Isolation and Characterization of the Gibberellin Biosynthetic Gene Cluster in Sphaceloma manihoticola

Gibberellins (GAs) are tetracyclic diterpenoid phytohormones that were first identified as secondary metabolites of the fungus Fusarium fujikuroi (teleomorph, Gibberella fujikuroi). GAs were also found in the cassava pathogen Sphaceloma manihoticola, but the spectrum of GAs differed from that in F. fujikuroi. In contrast to F. fujikuroi, the GA biosynthetic pathway has not been studied in detail in S. manihoticola, and none of the GA biosynthetic genes have been cloned from the species. Here, we present the identification of the GA biosynthetic gene cluster from S. manihoticola consisting of five genes encoding a bifunctional ent-copalyl/ent-kaurene synthase (CPS/KS), a pathway-specific geranylgeranyl diphosphate synthase (GGS2), and three cytochrome P450 monooxygenases. The functions of all of the genes were analyzed either by a gene replacement approach or by complementing the corresponding F. fujikuroi mutants. The cluster organization and gene functions are similar to those in F. fujikuroi. However, the two border genes in the Fusarium cluster encoding the GA₄ desaturase (DES) and the 13-hydroxylase (P450-3) are absent in the S. manihoticola GA gene cluster, consistent with the spectrum of GAs produced by this fungus. The close similarity between the two GA gene clusters, the identical gene functions, and the conserved intron positions suggest a common evolutionary origin despite the distant relatedness of the two fungi.     

Phylogenetic Diversity and In Vitro Susceptibility Profiles of Human Pathogenic Members of the <Emphasis Type="Italic">Fusarium fujikuroi</Emphasis> Species Complex Isolated from South India

Availability of molecular methods, gene sequencing, and phylogenetic species recognition have led to rare fungi being recognized as opportunistic pathogens. Fungal keratitis and onychomycosis are fairly common mycoses in the tropics, especially among outdoor workers and enthusiasts. The frequently isolated etiological agents belong to genera Candida, Aspergillus, and Fusarium. Within the genus Fusarium, known to be recalcitrant to prolonged antifungal treatment and associated with poor outcome, members of the Fusarium solani species complex are reported to be most common, followed by members of the Fusarium oxysporum SC and the Fusarium fujikuroi SC (FFSC). Morphological differentiation among the various members is ineffective most times. In the present study, we describe different species of the FFSC isolated from clinical specimen in south India. All twelve isolates were characterized up to species level by nucleic acid sequencing and phylogenetic analysis. The molecular targets chosen were partial regions of the internal transcribed spacer rDNA region, the panfungal marker and translation elongation factor-1α gene, the marker of choice for Fusarium speciation. Phylogenetic analysis was executed using the Molecular Evolutionary Genetics Analysis software (MEGA7). In vitro susceptibility testing against amphotericin B, voriconazole, posaconazole, natamycin, and caspofungin diacetate was performed following the CLSI M38-A2 guidelines for broth microdilution method. The twelve isolates of the FFSC were F. verticillioides (n = 4), F. sacchari (n = 3), F. proliferatum (n = 2), F. thapsinum (n = 1), F. andiyazi (n = 1), and F. pseudocircinatum (n = 1). To the best of our knowledge, this is the first report of F. andiyazi from India and of F. pseudocircinatum as a human pathogen worldwide. Natamycin and voriconazole were found to be most active agents followed by amphotericin B. Elderly outdoor workers figured more among the patients and must be recommended protective eye wear.     

Biochemical and genetic analysis of a unique poly(ADP-ribosyl) glycohydrolase (PARG) of the pathogenic fungus <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis>

The genome sequence of the plant pathogen Fusarium oxysporum f. sp. lycopersici contains a single gene encoding a predicted poly(ADP-ribose) glycohydrolase (FOXG_05947.2, PARG). Here, we assessed whether this gene has a role as a global regulator of DNA repair or in virulence as an ADP ribosylating toxin homologue of bacteria. The PARG protein was purified after expressing its encoding gene in Escherichia coli. Its inhibition by 6,9-diamino-2-ethoxyacridine lactate monohydrate and tannins was similar to its human orthologue that is involved in DNA repair. A deletion strain of F. oxysporum f. sp. lycopersici showed no growth defects and was not affected in pathogenicity. Together, our results indicate that the PARG protein of F. oxysporum f. sp. lycopersici is involved in DNA repair and does not act in pathogenicity as an effector.     

Heterologous expression of a novel <Emphasis Type="Italic">Poa pratensis</Emphasis> gibberellin 2-oxidase gene, <Emphasis Type="Italic">PpGA2ox</Emphasis>, caused dwarfism,late flowering,and increased chlorophyll accumulation in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Gibberellin 2-oxidases (GA2oxs) irreversibly convert bioactive gibberellins (GAs) and their immediate precursors into inactive GAs via 2-β hydroxylation and so regulate gibberellin content in plants. However, to the best of our knowledge, little has been known about the GA2oxs and its function in cool season turfgrass Poa pratensis. In this study, rapid amplification of cDNA end (RACE) was employed to isolate PpGA2ox from P. pratensis. The open reading frame of PpGA2ox was 1 047 bp in length, corresponding to 348 amino acids. PpGA2ox was localized in both nucleus and cytoplasm. The expression of PpGA2ox could be up-regulated by 10 μM gibberellic acid, 5 μM methyl jasmonate, or 10 μM indole-3-acetic acid. In addition, its native promoter could drive GUS expression in both leaf apex and shoot apical region. Moreover, overexpression of PpGA2ox in Arabidopsis led to GA-deficiency leading to dwarf phenotype, delayed flowering time, and increased chlorophyll content. Our study suggests that PpGA2ox could be a candidate gene for breeding new cultivars of P. pratensis.     

Diversity,regulation, and evolution of the gibberellin biosynthetic pathway in fungi compared to plants and bacteria

Bioactive gibberellins (GAs) are diterpene plant hormones that are biosynthesized through complex pathways and control diverse aspects of growth and development. GAs were first isolated as metabolites of a fungal rice pathogen, Gibberella fujikuroi, since renamed Fusarium fujikuroi. Although higher plants and the fungus produce structurally identical GAs, significant differences in their GA pathways, enzymes involved and gene regulation became apparent with the identification of GA biosynthetic genes in Arabidopsis thaliana and F. fujikuroi. Recent identifications of GA biosynthetic gene clusters in two other fungi, Phaeosphaeria spp. and Sphaceloma manihoticola, and the high conservation of GA cluster organization in these distantly related fungal species indicate that fungi evolved GA and other diterpene biosynthetic pathways independently from plants. Furthermore, the occurrence of GAs and recent identification of the first GA biosynthetic genes in the bacterium Bradyrhizobium japonicum make it possible to study evolution of GA pathways in general.In this review, we summarize our current understanding of the GA biosynthesis pathway, specifically the genes and enzymes involved as well as gene regulation and localization in the genomes of different fungi and compare it with that in higher and lower plants and bacteria.     

Loss of Gibberellin Production in Fusarium verticillioides (Gibberella fujikuroi MP-A) Is Due to a Deletion in the Gibberellic Acid Gene Cluster

Fusarium verticillioides (Gibberella fujikuroi mating population A [MP-A]) is a widespread pathogen on maize and is well-known for producing fumonisins, mycotoxins that cause severe disease in animals and humans. The species is a member of the Gibberella fujikuroi species complex, which consists of at least 11 different biological species, termed MP-A to -K. All members of this species complex are known to produce a variety of secondary metabolites. The production of gibberellins (GAs), a group of diterpenoid plant hormones, is mainly restricted to Fusarium fujikuroi (G. fujikuroi MP-C) and Fusarium konzum (MP-I), although most members of the G. fujikuroi species complex contain the GA biosynthesis gene cluster or parts of it. In this work, we show that the inability to produce GAs in F. verticillioides (MP-A) is due to the loss of a majority of the GA gene cluster as found in F. fujikuroi. The remaining part of the cluster consists of the full-length F. verticillioides des gene (Fvdes), encoding the GA₄ desaturase, and the coding region of FvP450-4, encoding the ent-kaurene oxidase. Both genes share a high degree of sequence identity with the corresponding genes of F. fujikuroi. The GA production capacity of F. verticillioides was restored by transforming a cosmid with the entire GA gene cluster from F. fujikuroi, indicating the existence of an active regulation system in F. verticillioides. Furthermore, the GA₄ desaturase gene des from F. verticillioides encodes an active enzyme which was able to restore the GA production in a corresponding des deletion mutant of F. fujikuroi.     

Detection of pea wilt pathogen <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">pisi</Emphasis> using DNA-based markers

Identification of the fungus Fusarium oxysporum f. sp. pisi (Fop), the causal organism of wilt disease of pea, is a time consuming and arduous task. Diagnosis of Fop by traditional means requires more than 2 months and involves two steps, identification of species using morphological characters and formae specialis ‘pisi’ using pathogenicity assays. The ambiguous morphological differences between F. solani and F. oxysporum further complicate the diagnosis of F. oxysporum. A polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) based method was developed to detect Fop from India. A PCR–RFLP marker, HPACAPS1₃₈₀, generated after restriction of 28S rDNA region with enzyme MvaI, detected accurately the Fop among several other fungi with detection sensitivity of 5 fg of Fop genomic DNA. In a mixture of Fop and pea DNA, the sensitivity was 500 pg of Fop DNA in 50 ng of pea DNA. The assay was further refined to detect the Fop from infected tissues and infested soil. The current assay can detect Fop from culture, plant tissues and soil in a considerably shorter period of time compared to traditional methods.     

Engineering gibberellin metabolism in <Emphasis Type="Italic">Solanum nigrum</Emphasis> L. by ectopic expression of gibberellin oxidase genes

Gibberellins (GAs) control many aspects of plant development, including seed germination, shoot growth, flower induction and growth and fruit expansion. Leaf explants of Solanum nigrum (Black Nightshade; Solanaceae) were used for Agrobacterium-mediated delivery of GA-biosynthetic genes to determine the influence of their encoded enzymes on the production of bioactive GAs and plant stature in this species. Constructs were prepared containing the neomycin phosphotransferase (nptII) gene for kanamycin resistance as a selectable marker, and the GA-biosynthetic genes, their expression under the control of the CaMV 35S promoter. The GA-biosynthetic genes comprised AtGA20ox1, isolated from Arabidopsis thaliana, the product from which catalyses the formation of C₁₉-GAs, and MmGA3ox1 and MmGA3ox2, isolated from Marah macrocarpus, which encode functionally different GA 3-oxidases that convert C₁₉-GAs to biologically active forms. Increase in stature was observed in plants transformed with AtGA20ox1, MmGA3ox2 and MmGA3ox1 + MmGA3ox2, their presence and expression being confirmed by PCR and RT-PCR, respectively, accompanied by an increase in GA₁ content. Interestingly, MmGA3ox1 alone did not induce a sustained increase in plant height, probably because of only a marginal increase in bioactive GA₁ content in the transformed plants. The results are discussed in the context of regulating plant stature, since this strategy would decrease the use of chemicals to promote plant growth.     

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.     

Polymorphism of mycotoxin biosynthetic genes among <Emphasis Type="Italic">Fusarium equiseti</Emphasis> isolates from Italy and Poland

Fusarium equiseti (Corda) Saccardo is a soil saprophyte and a weak pathogen, associated with several diseases of fruit and other crops in subtropical and tropical areas, but also in countries with temperate climate. A wide range of secondary metabolites has been identified among natural F. equiseti populations, with zearalenone (ZEA), fusarochromanone and fusarenon-X being the most common. In present study, the genetic diversity of strains from two populations (from Italy and Poland) was evaluated by analysing the translation elongation factor 1α (tef-1α) sequences, two polyketide synthases from the ZEA biosynthetic pathway (PKS13 and PKS4) and the TRI5 gene from the trichothecene biosynthetic pathway. ZEA was produced in rice cultures by 20 of the 27 tested isolates in concentrations ranging from 1.34 ng/g to 34,000 ng/g). The ability to produce enniatins and trichothecenes was evaluated in all strains by identifying esyn1, TRI13 and TRI4 genes. The presence of PKS4 and PKS13 genes was confirmed by polymerase chain reaction (PCR) in only some ZEA-producing isolates. Similarly, the TRI5 gene was found in 14 of the 27 isolates tested. This is likely to have been caused by the divergence of those genes between F. equiseti and F. graminearum (the latter species was used for the primers design) and can be exploited in phylogenetic studies. The analysis of the mycotoxin biosynthetic gene sequences can be used to differentiate the studied genotypes even more precisely than the analysis of the non-coding regions (like tef-1α).