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.     

玉蜀黍赤霉(Gibberella zeae)对多菌灵的抗药性遗传研究

根据在 0 5、1 4、5 0、10 0 μg ml等不同浓度的含药PSA平板上能否生长 ,将玉蜀黍赤霉田间菌株对多菌灵的敏感性划分为 :敏感 (S)、中抗 (MR)和高抗 (HR)等 3个水平 ,其中S菌株在 0 5 μg ml浓度下能生长 ,但在≥ 1 4μg ml浓度下生长受到完全抑制 ;R菌株在 1 4 μg ml浓度下能快速生长 ,在 5 0 μg ml浓度下能缓慢生长 ,但在≥ 10 0μg ml浓度下不能生长 ;HR在≥ 10 0 μg ml浓度下仍能生长。没有发现在 1 4 μg ml浓度下能快速生长 ,而在 5 0 μg ml浓度下能被完全抑制的田间抗性菌株。从 2 5个敏感菌株和 31个抗性菌株中随机挑选了 2个S、3个MR和 1个HR ,并以硝酸盐营养缺陷型突变体 (nit)作为另一个遗传标记 ,按照S×S、MR×MR、MR×S、HR×S、HR×MR等共设计了 7个杂交组合 ,对各杂交后代对多菌灵的敏感性测试发现 ,在所有杂交后代中均未出现除双亲表现型以外的重组型个体 ,MR×S、HR×S及HR×MR的杂交后代出现了 1∶1的分离比例。以上结果表明玉蜀黍赤霉田间菌株对多菌灵的抗药性是由单个孟德尔基因控制的 ,该基因发生不同点突变或同一点的不同等位基因发生突变可导致不同的抗性水平 ,抗药性不受修饰基因或胞质遗传因子的影响。     

Alignment of genetic and physical maps of Gibberella zeae

We previously published a genetic map of Gibberella zeae (Fusarium graminearum sensu lato) based on a cross between Kansas strain Z-3639 (lineage 7) and Japanese strain R-5470 (lineage 6). In this study, that genetic map was aligned with the third assembly of the genomic sequence of G. zeae strain PH-1 (lineage 7) using seven structural genes and 108 sequenced amplified fragment length polymorphism markers. Several linkage groups were combined based on the alignments, the nine original linkage groups were reduced to six groups, and the total size of the genetic map was reduced from 1,286 to 1,140 centimorgans. Nine supercontigs, comprising 99.2% of the genomic sequence assembly, were anchored to the genetic map. Eight markers (four markers from each parent) were not found in the genome assembly, and four of these markers were closely linked, suggesting that >150 kb of DNA sequence is missing from the PH-1 genome assembly. The alignments of the linkage groups and supercontigs yielded four independent sets, which is consistent with the four chromosomes reported for this fungus. Two proposed heterozygous inversions were confirmed by the alignments; otherwise, the colinearity of the genetic and physical maps was high. Two of four regions with segregation distortion were explained by the two selectable markers employed in making the cross. The average recombination rates for each chromosome were similar to those previously reported for G. zeae. Despite an inferred history of genetic isolation of lineage 6 and lineage 7, the chromosomes of these lineages remain homologous and are capable of recombination along their entire lengths, even within the inversions. This genetic map can now be used in conjunction with the physical sequence to study phenotypes (e.g., fertility and fitness) and genetic features (e.g., centromeres and recombination frequency) that do not have a known molecular signature in the genome.     

Occurrence of Gibberella zeae strains that produce both nivalenol and deoxynivalenol.

By single ascospore isolation, several sets of asci containing eight ascospores were isolated from perithecia of Gibberella zeae. Of these sets, seven were investigated for their ability to produce 8-ketotrichothecene mycotoxins on rice grains. Analyses were made with gas chromatography-mass spectrometry and gas chromatography with 63Ni electron capture detection. Of 56 total isolates, 11 produced nivalenol, 4-acetylnivalenol, and deoxynivalenol, 1 produced nivalenol and deoxynivalenol, 7 produced deoxynivalenol and 3-acetyldeoxynivalenol, 19 produced deoxynivalenol and 15-acetyldeoxynivalenol, and 6 produced deoxynivalenol and both 15- and 3-acetyldeoxynivalenol. The remaining 12 isolates produced nivalenol and 4-acetylnivalenol. All isolates of G. zeae that we examined could produce 8-ketotrichothecenes in this investigation. This report is the first to demonstrate the presence of G. zeae isolates producing both nivalenol and deoxynivalenol. In addition, differences in the production between 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol are discussed in relation to culture conditions.     

Gibberella zeae ascospore production and collection for microarray experiments

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain quality. F. graminearum also causes stalk and ear rots of maize and is a producer of mycotoxins such as the trichothecenes that contaminate grain and are harmful to humans and livestock (Goswami and Kistler, 2004). The fungus produces two types of spores. Ascospores, the propagules resulting from sexual reproduction, are the main source of primary infection. These spores are forcibly discharged from mature perithecia and dispersed by wind (Francl et al 1999). Secondary infections are mainly caused by macroconidia which are produced by asexual means on the plant surface. To study the developmental processes of ascospores in this fungus, a procedure for their collection in large quantity under sterile conditions was required. Our protocol was filmed in order to generate the highest level of information for understanding and reproducibility; crucial aspects when full genome gene expression profiles are generated and interpreted. In particular, the variability of ascospore germination and biological activity are dependent on the prior manipulation of the material. The use of video for documenting every step in ascospore production is proposed in order to increase standardization, complying with the increasingly stringent requirements for microarray analysis. The procedure requires only standard laboratory equipment. Steps are shown to prevent contamination and favor time synchronization of ascospores.     

Fusarenon X and nivalenol production by Gibberella zeae in liquid and rice cultures

Toxigenesis of Gibberella zeae strains NHL-F-1372 and NHL-F-1373 was compared in liquid and rice culture. Growth of both strains in glucose-yeast extract-peptone medium (GYEP) for 25 days resulted in peak levels of fusarenon X (FX) ranging from 40–200 mg/L with lower levels of nivalenol (NIV) (10 mg/L). Growth of these strains in modified Fries medium amended with 4% corn steep liquor (CSL) resulted in a much lower total 8-ketotrichothecene yield than in GYEP, with NIV being the primary compound produced. Although FX appeared initially in this latter medium, the toxin disappeared concurrently as the pH exceeded 8.0. Growth rates and total mycelial accumulation were lower in GYEP cultures than in the modified Fries with CSL cultures. Appearance of FX and NIV in modified Fries medium with CSL paralleled the order of appearance of these compounds in rice, but the total trichothecene yield in rice was much higher. In general, growth and toxigenesis by the nivalenol-producing fusaria in liquid and rice cultures was qualitatively similar to that previously found for deoxynivalenol-producing isolates.Michigan State University Agricultural Experiment Station Article No. 11700.     

Chemotaxonomy of Gibberella zeae with special reference to production of trichothecenes and zearalenone.

By adopting a single-spore isolation technique, 113 isolates of Gibberella zeae, the perfect stage of Fusarium graminearum, were isolated from rice stubbles in barley and wheat fields and tested for production of trichothecenes and zearalenone on rice grains. Of the isolates, 93% produced the trichothecenes, and they could be subdivided into two chemotaxonomic groups: nivalenol and fusarenon-X producers and deoxynivalenol and 3-acetyldeoxynivalenol producers. No cross production of these two types of trichothecenes was observed in these isolates. Zearalenone was detected in 68% of the isolates, but no clear relationship could be observed regarding its position with respect to the two chemotaxonomic groups.     

Occurrence of Gibberella zeae strains that produce both nivalenol and deoxynivalenol

By single ascospore isolation, several sets of asci containing eight ascospores were isolated from perithecia of Gibberella zeae. Of these sets, seven were investigated for their ability to produce 8-ketotrichothecene mycotoxins on rice grains. Analyses were made with gas chromatography-mass spectrometry and gas chromatography with 63Ni electron capture detection. Of 56 total isolates, 11 produced nivalenol, 4-acetylnivalenol, and deoxynivalenol, 1 produced nivalenol and deoxynivalenol, 7 produced deoxynivalenol and 3-acetyldeoxynivalenol, 19 produced deoxynivalenol and 15-acetyldeoxynivalenol, and 6 produced deoxynivalenol and both 15- and 3-acetyldeoxynivalenol. The remaining 12 isolates produced nivalenol and 4-acetylnivalenol. All isolates of G. zeae that we examined could produce 8-ketotrichothecenes in this investigation. This report is the first to demonstrate the presence of G. zeae isolates producing both nivalenol and deoxynivalenol. In addition, differences in the production between 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol are discussed in relation to culture conditions.     

A genetic map of Gibberella zeae (Fusarium graminearum)

We constructed a genetic linkage map of Gibberella zeae (Fusarium graminearum) by crossing complementary nitrate-nonutilizing (nit) mutants of G. zeae strains R-5470 (from Japan) and Z-3639 (from Kansas). We selected 99 nitrate-utilizing (recombinant) progeny and analyzed them for amplified fragment length polymorphisms (AFLPs). We used 34 pairs of two-base selective AFLP primers and identified 1048 polymorphic markers that mapped to 468 unique loci on nine linkage groups. The total map length is approximately 1300 cM with an average interval of 2.8 map units between loci. Three of the nine linkage groups contain regions in which there are high levels of segregation distortion. Selection for the nitrate-utilizing recombinant progeny can explain two of the three skewed regions. Two linkage groups have recombination patterns that are consistent with the presence of intercalary inversions. Loci governing trichothecene toxin amount and type (deoxynivalenol or nivalenol) map on linkage groups IV and I, respectively. The locus governing the type of trichothecene produced (nivalenol or deoxynivalenol) cosegregated with the TRI5 gene (which encodes trichodiene synthase) and probably maps in the trichothecene gene cluster. This linkage map will be useful in population genetic studies, in map-based cloning, for QTL (quantitative trait loci) analysis, for ordering genomic libraries, and for genomic comparisons of related species.     

Functional analyses of regulators of G protein signaling in Gibberella zeae

Regulators of G protein signaling (RGS) proteins make up a highly diverse and multifunctional protein family that plays a critical role in controlling heterotrimeric G protein signaling. In this study, seven RGS genes (FgFlbA, FgFlbB, FgRgsA, FgRgsB, FgRgsB2, FgRgsC, and FgGprK) were functionally characterized in the plant pathogenic fungus, Gibberella zeae. Mutant phenotypes were observed for deletion mutants of FgRgsA and FgRgsB in vegetative growth, FgFlbB and FgRgsB in conidia morphology, FgFlbA in conidia production, FgFlbA, FgRgsB, and FgRgsC in sexual development, FgFlbA and FgRgsA in spore germination and mycotoxin production, and FgFlbA, FgRgsA, and FgRgsB in virulence. Furthermore, FgFlbA, FgRgsA, and FgRgsB acted pleiotropically, while FgFlbB and FgRgsC deletion mutants exhibited a specific defect in conidia morphology and sexual development, respectively. Amino acid substitutions in Gα subunits and overexpression of the FgFlbA gene revealed that deletion of FgFlbA and dominant active GzGPA2 mutant, gzgpa2(Q207L), had similar phenotypes in cell wall integrity, perithecia formation, mycotoxin production, and virulence, suggesting that FgFlbA may regulate asexual/sexual development, mycotoxin biosynthesis, and virulence through GzGPA2-dependent signaling in G. zeae.     

Chemotaxonomy of Gibberella zeae with special reference to production of trichothecenes and zearalenone

By adopting a single-spore isolation technique, 113 isolates of Gibberella zeae, the perfect stage of Fusarium graminearum, were isolated from rice stubbles in barley and wheat fields and tested for production of trichothecenes and zearalenone on rice grains. Of the isolates, 93% produced the trichothecenes, and they could be subdivided into two chemotaxonomic groups: nivalenol and fusarenon-X producers and deoxynivalenol and 3-acetyldeoxynivalenol producers. No cross production of these two types of trichothecenes was observed in these isolates. Zearalenone was detected in 68% of the isolates, but no clear relationship could be observed regarding its position with respect to the two chemotaxonomic groups.     

Putative polyketide synthase and laccase genes for biosynthesis of aurofusarin in Gibberella zeae

Mycelia of Gibberella zeae (anamorph, Fusarium graminearum), an important pathogen of cereal crops, are yellow to tan with white to carmine red margins. We isolated genes encoding the following two proteins that are required for aurofusarin biosynthesis from G. zeae: a type I polyketide synthase (PKS) and a putative laccase. Screening of insertional mutants of G. zeae, which were generated by using a restriction enzyme-mediated integration procedure, resulted in the isolation of mutant S4B3076, which is a pigment mutant. In a sexual cross of the mutant with a strain with normal pigmentation, the pigment mutation was linked to the inserted vector. The vector insertion site in S4B3076 was a HindIII site 38 bp upstream from an open reading frame (ORF) on contig 1.116 in the F. graminearum genome database. The ORF, designated Gip1 (for Gibberella zeae pigment mutation 1), encodes a putative laccase. A 30-kb region surrounding the insertion site and Gip1 contains 10 additional ORFs, including a putative ORF identified as PKS12 whose product exhibits about 40% amino acid identity to the products of type I fungal PKS genes, which are involved in pigment biosynthesis. Targeted gene deletion and complementation analyses confirmed that both Gip1 and PKS12 are required for aurofusarin production in G. zeae. This information is the first information concerning the biosynthesis of these pigments by G. zeae and could help in studies of their toxicity in domesticated animals.     

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.     

玉蜀黍赤霉(Gibberella zeae)对多菌灵的室内抗药突变体的诱导及其抗药性遗传分析

通过紫外线照射和药剂驯化的方法均获得了玉蜀黍赤霉野生敏感菌株对多菌灵(carbendazim,MBC)的室内抗药突变体。这些抗药突变体一部分表现低抗(low resistance．LR),即能在临界致死剂量1．4μg／mL多菌灵浓度下生长,但不能在10μg／mL浓度以上生长,且对二氯苯胺甲酸甲酯(N-3,5-dichlorophenyl carbamate,MDPC)不表现负交互抗药性;另一部分表现高抗(high resistance．HR)．即能在100μg／mL多菌灵浓度下生长,并与田间高抗菌株一样,对MDPC表现负交互抗药性;没有获得类似田间的中抗(moderate resistance,MR)菌株,即能在10μg／mL多菌灵浓度下快速生长,在100μg／mL浓度以上被完全抑制的突变体。通过药剂驯化的方法还获得了田间中抗(MR)菌株的高抗(HR)突变体,但这些突变体与MR一样对MDPC仍然不表现负交互抗药性。抗药性遗传研究表明。在所研究的抗药突变体中,抗药性在自交和无性繁殖后代中能稳定遗传;室内抗药突变体和田间抗药菌株对多菌灵的抗药性由同一个主效基因控制,但它们发生突变的位点不同或者同一碱基位点发生了不同的突变;对MDPC的敏感性也是由单个基因控制的,该基因与控制多菌灵抗性的基因是等位基因,当该基因发生对MDPC的敏感性增加的突变时会使病菌对多菌灵产生高水平抗性。     

Putative Polyketide Synthase and Laccase Genes for Biosynthesis of Aurofusarin in Gibberella zeae

Mycelia of Gibberella zeae (anamorph, Fusarium graminearum), an important pathogen of cereal crops, are yellow to tan with white to carmine red margins. We isolated genes encoding the following two proteins that are required for aurofusarin biosynthesis from G. zeae: a type I polyketide synthase (PKS) and a putative laccase. Screening of insertional mutants of G. zeae, which were generated by using a restriction enzyme-mediated integration procedure, resulted in the isolation of mutant S4B3076, which is a pigment mutant. In a sexual cross of the mutant with a strain with normal pigmentation, the pigment mutation was linked to the inserted vector. The vector insertion site in S4B3076 was a HindIII site 38 bp upstream from an open reading frame (ORF) on contig 1.116 in the F. graminearum genome database. The ORF, designated Gip1 (for Gibberella zeae pigment mutation 1), encodes a putative laccase. A 30-kb region surrounding the insertion site and Gip1 contains 10 additional ORFs, including a putative ORF identified as PKS12 whose product exhibits about 40% amino acid identity to the products of type I fungal PKS genes, which are involved in pigment biosynthesis. Targeted gene deletion and complementation analyses confirmed that both Gip1 and PKS12 are required for aurofusarin production in G. zeae. This information is the first information concerning the biosynthesis of these pigments by G. zeae and could help in studies of their toxicity in domesticated animals.     

Bacterial artificial chromosome-based physical map of Gibberella zeae (Fusarium graminearum).

Fusarium graminearum is the primary causal pathogen of Fusarium head blight of wheat and barley. To accelerate genomic analysis of F. graminearum, we developed a bacterial artificial chromosome (BAC)-based physical map and integrated it with the genome sequence and genetic map. One BAC library, developed in the HindIII restriction enzyme site, consists of 4608 clones with an insert size of approximately 107 kb and covers about 13.5 genome equivalents. The other library, developed in the BamHI restriction enzyme site, consists of 3072 clones with an insert size of approximately 95 kb and covers about 8.0 genome equivalents. We fingerprinted 2688 clones from the HindIII library and 1536 clones from the BamHI library and developed a physical map of F. graminearum consisting of 26 contigs covering 39.2 Mb. Comparison of our map with the F. graminearum genome sequence showed that the size of our physical map is equivalent to the 36.1 Mb of the genome sequence. We used 31 sequence-based genetic markers, randomly spaced throughout the genome, to integrate the physical map with the genetic map. We also end-sequenced 17 BamHI BAC clones and identified 4 clones that spanned gaps in the genome sequence. Our new integrated map is highly reliable and useful for a variety of genomics studies.     

Shifting fungal reproductive mode by manipulation of mating type genes: obligatory heterothallism of Gibberella zeae

Fungi capable of sexual reproduction use heterothallic (self-sterile) or homothallic (self-fertile) mating strategies. In most ascomycetes, a single mating type locus, MAT, with two alternative forms (MAT1-1 and MAT1-2) called idiomorphs, controls mating ability. In heterothallic ascomycetes, these alternative idiomorphs reside in different nuclei. In contrast, most homothallic ascomycetes carry both MAT1-1 and MAT1-2 in a single nucleus, usually closely linked. An example of the latter is Gibberella zeae, a species that is capable of both selfing and outcrossing. G. zeae is a devastating cereal pathogen of ubiquitous geographical distribution, and also a producer of mycotoxins that threaten human and animal health. We asked whether G. zeae could be made strictly heterothallic by manipulation of MAT. Targeted gene replacement was used to differentially delete MAT1-1 or MAT1-2 from a wild-type haploid MAT1-1; MAT1-2 strain, resulting in MAT1-1; mat1-2, mat1-1; MAT1-2 strains that were self-sterile, yet able to cross to wild-type testers and, more importantly, to each other. These results indicated that differential deletion of MAT idiomorphs eliminates selfing ability of G. zeae, but the ability to outcross is retained. They also indicated that both MAT idiomorphs are required for self-fertility. To our knowledge, this is the first report of complete conversion of fungal reproductive strategy from homothallic to heterothallic by targeted manipulation of MAT. Practically, this approach opens the door to simple and efficient procedures for obtaining sexual recombinants of G. zeae that will be useful for genetic analyses of pathogenicity and other traits, such as the ability to produce mycotoxins.     

A novel F-box protein involved in sexual development and pathogenesis in Gibberella zeae

Gibberella zeae is an ascomyceteous fungus that causes serious diseases in cereal crops. Severe epidemics require strains that are virulent and that can reproduce sexually. We characterized an insertional mutant (designated ZH436) with a pleiotropic defect in both traits, and identified a novel F-box protein gene encoding FBP1 (F-box protein 1) that is similar to fungal F-box proteins including Saccharomyces cerevisiae Grr1, a well-characterized component of the Skp1-Cullin-F-box protein (SCF(Grr1)) E3 ligase complex required for protein degradation. FBP1 also can bind both S. cerevisiae Skp1 protein, the other component of the SCF(Grr1) complex, and its G. zeae sequence homologue SKP1. Two putative protein interacting domains in FBP1 are essential for in vivo function. FBP1 and ScGRR1 are not so interchangeable between S. cerevisiae and G. zeae, but FBP1 can partially complement several defects of a yeast grr1 deletion mutant. Functional analyses confirmed that FBP1 is required for several phenotypes including both sexual development and virulence in G. zeae; the phenotype of DeltaFBP1 strains is different from those of null mutants for F-box proteins in other filamentous fungi as well as from S. cerevisiae grr1Delta strains. Thus, FBP1 is a versatile F-box protein that presumably participates in the formation of the SCF(FBP1) complex that probably controls the ubiquitin-mediated degradation of proteins involved in sexual reproduction and virulence important for disease development by G. zeae.