disease resistance in tobacco and rice, inactivation of BR might affect pathogenicity during the infection of host plants by trichothecene-producing fungi.
Key words: Brassinosteroid, defence response, dwarf, morphology, phytotoxin, trichothecene
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
The trichothecene family of phytotoxins is produced by necrotrophic fungal phytopathogenic Fusarium species (e.g. F. graminearum) and is a group of sesquiterpenoid secondary metabolites (Desjardins et al., 1993; Kimura et al., 2006). Trichothecenes contain numerous molecular species and are classified into four major groups by their chemical structures (Sudakin, 2003). Type A trichothecenes [e.g. T-2 toxin, HT-2 toxin, and diacetoxyscirpenol (DAS)] are highly toxic at low concentrations in eukaryotic cells; type B, for example, nivalenol and deoxynivalenol (DON)-contaminated cereal crops and processed grains are most frequently reported (Sudakin, 2003; Fig. 1). Type A trichothecenes and type B trichothecenes are distinguished by the absence or the presence of a carbonyl group at the C8 position (Desjardins et al., 1993). Trichothecene-producing Fusarium species have strain-specific trichothecene metabolite profiles (Ward et al., 2002; Kimura et al., 2006)). It is thought that trichothecene chemotypes play a role in the phytopathogenicity of individual Fusarium strains.
|
Trichothecenes are known to act as translational inhibitors in eukaryotic ribosomes (McLaughlin et al., 1977). In addition, trichothecenes are thought to be a virulent factor in the infection of wheat plants with their producing fungi (Proctor et al., 1995; Bai et al., 2002). Therefore, it had generally been thought that trichothecenes suppress the defence response in host plants. However, our recent study revealed that some type A trichothecenes such as T-2 toxin have an elicitor-like activity when infiltrated into the leaves of Arabidopsis (Nishiuchi et al., 2006). By contrast, DON is capable of inhibiting the translation in Arabidopsis cells without the induction of an elicitor-like signalling pathway, implying that DON-producing Fusarium species may affect translational systems in host plants without the induction of a defence response. It may be that the role of type B trichothecenes in the virulence of fungi that produce them is different from that of type A trichothecenes. On the other hand, the phytotoxic action by trichothecene molecular species has not been systematically studied. Although it was reported that trichothecenes inhibit seed germination and cause growth defects of monocotyledonous and dicotyledonous plants (Wakulinski, 1989; Packa, 1991; Cossette and Miller, 1995; Muhitch et al., 2000; Poppenberger et al., 2003; Ohsato et al., 2007), many reports have investigated the individual molecules.
To perform a comparative analysis of the phytotoxic action of representative trichothecenes in host plants, the growth and morphology of Fusarium-susceptible Arabidopsis seedlings grown on agar media containing these compounds were investigated. Although all trichothecenes examined caused growth defects of both shoots and roots in Arabidopsis seedlings, their effects differed significantly among their molecular species. Both DON and DAS preferentially inhibited root elongation, whereas T-2 toxin caused dwarfism with an aberrant morphology. Furthermore, microarray analyses suggested that a defence response, inactivation of BR, and ROS production occurred in T-2 toxin-treated Arabidopsis shoots.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Plant materials and growth conditions
Arabidopsis thaliana (ecotype Columbia; Col-0) plants and wheat plants were gown in a growth chamber (16/8 h light/dark cycle at 22 °C) after a 3 d vernalization period. Surface-sterilized Arabidopsis seeds were sown on Murashige and Skoog growth agar medium supplemented with 3% (w/v) sucrose, with or without trichothecenes in plastic Petri dishes. Sterilized wheat (Triticum aestivum L. cv. Nourin 61) seeds were sown on LS (Linsmaier and Skoog) agar medium with or without DON in plastic plates. Alternatively, 7-d-old Arabidopsis seedlings growing on MS medium without trichothecenes were transferred to MS medium containing trichothecenes. Arabidopsis Col-0 NahG transgenic plants (Lawton et al., 1996) were obtained from Dr Leslie Friedrich (Syngenta-Biotechnology, NC, USA).
Seed germination assay
Between 150 and 200 seeds were sown onto each medium, and were incubated for 3 d in the dark at 4 °C. Then plates were incubated as stated above, and the germination rate of the seeds was scored up to the 3rd day after incubation.
Microscopic analysis
For microscopic analysis of the DON-treated roots, roots from 21-d-old seedlings were harvested and fixed as previously described (Tsuge et al., 1996). Transverse sections of primary roots were cut in the area of the differential zone. For differential interference contrast (DIC) imaging, samples of leaves were cleared with a saturated chloral hydrate solution. DIC imaging of mesophyll cells of cleared leaves was performed as previously described (Ruzin, 1999).
RNA blotting analysis
RNA blotting analysis was performed as previously described (Nishiuchi et al., 2006). Total RNA (5 µg) was denatured, separated by electrophoresis on formaldehyde–agarose gels, and then transferred to a nylon membrane (Hybond-N+; Amersham Biosciences). The RNA blots were hybridized to 32P-labelled probes specific for PR-1 (Rogers and Ausubel, 1997). Probes for T-2 toxin-inducible genes were prepared by amplification of appropriate sequences from cDNA by polymerase chain reaction (PCR).
Microarray analysis
Microarray analyses were performed according to the manufacturer's protocol (Affymetrix, Santa Clara, CA). Seven-day-old Arabidopsis seedlings growing on MS medium without T-2 toxin were transferred to medium with or without 1 µM T-2 toxin, then grown for 3 weeks. Total RNAs were prepared from T-2 toxin-treated or untreated Arabidopsis shoots using a guanidine hydrochloride–phenol–chloroform extraction, as described in a previous study (Nishiuchi et al., 2006). Double-strand cDNAs were synthesized from total RNA using oligo(dT)24 primer containing 5'-T7 RNA polymerase promoter sequence and using SuperScript II (Invitrogen Corp., Carlsbad, CA). Biotinylated cRNAs were transcribed from synthesized cDNA using T7 RNA polymerase (ENZO Biochem Inc., New York). They were subsequently purified with the use of an RNeasy RNA purification Kit (Qiagen Inc.). Then, 15 µg cRNA of each cRNA sample was fragmented at 94 °C for 35 min in fragmentation buffer and then 300 µl of hybridization mixture was prepared with control cRNA. A portion (200 µl) of each mixture was subjected to hybridization with the Arabidopsis GeneChip array containing probe sets for approximately 8100 Arabidopsis genes for 16 h at 45 °C with rotation at 60 rpm. After hybridization, the arrays were washed with non-stringent wash buffer and then with stringent wash buffer. Subsequently, arrays were stained with streptavidin-phycoerythrin, biotinylated antibodies to streptavidin once, and streptavidin-phycoerythrin, after which each array was washed again with non-stringent buffer. The whole procedure of washing and staining was carried out using a GeneChip Fluidics Station 400 (Affymetrix). Each array was scanned using a GeneArray Scanner (HP and Affymetrix). Analyses of differential gene expression were performed using GeneChip software (Microarray Suite; Affymetrix). Three independent experiments were carried out using different plant samples to achieve good reproducibility of microarray analyses. Up-regulated genes were determined by a greater than three-fold difference in their AvDf values in a comparative analysis (control versus T-2 toxin treated) in all three experiments. Classifications of the functional category in T-2 toxin-induced genes were based on those of the MIPS (Munich Information Center for Protein Sequence).
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Comparative analysis of phytotoxic effects of trichothecene molecular species on the growth and morphology in Arabidopsis
To analyse the phytotoxic action of trichothecene molecular species in host plants systematically, seed germination, and the growth and morphology of Arabidopsis seedlings grown on agar media containing these compounds were investigated (Fig 1). Figure 2A showed that DON did not inhibit seed germination even at 10 µM after 3 d. One µM DAS failed to inhibit germination of seeds, whereas 10 µM DAS decreased the germination rate to approximately 40%. Similarly, 10 µM DAS apparently inhibited seed germination in tobacco (Muhitch et al., 2000). T-2 toxin also inhibited seed germination in a concentration-dependent way. Figure 2A also showed that complete inhibition of germination was observed in 10 µM CHX-containing media. Thus, the inhibition of germination of Arabidopsis seeds decreased in the following order: CHX>T-2 toxin>DAS>DON. Figure 2B revealed that shoot growth was inhibited by all trichothecenes examined in a concentration-dependent manner. Among these trichothecenes, severe inhibition of shoot growth was observed in T-2 toxin-treated plants at a low concentration. By contrast, 1 µM DON had only a minor effect on growth inhibition of Arabidopsis shoots. Growth inhibition of DAS-treated shoots was intermediate between that of T-2 toxin and DON (Fig. 2B). One µM CHX and 10 µM T-2 toxin completely blocked shoot development. Similarly, the inhibitory effects of trichothecenes on root growth also decreased in the same order. One µM DAS and 10 µM DON severely inhibited root growth, but not shoot growth (Fig. 2B, C), indicating that both DON and DAS preferentially inhibited root elongation, shown clearly in Fig. 2D. Such a feature was not observed in T-2 toxin-treated seedlings (Fig. 2B–D). Among trichothecenes examined, T-2 toxin most strongly inhibited shoot and root growth of Arabidopsis seedlings at low concentrations. Likewise, T-2 toxin significantly inhibited root and shoot growth in wheat seedlings (Wakulinski, 1989). The abaxial side of trichothecene-treated leaves was purple-red in colour (data not shown). In fact, anthocyanins accumulated in these shoots (data not shown). By contrast, chlorosis of leaves was observed in CHX-treated leaves.
|
On the other hand, it was previously reported that type A trichothecenes, including T-2 toxin and DAS, had an elicitor-like activity and caused necrotic lesions in Arabidopsis leaves infiltrated with them (Nishiuchi et al., 2006). In addition, necrotic lesions were also observed when Arabidopsis was grown on medium containing Fusarium phytotoxin fumonisin B1 (Stone et al., 2000). However, necrotic lesions were not observed in T-2 toxin-treated Arabidopsis, except for chlorosis of leaves caused by direct contact with media.
In contrast to an elicitor-like activity, the phytotoxic effects of DAS on the growth and morphology of Arabidopsis seedlings are similar to those of DON rather than those of T-2 toxin (Fig. 2B–D). Although the structure of DAS is similar to that of T-2 toxin, DAS and T-2 toxin are distinguished by the presence and absence, respectively, of an isovaleryl group at the C8 position. Phytotoxic effects of HT-2 toxin (with the isovaleryl group) are comparable with those of T-2 toxin, whereas T-2 tetraol (without the isovaleryl group) had only minor effects on growth inhibition at low concentrations (data not shown). It may be that the isovaleryl group at the C8 position affects not only phytotoxicity but also the mode of action of trichothecenes in host plants.
Preferential inhibition of root elongation by DON was commonly observed in Arabidopsis and wheat plants
As stated above, DON preferentially inhibited the root growth of Arabidopsis seedlings. In addition, Fig. 3A shows that the length of root hairs decreased markedly in roots of 10 µM DON-treated seedlings. In addition, abnormal morphology was observed in DON-treated roots. Such a morphological change was not observed in stunted roots of T-2 toxin-treated seedlings, although T-2 toxin also inhibited the elongation of roots and root hairs (data not shown). Microscopic analyses revealed, through cross-sections, that DON-treated roots were less organized than control roots (Fig. 3B). In Medicago truncatula, silencing of calcium-dependent protein kinase 1 (CDPK1) also resulted in short roots and root hairs (Ivashuta et al., 2005). Furthermore, microarray analysis revealed that most up-regulated genes in CDPK1-silenced roots were related to cell wall biosynthesis and/or defence response (such as glucanase and peroxidase), suggesting that induction of these genes affects root development. This report raised the possibility that a defence response also occurred in DON-treated Arabidopsis roots. Although DON activated the elicitor-like signalling pathway only at 100 µM, in leaves infiltrated by this trichothecene (Nishiuchi et al., 2006), apparent inhibition of root development by DON was observed at low concentrations (Figs 2C, D, 3A, B). It may be that sensitivity to DON differed between shoots and roots.
|
The phytotoxic effects of DON on growth were also investigated in DON-producing Fusarium-susceptible wheat plants. As shown in Fig. 3C, DON markedly inhibited root elongation of wheat plants at concentrations above 15 µM, indicating that preferential inhibition of root growth by DON was commonly observed in Arabidopsis and wheat. Such a preferential inhibition of root elongation was not observed in wheat seedlings treated with T-2 toxin (data not shown). It was reported that DON production by F. graminearum is important for disease spread in wheat spikes (Bai et al., 2002). In addition, DON production was also detected in Fusarium-infected Arabidopsis (Urban et al., 2002). Therefore, it may be that phytotoxic effects of DON on root growth were related to the pathogenicity of the fungi producing them.
T-2 toxin-treated Arabidopsis plants showed retarded growth with aberrant morphological change
Dwarfism and short petioles were observed in 12-d-old T-2 toxin-treated seedlings (Fig. 2D). As shown in Fig. 2A, T-2 toxin affected seed germination. To investigate the phytotoxic effects of T-2 toxin on growth and morphology in detail, 7-d-old Arabidopsis seedlings growing on MS medium without trichothecenes were transferred to MS medium containing T-2 toxin. Thereafter, morphological change was observed in 28-d-old plants growing on media containing T-2 toxin at concentrations above 0.5 µM. As shown in Fig. 4A, representatives of these plants exhibited short petioles, with curled and dark-green leaves. These characteristics are at least partially similar to those of BR-related mutants (Altmann, 1998). Similar morphological phenotypes were observed in HT-2 toxin-treated plants, but not in other trichothecene-treated plants, suggesting that the isovaleryl group at the C8 position affected trichothecene-induced morphological change in Arabidopsis. Figure 4B shows that the cell size of rosette leaves was visibly reduced in T-2 toxin-treated plants, suggesting that dwarfism of their plants is caused by inhibition of cell extension.
|
Analysis of global gene expression in dwarfed shoots by T-2 toxin
Although preferential inhibition of root growth by DON was commonly observed in Arabidopsis and wheat, it had previously been reported that defence-related compounds such as JA and coronatine phytotoxin often inhibited root growth in plants (Staswick et al., 1992; Feys et al., 1994). Therefore, in order to analyse the site of action of trichothecenes, the focus was on the T-2 toxin-induced morphological change.
Microarray analysis was performed with probe sets for approximately 8100 genes to obtain gene expression profiles in T-2 toxin-treated plants. To eliminate false positive as much as possible, T-2 toxin-inducible genes were designated as those with greater than 3-fold induction in all three experiments. Table 1 lists 35 genes that are up-regulated in T-2 toxin-treated shoots. Of these 35 genes, the largest category (six genes) was annotated for metabolism. Putative indole-3-acetic acid (IAA) UDP-glucosyltransferase (UGT74E2) was also included in this category (Li et al., 2001). Overexpression of another IAA glucosyltransferase (UGT84B1) caused growth inhibition of Arabidopsis shoots with an aberrant morphology (Jackson et al., 2002), suggesting that induction of UGT74E2 plays some role(s) in growth retardation by T-2 toxin. In addition, another UGT72E gene was also observed in this table. Poppenberger et al. (2003) reported that another UDP-glycosyltransferase (UGT73C5) gene specifically inactivated DON and 15-acetyl DON in vivo and in vitro. By contrast, expression of the UGT73C5 gene failed to detoxify the T-2 toxin in yeast (Poppenberger et al., 2003). On the other hand, the UGT73C5 gene inactivated BR (Poppenberger et al., 2005). In fact, transgenic plants overexpressing the UGT73C5 gene exhibited BR-deficient phenotypes and contained reduced amounts of BR (Poppenberger et al., 2005). RNA blotting analysis showed that the UGT73C5 gene is weakly up-regulated in T-2 toxin-treated plants (Fig. 5A). It may be that inactivation of BR by UGT73C5 expression was involved in T-2 toxin-induced growth defects to some extent.
|
|
This table also contains four genes that are putatively involved in transcriptional regulation. Each of the four genes belongs to a different class of transcription factor families (bZIP, Nac, HD-ZIP, WRKY family proteins). The WRKY transcription factors are known to play pivotal roles in plants' defence responses (Eulgem et al., 2000). Expression of the WRKY25 gene is induced by oxidative stresses from both H2O2 and paraquat treatment (Rizhsky et al., 2004). Since three peroxidase genes were also contained in this table, ROS generation might occur in the presence of trichothecene. Delessert et al. (2005) reported that both SA and MeJA application in Arabidopsis plants induced the ATAF2 gene. Overexpression of the ATB2 gene caused dwarfism of Arabidopsis plants (Wiese et al., 2005). Therefore, induction of the ATB2 gene may be involved in dwarfism of Arabidopsis by T-2 toxin. In addition, an AtNFXL1 gene encodes a member of an evolutionarily conserved family of putative transcription factors that includes human NF-X1, Drosophila melanogaster STC, and yeast FAP1 (Kunz et al., 2000; Lisso et al., 2006). These proteins contained one RING finger and 7–9 NF-X1 type zinc-finger motifs. Moreover, the human NF-X1 gene functioned as a transcriptional repressor in the regulation of the human major histocompatibility complex (MHC) class II gene (Song et al., 1994). Recently, the AtNFXL1 gene was shown to play a role in salt stress (Lisso et al., 2006). This novel zinc-finger protein may also regulate as a trichothecene-inducible defence response in Arabidopsis.
Table 1 also contains other defence-related genes such as the Aox1a and AtST1 genes. Expression of an alternative oxidase 1a (Aox1a) gene was induced by infection of both virulent and avirulent pathogens (Simons et al., 1999). In addition, the AtST1 gene was also reported as the RaR047 gene that was induced by SA treatment and pathogen infection (Lacomme and Roby, 1996; Rouleau et al., 1999). Since it has recently been reported that trichothecene has an elicitor-like activity when infiltrated into the leaves of Arabidopsis (Nishiuchi et al., 2006), trichothecene also activated the defence response in plants growing on medium containing it. The defence response by T-2 toxin might play some role(s) in inducing dwarfism because growth defects were frequently observed in constitutive defence response mutants such as cpr1 (Bowling et al., 1994). On the other hand, the AtST1 (RaR047) gene is homologous to the Brassica napus BR sulphotransferase 3 gene (BnST3), which is involved in inactivation of brassinosteroid biological activity (Rouleau et al., 1999). As stated above, trichothecene-treated shoots resemble BR-related mutants. Therefore, it is likely that induction of the AtST1 gene by T-2 toxin plays a role in growth defects. Moreover, because expression of the AtST1 genes was also induced by defence-related signals (Lacomme and Roby, 1996; Rouleau et al. 1999), they might be also involved in dwarfism of constitutive defence response mutants. The AtHB5 gene was down-regulated by application of BR in both wild types and the det2 mutant (Goda et al., 2002), whereas the ATHB5 gene was up-regulated in T-2 toxin-treated shoots (Table 1). This result also supports BR inactivation in T-2 toxin-treated shoots. However, expression of other BR-responsive genes in this array did not change significantly. On the other hand, exposure of plants to brassinolide and brassinazole, which is a specific inhibitor of BR biosynthesis, elicited opposite effects on gene expression of many BR-regulated genes (Goda et al., 2002). It may be that a pleiotropic action of T-2 toxin affects the expression of these genes. Although this array contained DWF1, DWF7, DET2, DWF4, and CPD genes that are involved in BR biosynthesis, expression of these genes did not change markedly following T-2 toxin treatments (data not shown). Nakashita et al. (2003) reported that BR played an important role in a broad range of disease resistance in tobacco and rice. Therefore, inactivation of BR might affect the pathogenicity in the infection of host plants by trichothecene-producing fungi.
Table 1 also includes many stress-responsive genes such as pEARL1 1 and pEARL1 1-like genes encoding protease inhibitor/seed storage/lipid transfer protein (LTP) family proteins. Moreover, other stress-responsive genes such as Di21 and HSP70 genes also contained T-2 toxin-inducible genes. The pEARL 1 gene is reported as an early Arabidopsis aluminium-induced gene (Richards et al., 1998). Since aluminium induced the expression of oxidative stress-responsive genes (Richards et al., 1998), T-2 toxin might cause oxidative stress in Arabidopsis. Correspondingly, it was reported that ROS production was observed in T-2 toxin-injected Arabidopsis leaves (Nishiuchi et al., 2006).
Down-regulated genes in T-2 toxin-treated shoots were also identified (data not shown). By contrast with up-regulated genes, many down-regulated genes were categorized into unclassified proteins or unknown proteins. In fact, few genes were related to phenotypes of the T-2 toxin-treated shoots.
These identified genes by microarray analysis were constantly expressed in T-2 toxin-treated shoots
To confirm whether these identified genes are actually up-regulated in T-2 toxin-treated plants, RNA blotting analyses were performed. Figure 5A shows that all four genes were up-regulated in T-2 toxin-treated shoots throughout the experimental period. The levels of mRNAs for these genes increased markedly after 1 d; they fell within 3 d, but remained at higher levels throughout the experimental period. Thus, all four genes examined were constantly expressed in T-2 toxin-treated shoots.
As stated above, AtST1 expression was induced by SA treatment (Rouleau et al., 1999). It was investigated whether or not the T-2 toxin-inducible expression of the AtST1 gene is regulated by an SA-dependent signalling pathway. It is well known that expression of the PR-1 gene is controlled by the SA signalling pathway (Rogers and Ausubel, 1997). Figure 5B shows that the mRNA level for PR-1 increased only slightly within 1 d in the T-2 toxin-treated wild type, peaked after 7 d, then decreased after 21 d. Induction of the PR-1 gene was not observed in SA-depleted NahG transgenic plants, implying that the SA-dependent signalling pathway is transiently activated by the application of T-2 toxin. It is likely that T-2 toxin transiently induces the accumulation of SA. By contrast, T-2 toxin-induced expression of the AtST1 gene, which was also observed in the NahG plants, suggesting that the SA-independent signalling pathway regulates T-2 toxin-inducible expression of the AtST1 gene.
Comparative analysis revealed that phytotoxic effects of trichothecene on growth and morphology differed significantly among their molecular species in Arabidopsis (Fig. 2A–D). These results imply that different trichothecene molecular species not only affect the phytotoxic potency but also the site of action in host plants. Among these trichothecenes, T-2 toxin-treated seedlings have several interesting features such as dwarfed shoots, petiole shortening, and curled dark-green leaves. Microarray analysis of T-2 toxin-treated dwarfed shoots suggested that T-2 toxin caused defence response, BR inactivation, and ROS generation. In addition, T-2 toxin-inducible genes contained the five different classes of transcription factor genes. Since some of these genes were involved in stress response and growth control (Wiese et al., 2004; Delessert et al., 2005; Lisso et al., 2006), the genes identified here might contain important regulators in the action of trichothecenes in host plants. In fact, the artificial modulation of expression of some of these genes significantly affected the sensitivity to trichothecenes in Arabidopsis plants (T Asano et al., unpublished results). Further analysis of function of the genes identified may help to clarify the molecular mechanism of the phytotoxic action of trichothecenes in host plants.
|
Acknowledgements |
|---|
We thank Dr Leslie Friedrich (Syngenta-Biotechnology) for Arabidopsis Col-0 NahG transgenic seeds.
|
Footnotes |
|---|
* Present address: Presidential Office, Agricultural Chemicals Inspection Station (ACIS), 2–772 Suzuki-cho, Kodaira, Tokyo 187-0011, Japan.
|
Abbreviations |
|---|
BR, brassinosteroids; CHX, cycloheximide; DAS, diacetoxyscirpenol; DON, deoxynivalenol; ROS, reactive oxygen species; UGT, UDP-glucosyltransferase.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Altmann T. A tale of dwarfs and drugs: brassinosteroids to the rescue. Trends in Genetics (1998) 14:490–495.[CrossRef][ISI][Medline]
Bai GH, Desjardins AE, Plattner RD. Deoxynivalenol-nonproducing Fusarium graminearum causes initial infection, but does not cause disease spread in wheat spikes. Mycopathologia (2002) 153:91–98.[CrossRef][ISI][Medline]
Bowling SA, Guo A, Cao H, Gordon AS, Klessig DF, Dong X. A mutation in Arabidopsis that leads to constitutive expression of systemic acquired resistance. The Plant Cell (1994) 6:1845–1857.
Cossette F, Miller JD. Phytotoxic effect of deoxynivalenol and gibberella ear rot resistance of corn. Natural Toxins (1995) 3:383–388.[CrossRef][Medline]
Delessert C, Kazan K, Wilson IW, Van Der Straeten D, Manners J, Dennis ES, Dolferus R. The transcription factor ATAF2 represses the expression of pathogenesis-related genes in Arabidopsis. The Plant Journal (2005) 43:745–757.[CrossRef][ISI][Medline]
Desjardins AE, Hohn TM, McCormick SP. Trichothecene biosynthesis in Fusarium species: chemistry, genetics, and significance. Microbiology Review (1993) 57:595–604.
Eulgem T, Rushton PJ, Robatzek S, Somssich IE. The WRKY superfamily of plant transcription factors. Trends in Plant Science (2000) 5:199–206.[CrossRef][ISI][Medline]
Feys B, Benedetti CE, Penfold CN, Turner JG. Arabidopsis mutants selected for resistance to the phytotoxin coronatine are male sterile, insensitive to methyl jasmonate, and resistant to a bacterial pathogen. The Plant Cell (1994) 6:751–759.
Goda H, Shimada Y, Asami T, Fujioka S, Yoshida S. Microarray analysis of brassinosteroid-regulated genes in Arabidopsis. Plant Physiology (2002) 130:1319–1334.
Ivashuta S, Liu J, Liu J, Lohar DP, Haridas S, Bucciarelli B, VandenBosch KA, Vance CP, Harrison MJ, Gantt JS. RNA interference identifies a calcium-dependent protein kinase involved in Medicago truncatula root development. The Plant Cell (2005) 17:2911–2921.
Jackson RG, Kowalczyk M, Li Y, Higgins G, Ross J, Sandberg G, Bowles DJ. Over-expression of an Arabidopsis gene encoding a glucosyltransferase of indole-3-acetic acid: phenotypic characterization of transgenic lines. The Plant Journal (2002) 32:573–583.[CrossRef][ISI][Medline]
Kimura M, Takahashi-Ando N, Nishiuchi T, Ohsato S, Tokai T, Ochiai N, Fujimura M, Kudo T, Hamamoto H, Yamaguchi I. Molecular biology and biotechnology for reduction of Fusarium mycotoxin contamination. Pesticide Biochemistry and Physiology (2006) 86:117–123.[CrossRef][ISI]
Kunz J, Loeschmann A, Deuter-Reinhard M, Hall MN. FAP1, a homologue of human transcription factor NF-X1, competes with rapamycin for binding to FKBP12 in yeast. Molecular Microbiology (2000) 37:1480–1493.[CrossRef][ISI][Medline]
Lacomme C, Roby D. Molecular cloning of a sulfotransferase in Arabidopsis thaliana and regulation during development and in response to infection with pathogenic bacteria. Plant Molecular Biology (1996) 30:995–1008.[CrossRef][ISI][Medline]
Lawton KA, Friedrich L, Hunt M, Weymann K, Delaney T, Kessmann H, Staub T, Ryals J. Benzothiadiazole induces disease resistance in Arabidopsis by activation of the systemic acquired resistance signal transduction pathway. The Plant Journal (1996) 10:71–82.[CrossRef][ISI][Medline]
Li Y, Baldauf S, Lim EK, Bowles DJ. Phylogenetic analysis of the UDP-glycosyltransferase multigene family of Arabidopsis thaliana. Journal of Biological Chemistry (2001) 276:4338–4343.
Lisso J, Altmann T, Mussig C. The AtNFXL1 gene encodes a NF-X1 type zinc finger protein required for growth under salt stress. FEBS Letters (2006) 580:4851–4856.[CrossRef][ISI][Medline]
McLaughlin CS, Vaughan MH, Campbell IM, Wei CM, Stafford ME, Hansen BS. Inhibition of protein synthesis by trichothecenes. In: Mycotoxins in human and animal health—Rodericks JV, Hesseltine CW, Mehlman MA, eds. (1977) Park Forest Sound, IL: Pathotox Publishers. 263–273.
Muhitch MJ, McCormick SP, Alexander NJ, Hohn TM. Transgenic expression of the TRI101 or PDR5 gene increases resistance of tobacco to the phytotoxic effects of the trichothecene 4,15-diacetoxyscirpenol. Plant Science (2000) 157:201–207.[Medline]
Nakashita H, Yasuda M, Nitta T, Asami T, Fujioka S, Arai Y, Sekimata K, Takatsuto S, Yamaguchi I, Yoshida S. Brassinosteroid functions in a broad range of disease resistance in tobacco and rice. The Plant Journal (2003) 33:887–898.[CrossRef][ISI][Medline]
Nishiuchi T, Masuda D, Nakashita H, Ichimura K, Shinozaki K, Yoshida S, Kimura M, Yamaguchi I, Yamaguchi K. Fusarium phytotoxin trichothecenes have an elicitor-like activity in Arabidopsis thaliana, but its activity differed significantly among their molecular species. Molecular Plant–Microbe Interaction (2006) 19:512–520.
Ohsato S, Ochiai-Fukuda T, Nishiuchi T, Takahashi-Ando N, Koizumi S, Hamamoto H, Kudo T, Shinozaki K, Yamaguchi I, Kimura M. Transgenic inactivation of the Fusarium mycotoxin deoxynivalenol in rice plants as a model of cereal crops. Plant Cell Reports (2001) (in press).
Packa D. Cytogenetic changes in plant cells as influenced by mycotoxins. Mycotoxin Research (1991) 7:150–155.
Poppenberger B, Berthiller F, Lucyshyn D, Sieberer T, Schuhmacher R, Krska R, Kuchler K, Glossl J, Luschnig C, Adam G. Detoxification of the Fusarium mycotoxin deoxynivalenol by a UDP-glucosyltransferase from Arabidopsis thaliana. Journal of Biological Chemistry (2003) 278:47905–47914.
Poppenberger B, Fujioka S, Soeno K, et al. The UGT73C5 of Arabidopsis thaliana glucosylates brassinosteroids. Proceedings of the National Academy of Sciences, USA (2005) 102:15253–15258.
Proctor RH, Hohn TM, McCormick SP. Reduced virulence of Gibberella zeae caused by disruption of a trichothecene toxin biosynthetic gene. Molecular Plant–Microbe Interaction (1995) 8:593–601.
Richards KD, Schott EJ, Sharma YK, Davis KR, Gardner RC. Aluminum induces oxidative stress genes in Arabidopsis thaliana. Plant Physiology (1998) 116:409–418.
Rizhsky L, Davletova S, Liang H, Mittler R. The zinc finger protein Zat12 is required for cytosolic ascorbate peroxidase expression during oxidative stress in Arabidopsis. Journal of Biological Chemistry (2004) 279:11736–11743.
Rogers EE, Ausubel FM. Arabidopsis enhanced disease susceptibility mutants exhibit enhanced susceptibility to several bacterial pathogens and alterations in PR-1 gene expression. The Plant Cell (1997) 9:305–316.[Abstract]
Rouleau M, Marsolais F, Richard M, Nicolle L, Voigt B, Adam G, Varin L. Inactivation of brassinosteroid biological activity by a salicylate-inducible steroid sulfotransferase from Brassica napus. Journal of Biological Chemistry (1999) 274:20925–20930.
Ruzin SE. Tissue clearing. In: In: Plant microtechnique and microscopy (1999) Oxford: Oxford University Press. 127–136.
Simons BH, Millenaar FF, Mulder L, van Loon LC, Lambers H. Enhanced expression and activation of the alternative oxidase during infection of Arabidopsis with Pseudomonas syringae pv. tomato. Plant Physiology (1999) 120:529–538.
Song Z, Krishna S, Thanos D, Strominger JL, Ono SJ. A novel cysteine-rich sequence-specific DNA-binding protein interacts with the conserved X-box motif of the human major histocompatibility complex class II genes via a repeated Cys-His domain and functions as a transcriptional repressor. Journal of Experimental Medicine (1994) 180:1763–1774.
Staswick PE, Su W, Howell SH. Methyl jasmonate inhibition of root growth and induction of a leaf protein are decreased in an Arabidopsis thaliana mutant. Proceedings of the National Academy of Sciences, USA (1992) 89:6837–6840.
Stone JM, Heard JE, Asai T, Ausubel FM. Simulation of fungal-mediated cell death by fumonisin B1 and selection of fumonisin B1-resistant (fbr) Arabidopsis mutants. The Plant Cell (2000) 12:1811–1822.
Sudakin DL. Trichothecenes in the environment: relevance to human health. Toxicology Letters (2003) 143:97–107.[CrossRef][ISI][Medline]
Tsuge T, Tsukaya H, Uchimiya H. Two independent and polarized processes of cell elongation regulate leaf blade expansion in Arabidopsis thaliana (L.) Heynh. Development (1996) 122:1589–1600.[Abstract]
Urban M, Daniels S, Mott E, Hammond-Kosack K. Arabidopsis is susceptible to the cereal ear blight fungal pathogens Fusarium graminearum and Fusarium culmorum. The Plant Journal (2002) 32:961–973.[CrossRef][ISI][Medline]
Wakulinski W. Phytotoxicity of the secondary metabolites of fungi causing wheat head fusariosis (head blight). Acta Physiologia Plantarum (1989) 11:301–306.[ISI]
Ward TJ, Bielawski JP, Kistler HC, Sullivan E, O'Donnell K. Ancestral polymorphism and adaptive evolution in the trichothecene mycotoxin gene cluster of phytopathogenic Fusarium. Proceedings of the National Academy of Sciences, USA (2002) 99:9278–9283.
Wiese A, Elzinga N, Wobbes B, Smeekens S. A conserved upstream open reading frame mediates sucrose-induced repression of translation. The Plant Cell (2004) 16:1717–1729.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||