The linear mitochondrial plasmid pClK1 of the phytopathogenic fungus Claviceps purpurea may code for a DNA polymerase and an RNA polymerase

Summary Plasmid pClK1, a linear mitochondrial plasmid of Claviceps purpurea, was completely sequenced. The sequence contains two long open reading frames (ORF1, 3291 bp; ORF2, 2910 bp), and at least four smaller ORFs. The potential polypeptide derived from ORF1 shows homology to the family B type DNA polymerases. The product of ORF2 has significant homology to the mitochondrial RNA polymerase of yeast and RNA polymerases from bacteriophages. ORF1 and ORF2 show homology to URF3 and URF1 of the maize plasmids S1 and S2, respectively. No homology to any published protein sequence was found for the smaller ORFs. The origin of the terminal protein attached to the 5 ends of pClK1 remains open; several alternatives for its origin are discussed. The sequence data as a whole confirm the virus-like character of pClK1 already postulated from structural properties. Thus pClK1 together with S plasmids of maize and several other linear plasmids make up a distinct class of DNA species of plants and fungi probably derived from a common virus-like ancestor.     

Genetic evidence for natural product-mediated plant-plant allelopathy in rice (Oryza sativa)

? There is controversy as to whether specific natural products play a role in directly mediating antagonistic plant-plant interactions - that is, allelopathy. If proved to exist, such phenomena would hold considerable promise for agronomic improvement of staple food crops such as rice (Oryza sativa). ? However, while substantiated by the presence of phytotoxic compounds at potentially relevant concentrations, demonstrating a direct role for specific natural products in allelopathy has been difficult because of the chemical complexity of root and plant litter exudates. This complexity can be bypassed via selective genetic manipulation to ablate production of putative allelopathic compounds, but such an approach previously has not been applied. ? The rice diterpenoid momilactones provide an example of natural products for which correlative biochemical evidence has been obtained for a role in allelopathy. Here, we apply reverse genetics, using knock-outs of the relevant diterpene synthases (copalyl diphosphate synthase 4 (OsCPS4) and kaurene synthase-like 4 (OsKSL4)), to demonstrate that rice momilactones are involved in allelopathy, including suppressing growth of the widespread rice paddy weed, barnyard grass (Echinochloa crus-galli). ? Thus, our results not only provide novel genetic evidence for natural product-mediated allelopathy, but also furnish a molecular target for breeding and metabolic engineering of this important crop plant.     

Characterization of the Fungal Gibberellin Desaturase as a 2-Oxoglutarate-Dependent Dioxygenase and Its Utilization for Enhancing Plant Growth

The biosynthesis of gibberellic acid (GA₃) by the fungus Fusarium fujikuroi is catalyzed by seven enzymes encoded in a gene cluster. While four of these enzymes are characterized as cytochrome P450 monooxygenases, the nature of a fifth oxidase, GA₄ desaturase (DES), is unknown. DES converts GA₄ to GA₇ by the formation of a carbon-1,2 double bond in the penultimate step of the pathway. Here, we show by expression of the des complementary DNA in Escherichia coli that DES has the characteristics of a 2-oxoglutarate-dependent dioxygenase. Although it has low amino acid sequence homology with known 2-oxoglutarate-dependent dioxygenases, putative iron- and 2-oxoglutarate-binding residues, typical of such enzymes, are apparent in its primary sequence. A survey of sequence databases revealed that homologs of DES are widespread in the ascomycetes, although in most cases the homologs must participate in non-gibberellin (GA) pathways. Expression of des from the cauliflower mosaic virus 35S promoter in the plant species Solanum nigrum, Solanum dulcamara, and Nicotiana sylvestris resulted in substantial growth stimulation, with a 3-fold increase in height in S. dulcamara compared with controls. In S. nigrum, the height increase was accompanied by a 20-fold higher concentration of GA₃ in the growing shoots than in controls, although GA₁ content was reduced. Expression of des was also shown to partially restore growth in plants dwarfed by ectopic expression of a GA 2-oxidase (GA-deactivating) gene, consistent with GA₃ being protected from 2-oxidation. Thus, des has the potential to enable substantial growth increases, with practical implications, for example, in biomass production.The GAs are a class of diterpenoid hormones that regulate many aspects of growth and development in plants, including stem extension (Thomas and Hedden, 2006). Despite being ubiquitous in higher plants, they were first discovered as secondary metabolites of the plant pathogenic fungus Gibberella fujikuroi, the causative agent of the bakanae disease of rice (Oryza sativa; Phinney, 1983). This fungus is now known to comprise a group of reproductively isolated species or mating populations, the rice pathogen belonging to mating group C and assigned the name Fusarium fujikuroi (Leslie and Summerell, 2006; Kvas et al., 2009). Details of the GA biosynthetic pathways in both plants and the fungus are known in considerable detail and have revealed that, although they give rise to common metabolites, the pathways utilize different types of enzymes for several steps and appear to have evolved independently (Hedden et al., 2001; Bömke and Tudzynski, 2009).Higher plants differ from the GA-producing fungi by possessing the means for GA inactivation, which is necessary to allow precise regulation of their GA concentration. In contrast, the fungi are not dependent on GAs for their development but produce and secrete large quantities of the compounds to modify the behavior of their hosts. It has been shown that GAs interfere with plant defense by suppressing jasmonate signaling and may thus compromise the host’s ability to evade fungal infection (Navarro et al., 2008; Hou et al., 2010). An apparent ubiquitous inactivation mechanism involves 2β-hydroxylation (Thomas et al., 1999), the effect of which reduces binding of the GA within the active site of the GID1 receptor (Murase et al., 2008). However, GAs such as GA₃ and GA₅, which are unsaturated on C-2, are protected from 2β-hydroxylation and, as a consequence, would be expected to be turned over more slowly than their saturated analogs (King et al., 2008). In accordance with the requirement to regulate GA content, shoots of higher plants contain relatively little 1,2-unsaturated GAs, although developing seeds of some species contain substantial quantities. They are produced in a two-step reaction via a 2,3-dehydro intermediate, which is then hydroxylated on C-3β with rearrangement of the double bond from C-2,3 to C-1,2 (Albone et al., 1990). The reactions are catalyzed by GA 3-oxidase-type enzymes, with a single enzyme catalyzing both reactions in cereal shoots to produce GA₃ from GA₂₀ as a minor by-product of GA₁ biosynthesis (Itoh et al., 2001; Appleford et al., 2006; Fig. 1). In developing seeds of Marah macrocarpus, which contain high concentrations of the 1,2-unsaturated GA, GA₇, the formation of this GA from GA₉ requires the activities of two functionally different GA 3-oxidases acting sequentially (Ward et al., 2010). However, direct formation of GA₇ from GA₄, such as occurs in F. fujikuroi, is not usual in higher plants.Open in a separate windowFigure 1.The GA biosynthetic pathway in plants and F. fujikuroi. The fungal pathway to GA₃ is indicated by the thick gray arrow. DES catalyzes the conversion of GA₄ to GA₇.While the late stages of GA biosynthesis in higher plants, including desaturation when it occurs and 2β-hydroxylation, are catalyzed by 2-oxoglutarate-dependent dioxygenases (ODDs), these enzymes have not been shown to be involved in GA biosynthesis in fungi. F. fujikuroi contains a cluster of seven genes for GA biosynthesis, including a geranylgeranyl diphosphate synthase that is specific to the GA pathway and a bifunctional terpene cyclase that converts geranylgeranyl diphosphate to ent-kaurene in two steps via ent-copalyl diphosphate (for review, see Hedden et al. [2001]; Bömke and Tudzynski [2009]). The formation of GA₃ from ent-kaurene requires the activity of five oxidases (Fig. 1), four of which are cytochrome P450 monooxygenases: P450-4 (ent-kaurene oxidase) oxidizes ent-kaurene to ent-kaurenoic acid (Tudzynski et al., 2001), which is converted to GA₁₄ by P450-1 (GA₁₄ synthase; Rojas et al., 2001); P450-2 functions as a GA 20-oxidase, converting GA₁₄ to GA₄ (Tudzynski et al., 2002), while, in the final step of the pathway, P450-3 13-hydroxylates GA₇ to form GA₁₃ (Tudzynski et al., 2003). However, the nature of the desaturase (DES), which converts GA₄ to GA₇ (Fig. 1), is unknown. When first described, it was found to have closest, albeit weak, homology to a component of the 7α-cephem-methoxylase from Nocardia lactamdurans, giving little indication of its mechanism (Tudzynski et al., 2003). Besides F. fujikuroi, several other ascomycetes, including Sphaceloma manihoticola (Bömke et al., 2008), Phaeosphaeria spp. (Kawaide, 2006), and two other species of the G. fujikuroi species complex, Fusarium konzum (Malonek et al., 2005) and Fusarium sacchari (Troncoso et al., 2010), have been shown to synthesize GAs, although the first two species do not carry out the desaturation step and do not contain a desaturase gene.The promotion of vegetative growth offers potential benefits, for example, in biomass production (Demura and Ye, 2010). In order to test the hypothesis that growth could be stimulated by increasing the shoot concentrations of GAs that are unsaturated on C-2 and therefore resistant to 2β-hydroxylation, we introduced the fungal desaturase gene into plants. The feasibility of this approach was reinforced by the demonstration that DES has the characteristics of an ODD and, therefore, would be expected to function in higher plants.     

In vitro pathogenicity assay for the ergot fungus Claviceps purpurea

The pathogenic development of the biotrophic ergot fungus Claviceps purpurea is strictly limited to the ovary of grasses. Early colonization stages occur within a defined spatio-temporal course of events, including the directed growth to the vascular tissue for nutrient supply. To characterize mutant strains with putative defects in pathogenicity, the close observation of the infection pathway is therefore indispensable. Here, we describe the establishment of a new pathogenicity assay, based on the in vitro cultivation of isolated rye ovaries. The pathogenic development of a wild-type strain of C. purpurea was compared with the infection of mature rye flowers on whole plants. Up to the sixth day post inoculation, the route of infection within the isolated ovaries was maintained and temporally equal to that seen in mature flowers. Therefore, the in vitro pathogenicity assay is an effective alternative to the whole-plant infection tests, and suitable for detailed infection studies and screening high numbers of mutants for defects in early pathogenesis.     

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.     

Identification of Botrytis cinerea genes up-regulated during infection and controlled by the Galpha subunit BCG1 using suppression subtractive hybridization (SSH)

The Galpha subunit BCG1 plays an important role during the infection of host plants by Botrytis cinerea. Delta bcg1 mutants are able to conidiate, penetrate host leaves, and produce small primary lesions. However, in contrast to the wild type, the mutants completely stop invasion of plant tissue at this stage; secondary lesions have never been observed. Suppression subtractive hybridization (SSH) was used to identify fungal genes whose expression on the host plant is specifically affected in bcg1 mutants. Among the 22 differentially expressed genes, we found those which were predicted to encode proteases, enzymes involved in secondary metabolism, and others encoding cell wall-degrading enzymes. All these genes are highly expressed during infection in the wild type but not in the mutant. However, the genes are expressed in both the wild type and the mutant under certain conditions in vitro. Most of the BCG1-controlled genes are still expressed in adenylate cyclase (bac) mutants in planta, suggesting that BCG1 is involved in at least one additional signaling cascade in addition to the cAMP-depending pathway. In a second SSH approach, 1,500 clones were screened for those that are specifically induced by the wild type during the infection of bean leaves. Of the 22 BCG1-controlled genes, 11 also were found in the in planta SSH library. Therefore, SSH technology can be successfully applied to identify target genes of signaling pathways and differentially expressed genes in planta.     

Characterization of the final two genes of the gibberellin biosynthesis gene cluster of Gibberella fujikuroi: des and P450-3 encode GA4 desaturase and the 13-hydroxylase,respectively

Recently, six genes of the gibberellin (GA) biosynthesis gene cluster in Gibberella fujikuroi were cloned and the functions of five of these genes were determined. Here we describe the function of the sixth gene, P450-3, and the cloning and functional analysis of a seventh gene, orf3, located at the left border of the gene cluster. We have thereby defined the complete GA biosynthesis gene cluster in this fungus. The predicted amino acid sequence of orf3 revealed no close homology to known proteins. High performance liquid chromatography and gas chromatography-mass spectrometry analyses of the culture fluid of knock-out mutants identified GA1 and GA4, rather than GA3 and GA7, as the major C19-GA products, suggesting that orf3 encodes the GA4 1,2-desaturase. This was confirmed by transformation of the SG139 mutant, which lacks the GA biosynthesis gene cluster, with the desaturase gene renamed des. The transformants converted GA4 to GA7, and also metabolized GA9 (3-deoxyGA4) to GA120 (1,2-didehydroGA9), but the 2alpha-hydroxylated compound GA40 was the major product in this case. We demonstrate also by gene disruption that P450-3, one of the four cytochrome P450 monooxygenase genes in the GA gene cluster, encodes the 13-hydroxylase, which catalyzes the conversion of GA7 to GA3, in the last step of the pathway. This enzyme also catalyzes the 13-hydroxylation of GA4 to GA1. Disruption of the des gene in an UV-induced P450-3 mutant produced a double mutant lacking both desaturase and 13-hydroxylase activities that accumulated high amounts of the commercially important GA4. The des and P450-3 genes differ in their regulation by nitrogen metabolite repression. In common with the other five GA biosynthesis genes, expression of the desaturase gene is repressed by high amounts of nitrogen in the culture medium, whereas P450-3 is the only gene in the cluster not repressed by nitrogen.     

The biotrophic,non-appressorium-forming grass pathogen Claviceps purpurea needs a Fus3/Pmk1 homologous mitogen-activated protein kinase for colonization of rye ovarian tissue

Claviceps purpurea is a common pathogen of a wide range of grasses and cereals that is able to establish a stable, balanced interaction with its host plant and is considered a biotroph. It does not form special penetration structures such as appressoria. To study the signaling processes involved in this special host-pathogen interaction, we have cloned a gene, cpmk1, encoding a mitogen-activated protein (MAP) kinase that shows significant homology to Fus3 of Saccharomyces cerevisiae and to pmk1 of Magnaporthe grisea. Using a gene-replacement approach, we isolated a Acpmk1 mutant and characterized it in detail. Loss of CPMK1 has no obvious effect on vegetative properties (such as growth rate, morphology, and conidia formation); however, infection tests on rye show that the mutant is unable to colonize rye tissue, i.e., it appears to be completely nonpathogenic. Complementation of the mutant with a wild-type copy of cpmk1 fully restores its pathogenicity, confirming that this MAP kinase is essential for infection of rye by C. purpurea. Transformation of the delta pmk1 mutant of M. grisea with a complete copy of cpmk1 (including the C. purpurea promoter) fully restored its ability to form appressoria and its pathogenicity on barley. Although both fungi drastically differ in their pathogenic strategies, this result indicates that the signal pathway involving CPMK1 is highly conserved.