Stuart B. Krasnoff

NPS6, Encoding a Nonribosomal Peptide Synthetase Involved in Siderophore-Mediated Iron Metabolism, Is a Conserved Virulence Determinant of Plant Pathogenic Ascomycetes

NPS6, encoding a nonribosomal peptide synthetase, is a virulence determinant in the maize (Zea mays) pathogen Cochliobolus heterostrophus and is involved in tolerance to H2O2. Deletion of NPS6 orthologs in the rice (Oryza sativa) pathogen, Cochliobolus miyabeanus, the wheat (Triticum aestivum) pathogen, Fusarium graminearum, and the Arabidopsis thaliana pathogen, Alternaria brassicicola, resulted in reduced virulence and hypersensitivity to H2O2. Introduction of the NPS6 ortholog from the saprobe Neurospora crassa to the Δnps6 strain of C. heterostrophus restored wild-type virulence to maize and tolerance to H2O2, demonstrating functional conservation in filamentous ascomycete phytopathogens and saprobes. Increased sensitivity to iron depletion was identified as a conserved phenotype of Δnps6 strains. Exogenous application of iron enhanced the virulence of Δnps6 strains of C. heterostrophus, C. miyabeanus, F. graminearum, and A. brassicicola to each host. NPS6 is responsible for the biosynthesis of extracellular siderophores by C. heterostrophus, F. graminearum, and A. brassicicola. Application of the extracellular siderophore of A. brassicicola restored wild-type virulence of the ΔAbnps6 strain to Arabidopsis. It is proposed that the role of extracellular siderophores in fungal virulence to plants is to supply an essential nutrient, iron, to their producers in planta and not to act as phytotoxins, depriving their hosts of iron.

The txtAB genes of the plant pathogen Streptomyces acidiscabies encode a peptide synthetase required for phytotoxin thaxtomin A production and pathogenicity.

Four Streptomyces species have been described as the causal agents of scab disease, which affects economically important root and tuber crops worldwide. These species produce a family of cyclic dipeptides, the thaxtomins, which alone mimic disease symptomatology. Structural considerations suggest that thaxtomins are synthesized non‐ribosomally. Degenerate oligonucleotide primers were used to amplify conserved portions of the acyladenylation module of peptide synthetase genes from genomic DNA of representatives of the four species. Pairwise Southern hybridizations identified a peptide synthetase acyladenylation module conserved among three species. The complete nucleotide sequences of two peptide synthetase genes (txtAB) were determined from S. acidiscabies 84.104 cosmid library clones. The organization of the deduced TxtA and TxtB peptide synthetase catalytic domains is consistent with the formation of N‐methylated cyclic dipeptides such as thaxtomins. Based on high‐performance liquid chromatography (HPLC) analysis, thaxtomin A production was abolished in txtA gene disruption mutants. Although the growth and morphological characteristics of the mutants were identical to those of the parent strain, txtA mutants were avirulent on potato tubers. Moreover, introduction of the thaxtomin synthetase cosmid into a txtA mutant restored both pathogenicity and thaxtomin A production, demonstrating a critical role for thaxtomins in pathogenesis.

Secondary metabolites from entomopathogenic Hypocrealean fungi

Covering: up to the end of February 2010 This review surveys the natural products described from entomopathogenic Hypocrealean fungi, including their structures, biological activities, potential utilities in medicine, roles in entomopathogenesis, and known or predicted biosynthetic pathways.

Cytochrome P450–catalyzed L-tryptophan nitration in thaxtomin phytotoxin biosynthesis

Thaxtomin phytotoxins produced by plant-pathogenic Streptomyces species contain a nitro group that is essential for phytotoxicity. The N,N’-dimethyldiketopiperazine core of thaxtomins is assembled from L-phenylalanine and L-4-nitrotryptophan by a nonribosomal peptide synthetase and nitric oxide synthase-generated NO is incorporated into the nitro group, but the biosynthesis of the non-proteinogenic amino acid L-4-nitrotryptophan is unclear. Here we report that TxtE, a unique cytochrome P450, catalyzes L-tryptophan nitration using NO and O2.

Intracellular siderophores are essential for ascomycete sexual development in heterothallic Cochliobolus heterostrophus and homothallic Gibberella zeae.

ABSTRACT Connections between fungal development and secondary metabolism have been reported previously, but as yet, no comprehensive analysis of a family of secondary metabolites and their possible role in fungal development has been reported. In the present study, mutant strains of the heterothallic ascomycete Cochliobolus heterostrophus, each lacking one of 12 genes (NPS1 to NPS12) encoding a nonribosomal peptide synthetase (NRPS), were examined for a role in sexual development. One type of strain (Δnps2) was defective in ascus/ascospore development in homozygous Δnps2 crosses. Homozygous crosses of the remaining 11 Δnps strains showed wild-type (WT) fertility. Phylogenetic, expression, and biochemical analyses demonstrated that the NRPS encoded by NPS2 is responsible for the biosynthesis of ferricrocin, the intracellular siderophore of C. heterostrophus. Functional conservation of NPS2 in both heterothallic C. heterostrophus and the unrelated homothallic ascomycete Gibberella zeae was demonstrated. G. zeae Δnps2 strains are concomitantly defective in intracellular siderophore (ferricrocin) biosynthesis and sexual development. Exogenous application of iron partially restored fertility to C. heterostrophus and G. zeae Δnps2 strains, demonstrating that abnormal sexual development of Δnps2 strains is at least partly due to their iron deficiency. Exogenous application of the natural siderophore ferricrocin to C. heterostrophus and G. zeae Δnps2 strains restored WT fertility. NPS1, a G. zeae NPS gene that groups phylogenetically with NPS2, does not play a role in sexual development. Overall, these data demonstrate that iron and intracellular siderophores are essential for successful sexual development of the heterothallic ascomycete C. heterostrophus and the homothallic ascomycete G. zeae.

Dihydropyrrolizine attractants for arctiid moths that visit plants containing pyrrolizidine alkaloids

Adults of three species of arctiid moths (Cisseps fulvicollis, Ctenucha virginia, andHalysidota tessellaris) are attracted to plants that contain pyrrolizidine alkaloids (PAs). The moths use olfactory cues to locate these plants, then feed on leaves, flowers, and roots with the proboscis. To investigate the chemical basis of attraction, sticky traps were baited with roots of a PA-containing plant,Eupatorium maculatum, alkaloids ofE. maculatum, and several derivatives of these alkaloids. Volatile derivatives of the bicyclic pyrrolizidine skeleton attracted all three arctiid species. The dihydropyrrolizines, (S)-(+)-hydroxydanaidal and (R)-(−)-hydroxydanaidal, proved to be the most attractive compounds tested, accounting for over 70% of the moths captured. Different alkaloid derivatives attracted different proportions of male and femaleCisseps. Both (S)-(+)-hydroxydanaidal (52% male) and (R)-(−)-hydroxydanaidal (71% male) attracted a significantly lower percentage ofCisseps males thanE. maculatum roots (87% male).Cisseps males possess eversible scent organs (coremata) that are displayed during courtship. Analysis of corematal extracts revealed the presence of hydroxydanaidal.Cisseps moths thus resemble danaine and ithomiine butterflies, both in their attraction to PA sources and in the presence of PA derivatives in the male scent organs.

Tolypocladium inflatum is the anamorph of Cordyceps subsessilis

A collection of Cordyceps subsessilis is doc- umented. Axenic cultures of single part ascospores produced an anamorph attributable to the common soil hyphomycete Tolypocladium inflatum (= T ni- veum). Efrapeptins were identified in culture filtrates of the anamorph. The efrapeptin profile of the C. subsessilis anamorph was found to be similar to that

Loss of Plastoglobule Kinases ABC1K1 and ABC1K3 Causes Conditional Degreening, Modified Prenyl-Lipids, and Recruitment of the Jasmonic Acid Pathway

ABC1K1 and ABC1K3, two of the six plastoglobule ABC1K kinases, physically interact, and the double mutant displays a conditional senescent-like phenotype. ABC1K1 and 3 regulate prenyl-lipid metabolism and phosphorylation targets likely include tocopherol cyclase, VTE1. Plastoglobules function as specialized thylakoid microdomains with local enrichment of selected proteins and metabolites. Plastoglobules (PGs) are plastid lipid-protein particles. This study examines the function of PG-localized kinases ABC1K1 and ABC1K3 in Arabidopsis thaliana. Several lines of evidence suggested that ABC1K1 and ABC1K3 form a protein complex. Null mutants for both genes (abc1k1 and abc1k3) and the double mutant (k1 k3) displayed rapid chlorosis upon high light stress. Also, k1 k3 showed a slower, but irreversible, senescence-like phenotype during moderate light stress that was phenocopied by drought and nitrogen limitation, but not cold stress. This senescence-like phenotype involved degradation of the photosystem II core and upregulation of chlorophyll degradation. The senescence-like phenotype was independent of the EXECUTER pathway that mediates genetically controlled cell death from the chloroplast and correlated with increased levels of the singlet oxygen–derived carotenoid β-cyclocitral, a retrograde plastid signal. Total PG volume increased during light stress in wild type and k1 k3 plants, but with different size distributions. Isolated PGs from k1 k3 showed a modified prenyl-lipid composition, suggesting reduced activity of PG-localized tocopherol cyclase (VTE1), and was consistent with loss of carotenoid cleavage dioxygenase 4. Plastid jasmonate biosynthesis enzymes were recruited to the k1 k3 PGs but not wild-type PGs, while pheophytinase, which is involved in chlorophyll degradation, was induced in k1 k3 and not wild-type plants and was localized to PGs. Thus, the ABC1K1/3 complex contributes to PG function in prenyl-lipid metabolism, stress response, and thylakoid remodeling.

Agrobacterium-Mediated Disruption of a Nonribosomal Peptide Synthetase Gene in the Invertebrate Pathogen Metarhizium anisopliae Reveals a Peptide Spore Factor

ABSTRACT Numerous secondary metabolites have been isolated from the insect pathogenic fungus Metarhizium anisopliae, but the roles of these compounds as virulence factors in disease development are poorly understood. We targeted for disruption by Agrobacterium tumefaciens-mediated transformation a putative nonribosomal peptide synthetase (NPS) gene, MaNPS1. Four of six gene disruption mutants identified were examined further. Chemical analyses showed the presence of serinocyclins, cyclic heptapeptides, in the extracts of conidia of control strains, whereas the compounds were undetectable in ΔManps1 mutants treated identically or in other developmental stages, suggesting that MaNPS1 encodes a serinocyclin synthetase. Production of the cyclic depsipeptide destruxins, M. anisopliae metabolites also predicted to be synthesized by an NPS, was similar in ΔManps1 mutant and control strains, indicating that MaNPS1 does not contribute to destruxin biosynthesis. Surprisingly, a MaNPS1 fragment detected DNA polymorphisms that correlated with relative destruxin levels produced in vitro, and MaNPS1 was expressed concurrently with in vitro destruxin production. ΔManps1 mutants exhibited in vitro development and responses to external stresses comparable to control strains. No detectable differences in pathogenicity of the ΔManps1 mutants were observed in bioassays against beet armyworm and Colorado potato beetle in comparison to control strains. This is the first report of targeted disruption of a secondary metabolite gene in M. anisopliae, which revealed a novel cyclic peptide spore factor.

Identification of the antibiotic phomalactone from the entomopathogenic fungusHirsutella thompsonii var.synnematosa.

Dichloromethane extracts of culture broth from three strains of the entomopathogenic fungusHirsutella thompsonii var.synnematosa were toxic to two species of tephritid fruit fly and inhibited conidial germination in vitro in several other entomopathogenic fungi includingBeauveria bassiana, Tolypocladium spp., andMetarhizium anisopliae. A major metabolite, toxic to apple maggot,Rhagoletis pomonella, and inhibitory to conidial germination inB. bassiana, was isolated and identified as the antibiotic (+)-phomalactone, 6-(1-propenyl)-5,-6-dihydro-5-hydroxypyran-2-one. This is the first biologically active compound of low molecular weight isolated from the genusHirsutella.