FUM cluster divergence in fumonisins-producing Fusarium species

Fumonisins are polyketide-derived mycotoxins, produced by several Fusarium species, and its biosynthetic pathway is controlled by the FUM cluster--a group of genes exhibiting a common expression pattern during fumonisin biosynthesis. The most common are the B analogues with fumonisin B(1) (FB(1)) being the most prevalent. At least a part of the inter- and intraspecific variation in FBs synthesis level can be explained by the sequence differences inside FUM cluster. The aim of our study was to evaluate the toxin production and sequence variability in FUM genes and intergenic regions among thirty isolates of seven species reported as potential fumonisins producers: Fusarium anthophilum, Fusarium fujikuroi, Fusarium nygamai, Fusarium oxysporum, Fusarium proliferatum, Fusarium subglutinans and Fusarium verticillioides, particularly with respect to FBs synthesis. Fumonisins were produced in high amounts (over 1mg g(-1)) by one isolate of F. subglutinans, three of F. verticillioides and all F. proliferatum isolates except one, regardless of the host organism. The remaining isolates produced low amounts of FBs and two F. verticillioides isolates didn't produce it at all. The lowest variation in amount of toxin produced was found among F. proliferatum isolates. Based on the translation elongation factor 1α (tef-1α) sequence of F. fujikuroi, a species-specific marker was developed. The intergenic region presents similar opportunity for F. nygamai identification. The phylogenetic reconstruction based on FUM1 gene generally reflects the scenario presented by tef-1α sequences. Although the sequence similarities for intergenic regions were lower than in coding regions, there are clearly conserved patterns enabling separation of different subsets of species, including the non-producer species.     

Biological activity of the rolB-like 5' end of the A4-orf8 gene from the Agrobacterium rhizogenes TL-DNA

The iaaM gene from different plant-associated bacteria encodes a tryptophan monooxygenase (IaaM) that catalyzes the synthesis of indole-3-acetamide (IAM), a precursor of indole-3-acetic acid (IAA). Unlike the IaaM proteins from other bacteria, Agrobacterium spp. T-DNA-encoded IaaM proteins carry a 200 amino acid N-terminal extension with low homology to various members of the RolB protein family. This family is composed of 18 highly divergent T-DNA-encoded proteins, the basic functions of which are still largely undetermined. Deletion of the 5' rolB-like extension of the iaaM gene from Agrobacterium tumefaciens strain Ach5 did not lead to a reduction in IAM synthesis in plants. When expressed in tobacco, the rolB-like fragment did not affect growth or morphology. An iaaM homolog (A4-orf8) from the TL-DNA of Agrobacterium rhizogenes strain A4 also was investigated. Neither the full-size A4-orf8 gene nor the 5'-truncated form induced detectable IAM synthesis. Plants expressing the rolB-like part of the A4-orf8 gene, however, were dwarfed and mottled to various extents and synthesized abnormally high amounts of glucose, fructose, sucrose, and starch.     

A fumonisin biosynthetic gene cluster in Fusarium oxysporum strain O-1890 and the genetic basis for B versus C fumonisin production

Most species of Fusarium that produce fumonisin mycotoxins produce predominantly B fumonisins (FBs). However, Fusarium oxysporum strain O-1890 produces predominantly C fumonisins (FCs). In this study, the nucleotide sequence of the fumonisin biosynthetic gene (FUM) cluster in strain O-1890 was determined. The order and orientation of FUM genes were the same as in the previously described clusters in Fusarium verticillioides and Fusarium proliferatum. Coding regions of F. oxysporum and F. verticillioides FUM genes were 88-92% identical, but regions flanking the clusters did not share significant identity. The FUM cluster gene FUM8 encodes an alpha-oxoamine synthase, and fum8 mutants of F. verticillioides do not produce fumonisins. Complementation of a fum8 mutant with the F. verticillioidesFUM8 restored FB production. Complementation with F. oxysporumFUM8 also restored production, but the fumonisins produced were predominantly FCs. These data indicate that different orthologues of FUM8 determine whether Fusarium produces predominantly FBs or FCs.     

利用大肠杆菌细胞工厂生产吲哚-3-乙酸的研究

目的:利用重组大肠杆菌全细胞转化色氨酸生产IAA.方法:在大肠杆菌胞内构建两条全新的IAA合成途径,即吲哚-3-乙酰胺(indole-3-acetamide,IAM)途径和色胺(tryptamine,TRP)途径.结果:IAM途径涉及两个酶,分别是色氨酸-2-单加氧酶(IAAM)和酰胺酶(AMI1),构建好的重组大肠杆...     

In Planta Production of Indole-3-Acetic Acid by Colletotrichum gloeosporioides f. sp. aeschynomene

The plant pathogenic fungus Colletotrichum gloeosporioides f. sp. aeschynomene utilizes external tryptophan to produce indole-3-acetic acid (IAA) through the intermediate indole-3-acetamide (IAM). We studied the effects of tryptophan, IAA, and IAM on IAA biosynthesis in fungal axenic cultures and on in planta IAA production by the fungus. IAA biosynthesis was strictly dependent on external tryptophan and was enhanced by tryptophan and IAM. The fungus produced IAM and IAA in planta during the biotrophic and necrotrophic phases of infection. The amounts of IAA produced per fungal biomass were highest during the biotrophic phase. IAA production by this plant pathogen might be important during early stages of plant colonization.     

Production of gibberellic acids by an orchid-associated Fusarium proliferatum strain

The rice pathogen Fusarium fujikuroi is well known for its ability to produce the plant hormones gibberellins (GAs). However, the majority of closely related Fusarium species is unable to produce GAs although the GA gene cluster is present in their genomes. In this study, we analyzed five orchid-associated Fusarium isolates for their capacity to produce GAs. Four of them did not produce any GAs and were shown not to contain any GA biosynthetic genes. However, the fifth isolate, which has been identified as F. proliferatum based on five molecular markers, produced significant amounts of GAs in contrast to previously characterized F. proliferatum strains. We focused on the molecular characterization of two GA-specific genes, ggs2 and cps/ks, both inactive in F. proliferatum strain D-02945. Complementation of a F. fujikuroi Deltaggs2 mutant with the ET1 ggs2 gene fully restored GA biosynthesis, confirming that the orchid-associated isolate contains an active gene copy. A possible correlation between GA production and their role in plant-fungal interactions is discussed.     

A single amino acid substitution in highly similar endo-PGs from Fusarium verticillioides and related Fusarium species affects PGIP inhibition.

Endo-polygalacturonase (PG) may be a critical virulence factor secreted by several fungi upon plant invasion. The single-copy gene encoding PG in Fusarium verticillioides and in eight other species of the Gibberella fujikuroi complex (F. sacchari, F. fujikuroi, F. proliferatum, F. subglutinans, F. thapsinum, F. nygamai, F. circinatum, and F. anthophilum) was functionally analyzed in this paper. Both the nucleotide and amino acid sequences were highly similar among the 12 strains of F. verticillioides analyzed, as well as among those from the G. fujikuroi complex. The PGs were not inhibited by the polygalacturonase-inhibiting proteins (PGIPs) from the monocot asparagus and leek plants, but were inhibited to variable extents by bean PGIP. PGs from F. verticillioides, F. nygamai and one strain of F. proliferatum were barely inhibited. Residue 97 within PG was demonstrated to contribute to the different levels of inhibition. Together these findings provide new insights into the structural and functional relationships between the PG from the species of the G. fujikuroi complex and the plant PGIP.     

A single amino acid substitution in highly similar endo-PGs from Fusarium verticillioides and related Fusarium species affects PGIP inhibition

Endo-polygalacturonase (PG) may be a critical virulence factor secreted by several fungi upon plant invasion. The single-copy gene encoding PG in Fusarium verticillioides and in eight other species of the Gibberella fujikuroi complex (F. sacchari, F. fujikuroi, F. proliferatum, F. subglutinans, F. thapsinum, F. nygamai, F. circinatum, and F. anthophilum) was functionally analyzed in this paper. Both the nucleotide and amino acid sequences were highly similar among the 12 strains of F. verticillioides analyzed, as well as among those from the G. fujikuroi complex. The PGs were not inhibited by the polygalacturonase-inhibiting proteins (PGIPs) from the monocot asparagus and leek plants, but were inhibited to variable extents by bean PGIP. PGs from F. verticillioides, F. nygamai and one strain of F. proliferatum were barely inhibited. Residue 97 within PG was demonstrated to contribute to the different levels of inhibition. Together these findings provide new insights into the structural and functional relationships between the PG from the species of the G. fujikuroi complex and the plant PGIP.     

In planta production of indole-3-acetic acid by Colletotrichum gloeosporioides f. sp. aeschynomene

The plant pathogenic fungus Colletotrichum gloeosporioides f. sp. aeschynomene utilizes external tryptophan to produce indole-3-acetic acid (IAA) through the intermediate indole-3-acetamide (IAM). We studied the effects of tryptophan, IAA, and IAM on IAA biosynthesis in fungal axenic cultures and on in planta IAA production by the fungus. IAA biosynthesis was strictly dependent on external tryptophan and was enhanced by tryptophan and IAM. The fungus produced IAM and IAA in planta during the biotrophic and necrotrophic phases of infection. The amounts of IAA produced per fungal biomass were highest during the biotrophic phase. IAA production by this plant pathogen might be important during early stages of plant colonization.     

Restoration of gibberellin production in Fusarium proliferatum by functional complementation of enzymatic blocks

Nine biological species, or mating populations (MPs), denoted by letters A to I, and at least 29 anamorphic Fusarium species have been identified within the Gibberella fujikuroi species complex. Members of this species complex are the only species of the genus Fusarium that contain the gibberellin (GA) biosynthetic gene cluster or at least parts of it. However, the ability of fusaria to produce GAs is so far restricted to Fusarium fujikuroi, although at least six other MPs contain all the genes of the GA biosynthetic gene cluster. Members of Fusarium proliferatum, the closest related species, have lost the ability to produce GAs as a result of the accumulation of several mutations in the coding and 5' noncoding regions of genes P450-4 and P450-1, both encoding cytochrome P450 monooxygenases, resulting in metabolic blocks at the early stages of GA biosynthesis. In this study, we have determined additional enzymatic blocks at the first specific steps in the GA biosynthesis pathway of F. proliferatum: the synthesis of geranylgeranyl diphosphate and the synthesis of ent-kaurene. Complementation of these enzymatic blocks by transferring the corresponding genes from GA-producing F. fujikuroi to F. proliferatum resulted in the restoration of GA production. We discuss the reasons for Fusarium species outside the G. fujikuroi species complex having no GA biosynthetic genes, whereas species distantly related to Fusarium, e.g., Sphaceloma spp. and Phaeosphaeria spp., produce GAs.     

Evidence that Indole-3-Acetic Acid is Not Synthesized Via the Indole-3-Acetamide Pathway in Pea Roots

The biosynthetic route of the key plant hormone, indole-3-acetic acid (IAA) has confounded generations of biologists. Evidence in higher plants has implicated two auxin intermediates with roles established in bacteria: indole-3-acetamide (IAM) and indole-3-pyruvic acid. Herein, the IAM pathway is investigated in pea (Pisum sativum), a model legume. The compound was not detected in pea tissue, although evidence was obtained for its presence in Arabidopsis, tobacco, and maize. Deuterium-labeled tryptophan was not converted to IAM in pea roots, despite being converted to IAA. After feeds of deuterium-labeled IAM, label was recovered in the IAA conjugate IAA-aspartate (IAAsp), although there was little or no labeling of IAA itself. Plants treated with IAM did not exhibit high-IAA phenotypes, and did not accumulate IAA. This evidence, taken together, indicates that although exogenous IAM may be converted to IAA (and further to IAAsp), the IAM pathway does not operate naturally in pea roots.     

The pathway of auxin biosynthesis in plants

The plant hormone auxin, which is predominantly represented by indole-3-acetic acid (IAA), is involved in the regulation of plant growth and development. Although IAA was the first plant hormone identified, the biosynthetic pathway at the genetic level has remained unclear. Two major pathways for IAA biosynthesis have been proposed: the tryptophan (Trp)-independent and Trp-dependent pathways. In Trp-dependent IAA biosynthesis, four pathways have been postulated in plants: (i) the indole-3-acetamide (IAM) pathway; (ii) the indole-3-pyruvic acid (IPA) pathway; (iii) the tryptamine (TAM) pathway; and (iv) the indole-3-acetaldoxime (IAOX) pathway. Although different plant species may have unique strategies and modifications to optimize their metabolic pathways, plants would be expected to share evolutionarily conserved core mechanisms for auxin biosynthesis because IAA is a fundamental substance in the plant life cycle. In this review, the genes now known to be involved in auxin biosynthesis are summarized and the major IAA biosynthetic pathway distributed widely in the plant kingdom is discussed on the basis of biochemical and molecular biological findings and bioinformatics studies. Based on evolutionarily conserved core mechanisms, it is thought that the pathway via IAM or IPA is the major route(s) to IAA in plants.     

Segregation of secondary metabolite biosynthesis in hybrids of Fusarium fujikuroi and Fusarium proliferatum

Fusarium fujikuroi and Fusarium proliferatum are two phylogenetically closely related species of the Gibberella fujikuroi species complex (GFC). In some cases, strains of these species can cross and produce a few ascospores. In this study, we analyzed 26 single ascospore isolates of an interspecific cross between F. fujikuroi C1995 and F. proliferatum D4854 for their ability to produce four secondary metabolites: gibberellins (GAs), the mycotoxins fusarin C and fumonisin B(1), and a family of red polyketides, the fusarubins. Both parental strains contain the biosynthetic genes for all four metabolites, but differ in their ability to produce these metabolites under certain conditions. F. fujikuroi C1995 produces GAs and fusarins, while F. proliferatum D4854 produces fumonisins and fusarubins. The segregation amongst the progeny of these traits is not the expected 1:1 Mendelian ratio. Only eight, six, three and three progeny, respectively, produce GAs, fusarins, fumonisin B(1) and fusarubins in amounts similar to those synthesized by the producing parental strain. Beside the eight highly GA(3)-producing progeny, some of the progeny produce small amounts of GAs, predominantly GA(1), although these strains contain the GA gene cluster of the non-GA-producing F. proliferatum parental strain. Some progeny had recombinant secondary metabolite profiles under the conditions examined indicating that interspecific crosses can yield secondary metabolite production profiles that are atypical of the parent species.     

Functional characterization of two cytochrome P450 monooxygenase genes, P450-1 and P450-4, of the gibberellic acid gene cluster in Fusarium proliferatum (Gibberella fujikuroi MP-D)

Gibberella fujikuroi is a species complex with at least nine different biological species, termed mating populations (MPs) A to I (MP-A to MP-I), known to produce many different secondary metabolites. So far, gibberellin (GA) production is restricted to Fusarium fujikuroi (G. fujikuroi MP-C), although at least five other MPs contain all biosynthetic genes. Here, we analyze the GA gene cluster and GA pathway in the closest related species, Fusarium proliferatum (MP-D), and demonstrate that the GA genes share a high degree of sequence homology with the corresponding genes of MP-C. The GA production capacity was restored after integration of the entire GA gene cluster from MP-C, indicating the existence of an active regulation system in F. proliferatum. The results further indicate that one reason for the loss of GA production is the accumulation of several mutations in the coding and 5' noncoding regions of the ent-kaurene oxidase gene, P450-4.     

香蕉上的镰孢菌种类及其系统发育关系(英文)

镰孢菌属真菌是香蕉上的重要病原菌,主要引起香蕉枯萎病以及香蕉冠腐病,在我国已明确引起香蕉枯萎病的病原为尖孢镰孢古巴专化型 Fusarium oxysporum f. sp. cubense(FOC)1号和4号生理小种,但是引起香蕉冠腐病的镰孢菌种类还未明确。为了解香蕉上镰孢菌在种间及种内水平上的多样性,2008–2011 年间作者从华南地区不同的水果市场及香蕉果园采集香蕉样品90份,分离得到143株镰孢菌。通过形态学观察及基于 EF-1α基因的系统进化分析鉴定出10种镰孢菌,即F. oxysporum、F. solani、F. camptoceras、F. pallidoroseum、F. stiloides、F. chlamydosporum、F.verticillioides、F. proliferatum、F. concentricum、F. sacchari,以及藤仓赤霉复合种(Gibberella fujikuroi species complex,GFC)中 3 个未定名的类群。轮纹镰孢 F. concentricum 及甘蔗镰孢 F.sacchari 是香蕉果实中最常见种,前菌为我国首次报道,后菌是首次报道与香蕉有关。对从香蕉上分离的藤仓赤霉复合种(GFC)及尖孢镰孢复合种(FOSC)的EF-1α序列进行了系统发育分析,其GFC中的27个菌株组成的单系群可分为7个不同的亚群,分别为 F.verticillioides、F. proliferatum、F. concentricum、F. sacchari 以及3个没有描述过的菌系 Fusarium sp. 1、Fusarium sp.2和 Fusarium sp.3;FOSC中的50个菌株形成2大分枝共12个谱系,分离自我国华南地区的21株尖孢镰孢形成7个谱系,其中 13株已知的香蕉枯萎病病原菌分布在3个谱系中,我国大陆的香蕉枯萎病病原菌菌株与来源于台湾地区及东南亚的菌株亲缘关系较近,FOC1号生理小种的遗传分化大于4号生理小种,FOC 1号生理小种与分离自香蕉果实上的尖孢镰孢菌的亲缘关系比与FOC 4号生理小种的亲缘关系更近。研究结果表明,我国香蕉上存在着丰富的镰孢菌种类,而且种内遗传多样性丰富。     

35S-β-glucuronidase gene blocks biological effects of cotransferred iaa genes

The iaaM and iaaH genes of Agrobacterium tumefaciens and Agrobacterium rhizogenes play an important role in crown gall and hairy root disease. The iaaM gene codes for tryptophan monooxygenase which converts tryptophan into indole-3-acetamide (IAM). IAM is converted into the auxin indole-3-acetic acid (IAA) by indoleacetamide hydrolase, encoded by the iaaH gene. In functional studies on the activity of the iaa genes of the TB region of the A. tumefaciens biotype III strain Tm4, the frequently used 35S--glucuronidase (35S-UidA or GUS) marker gene was found to inhibit IAA synthesis and root induction encoded by the TB iaa genes. To exert this inhibition, the 35S-UidA gene must be cotransferred with the iaaH gene. The 35S promoter alone is sufficient to cause the inhibitory effect.     

Differentiation of Fusarium subglutinans f. sp. pini by histone gene sequence data.

Fusarium subglutinans f. sp. pini (= F. circinatum) is a pathogen of pine and is one of eight mating populations (i.e., biological species) in the Gibberella fujikuroi species complex. This species complex includes F. thapsinum, F. moniliforme (= F. verticillioides), F. nygamai, and F. proliferatum, as well as F. subglutinans associated with sugarcane, maize, mango, and pineapple. Differentiating these forms of F. subglutinans usually requires pathogenicity tests, which are often time-consuming and inconclusive. Our objective was to develop a technique to differentiate isolates of F. subglutinans f. sp. pini from other isolates identified as F. subglutinans. We sequenced the histone H3 gene from a representative set of Fusarium isolates. The H3 gene sequence was conserved and contained two introns in all the isolates studied. From both the intron and the exon sequence data, we developed a PCR-restriction fragment length polymorphism technique that reliably distinguishes F. subglutinans f. sp. pini from the other biological species in the G. fujikuroi species complex.     

Biosynthesis of indole-3-acetic acid in Azospirillum brasilense. Insights from quantum chemistry.

Quantum chemical methods AM1 and PM3 and chromatographic methods were used to qualitatively characterize pathways of bacterial production of indole-3-acetic acid (IAA). The standard free energy changes (delta G(o)'sum) for the synthesis of tryptophan (Trp) from chorismic acid via anthranilic acid and indole were calculated, as were those for several possible pathways for the synthesis of IAA from Trp, namely via indole-3-acetamide (IAM), indole-3-pyruvic acid (IPyA), and indole-3-acetonitrile (IAN). The delta G(o)'sum for Trp synthesis from chorismic acid was -402 (-434) kJ.mol-1 (values in parentheses were calculated by PM3). The delta G(o)'sum for IAA synthesis from Trp were -565 (-548) kJ.mol-1 for the IAN pathway, -481 (-506) kJ.mol-1 for the IAM pathway, and -289 (-306) kJ.mol-1 for the IPyA pathway. By HPLC analysis, the possibility was assessed that indole, anthranilic acid, and Trp might be utilized as precursors for IAA synthesis by Azospirillum brasilense strain Sp 245. The results indicate that there is a high motive force for Trp synthesis from chorismic acid and for IAA synthesis from Trp, and make it unlikely that anthranilic acid and indole act as the precursors to IAA in a Trp-independent pathway.     

Detection of the IAA Biosynthetic Pathway from Tryptophan via Indole-3-Acetamide in Bradyrhizobium spp.

The IAA biosynthetic pathway of tryptophan to IAA via IAM wasdetected in Bradyrhizobium spp. (slow-growing Rhizobium) butnot in Rhizobium spp. (fast-growing Rhizobium). A simple methodusing rapid HPLC analysis to measure the conversion from NAMto NAA was developed to detect indole-3-acetamide hydrolaseactivity in cultures of bacteria. Most of the Bradyrhizobiumstrains produce large amounts of NAA converted from NAM underour assay conditions. In addition, GC/MS analysis of purifiedextracts from cultures of B.japonicum wild-type strain J1063,grown in a tryptophan-supplemented liquid medium, demonstratedthe presence of IAM and IAA. The results strongly suggest thatbiosynthesis of IAA in Bradyrhizobium spp. involves the samepathway as that operating in Pseudomonas savastanoi and Agrobacteriumtumefaciens. (Received December 25, 1988; Accepted May 18, 1988)