A Study on Auto-Catalysis and Product Inhibition: A Nucleophilic Aromatic Substitution Reaction Catalysed within the Cavity of an Octanuclear Coordination Cage.

The ability of an octanuclear cubic coordination cage to catalyse a nucleophilic aromatic substitution reaction on a cavity-bound guest was studied with 2,4-dinitrofluorobenzene (DNFB) as the guest/substrate. It was found that DNFB undergoes a catalysed reaction with hydroxide ions within the cavity of the cubic cage (in aqueous buffer solution, pH 8.6). The rate enhancement of kcat/kuncat was determined to be 22, with cavity binding of the guest being required for catalysis to occur. The product, 2,4-dinitrophenolate (DNP), remained bound within the cavity due to electrostatic stabilisation and exerts two apparently contradictory effects: it initially auto-catalyses the reaction when present at low concentrations, but at higher concentrations inhibits catalysis when a pair of DNP guests block the cavity. When encapsulated, the UV/Vis absorption spectrum of DNP is red-shifted when compared to the spectrum of free DNP in aqueous solution. Further investigations using other aromatic guests determined that a similar red-shift on cavity binding also occurred for 4-nitrophenolate (4NP) at pH 8.6. The red-shift was used to determine the stoichiometry of guest binding of DNP and 4NP within the cage cavity, which was confirmed by structural analysis with X-ray crystallography; and was also used to perform catalytic kinetic studies in the solution-state.

Imaging protein aggregates in the serum and cerebrospinal fluid in Parkinson's disease.

Aggregation of α-synuclein plays a key role in the development of Parkinson's disease. Soluble aggregates are present not only within human brain but also the CSF and blood. Characterizing the aggregates present in these biofluids may provide insights into disease mechanisms and also have potential for aiding diagnosis. We used two optical single-molecule imaging methods called aptamer DNA-PAINT and single-aggregate confocal fluorescence, together with high-resolution atomic force microscopy for specific detection and characterization of individual aggregates with intermolecular ß-sheet structure, present in the CSF and serum of 15 early stage Parkinson's disease patients compared to 10 healthy age-matched controls. We found aggregates ranging in size from 20 nm to 200 nm, in both CSF and serum. There was a difference in aggregate size distribution between Parkinson's disease and control groups with a significantly increased number of larger aggregates (longer than 150 nm) in the serum of patients with Parkinson's disease. To determine the chemical composition of the aggregates, we performed aptamer DNA-PAINT on serum following α-synuclein and amyloid-ß immunodepletion in an independent cohort of 11 patients with early stage Parkinson's disease and 10 control subjects. ß-Sheet aggregates in the serum of Parkinson's disease patients were found to consist of, on average, 50% α-synuclein and 50% amyloid-ß in contrast to 30% α-synuclein and 70% amyloid-ß in control serum [the differences in the proportion of these aggregates were statistically significant between diseased and control groups (P = 1.7 × 10-5 for each species)]. The ratio of the number of ß-sheet α-synuclein aggregates to ß-sheet amyloid-ß aggregates in serum extracted using our super-resolution method discriminated Parkinson's disease cases from controls with an accuracy of 98.2% (AUC = 98.2%, P = 4.3 × 10-5). Our data suggest that studying the protein aggregates present in serum can provide information about the disruption of protein homeostasis occurring in Parkinson's disease and warrants further investigation as a potential biomarker of disease.

On-Farm Evaluations of Anaerobic Soil Disinfestation and Grafting for Management of a Widespread Soilborne Disease Complex in Protected Culture Tomato Production.

Tomato production in Ohio protected culture systems is hindered by a soilborne disease complex consisting of corky root rot (Pyrenochaeta lycopersici), black dot root rot (Colletotrichum coccodes), Verticillium wilt (Verticillium dahliae), and root-knot (Meloidogyne hapla and M. incognita). In a survey of 71 high tunnels, C. coccodes was detected in 90% of high tunnels, and P. lycopersici (46%), V. dahliae (48%), and Meloidogyne spp. (45%) were found in nearly half of high tunnels. Anaerobic soil disinfestation (ASD) with wheat bran (20.2 Mg/ha) plus molasses (10.1 Mg/ha) and grafting onto 'Maxifort' or 'Estamino' rootstocks were evaluated in high tunnels on five farms. In post-ASD bioassays of trial soils, root and taproot rot severity were significantly reduced after ASD, and root-knot galling was also reduced by ASD. Soilborne pathogenic fungi were isolated less frequently from bioassay plants grown in ASD-treated soils than control soils. Similar results were observed in tomato plants grown in high tunnels. Root rot was significantly reduced by ASD in nearly all trials. Corky root rot severity was highest in nongrafted plants grown in nontreated soils, and the lowest levels of corky root rot were observed in 'Maxifort'-grafted plants. Black dot root rot severity was higher or equivalent in grafted plants compared with nongrafted plants. Root-knot severity was lower in plants grown in ASD-treated soils in high tunnels compared with plants grown in control soils, but grafting did not significantly decrease root-knot severity. However, soil treatment did not significantly affect yield, and grafting led to inconsistent impacts on yield.

One Guest or Two? A Crystallographic and Solution Study of Guest Binding in a Cubic Coordination Cage.

A crystallographic investigation of a series of host-guest complexes in which small-molecule organic guests occupy the central cavity of an approximately cubic M8 L12 coordination cage has revealed some unexpected behaviour. Whilst some guests form 1:1 H⋅G complexes as we have seen before, an extensive family of bicyclic guests-including some substituted coumarins and various saturated analogues-form 1:2 H⋅G2 complexes in the solid state, despite the fact that solution titrations are consistent with 1:1 complex formation, and the combined volume of the pair of guests significantly exceeds the Rebek 55±9 % packing for optimal guest binding, with packing coefficients of up to 87 %. Re-examination of solution titration data for guest binding in two cases showed that, although conventional fluorescence titrations are consistent with 1:1 binding model, alternative forms of analysis-Job plot and an NMR titration-at higher concentrations do provide evidence for 1:2 H⋅G2 complex formation. The observation of guests binding in pairs in some cases opens new possibilities for altered reactivity of bound guests, and also highlights the recently articulated difficulties associated with determining stoichiometry of supramolecular complexes in solution.

Coordination-Cage-Catalysed Hydrolysis of Organophosphates: Cavity- or Surface-Based?

The hydrophobic central cavity of a water-soluble M8 L12 cubic coordination cage can accommodate a range of phospho-diester and phospho-triester guests such as the insecticide "dichlorvos" (2,2-dichlorovinyl dimethyl phosphate) and the chemical warfare agent analogue di(isopropyl) chlorophosphate. The accumulation of hydroxide ions around the cationic cage surface due to ion-pairing in solution generates a high local pH around the cage, resulting in catalysed hydrolysis of the phospho-triester guests. A series of control experiments unexpectedly demonstrates that-in marked contrast to previous cases-it is not necessary for the phospho-triester substrates to be bound inside the cavity for catalysed hydrolysis to occur. This suggests that catalysis can occur on the exterior surface of the cage as well as the interior surface, with the exterior-binding catalysis pathway dominating here because of the small binding constants for these phospho-triester substrates in the cage cavity. These observations suggest that cationic but hydrophobic surfaces could act as quite general catalysts in water by bringing substrates into contact with the surface (via the hydrophobic effect) where there is also a high local concentration of anions (due to ion pairing/electrostatic effects).

One Guest or Two? A Crystallographic and Solution Study of Guest Binding in a Cubic Coordination Cage.

Invited for the cover of this issue is the group of Michael D. Ward at the University of Warwick. The image depicts structures of the host cage containing one guest or two guests. Read the full text of the article at 10.1002/chem.201905499.

Photophysics of Cage/Guest Assemblies: Photoinduced Electron Transfer between a Coordination Cage Containing Osmium(II) Luminophores, and Electron-Deficient Bound Guests in the Central Cavity.

A heterometallic octanuclear coordination cage [Os4Zn4(Lnap)12]X16 (denoted Os•Zn; X = perchlorate or chloride) has been prepared (Lnap is a bis-bidentate bridging ligand containing two pyrazolyl-pyridine chelating units separated by a 1,5-naphthalenediyl spacer group). The {Os(NN)3}2+ units located at four of the eight vertices of the cube have a long-lived, phosphorescent 3MLCT excited state which is a stronger electron donor than [Ru(bipy)3]2+. The chloride form of Os•Zn is water-soluble and binds in its central cavity the hydrophobic electron-accepting organic guests 1,2,4,5-tetracyanobenzene, 1,4-naphthoquinone and 1-nitronaphthalene, with binding constants in the range 103-104 M-1, resulting in quenching of the phosphorescence arising from the Os(II) units. A crystal structure of an isostructural Co8 cage containing one molecule of 1,2,4,5-tetracyanobenzene as a guest inside the cavity has been determined. Ultrafast transient absorption measurements show formation of a charge-separated Os(III)/guest•- state arising from cage-to-guest photoinduced electron transfer; this state is formed within 13-21 ps, and decays on a time scale of ca. 200 ps. In the presence of a competing guest with a large binding constant (cycloundecanone) which displaces each electron-accepting quencher from the cage cavity, the charge-separated state is no longer observed. Further, a combination of mononuclear {Os(NN)3}2+ model complexes with the same electron-accepting species showed no evidence for formation of charge-separated Os(III)/guest•- states. These two control experiments indicate that the {Os(NN)3}2+ chromophores need to be assembled into the cage structure to bind the electron-accepting guests, and for PET to occur. These results help to pave the way for use of photoactive coordination cages as hosts for photoredox catalysis reactions on bound guests.

Fabrication and Characterization of Reconstituted Silk Microgels for the Storage and Release of Small Molecules.

Silk fibroin is a natural protein obtained from the Bombyx mori silkworm. In addition to being the key structural component in silkworm cocoons, it also has the propensity to self-assemble in vitro into hierarchical structures with desirable properties such as high levels of mechanical strength and robustness. Furthermore, it is an appealing biopolymer due to its biocompatability, low immunogenicity, and lack of toxicity, making it a prime candidate for biomedical material applications. Here, it is demonstrated that nanofibrils formed by reconstituted silk fibroin can be engineered into supramolecular microgels using a soft lithography-based microfluidic approach. Building on these results, a potential application for these protein microgels to encapsulate and release small molecules in a controlled manner is illustrated. Taken together, these results suggest that the tailored self-assembly of biocompatible and biodegradable silk nanofibrils can be used to generate functional micromaterials for a range of potential applications in the biomedical and pharmaceutical fields.

Catalysis in a Cationic Coordination Cage Using a Cavity-Bound Guest and Surface-Bound Anions: Inhibition, Activation, and Autocatalysis.

The Kemp elimination (reaction of benzisoxazole with base to give 2-cyanophenolate) is catalyzed in the cavity of a cubic M8L12 coordination cage because of a combination of (i) benzisoxazole binding in the cage cavity driven by the hydrophobic effect, and (ii) accumulation of hydroxide ions around the 16+ cage surface driven by ion-pairing. Here we show how reaction of the cavity-bound guest is modified by the presence of other anions which can also accumulate around the cage surface and displace hydroxide, inhibiting catalysis of the cage-based reaction. Addition of chloride or fluoride inhibits the reaction with hydroxide to the extent that a new autocatalytic pathway becomes apparent, resulting in a sigmoidal reaction profile. In this pathway the product 2-cyanophenolate itself accumulates around the cationic cage surface, acting as the base for the next reaction cycle. The affinity of different anions for the cage surface is therefore 2-cyanophenolate (generating autocatalysis) > chloride > fluoride (which both inhibit the reaction with hydroxide but cannot deprotonate the benzisoxazole guest) > hydroxide (default reaction pathway). The presence of this autocatalytic pathway demonstrates that a reaction of a cavity-bound guest can be induced with different anions around the cage surface in a controllable way; this was confirmed by adding different phenolates to the reaction, which accelerate the Kemp elimination to different extents depending on their basicity. This represents a significant step toward the goal of using the cage as a catalyst for bimolecular reactions between a cavity-bound guest and anions accumulated around the surface.

Extrinsic Amyloid-Binding Dyes for Detection of Individual Protein Aggregates in Solution.

Protein aggregation is a key molecular feature underlying a wide array of neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. To understand protein aggregation in molecular detail, it is crucial to be able to characterize the array of heterogeneous aggregates that are formed during the aggregation process. We present here a high-throughput method to detect single protein aggregates, in solution, from a label-free aggregation reaction, and we demonstrate the approach with the protein associated with Parkinson's disease, α-synuclein. The method combines single-molecule confocal microscopy with a range of amyloid-binding extrinsic dyes, including thioflavin T and pentameric formylthiophene acetic acid, and we show that we can observe aggregates at low picomolar concentrations. The detection of individual aggregates allows us to quantify their numbers. Furthermore, we show that this approach also allows us to gain structural insights from the emission intensity of the extrinsic dyes that are bound to aggregates. By analyzing the time evolution of the aggregate populations on a single-molecule level, we then estimate the fragmentation rate of aggregates, a key process that underlies the multiplication of pathological aggregates. We additionally demonstrate that the method permits the detection of these aggregates in biological samples. The capability to detect individual protein aggregates in solution opens up a range of new applications, including exploiting the potential of this method for high-throughput screening of human biofluids for disease diagnosis and early detection.

Absolute Quantification of Amyloid Propagons by Digital Microfluidics.

The self-replicating properties of proteins into amyloid fibrils is a common phenomenon and underlies a variety of neurodegenerative diseases. Because propagation-active fibrils are chemically indistinguishable from innocuous aggregates and monomeric precursors, their detection requires measurements of their replicative capacity. Here we present a digital amyloid quantitative assay (d-AQuA) with insulin as model protein for the absolute quantification of single replicative units, propagons. D-AQuA is a microfluidics-based technology that performs miniaturized simultaneous propagon-induced amplification chain reactions within hundreds to thousands of picoliter-sized droplets. At limiting dilutions, the d-AQuA reactions follow a stochastic regime indicative of the detection of single propagons. D-AQuA thus enables absolute quantification of single propagons present in a given sample at very low concentrations. The number of propagons quantified by d-AQuA was similar to that of fibrillar insulin aggregates detected by atomic-force microscopy and to an equivalent microplate-based assay, providing independent evidence for the identity of insulin propagons with a subset of morphologically defined protein aggregates. The sensitivity, precision, and accuracy of d-AQuA enable it to be suitable for multiple biotechnological and medical applications.

An Enterotoxin-Like Binary Protein from Pseudomonas protegens with Potent Nematicidal Activity.

Soil microbes are a major food source for free-living soil nematodes. It is known that certain soil bacteria have evolved systems to combat predation. We identified the nematode-antagonistic Pseudomonas protegens strain 15G2 from screening of microbes. Through protein purification we identified a binary protein, designated Pp-ANP, which is responsible for the nematicidal activity. This binary protein inhibits Caenorhabditis elegans growth and development by arresting larvae at the L1 stage and killing older-staged worms. The two subunits, Pp-ANP1a and Pp-ANP2a, are active when reconstituted from separate expression in Escherichia coli The binary toxin also shows strong nematicidal activity against three other free-living nematodes (Pristionchus pacificus, Panagrellus redivivus, and Acrobeloides sp.), but we did not find any activity against insects and fungi under test conditions, indicating specificity for nematodes. Pp-ANP1a has no significant identity to any known proteins, while Pp-ANP2a shows ∼30% identity to E. coli heat-labile enterotoxin (LT) subunit A and cholera toxin (CT) subunit A. Protein modeling indicates that Pp-ANP2a is structurally similar to CT/LT and likely acts as an ADP-ribosyltransferase. Despite the similarity, Pp-ANP shows several characteristics distinct from CT/LT toxins. Our results indicate that Pp-ANP is a new enterotoxin-like binary toxin with potent and specific activity to nematodes. The potency and specificity of Pp-ANP suggest applications in controlling parasitic nematodes and open an avenue for further research on its mechanism of action and role in bacterium-nematode interaction.IMPORTANCE This study reports the discovery of a new enterotoxin-like binary protein, Pp-ANP, from a Pseudomonas protegens strain. Pp-ANP shows strong nematicidal activity against Caenorhabditis elegans larvae and older-staged worms. It also shows strong activity on other free-living nematodes (Pristionchus pacificus, Panagrellus redivivus, and Acrobeloides sp.). The two subunits, Pp-ANP1a and Pp-ANP2a, can be expressed separately and reconstituted to form the active complex. Pp-ANP shows some distinct characteristics compared with other toxins, including Escherichia coli enterotoxin and cholera toxin. The present study indicates that Pp-ANP is a novel binary toxin and that it has potential applications in controlling parasitic nematodes and in studying toxin-host interaction.

A Quantitative Study of the Effects of Guest Flexibility on Binding Inside a Coordination Cage Host.

We have performed a systematic investigation of the effects of guest flexibility on their ability to bind in the cavity of a coordination cage host in water, using two sets of isomeric aliphatic ketones that differ only in the branching patterns of their alkyl chains. Apart from the expected increase in binding strength for C9 over C7 ketones associated with their greater hydrophobic surface area, within each isomeric set there is a clear inverse correlation between binding free energy and guest flexibility, associated with loss of conformational entropy. This can be parameterized by the number of rotatable C-C bonds in the guest, with each additional rotatable bond resulting in a penalty of around 2 kJ mol-1 in the binding free energy, in good agreement with values obtained from protein/ligand binding studies. We used the binding data for the new flexible guests to improve the scoring function that we had previously developed that allowed us to predict binding constants of relatively rigid guests in the cage cavity using the molecular docking programme GOLD (Genetic Optimisation of Ligand Docking). This improved scoring function resulted in a significant improvement in the ability of GOLD to predict binding constants for flexible guests, without any detriment to its ability to predict binding for more rigid guests.

Coordination-cage binding and catalysed hydrolysis of organophosphorus chemical warfare agent simulants.

The use of organophosphorus chemical warfare agents still remains an ongoing global threat. Here we investigate the binding of small-molecule organic guests including phosphate esters, sulfonate esters, carbonate esters and a sulfite ester - some of which act as simulants for organophosphorus chemical warfare agents - in the cavity of a water-soluble coordination cage. For several of these guest species, binding constants in the range 102 to 103 M-1 were determined in water/DMSO (98 : 2 v/v) solution, through a combination of fluorescence and 1H NMR spectroscopy, and subsequent fitting of titration data to a 1 : 1 binding isotherm model. For three cage/guest complexes crystallographic structure determinations were possible: in two cases (with guests phenyl methanesulfonate and phenyl propyl carbonate) the guest lies inside the cavity, forming a range of CH⋯O hydrogen-bonding interactions with the cage interior surface involving CH groups on the cationic cage surface that act as H-bond donors and O atoms on the guests that act as H-bond acceptors. In a third case, with the guest 4-nitrophenyl-methanesulfonate, the guest lies in the spaces outside a cage cavity between cages and forms weak CH⋯O interactions with the cage exterior surface: the cavity is occupied by a network of H-bonded water molecules, though this guest does show cavity binding in solution. For the isomeric guests 4-nitrophenyl-methanesulfonate and 4-nitrophenyl methyl sulfite, hydrolysis in water/DMSO (98 : 2 v/v) could be monitored colorimetrically via appearance of the 4-nitrophenolate anion; both showed accelerated hydrolysis rates in the presence of the host cage with second-order rate constants for the catalysed reactions in the range 10-3 to 10-2 M-1 s-1 at pH 9. The typical rate dependence on external pH and the increased reaction rates when chloride ions are present (which can bind inside the cavity and displace other cavity-bound guests) imply that the catalysed reaction actually occurs at the external surface of the cage rather than inside the cavity.

Chikungunya seroprevalence, force of infection, and prevalence of chronic disability after infection in endemic and epidemic settings: a systematic review, meta-analysis, and modelling study.

BACKGROUND: Chikungunya is an arboviral disease transmitted by Aedes aegypti and Aedes albopictus mosquitoes with a growing global burden linked to climate change and globalisation. We aimed to estimate chikungunya seroprevalence, force of infection (FOI), and prevalence of related chronic disability and hospital admissions in endemic and epidemic settings. METHODS: In this systematic review, meta-analysis, and modelling study, we searched PubMed, Ovid, and Web of Science for articles published from database inception until Sept 26, 2022, for prospective and retrospective cross-sectional studies that addressed serological chikungunya virus infection in any geographical region, age group, and population subgroup and for longitudinal prospective and retrospective cohort studies with data on chronic chikungunya or hospital admissions in people with chikungunya. We did a systematic review of studies on chikungunya seroprevalence and fitted catalytic models to each survey to estimate location-specific FOI (ie, the rate at which susceptible individuals acquire chikungunya infection). We performed a meta-analysis to estimate the proportion of symptomatic patients with laboratory-confirmed chikungunya who had chronic chikungunya or were admitted to hospital following infection. We used a random-effects model to assess the relationship between chronic sequelae and follow-up length using linear regression. The systematic review protocol is registered online on PROSPERO, CRD42022363102. FINDINGS: We identified 60 studies with data on seroprevalence and chronic chikungunya symptoms done across 76 locations in 38 countries, and classified 17 (22%) of 76 locations as endemic settings and 59 (78%) as epidemic settings. The global long-term median annual FOI was 0·007 (95% uncertainty interval [UI] 0·003-0·010) and varied from 0·0001 (0·00004-0·0002) to 0·113 (0·07-0·20). The highest estimated median seroprevalence at age 10 years was in south Asia (8·0% [95% UI 6·5-9·6]), followed by Latin America and the Caribbean (7·8% [4·9-14·6]), whereas median seroprevalence was lowest in the Middle East (1·0% [0·5-1·9]). We estimated that 51% (95% CI 45-58) of people with laboratory-confirmed symptomatic chikungunya had chronic disability after infection and 4% (3-5) were admitted to hospital following infection. INTERPRETATION: We inferred subnational heterogeneity in long-term average annual FOI and transmission dynamics and identified both endemic and epidemic settings across different countries. Brazil, Ethiopia, Malaysia, and India included both endemic and epidemic settings. Long-term average annual FOI was higher in epidemic settings than endemic settings. However, long-term cumulative incidence of chikungunya can be similar between large outbreaks in epidemic settings with a high FOI and endemic settings with a relatively low FOI. FUNDING: International Vaccine Institute.

The melon Fom-1-Prv resistance gene pair: Correlated spatial expression and interaction with a viral protein.

The head-to-head oriented pair of melon resistance genes, Fom-1 and Prv, control resistance to Fusarium oxysporum races 0 and 2 and papaya ringspot virus (PRSV), respectively. They encode, via several RNA splice variants, TIR-NBS-LRR proteins, and Prv has a C-terminal extra domain with a second NBS homologous sequence. In other systems, paired R-proteins were shown to operate by "labor division," with one protein having an extra integrated domain that directly binds the pathogen's Avr factor, and the second protein executing the defense response. We report that the expression of the two genes in two pairs of near-isogenic lines was higher in the resistant isoline and inducible by F. oxysporum race 2 but not by PRSV. The intergenic DNA region separating the coding sequences of the two genes acted as a bi-directional promoter and drove GUS expression in transgenic melon roots and transgenic tobacco plants. Expression of both genes was strong in melon root tips, around the root vascular cylinder, and the phloem and xylem parenchyma of tobacco stems and petioles. The pattern of GUS expression suggests coordinated expression of the two genes. In agreement with the above model, Prv's extra domain was shown to interact with the cylindrical inclusion protein of PRSV both in yeast cells and in planta.

Surface effects on functional amyloid formation.

Functional amyloids formed by the protein FapC in Pseudomonas bacteria are key structural components of Pseudomonas biofilms, which mediate chronic infections and also contribute to antimicrobial resistance. Here, we combine kinetic experiments with mechanistic modelling to probe the role of surfaces in FapC functional amyloid formation. We find that nucleation of new fibrils is predominantly heterogeneous in vitro, being catalysed by reaction vessel walls but not by the air/water interface. Removal of such interfaces by using microdroplets greatly slows heterogeneous nucleation and reveals a hitherto undetected fibril surface-catalysed "secondary nucleation" reaction step. We tune the degree of catalysis by varying the interface chemistry of the reaction vessel and by adding nanoparticles with tailored surface properties that catalyse fibril nucleation. In so doing, we discover that the rate of nucleation is controlled predominantly by the strength with which FapC binds to the catalytic sites on the interface, and by its surface area. Surprisingly, neither primary nucleation rate nor catalytic site binding strength appear closely correlated to the charge and hydrophilicity of the interface. This indicates the importance of considering experimental design in terms of surface chemistry of the reaction container while also highlighting the notion that fibril nucleation during protein aggregation is a heterogeneous process.

The amino acid permeases AAP3 and AAP6 are involved in root-knot nematode parasitism of Arabidopsis.

The root-knot nematode, Meloidogyne incognita, is an obligate parasite which depends entirely on the host plant for its nutrition. Root-knot nematodes induce the formation of a highly specialized feeding site consisting of several giant cells surrounded by a network of vascular tissues. Nutrients, including amino acids and sugars, are transferred apoplastically from the vascular tissues to the feeding site. Using Arabidopsis thaliana lacking the vascular-expressed amino acid permeases (AAP) AAP3 or AAP6, we demonstrate that disruption of amino acid transport can affect nematode parasitism. Nematode infestation levels are significantly reduced on the aap3 and aap6 mutants. AAP3 and AAP6 act distinctly in the transport of amino acids to the feeding site, as demonstrated by differences in their carrying capacity profiles. Furthermore, analyses of promoter: ß-glucuronidase lines show different expression patterns for AAP3 and AAP6 in infected roots. In the aap3-3 mutant, part of the decrease in infestation is connected to a defect in early infection, where juveniles enter but then leave the root. Both aap3-3 and aap6-1 produce fewer females and produce more adult male nematodes. Additionally, detrimental effects are observed in the nematodes harvested from aap3-3 and aap6-1 mutants, including decreased egg hatching and infectivity and lower levels of lipid reserves. The transport of amino acids by AAP3 and AAP6 is important for nematode infection and success of the progeny.

Functional genomics of a generalist parasitic plant: laser microdissection of host-parasite interface reveals host-specific patterns of parasite gene expression.

BACKGROUND: Orobanchaceae is the only plant family with members representing the full range of parasitic lifestyles plus a free-living lineage sister to all parasitic lineages, Lindenbergia. A generalist member of this family, and an important parasitic plant model, Triphysaria versicolor regularly feeds upon a wide range of host plants. Here, we compare de novo assembled transcriptomes generated from laser micro-dissected tissues at the host-parasite interface to uncover details of the largely uncharacterized interaction between parasitic plants and their hosts. RESULTS: The interaction of Triphysaria with the distantly related hosts Zea mays and Medicago truncatula reveals dramatic host-specific gene expression patterns. Relative to above ground tissues, gene families are disproportionally represented at the interface including enrichment for transcription factors and genes of unknown function. Quantitative Real-Time PCR of a T. versicolor ß-expansin shows strong differential (120x) upregulation in response to the monocot host Z. mays; a result that is concordant with our read count estimates. Pathogenesis-related proteins, other cell wall modifying enzymes, and orthologs of genes with unknown function (annotated as such in sequenced plant genomes) are among the parasite genes highly expressed by T. versicolor at the parasite-host interface. CONCLUSIONS: Laser capture microdissection makes it possible to sample the small region of cells at the epicenter of parasite host interactions. The results of our analysis suggest that T. versicolor's generalist strategy involves a reliance on overlapping but distinct gene sets, depending upon the host plant it is parasitizing. The massive upregulation of a T. versicolor ß-expansin is suggestive of a mechanism for parasite success on grass hosts. In this preliminary study of the interface transcriptomes, we have shown that T. versicolor, and the Orobanchaceae in general, provide excellent opportunities for the characterization of plant genes with unknown functions.

14-3-3 proteins SGF14c and SGF14l play critical roles during soybean nodulation.

The soybean (Glycine max) genome contains 18 members of the 14-3-3 protein family, but little is known about their association with specific phenotypes. Here, we report that the Glyma0529080 Soybean G-box Factor 14-3-3c (SGF14c) and Glyma08g12220 (SGF14l) genes, encoding 14-3-3 proteins, appear to play essential roles in soybean nodulation. Quantitative reverse transcription-polymerase chain reaction and western-immunoblot analyses showed that SGF14c mRNA and protein levels were specifically increased in abundance in nodulated soybean roots 10, 12, 16, and 20 d after inoculation with Bradyrhizobium japonicum. To investigate the role of SGF14c during soybean nodulation, RNA interference was employed to silence SGF14c expression in soybean roots using Agrobacterium rhizogenes-mediated root transformation. Due to the paleopolyploid nature of soybean, designing a specific RNA interference sequence that exclusively targeted SGF14c was not possible. Therefore, two highly similar paralogs (SGF14c and SGF14l) that have been shown to function as dimers were silenced. Transcriptomic and proteomic analyses showed that mRNA and protein levels were significantly reduced in the SGF14c/SGF14l-silenced roots, and these roots exhibited reduced numbers of mature nodules. In addition, SGF14c/SGF14l-silenced roots contained large numbers of arrested nodule primordia following B. japonicum inoculation. Transmission electron microscopy further revealed that the host cytoplasm and membranes, except the symbiosome membrane, were severely degraded in the failed nodules. Altogether, transcriptomic, proteomic, and cytological data suggest a critical role of one or both of these 14-3-3 proteins in early development stages of soybean nodules.