Vigorous hyphal swelling during growth in putrescine medium is seen to a lesser extent in Δ<i>fgp1</i>.

<p>Mycelium of wild type, a <i>FGP1</i> deletion strain and a complemented strain growing in control minimal medium (upper panel) or putrescine (middle panel) at 40 HPI. A representative area of the mycelium observed under the microscope is given for each strain. Mycelium of wild type PH-1 and the complemented strain show branching and numerous hyphae with bulbous structures when compared to mycelium grown in control minimal medium. Mycelium of the <i>FGP1</i> deletion strain also shows branching but fewer hyphae with bulbous structures. Greater magnifications of the boxed frames from the middle pictures (lower panel) show that in the mycelium of the wild type and complemented strains many bulbous structures are formed (left and right pictures in the lower panel). The mycelium of the Δ<i>fgp1</i> strain shows some bulbous structures but less when compared to with wild type and complemented strain (middle picture in the lower panel). Bulbous structures are indicated by arrows in the middle and lower panels.</p

Fgp1 mediates expression of genes in several gene clusters.

<p>Histograms represent mean relative expression levels from three replicate experiments. Error bars represent standard deviation. A) Expression means for genes from the <i>TRI</i> cluster in wild type PH-1 grown in putrescine (black bars) or during wheat head infection (dark gray bars) and in a <i>FGP1</i> deletion strain grown in putrescine (white bars) or during wheat head infection (light gray bars). B) Expression means for genes from the butenolide biosynthetic cluster in wild type PH-1 during wheat head infection (black bars) and in a <i>FGP1</i> deletion strain during wheat head infection (white bars). C) Expression means for genes from the aurofusarin biosynthetic cluster in wild type PH-1 grown in putrescine medium (black bars) or for a <i>FGP1</i> deletion strain grown in putrescine medium (white bars). D) Expression means for genes from the NPS8 cluster in wild type PH-1 grown in putrescine medium (black bars) or a <i>FGP1</i> deletion strain grown in putrescine medium (white bars).</p

<i>FGP1</i> is required for pathogenicity and trichothecene accumulation but <i>FGP2</i> is not.

<p>A) Average number of diseased spikelets of 30 plants after mock inoculated with H₂O, inoculated with four independent <i>FGP1</i> deletion mutants, with wild type PH-1 or with four independent <i>FGP1</i> complemented transformants. Wheat heads were point inoculated and disease spread through adjacent spikelets was enumerated after 14 days. Error bars indicate standard deviation. B) Photographs from wild type inoculated wheat head (lower head on the left), wheat heads inoculated with the four <i>FGP1</i> deletion mutants (upper four heads on the left) and wheat heads inoculated with the four <i>FGP1</i> complemented transformants (four heads on the right). Arrows indicate the inoculated spikelet in each head photographed. C) DON and 15-ADON concentrations in the inoculated spikelet 14 days after inoculation with H₂O, four independent <i>FGP1</i> deletion mutants, wild type PH-1 or four <i>FGP1</i> complemented transformants. Asterisks mean no toxin was detected. D) Average number of diseased spikelets of 30 plants after mock inoculated with H₂O or inoculated with four independent <i>FGP2</i> deletion mutants or wild type PH-1. Wheat heads were point inoculated and spread through adjacent spikelets was enumerated after 14 days. Error bars indicate standard deviation. E) DON and 15-ADON concentrations in the inoculated spikelet 14 days after inoculation with H₂O, four independent <i>FGP2</i> deletion mutants or wild type PH-1. Asterisk means no toxin was detected.</p

Fgp1 is involved in conidiogenesis as well as in ascospore formation.

<p>A) Macroconidia production was assessed in three experiments each in two replicas on mung bean agar (MBA) plates and counted in a haemocytometer. B) Average macroconidium length was determined by measuring 30 macroconidia of each strain. C) Photos of a representative wild type PH-1 macroconidium (left), three representative conidia formed by the <i>FGP1</i> deletion mutant (middle) and a representative <i>FGP1</i> complementation strain macroconidium (right). D) Ascospore production by wild type PH-1, two <i>FGP1</i> deletion mutants and two <i>FGP1</i> complementation strains was assessed in four replicas for each strain on carrot agar plates two weeks after the mycelium was knocked down using a 2.5% Tween-60 solution. Ascospores were counted with a haemocytometer.</p

Alignment of four Wor1-like <i>Fusarium</i> orthologs.

<p>A) Protein sequence alignment of four <i>Fusarium</i> Wor1-like proteins: Fo: Sge1 (FOXG_10510) from <i>F. oxysporum</i>, FVEG_09150 from <i>F. verticillioides</i>, Fg: Fgp1 (FGSG_12164) from <i>F. graminearum</i> and Fs: Fs_81912 from <i>F. solani</i> (<i>Nectria haematococca</i>). Conserved and similar residues are shaded grey. The boxed threonine residue at position 68 is a conserved putative phosphorylation site. The solid black line represents the WOPRa box and the dashed black line the WOPRb box. The C-terminal part is indicated by grey background. The protein alignment was created using MacVector version 10.6.0. B) Table in which the percentages of similarities are given for each of the Wor1-like ortholog (Fg: Fgp1 (FGSG_12164) from <i>F. graminearum</i>, Fv: FVEG_09150 from <i>F. verticillioides</i>, Fo: Sge1 (FOXG_10510) from <i>F. oxysporum</i> and Fs: Fs_81912 from <i>F. solani</i> (<i>Nectria haematococca</i>)) to each of the other protein in the alignment presented in A. Different percentages are presented for the N-terminal portion (white cells) and the C-terminal portion (grey cells).</p

The Δ<i>fgp1</i> strain is unable to pass through the rachis node.

<p>The infection behavior of the <i>FGP1</i> and <i>FGP2</i> deletion mutants in the wheat head was determined by light and fluorescence microscopy. Spikelets were inoculated and assessed after two to three days for fungal spread within the floral tissue. A) The palea and lemma of a flower inoculated with Δ<i>fgp1</i> show browning. The glume remains green, as no fungal colonization occurs within this portion of the flower. B) The palea, lemma and glume of a flower inoculated with Δ<i>fgp2</i> all show browning. C) The GFP expressing Δ<i>fgp1</i> strain grows inside the flower but does not penetrate the rachis node. Patches of GFP-expressing fungal mycelium are observed along palea and lemma inside the flower (arrow) but no GFP is seen in the rachis node (red circle) or beyond into the rachis. D) The GFP expressing virulent Δ<i>fgp2</i> strain grows in the flower and penetrates the rachis node. Patches of GFP expressing fungal mycelium are observed along palea and lemma inside the flower and GFP is seen within the rachis node (red circle), the rachis and beyond.</p

The <i>FGP1</i> deletion strain does not produce trichothecene toxins in putrescine medium.

<p>A) Histogram of toxin concentration (parts per million - ppm) of DON and 15-ADON measured in putrescine medium after one week of growth of wild type, four Δ<i>fgp1</i> strains and two complemented strains. Asterisks indicate no toxin was detected. B) Histogram of toxin concentration (parts per million - ppm) of DON, 15-ADON and 3-ADON measured in putrescine medium with samples taken at 8, 16, 24, 32, 40, and 48 hours post inoculation (HPI) with wild type PH-1 or a <i>FGP1</i> deletion strain. Detectable toxin concentrations are found at 32, 40 and 48 HPI in the wild type PH-1 but not in the <i>FGP1</i> deletion strain at any time point. Asterisks indicate no toxin was detected. A northern blot of RNA obtained from the same samples shows expression of the <i>TRI</i> gene, <i>TRI14</i>, after 32, 40 and 48 HPI in the wild type PH-1 but not in the <i>FGP1</i> deletion strain at any time point. The loading control gene <i>ACTIN</i> is expressed equally at all time points for both wild type and the <i>FGP1</i> deletion strain.</p

Steered molecular dynamics for studying ligand unbinding of ecdysone receptor

<p>Ecdysone receptor (EcR) is an important target for pesticide design. Ligand binding regulates EcR transcriptional activity similar to other nuclear receptors; however, the pathways by which ligands enter and leave the EcR remain poorly understood. Here, we performed computational studies to identify unbinding pathways of an ecdysone agonist [the selective ecdysone agonist, BYI06830] from the EcR ligand binding domain (EcR LBD). BYI06830 can dissociate from EcR LBD via four different pathways with little effect on receptor structure. By comparing the potential of mean force (PMF) of four pathways, path 2 was considered to be the most likely exit path for BYI06830, which was located in the cleft formed by the H3-H4 loop, H6-H7 loop, and the H11 C-terminus. Furthermore, structural features along path 2 were analyzed and the structural snapshots of the metastable and transition states were isolated to illustrate the unbinding mechanism of ecdysone agonist from EcR LBD.</p

Structural, EPR, and Mössbauer Characterization of (μ-Alkoxo)(μ-Carboxylato)Diiron(II,III) Model Complexes for the Active Sites of Mixed-Valent Diiron Enzymes

To obtain structural and spectroscopic models for the diiron­(II,III) centers in the active sites of diiron enzymes, the (μ-alkoxo)­(μ-carboxylato)­diiron­(II,III) complexes [Fe^IIFe^III(<i>N</i>-Et-HPTB)­(O₂CPh)­(NCCH₃)₂]­(ClO₄)₃ (<b>1</b>) and [Fe^IIFe^III(<i>N</i>-Et-HPTB)­(O₂CPh)­(Cl)­(HOCH₃)]­(ClO₄)₂ (<b>2</b>) (<i>N</i>-Et-HPTB = <i>N,N,N</i>′<i>,N</i>′-tetrakis­(2-(1-ethyl-benzimidazolylmethyl))-2-hydroxy-1,3-diaminopropane) have been prepared and characterized by X-ray crystallography, UV–visible absorption, EPR, and Mössbauer spectroscopies. Fe1–Fe2 separations are 3.60 and 3.63 Å, and Fe1–O1–Fe2 bond angles are 128.0° and 129.4° for <b>1</b> and <b>2</b>, respectively. Mössbauer and EPR studies of <b>1</b> show that the Fe^III (<i>S</i>_A = 5/2) and Fe^II (<i>S</i>_B = 2) sites are antiferromagnetically coupled to yield a ground state with <i>S</i> = 1/2 (<i>g</i> <b>=</b> 1.75, 1.88, 1.96); Mössbauer analysis of solid <b>1</b> yields <i>J</i> = 22.5 ± 2 cm^–1 for the exchange coupling constant (H = <i>J</i><b>S</b>_A·<b>S</b>_B convention). In addition to the <i>S</i> = 1/2 ground-state spectrum of <b>1</b>, the EPR signal for the <i>S</i> = 3/2 excited state of the spin ladder can also be observed, the first time such a signal has been detected for an antiferromagnetically coupled diiron­(II,III) complex. The anisotropy of the ⁵⁷Fe magnetic hyperfine interactions at the Fe^III site is larger than normally observed in mononuclear complexes and arises from admixing <i>S</i> > 1/2 excited states into the <i>S</i> = 1/2 ground state by zero-field splittings at the two Fe sites. Analysis of the “<i>D</i>/<i>J</i>” mixing has allowed us to extract the zero-field splitting parameters, local <i>g</i> values, and magnetic hyperfine structural parameters for the individual Fe sites. The methodology developed and followed in this analysis is presented in detail. The spin Hamiltonian parameters of <b>1</b> are related to the molecular structure with the help of DFT calculations. Contrary to what was assumed in previous studies, our analysis demonstrates that the deviations of the <i>g</i> values from the free electron value (<i>g</i> = 2) for the antiferromagnetically coupled diiron­(II,III) core in complex <b>1</b> are predominantly determined by the anisotropy of the effective <i>g</i> values of the ferrous ion and only to a lesser extent by the admixture of excited states into ground-state ZFS terms (<i>D</i>/<i>J</i> mixing). The results for <b>1</b> are discussed in the context of the data available for diiron­(II,III) clusters in proteins and synthetic diiron­(II,III) complexes

Structural, EPR, and Mössbauer Characterization of (μ-Alkoxo)(μ-Carboxylato)Diiron(II,III) Model Complexes for the Active Sites of Mixed-Valent Diiron Enzymes

To obtain structural and spectroscopic models for the diiron­(II,III) centers in the active sites of diiron enzymes, the (μ-alkoxo)­(μ-carboxylato)­diiron­(II,III) complexes [Fe^IIFe^III(<i>N</i>-Et-HPTB)­(O₂CPh)­(NCCH₃)₂]­(ClO₄)₃ (<b>1</b>) and [Fe^IIFe^III(<i>N</i>-Et-HPTB)­(O₂CPh)­(Cl)­(HOCH₃)]­(ClO₄)₂ (<b>2</b>) (<i>N</i>-Et-HPTB = <i>N,N,N</i>′<i>,N</i>′-tetrakis­(2-(1-ethyl-benzimidazolylmethyl))-2-hydroxy-1,3-diaminopropane) have been prepared and characterized by X-ray crystallography, UV–visible absorption, EPR, and Mössbauer spectroscopies. Fe1–Fe2 separations are 3.60 and 3.63 Å, and Fe1–O1–Fe2 bond angles are 128.0° and 129.4° for <b>1</b> and <b>2</b>, respectively. Mössbauer and EPR studies of <b>1</b> show that the Fe^III (<i>S</i>_A = 5/2) and Fe^II (<i>S</i>_B = 2) sites are antiferromagnetically coupled to yield a ground state with <i>S</i> = 1/2 (<i>g</i> <b>=</b> 1.75, 1.88, 1.96); Mössbauer analysis of solid <b>1</b> yields <i>J</i> = 22.5 ± 2 cm^–1 for the exchange coupling constant (H = <i>J</i><b>S</b>_A·<b>S</b>_B convention). In addition to the <i>S</i> = 1/2 ground-state spectrum of <b>1</b>, the EPR signal for the <i>S</i> = 3/2 excited state of the spin ladder can also be observed, the first time such a signal has been detected for an antiferromagnetically coupled diiron­(II,III) complex. The anisotropy of the ⁵⁷Fe magnetic hyperfine interactions at the Fe^III site is larger than normally observed in mononuclear complexes and arises from admixing <i>S</i> > 1/2 excited states into the <i>S</i> = 1/2 ground state by zero-field splittings at the two Fe sites. Analysis of the “<i>D</i>/<i>J</i>” mixing has allowed us to extract the zero-field splitting parameters, local <i>g</i> values, and magnetic hyperfine structural parameters for the individual Fe sites. The methodology developed and followed in this analysis is presented in detail. The spin Hamiltonian parameters of <b>1</b> are related to the molecular structure with the help of DFT calculations. Contrary to what was assumed in previous studies, our analysis demonstrates that the deviations of the <i>g</i> values from the free electron value (<i>g</i> = 2) for the antiferromagnetically coupled diiron­(II,III) core in complex <b>1</b> are predominantly determined by the anisotropy of the effective <i>g</i> values of the ferrous ion and only to a lesser extent by the admixture of excited states into ground-state ZFS terms (<i>D</i>/<i>J</i> mixing). The results for <b>1</b> are discussed in the context of the data available for diiron­(II,III) clusters in proteins and synthetic diiron­(II,III) complexes