A gain-of-function mutation in the Arabidopsis pleiotropic drug resistance transporter PDR9 confers resistance to auxinic herbicides

Arabidopsis (Arabidopsis thaliana) contains 15 genes encoding members of the pleiotropic drug resistance (PDR) family of ATP-binding cassette transporters. These proteins have been speculated to be involved in the detoxification of xenobiotics, however, little experimental support of this hypothesis has been obtained to date. Here we report our characterization of the Arabidopsis PDR9 gene. We isolated a semidominant, gain-of-function mutant, designated pdr9-1, that exhibits increased tolerance to the auxinic herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). Reciprocally, loss-of-function mutations in PDR9 confer 2,4-D hypersensitivity. This altered auxin sensitivity defect of pdr9 mutants is specific for 2,4-D and closely related compounds as these mutants respond normally to the endogenous auxins indole-3-acetic acid and indole-butyric acid. We demonstrate that 2,4-D, but not indole-3-acetic acid transport is affected by mutations in pdr9, suggesting that the PDR9 transporter specifically effluxes 2,4-D out of plant cells without affecting endogenous auxin transport. The semidominant pdr9-1 mutation affects an extremely highly conserved domain present in all known plant PDR transporters. The single amino acid change results in increased PDR9 abundance and provides a novel approach for elucidating the function of plant PDR proteins.     

PDR16-mediated azole resistance in Candida albicans

Many Candida albicans azole-resistant (AR) clinical isolates overexpress the CDR1 and CDR2 genes encoding homologous multidrug transporters of the ATP-binding cassette family. We show here that these strains also overexpress the PDR16 gene, the orthologue of Saccharomyces cerevisiae PDR16 encoding a phosphatidylinositol transfer protein of the Sec14p family. It has been reported that S. cerevisiae pdr16Delta mutants are hypersusceptible to azoles, suggesting that C. albicans PDR16 may contribute to azole resistance in these isolates. To address this question, we deleted both alleles of PDR16 in an AR clinical strain overexpressing the three genes, using the mycophenolic acid resistance flipper strategy. Our results show that the homozygous pdr16Delta/pdr16Delta mutant is approximately twofold less resistant to azoles than the parental strain whereas reintroducing a copy of PDR16 in the mutant restored azole resistance, demonstrating that this gene contributes to the AR phenotype of the cells. In addition, overexpression of PDR16 in azole-susceptible (AS) C. albicans and S. cerevisiae strains increased azole resistance by about twofold, indicating that an increased dosage of Pdr16p can confer low levels of azole resistance in the absence of additional molecular alterations. Taken together, these results demonstrate that PDR16 plays a role in C. albicans azole resistance.     

Bioprospecting for trichothecene 3-O-acetyltransferases in the fungal genus Fusarium yields functional enzymes with different abilities to modify the mycotoxin deoxynivalenol

The trichothecene mycotoxin deoxynivalenol (DON) is a common contaminant of small grains, such as wheat and barley, in the United States. New strategies to mitigate the threat of DON need to be developed and implemented. TRI101 and TRI201 are trichothecene 3-O-acetyltransferases that are able to modify DON and reduce its toxicity. Recent work has highlighted differences in the activities of TRI101 from two different species of Fusarium (F. graminearum and F. sporotrichioides), but little is known about the relative activities of TRI101/TRI201 enzymes produced by other species of Fusarium. We cloned TRI101 or TRI201 genes from seven different species of Fusarium and found genetic identity between sequences ranging from 66% to 98%. In vitro feeding studies using transformed yeast showed that all of the TRI101/TRI201 enzymes tested were able to acetylate DON; conversion of DON to 3-acetyl-deoxynivalenol (3ADON) ranged from 50.5% to 100.0%, depending on the Fusarium species from which the gene originated. A time course assay showed that the rate of acetylation varied from species to species, with the gene from F. sporotrichioides having the lowest rate. Steady-state kinetic assays using seven purified enzymes produced catalytic efficiencies for DON acetylation ranging from 6.8 × 10(4) M(-1)·s(-1) to 4.7 × 10(6) M(-1)·s(-1). Thermostability measurements for the seven orthologs ranged from 37.1°C to 43.2°C. Extended sequence analysis of portions of TRI101/TRI201 from 31 species of Fusarium (including known trichothecene producers and nonproducers) suggested that other members of the genus may contain functional TRI101/TRI201 genes, some with the potential to outperform those evaluated in the present study.     

Tri13 and Tri7 determine deoxynivalenol- and nivalenol-producing chemotypes of Gibberella zeae

Gibberella zeae, a major cause of cereal scab, can be divided into two chemotypes based on production of the 8-ketotrichothecenes deoxynivalenol (DON) and nivalenol (NIV). We cloned and sequenced a Tri13 homolog from each chemotype. The Tri13 from a NIV chemotype strain (88-1) is located in the trichothecene gene cluster and carries an open reading frame similar to that of Fusarium sporotrichioides, whereas the Tri13 from a DON chemotype strain (H-11) carries several mutations. To confirm the roles of the Tri13 and Tri7 genes in trichothecene production by G. zeae, we genetically altered toxin production in 88-1 and H-11. In transgenic strains, the targeted deletion of Tri13 from the genome of 88-1 caused production of DON rather than NIV. Heterologous expression of the 88-1 Tri13 gene alone or in combination with the 88-1 Tri7 gene conferred on H-11 the ability to synthesize NIV; in the latter case, 4-acetylnivalenol (4-ANIV) also was produced. These results suggest that Tri13 and Tri7 are required for oxygenation and acetylation of the oxygen at C-4 during synthesis of NIV and 4-ANIV in G. zeae. These functional analyses of the Tri13 and Tri7 genes provide the first clear evidence for the genetic basis of the DON and NIV chemotypes in G. zeae.     

Variation in 8-ketotrichothecenes and zearalenone production by Fusarium graminearum isolates from corn and barley in Korea

A total of 214 Fusarium graminearum isolates were obtained from corn and barley which were collected from Kangwon province and the southern part of Korea, respectively, and were tested for 8-ketotrichothecenes and zearalenone (ZEA) production on rice grains. The incidences of trichothecene production by 105 isolates of F. graminearum from corn were 59.0% for deoxynivalenol (DON), 37.1% for 15-acetyldeoxynivalenol(15-ADON), 13.3% for 3-acetyldeoxynivalenol (3-ADON), 7.6% for 3,15-diacetyldeoxynivalenol (3,15-DADON), 20.0% for nivalenol (NIV), 6.7% for 4-acetylnivalenol (4-ANIV), and 1.0% for 4,15-diacetylnivalenol (4,15-DANIV). DON chemotypes frequently produced 15-ADON as the major isomer rather than 3-ADON and 9 of the 61 DON chemotypes produced low levels of NIV. On the other hand, the incidences of trichothecene production of 109 isolates by F. graminearum from barley were 24.8% for DON, 72.5% for NIV, 62.4% for 4-ANIV, and 10.1% for 4,15-DANIV. Of these isolates, 78 were NIV chemotypes and only one isolate produced DON and 3-ADON as major toxins. In addition, 26 of the 78 NIV chemotypes produced low levels of DON. ZEA was frequently produced by the trichothecene-producing isolates and the incidences of ZEA were 51.4% and 31.2% for the isolates from corn and barley, respectively. There was a great regional difference in trichothecene production by F. graminearum isolates between corn- and barley-producing areas in Korea.     

A genetic and biochemical approach to study trichothecene diversity in Fusarium sporotrichioides and Fusarium graminearum

The trichothecenes T-2 toxin and deoxynivalenol (DON) are natural fungal products that are toxic to both animals and plants. Their importance in the pathogenicity of Fusarium spp. on crop plants has inspired efforts to understand the genetic and biochemical mechanisms leading to trichothecene synthesis. In order to better understand T-2 toxin biosynthesis by Fusarium sporotrichioides and DON biosynthesis by F. graminearum, we compared the nucleotide sequence of the 23-kb core trichothecene gene cluster from each organism. This comparative genetic analysis allowed us to predict proteins encoded by two trichothecene genes, TRI9 and TRI10, that had not previously been described from either Fusarium species. Differences in gene structure also were correlated with differences in the types of trichothecenes that the two species produce. Gene disruption experiments showed that F. sporotrichioides TRI7 (FsTRI7) is required for acetylation of the oxygen on C-4 of T-2 toxin. Sequence analysis indicated that F. graminearum TRI7 (FgTRI7) is nonfunctional. This is consistent with the fact that the FgTRI7 product is not required for DON synthesis in F. graminearum because C-4 is not oxygenated.     

Effects of different carbon sources on trichothecene production and Tri gene expression by Fusarium graminearum in liquid culture

Fusarium head blight caused by Fusarium graminearum is a disease of cereal crops that not only reduces crop yield and quality but also results in contamination with trichothecenes such as nivalenol and deoxynivalenol (DON). To analyze the trichothecene induction mechanism, effects of 12 carbon sources on the production of DON and 3-acetyldexynivalenol (3ADON) were examined in liquid cultures incubated with nine strains of 3ADON-producing F. graminearum. Significantly high levels of trichothecene (DON and 3ADON) production by sucrose, 1-kestose and nystose were commonly observed among all of the strains tested. On the other hand, the levels of trichothecene biosynthesis induced by the other carbon sources were strain-specific. Tri4 and Tri5 expressions were up-regulated in the sucrose-containing medium but not in glucose. Trichothecene accumulation in the sucrose-containing medium was not repressed by the addition of glucose, indicating that trichothecene production was not regulated by carbon catabolite repression. These findings suggest that F. graminearum recognizes sucrose molecules, activates Tri gene expression and induces trichothecene biosynthesis.     

Fusarium phytotoxin trichothecenes have an elicitor-like activity in Arabidopsis thaliana, but the activity differed significantly among their molecular species

Phytopathogenic fungi such as Fusarium spp. synthesize trichothecene family phytotoxins. Although the type B trichothecene, deoxynivalenol (DON), is thought to be a virulence factor allowing infection of plants by their trichothecene-producing Fusarium spp., little is known about effects of trichothecenes on the defense response in host plants. Therefore, in this article, we investigated these effects of various trichothecenes in Fusarium-susceptible Arabidopsis thaliana. Necrotic lesions were observed in Arabidopsis leaves infiltrated by 1 microM type A trichothecenes such as T-2 toxin. Trichothecene-induced lesions exhibited dead cells, callose deposition, generation of hydrogen peroxide, and accumulation of salicylic acids. Moreover, infiltration by trichothecenes caused rapid and prolonged activation of two mitogen-activated protein kinases and induced expression of both PR-1 and PDF1.2 genes. Thus, type A trichothecenes trigger the cell death by activation of an elicitor-like signaling pathway in Arabidopsis. Although DON did not have such an activity even at 10 microM, translational inhibition by DON was observed at concentrations above 5 microM. These results suggested that DON is capable of inhibiting translation in Arabidopsis cells without induction of the elicitor-like signaling pathway.     

Molecular mechanisms of deoxynivalenol resistance in the yeast<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The production of trichothecene toxins is a suspected virulence mechanism of several plant pathogenic fungi. This hypothesis has been confirmed forGibberella zeae (Fusarium graminearum) by gene disruption experiments, suggesting in turn, that resistance against the fungal toxin is a relevant component ofFusarium resistance of the host plant. Our goal is therefore to identify molecular mechanisms of trichothecene resistance. Using yeast as a model system we have found the following resistance mechanisms and genes: a) reduced uptake of deoxynivalenol (PDR5), b) toxin modification and reduction of toxicity (AYT1), and c) formation of a resistant toxin target (RPL3). Homologous plant genes exist and are attractive candidates forFusarium resistance genes.