Parasitic dodder expresses an arsenal of secreted cellulases with multi-substrate specificity during host invasion.

Cuscuta campestris is a common and problematic parasitic plant which relies on haustoria to connect to and siphon nutrients from host plants. Glycoside hydrolase family 9 (GH9) cellulases (EC 3.2.1.4) play critical roles in plant cell wall biosynthesis and disassembly, but their roles during Cuscuta host invasion remains underexplored. In this study, we identified 22 full-length GH9 cellulase genes in C. campestris genome, which encoded fifteen secreted and seven membrane-anchored cellulases that showed distinct phylogenetic relationships. Expression profiles suggested that some of the genes are involved in biosynthesis and remodeling of the parasite's cell wall during haustoriogenesis, while other genes encoding secreted B- and C-type cellulases are tentatively associated with degrading host cell walls during invasion. Transcriptomic data in a host-free system and in the presence of susceptible or partially resistant tomato hosts, showed for especially GH9B7, GH9B11 and GH9B12 a shift in expression profiles in the presence of hosts, being more highly expressed during host attachment, indicating that Cuscuta can tune cellulase expression in response to a host. Functional analyses of recombinant B- and C-type cellulases showed endoglucanase activities over wide pH and temperature conditions, and activities towards multiple cellulose and hemicellulose substrates. These findings improve our understanding of host cell wall disassembly by Cuscuta, and cellulase activity towards broad substrate range potentially explain its wide host range. This is the first study to provide a broad biochemical insight into Cuscuta GH9 cellulases, which based on our study may have potential applications in industrial bioprocessing.

Cuscuta chinensis Lam. Flavonoids (CCLF) alleviate the symptoms of sepsis-associated encephalopathy via PI3K/Nrf2 pathway.

Sepsis-associated encephalopathy (SAE) frequently encounters patients who are in intensive care units and ∼70% of patients with severe systemic infection. However, due to the unclear pathological mechanisms of SAE, the desease-modifying drug is still lack. Here, we aimed to explore whether the flavonoid components extracted from CCL (CCLF) seeds possess protective effects on SAE animals, and systematically evaluate the transcriptomic alteration (in the hippocampus) after CCLF treatment on SAE animals employing RNA sequencing. We observed that CCLF improved the brain's learning and memory abilities and the structural integrity of BBB using cecal ligation and puncture (CLP)-induced SAE animal models, evaluated by behavioral test and tissue examination of animals respectively. RNA sequencing results showed that CCLF treatment reverses SAE-induced transcriptomic alteration in the hippocampus. Moreover, CCLF also dramatically relieved inflammatory (such as TNF-α, IL-2, and IL-6) and oxidative (MDA and SOD activity) stresses, and inhibited SAE-induced neuron apoptosis in brain tissues. More importantly, CCLF restored the PI3K/AKT signaling pathway and then induced the Nrf2 nuclear translocation to drive HO-1 expression both in vitro and in vivo. LY294002, an inhibitor of PI3K, obviously blocked CCLF's functions on anti-apoptosis, anti-inflammation, and anti-oxidation in vivo, demonstrating that CCLF achieves its bioactivities in a PI3K/AKT signaling dependent manner. Altogether, CCLF exhibits remarkable neuro-protective function and may be a promising candidate for further clinical trials for SAE treatment.

Trans-species microRNA loci in the parasitic plant Cuscuta campestris have a U6-like snRNA promoter.

Small regulatory RNAs can move between organisms and regulate gene expression in the recipient. Whether the trans-species small RNAs being exported are distinguished from the normal endogenous small RNAs of the source organism is not known. The parasitic plant Cuscuta campestris (dodder) produces many microRNAs that specifically accumulate at the host-parasite interface, several of which have trans-species activity. We found that induction of C. campestris interface-induced microRNAs is similar regardless of host species and occurs in C. campestris haustoria produced in the absence of any host. The loci-encoding C. campestris interface-induced microRNAs are distinguished by a common cis-regulatory element. This element is identical to a conserved upstream sequence element (USE) used by plant small nuclear RNA loci. The properties of the interface-induced microRNA primary transcripts strongly suggest that they are produced via U6-like transcription by RNA polymerase III. The USE promotes accumulation of interface-induced miRNAs (IIMs) in a heterologous system. This promoter element distinguishes C. campestris IIM loci from other plant small RNAs. Our data suggest that C. campestris IIMs are produced in a manner distinct from canonical miRNAs. All confirmed C. campestris microRNAs with documented trans-species activity are interface-induced and possess these features. We speculate that RNA polymerase III transcription of IIMs may allow these miRNAs to be exported to hosts.

Cuscuta epithymum Murr. crude extract pre-conditioning protects C6 cells from L-glutamate-induced neurotoxicity.

BACKGROUND: Cuscuta epithymum Murr. (C. epithymum), as an herbal medicine, has played an anti-cancerous role in various studies; however, its possible neuroprotective effects have been neglected. Here, we aimed to investigate the protective effects of C. epithymum seeds crude extract and different fractions on rat glioblastoma cells (C6) in L-glutamate oxidative condition. METHODS: Initially, the total phenolic content of C. epithymum crude extract and the fractions (all produced by maceration method) was determined. Subsequently, C6 cells were pre-treated with the various concentrations of crude extract and fractions 24 h before L-glutamate exposure. Likewise, C6 cells were treated with the same concentrations of crude extract and fractions 24 h after exposure to L-glutamate. The cell viability and morphology were compared in crude extract and fractions groups, then superoxide dismutase (SODs) activity, reactive oxygen species (ROS), and malondialdehyde (MDA) levels were measured. The flow cytometry test was used to study C. epithymum crude extract's effects on the cell cycle and also to quantify the apoptosis, necrosis, and live cells population in different groups. RESULTS: C. epithymum crude extract and fractions (hexanoic, dichloromethanolic, and methanolic) had concentration-dependent cytotoxicity (IC50:126.47, 2101.96, 140.97, and 218.96 µg/ml, respectively). The crude extract and methanolic fraction contained phenolic compounds (55.99 ± 2.795 and 50.80 ± 2.969 mg gallic acid/g extract), while in hexanoic and dichloromethanolic fractions, the phenolic content was undetectable. In the cell viability assay, in comparison to fractions, the crude extract showed a more protective effect against glutamate-induced oxidative condition (P < 0.0001). The crude extract increased the SODs activity (P < 0.001) and decreased MDA and ROS levels (P < 0.0001) in comparison to the glutamate group. The crude extract significantly increased the population of cells in G1 (from 63.04 to 76.29) and decreased the percentage of cells in G2 (from 11.56 to 6.7) and S phase (from 25.4 to 17.01). In addition, it decreased the apoptotic and necrotic cell populations (from 34 to 17.1) and also increased the percentage of live cells (from 66.8 to 83.4 percent) in the flow cytometry test. CONCLUSION: C. epithymum crude extract plays a neuroprotective role by activating the defense mechanisms in cell against the oxidative condition.

Metabolic changes induced by Cuscuta campestris Yunck in the host species Artemisia campestris subsp. variabilis (Ten.) Greuter as a strategy for successful parasitisation.

MAIN CONCLUSIONS: C. campestris parasitisation increases internal host defences at the expense of environmentally directed ones in the host species A. campestris, thus limiting plant defence against progressive parasitisation. Cuscuta campestris Yunck is a holoparasitic species that parasitises wild species and crops. Among their hosts, Artemisia campestris subsp. variabilis (Ten.) Greuter is significantly affected in natural ecosystems. Limited information is available on the host recognition mechanism and there are no data on the interactions between these species and the effects on the primary and specialised metabolism in response to parasitisation. The research aims at evaluating the effect of host-parasite interactions, through a GC-MS untargeted metabolomic analysis, chlorophyll a fluorescence, ionomic and δ13C measurements, as well as volatile organic compound (VOC) fingerprint in A. campestris leaves collected in natural environment. C. campestris parasitisation altered plant water status, forcing stomatal opening, stimulating plant transpiration, and inducing physical damages to the host antenna complex, thus reducing the efficiency of its photosynthetic machinery. Untargeted-metabolomics analysis highlighted that the parasitisation significantly perturbed the amino acids and sugar metabolism, inducing an increase in the production of osmoprotectants, which generally accumulate in plants as a protective strategy against oxidative stress. Notably, VOCs analysis highlighted a reduction in sesquiterpenoids and an increase in monoterpenoids levels; involved in plant defence and host recognition, respectively. Moreover, C. campestris induced in the host a reduction in 3-hexenyl-acetate, a metabolite with known repellent activity against Cuscuta spp. We offer evidences that C. campestris parasitisation increases internal host defences via primary metabolites at the expense of more effective defensive compounds (secondary metabolites), thus limiting A. campestris defence against progressive parasitisation.

The novel anti-neuroinflammatory functional food CCL01, a mixture of Cuscuta seed extracts and Lactobacillus paracasei NK112.

Neuroinflammation, which occurs due to microglia, is related to the pathogenesis of neurodegenerative disorders. Recently, the development of functional foods that down-regulate over-activated microglial cells to prevent the progression of neurodegenerative disorders has been proposed, since over-activated microglia induce a chronic source of neurotoxic factors and reduce neuronal survival. Thus, the anti-neuroinflammatory effects of a functional food mixture (CCL01) including Cuscuta seeds and Lactobacillus paracasei NK112 on lipopolysaccharide (LPS)-induced experimental models were investigated. In LPS-induced in vitro models, the expression levels of inflammatory mediators (e.g., inducible nitric oxide synthase, cyclooxygenase-2, nitric oxide, and prostaglandin E2) and pro-inflammatory cytokines (e.g., tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6) were decreased upon CCL01 treatment. CCL01 showed an anti-neuroinflammatory effect in LPS-induced microglial cells via the inhibition of the mitogen-activated protein kinase (MAPK)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway and the activation of the nuclear factor erythroid-2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. In the LPS-treated in vivo mouse models, the increased expression of ionized calcium binding adaptor molecule 1 (Iba-1), which indicates microglial activity, was markedly decreased upon treatment with CCL01 (50 and 200 mg kg-1) in the hippocampus and cortex areas of the mouse brains in comparison with the LPS-injected group. In addition, the groups to which CCL01 was administered had significantly decreased plasma levels of tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 in the LPS-injected mouse models. Our data suggest that CCL01 may be a potential anti-neuroinflammatory agent that can prevent microglia overactivation, and it could be useful for developing functional foods.

Protective effect of polysaccharides isolated from the seeds of Cuscuta chinensis Lam. on 5-fluorouracil-induced intestinal mucositis in mice.

PURPOSE: To evaluate the protective effect of Cuscuta chinensis Lam. polysaccharides (PCCL) on 5-fluorouracil-(5-FU)-induced intestinal mucositis (IM) in mice. METHODS: PCCL was orally administered at a dose of 20 mg·kg-1 for 7 days and its protective effect on 5-FU-induced IM (5-FU, 50 mg·kg-1 for 5 days) was evaluated by monitoring changes in body weight, degree of diarrhea, levels of tissue inflammatory factors (tumor necrosis factor α, interleukin 6, and interleukin 1ß levels), apoptosis rates, and the expression levels of caspase-3, Bax and Bcl-2. RESULTS: The severity of mucosal injury (as reflected by body weight changes, degree of diarrhea, height of villi, and damage to crypts) was significantly attenuated by PCCL administration. PCCL also reduced the levels of tissue inflammatory factors, the apoptosis rate, and the expression of caspase-3 and Bax, and increased Bcl-2 expression. CONCLUSIONS: PCCL administration may be significantly protective against 5-FU-induced IM by inhibiting apoptosis and regulating the abnormal inflammation associated with it.

Functional Characterization of the Photosynthetic Machinery in Smicronix Galls on the Parasitic Plant Cuscuta campestris by JIP-Test.

Members of the genus Cuscuta are generally considered to be non-photosynthetic, stem-holoparasitic flowering plants. Under certain circumstances, at least some members of the genus are capable of limited photosynthesis. The galls of the Smicronyx weevils formed on Cuscuta campestris are particularly rich in chlorophylls compared to the stem of the parasitic plant. In the present study, we aimed to characterize the photosynthetic activity in the inner and outer gall cortices in comparison to the non-photosynthetic stems and a reference plant (Arabidopsis thaliana). The recorded prompt chlorophyll fluorescence transients were analyzed using JIP test. Detailed analysis of the chlorophyll fluorescence confirmed the presence of actively functioning photosynthetic machinery, especially in the inner cortex of the galls. This photosynthesis, induced by the insect larvae, did not reach the levels of the photosynthetic activity in Arabidopsis thaliana plants. Thylakoid protein complexes were identified by separation with two-dimensional Blue Native/SDS PAGE. It appeared that some of the complexes presented in A. thaliana are missing in C. campestris. We hypothesize that the insect-triggered transition from non-photosynthetic to photosynthetic tissue in the gall is driven by the increased requirements for nutrients related to the larval nutrition.

Efficient carbon recycling and modulation of antioxidants involved in elongation of the parasitic plant dodder (Cuscuta spp.) in vitro.

Dodder is a holoparasitic flowering plant that re-establishes parasitism with the host when broken off from the host. However, how in vitro dodder shoots recycle stored nutrients to maintain growth for reparasitizing hosts is not well characterized. Here, the spatial and temporal distribution characteristics of carbohydrates and reactive oxygen species (ROS) were analysed to explore the mechanism of recycling stored nutrients in dodder shoots in vitro. Our results showed that in vitro dodder shoots grew actively for more than 10 d, while dry mass decreased continuously. During this process, the transcript levels and activities of amylases gradually increased until 2 d and then declined in basal stems, which induced starch degradation at the tissue, cellular and subcellular levels. Additionally, the distribution characteristics of H2O2 and the activities and transcript levels of antioxidant enzymes indicated that shoot tips exhibited more robust ROS-scavenging capacity, and basal stems maintained higher ROS accumulation. Comparative proteomics analysis revealed that starch in basal stems acted as an energy source, and the glycolysis, TCA cycle and pentose phosphate pathway represented the energy supply for shoot tip elongation with time. These results indicated that efficient nutrient recycling and ROS modulation facilitated the parasitism of dodder grown in vitro by promoting shoot elongation growth to reach the host.

To study the mechanism of Cuscuta chinensis Lam. And Lycium barbarum L. in the treatment of asthenospermia based on network pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Cuscuta chinensis Lam. and Lycium barbarum L. (SC-FL) is a commonly used kidney tonic Chinese medicine combination that is widely used in the clinical treatment of oligoasthenospermia.However, its specific mechanism remains unclear and requires in-depth study. AIM OF THE STUDY: To explore the potential targets of SC-FL in the treatment of oligoasthenospermia using network pharmacology, and to verify the results with in vivo and in vitro experiments. MATERIALS AND METHODS: A herb-compound-target-disease network and PPI network were constructed with Cytoscape software. The targets of SC-FL for the treatment of male sterility were introduced into a bioinformatics annotation database, and the GO and KEGG databases were used for pathway enrichment analysis. Subsequently, Tripterygium wilfordii Hook. f. (GTW) polyglycoside was used to induce a spermatogenic dysfunction model in GC-1 spg cells and SD male rats in in vitro and in vivo experiments, respectively. The SC-FL and PI3K pathway inhibitor LY294002 was used to intervene in the spermatogenic dysfunction model to detect the expression of proteins and mRNA related to the PI3K pathway and to detect the indicators related to proliferation and apoptosis. RESULTS: In in vitro experiments, the percentage of spermatogenic cells and the proportion of GC-1 spg cells at G0/G1 and G2/M stages in the model group (GTW group) and the inhibitor group (LY group) were significantly decreased (P < 0.01) compared with the blank control group (NC group). The apoptosis rate of the GTW group was significantly increased (P < 0.01). The ultrastructures of GC-1 spg cells in the GTW group and LY group were obviously destroyed. Compared with the GTW group, the SC-FL group had a significantly reduced apoptosis rate of GC-1 spg cells, reduced percentage of cells in S phase, and a significantly improved mitochondrial membrane potential. SC-FL can repair the ultrastructure of GC-1 spg cells damaged by GTW. The above effects of SC-FL are closely related to up-regulation of GFRa1, RET, PI3K, p-AKT, and Bcl-2 and down-regulation of BAD and BAX proteins and mRNA expression. In vivo, compared with the GTW group, the body mass, testicular mass, and epididymal weight of the GTW + SC-FL group were significantly increased (P < 0.01). Sperm concentrations and the PR + NP of GTW + SC-FL were significantly higher than in the GTW group (P < 0.01 or P < 0.05). FSH, LH, and T levels in the GTW + SC-FL and LY + SC-FL groups were significantly higher than those in the GTW and LY group (P < 0.01 or P < 0.05). HE staining results showed that the morphology of testicular tissue in the GTW + SC-FL and LY + SC-FL groups was superior to that in the GTW and LY group. The above effects of SC-FL are closely related to the up-regulation of proteins and mRNA expression of PI3K, p-AKT, and Bcl-2. CONCLUSION: Through the PI3K/Akt signaling pathway, SC-FL up-regulates GFRa1, RET, PI3K, p-AKT, and Bcl-2, and down-regulates the expression of BAD and BAX proteins and mRNA, thus reducing the percentage of GC-1 spg cells in S-phase, significantly increasing the mitochondrial membrane potential, significantly reducing cell apoptosis, and improving sperm counts and viability.

The tomato receptor CuRe1 senses a cell wall protein to identify Cuscuta as a pathogen.

Parasitic plants of the genus Cuscuta penetrate shoots of host plants with haustoria and build a connection to the host vasculature to exhaust water, solutes and carbohydrates. Such infections usually stay unrecognized by the host and lead to harmful host plant damage. Here, we show a molecular mechanism of how plants can sense parasitic Cuscuta. We isolated an 11 kDa protein of the parasite cell wall and identified it as a glycine-rich protein (GRP). This GRP, as well as its minimal peptide epitope Crip21, serve as a pathogen-associated molecular pattern and specifically bind and activate a membrane-bound immune receptor of tomato, the Cuscuta Receptor 1 (CuRe1), leading to defense responses in resistant hosts. These findings provide the initial steps to understand the resistance mechanisms against parasitic plants and further offer great potential for protecting crops by engineering resistance against parasitic plants.

Genome-wide codon usage pattern analysis reveals the correlation between codon usage bias and gene expression in Cuscuta australis.

The protein-coding genes and pseudogenes of Cuscuta australis had the diverse contribution to the formation and evolution of parasitism. The codon usage pattern analysis of these two type genes could be used to understand the gene transcription and translation. In this study, we systematically analyzed the codon usage patterns of protein-coding sequences and pseudogenes sequences in C. australis. The results showed that the high frequency codons of protein coding sequences and pseudogenes had the same A/U bias in the third position. However, these two sequences had converse bias at the third base in optimal codons: the protein coding sequences preferred G/C-ending codons while pseudogene sequences preferred A/U-ending codons. Neutrality plot and effective number of codons plot revealed that natural selection played a more important role than mutation pressure in two sequences codon usage bias. Furthermore, the gene expression level had a significant positive correlation with codon usage bias in C. australis. Highly-expressed protein coding genes exhibited a higher codon bias than lowly-expressed genes. Meanwhile, the high-expression genes tended to use G/C-ending synonymous codons. This result further verified the optimal codons usage bias and its correlation with the gene expression in C. australis.

Selective mineral transport barriers at Cuscuta-host infection sites.

The uptake of inorganic nutrients by rootless parasitic plants, which depend on host connections for all nutrient supplies, is largely uncharted. Using X-ray fluorescence spectroscopy (XRF), we analyzed the element composition of macro- and micronutrients at infection sites of the parasitic angiosperm Cuscuta reflexa growing on hosts of the genus Pelargonium. Imaging methods combining XRF with 2-D or 3-D (confocal) microscopy show that most of the measured elements are present at similar concentrations in the parasite compared to the host. However, calcium and strontium levels drop pronouncedly at the host/parasite interface, and manganese appears to accumulate in the host tissue surrounding the interface. Chlorine is present in the haustorium at similar levels as in the host tissue but is decreased in the stem of the parasite. Thus, our observations indicate a restricted uptake of calcium, strontium, manganese and chlorine by the parasite. Xylem-mobile dyes, which can probe for xylem connectivity between host and parasite, provided evidence for an interspecies xylem flow, which in theory would be expected to carry all of the elements indiscriminately. We thus conclude that inorganic nutrient uptake by the parasite Cuscuta is regulated by specific selective barriers whose existence has evaded detection until now.

Dodder-transmitted mobile signals prime host plants for enhanced salt tolerance.

The dodders (Cuscuta spp.) are a genus of shoot parasites. In nature, a dodder often simultaneously parasitizes two or more neighboring hosts. Salt stress is a common abiotic stress for plants. It is unclear whether dodder transmits physiologically relevant salt stress-induced systemic signals among its hosts and whether these systemic signals affect the hosts' tolerance to salt stress. Here, we simultaneously parasitized two or more cucumber plants with dodder. We found that salt treatment of one host highly primed the connected host, which showed strong decreases in the extent of leaf withering and cell death in response to subsequent salt stress. Transcriptomic analysis indicated that 24 h after salt treatment of one cucumber, the transcriptome of the other dodder-connected cucumber largely resembled that of the salt-treated one, indicating that inter-plant systemic signals primed these dodder-connected cucumbers at least partly through transcriptomic reconfiguration. Furthermore, salt treatment of one of the cucumbers induced physiological changes, including altered proline contents, stomatal conductance, and photosynthetic rates, in both of the dodder-connected cucumbers. This study reveals a role of dodder in mediating salt-induced inter-plant signaling among dodder-connected hosts and highlights the physiological function of these mobile signals in plant-plant interactions under salt stress.

Extensive Inter-plant Protein Transfer between Cuscuta Parasites and Their Host Plants.

Cuscuta species (dodders) are holoparasites that totally rely on host plants to survive. Although various mobile proteins have been identified to travel within a plant, whether and to what extent protein transfer between Cuscuta and host plants remain unclear. We found that hundreds to more than 1500 proteins were transferred between Cuscuta and the host plants Arabidopsis and soybean, and hundreds of inter-plant mobile proteins were even detected in the seeds of Cuscuta and the host soybean. Different hosts bridge-connected by dodder were also found to exchange hundreds of proteins. Quantitatively, the mobile proteins represent a few to more than 10% of the proteomes of foreign plants. Using Arabidopsis plants expressing different reporter proteins, we further showed that these reporter proteins could travel between plants and, importantly, retained their activity in the foreign plants. Comparative analysis between the inter-plant mobile proteins and mRNAs indicated that the majority of mobile proteins were not de novo synthesized from the translocated mRNAs, but bona fide mobile proteins. We propose that large-scale inter-plant protein translocation may play an important role in the interactions between host plants and dodder and even among the dodder bridge-connected hosts.

Compensatory sequence variation between trans-species small RNAs and their target sites.

Trans-species small regulatory RNAs (sRNAs) are delivered to host plants from diverse pathogens and parasites and can target host mRNAs. How trans-species sRNAs can be effective on diverse hosts has been unclear. Multiple species of the parasitic plant Cuscuta produce trans-species sRNAs that collectively target many host mRNAs. Confirmed target sites are nearly always in highly conserved, protein-coding regions of host mRNAs. Cuscuta trans-species sRNAs can be grouped into superfamilies that have variation in a three-nucleotide period. These variants compensate for synonymous-site variation in host mRNAs. By targeting host mRNAs at highly conserved protein-coding sites, and simultaneously expressing multiple variants to cover synonymous-site variation, Cuscuta trans-species sRNAs may be able to successfully target multiple homologous mRNAs from diverse hosts.

Metabolic and functional distinction of the Smicronyx sp. galls on Cuscuta campestris.

MAIN CONCLUSION: The weevil gall contains two distinct regions, differing in hydrolytic and antioxidant enzymes activity and profiles, which is also functionally distinct from the non-infected Cuscuta stems. Weevils of the genus Smicronyx are gall-forming insects, widely distributed on parasitic flowering plants of the genus Cuscuta. Thus, they are considered epiparasites and potential method for biological control of their agriculturally harmful hosts. Although several reports on gall formation in Cuscuta spp. exist, the metabolic and functional changes, occurring in the gall, remained largely unknown. Smicronyx sp. galls, collected from a wild Cuscuta campestris population, were dissected into two distinct regions, inner and outer cortex, defined by the higher chlorophyll content of the inner cortex. Based on hydrolytic and antioxidant enzymes activity and isoenzymatic profiles as analyzed after electrophoretic separation, we suggested that the gall differs in its metabolic activity from the non-infected plant tissue. While the outer cortex serves as a region of nutrient storage and mobilization, the inner cortex is directly involved in larvae nutrition. The increase in metabolic activity resulted in significantly increased superoxide dismutase activity in the gall, while several other antioxidant enzymes diminished. The present research offers new insights into the functionally differing regions of Smicronyx galls and the metabolic changes, induced in C. campestris in result of the gall formation.

Sesamin from Cuscuta palaestina natural plant extracts: Directions for new prospective applications.

The aim of this study is to disclose the potential bioactive components of Cuscuta palaestina, a native parasitic natural plant of flora palaestina and to open direction towards new prospective application. GC-MS analysis identified 18 components in the methanolic extract of C. palaestina for the first time. The most appealing among them are Sesamin and two other phytosterols (Campesterol and Stigmasterol), all of which are documented in the scientific literature for their anticancer activity. Quantitation of Sesamin extracted from C. palaestina by HPLC-PDA with the use of three organic solvents showed that the Sesamin content in the methanolic extract was the highest. Following the disclosure of Sesamin presence in C. palaestina, we raised the question of whether it is produced naturally in C. palaestina or acquired from the host plant. The quantitation of Sesamin in C. palaestina was performed while being with five different host plants, and was compared with the amount of Sesamin in C. palaestina grown alone. The findings reveal that Sesamin is an endogenous secondary metabolite in C. palaestina. Thus, further studies are required to prove if C. palaestina can be used as an alternative source of anticancer phytochemicals, mainly Sesamin, and if proteins in the Sesamin production pathway could be valid biological targets for the development of novel and selective pesticides for control/ eradication of C. palaestina and maybe some other Cuscuta species. As well, the findings from this study raise a big question of whether inferring Sesamin production in C. palaestina could reduce its attack ability to host plants.

Expression of active chimeric-tissue plasminogen activator in tobacco hairy roots, identification of a DNA aptamer and purification by aptamer functionalized-MWCNTs chromatography.

Tissue-type plasminogen activator (tPA) is a serine protease that plays a crucial role in the fibrinolytic system. We increased the activity of tPA by splicing the active site of dodder-cuscutain gene to human tPA. The chimeric cDNA of tPA was constructed by Splicing by Overlap Extension Polymerase Chain Reaction (SOEing-PCR) method and transferred to the hairy roots of tobacco using different strains of Agrobacterium rhizogenes. Chimeric-tPA was purified by lysine-sepharose chromatography and specific aptamers were designed using SELEX method. Multi wall carbon nanotubes were functionalized with selected aptamers, packed in a column, and used for purification. The results demonstrated that selected aptamer having KD values of 0.320 nM and IC50 of 28.9 nM possessed good affinity to tPA, and the chimeric-tPA was properly purified by aptamer-chromatography. Hairy roots expressing chimeric-tPA and normal-tPA produced 900 and 450 ngmg-1 of total protein, respectively. The activities of chimeric-tPA and normal-tPA were 90 and 60 IUml-1, respectively. Compared to the normal-tPA, chimeric-tPA showed more activity.

Arabinogalactan Proteins Accumulate in the Cell Walls of Searching Hyphae of the Stem Parasitic Plants, Cuscuta campestris and Cuscuta japonica.

Stem parasitic plants (Cuscuta spp.) develop a specialized organ called a haustorium to penetrate their hosts' stem tissues. To reach the vascular tissues of the host plant, the haustorium needs to overcome the physical barrier of the cell wall, and the parasite-host interaction via the cell wall is a critical process. However, the cell wall components responsible for the establishment of parasitic connections have not yet been identified. In this study, we investigated the spatial distribution patterns of cell wall components at a parasitic interface using parasite-host complexes of Cuscuta campestris-Arabidopsis thaliana and Cuscuta japonica-Glycine max. We focused on arabinogalactan proteins (AGPs), because AGPs accumulate in the cell walls of searching hyphae of both C. campestris and C. japonica. We found more AGPs in elongated haustoria than in pre haustoria, indicating that AGP accumulation is developmentally regulated. Using in situ hybridization, we identified five genes in C. campestris that encode hyphal-expressed AGPs that belong to the fasciclin-like AGP (FLA) family, which were named CcFLA genes. Three of the five CcFLA genes were expressed in the holdfast, which develops on the Cuscuta stem epidermis at the attachment site for the host's stem epidermis. Our results suggest that AGPs are involved in hyphal elongation and adhesion to host cells, and in the adhesion between the epidermal tissues of Cuscuta and its host.