Lactucin Synthase Inactivation Boosts the Accumulation of Anti-inflammatory 8-Deoxylactucin and Its Derivatives in Chicory (Cichorium intybus L.).

For several sesquiterpene lactones (STLs) found in Asteraceae plants, very interesting biomedical activities have been demonstrated. Chicory roots accumulate the guaianolide STLs 8-deoxylactucin, lactucin, and lactucopicrin predominantly in oxalated forms in the latex. In this work, a supercritical fluid extract fraction of chicory STLs containing 8-deoxylactucin and 11ß,13-dihydro-8-deoxylactucin was shown to have anti-inflammatory activity in an inflamed intestinal mucosa model. To increase the accumulation of these two compounds in chicory taproots, the lactucin synthase that takes 8-deoxylactucin as the substrate for the regiospecific hydroxylation to generate lactucin needs to be inactivated. Three candidate cytochrome P450 enzymes of the CYP71 clan were identified in chicory. Their targeted inactivation using the CRISPR/Cas9 approach identified CYP71DD33 to have lactucin synthase activity. The analysis of the terpene profile of the taproots of plants with edits in CYP71DD33 revealed a nearly complete elimination of the endogenous chicory STLs lactucin and lactucopicrin and their corresponding oxalates. Indeed, in the same lines, the interruption of biosynthesis resulted in a strong increase of 8-deoxylactucin and its derivatives. The enzyme activity of CYP71DD33 to convert 8-deoxylactucin to lactucin was additionally demonstrated in vitro using yeast microsome assays. The identified chicory lactucin synthase gene is predominantly expressed in the chicory latex, indicating that the late steps in the STL biosynthesis take place in the latex. This study contributes to further elucidation of the STL pathway in chicory and shows that root chicory can be positioned as a crop from which different health products can be extracted.

CRISPR/Cas9 targeted inactivation of the kauniolide synthase in chicory results in accumulation of costunolide and its conjugates in taproots.

Chicory taproots accumulate sesquiterpene lactones lactucin, lactucopicrin, and 8-deoxylactucin, predominantly in their oxalated forms. The biosynthetic pathway for chicory sesquiterpene lactones has only partly been elucidated; the enzymes that convert farnesyl pyrophosphate to costunolide have been described. The next biosynthetic step of the conversion of costunolide to the tricyclic structure, guaianolide kauniolide, has so far not been elucidated in chicory. In this work three putative kauniolide synthase genes were identified in chicory named CiKLS1, CiKLS2, and CiKLS3. Their activity to convert costunolide to kauniolide was demonstrated in vitro using yeast microsome assays. Next, introduction of CRISPR/Cas9 reagents into chicory protoplasts was used to inactivate multiple chicory KLS genes and several chicory lines were successfully regenerated. The inactivation of the kauniolide synthase genes in chicory by the CRISPR/Cas9 approach resulted in interruption of the sesquiterpene lactone biosynthesis in chicory leaves and taproots. In chicory taproots, but not in leaves, accumulation of costunolide and its conjugates was observed to high levels, namely 1.5 mg/g FW. These results confirmed that all three genes contribute to STL accumulation, albeit to different extent. These observations demonstrate that three genes oriented in tandem on the chicory genome encode kauniolide synthases that initiate the conversion of costunolide toward the sesquiterpene lactones in chicory.

Identification of a defense response gene involved in signaling pathways against PVA and PVY in potato.

Potato is the most important non-grain food crop in the world. Viruses, particularly potato virus Y (PVY) and potato virus A (PVA), are among the major agricultural pathogens causing severe reduction in potato yield and quality worldwide. Virus infection induces host factors to interfere with its infection cycle. Evaluation of these factors facilitates the development of intrinsic resistance to plant viruses. In this study, a small G-protein as one of the critical signaling factors was evaluated in plant response to PVY and PVA to enhance resistance. For this purpose, the gene expression dataset of G-proteins in potato plant under five biotic (viruses, bacteria, fungi, nematodes, and insects) and four abiotic (cold, heat, salinity, and drought) stress conditions were collected from gene expression databases. We reduced the number of the selected G-proteins to a single protein, StSAR1A, which is possibly involved in virus inhibition. StSAR1A overexpressed transgenic plants were created via the Agrobacterium-mediated method. Real-time PCR and Enzyme-linked immunosorbent assay tests of transgenic plants mechanically inoculated with PVY and PVA indicated that the overexpression of StSAR1A gene enhanced resistance to both viruses. The virus-infected transgenic plants exhibited a greater stem length, a larger leaf size, a higher fresh/dry weight, and a greater node number than those of the wild-type plants. The maximal photochemical efficiency of photosystem II, stomatal conductivity, and net photosynthetic rate in the virus-infected transgenic plants were also obviously higher than those of the control. The present study may help to understand aspects of resistance against viruses.

Alternaria Brassicae Induces Systemic Jasmonate Responses in Arabidopsis Which Travel to Neighboring Plants via a Piriformsopora Indica Hyphal Network and Activate Abscisic Acid Responses.

Stress information received by a particular local plant tissue is transferred to other tissues and neighboring plants, but how the information travels is not well understood. Application of Alternaria Brassicae spores to Arabidopsis leaves or roots stimulates local accumulation of jasmonic acid (JA), the expression of JA-responsive genes, as well as of NITRATE TRANSPORTER (NRT)2.5 and REDOX RESPONSIVE TRANSCRIPTION FACTOR1 (RRTF1). Infection information is systemically spread over the entire seedling and propagates radially from infected to non-infected leaves, axially from leaves to roots, and vice versa. The local and systemic NRT2.5 responses are reduced in the jar1 mutant, and the RRTF1 response in the rbohD mutant. Information about A. brassicae infection travels slowly to uninfected neighboring plants via a Piriformospora Indica hyphal network, where NRT2.5 and RRTF1 are up-regulated. The systemic A. brassicae-induced JA response in infected plants is converted to an abscisic acid (ABA) response in the neighboring plant where ABA and ABA-responsive genes are induced. We propose that the local threat information induced by A. brassicae infection is spread over the entire plant and transferred to neighboring plants via a P. indica hyphal network. The JA-specific response is converted to a general ABA-mediated stress response in the neighboring plant.

Bactericidal, quorum quenching and anti-biofilm nanofactories: a new niche for nanotechnologists.

Despite several conventional potent antibacterial therapies, bacterial infections pose a significant threat to human health because they are emerging as the leading cause of death worldwide. Due to the development of antibiotic resistance in bacteria, there is a pressing demand to discover novel approaches for developing more effective therapies to treat multidrug-resistant bacterial strains and biofilm-associated infections. Therefore, attention has been especially devoted to a new and emerging branch of science "nanotechnology" to design non-conventional antimicrobial chemotherapies. A range of nanomaterials and nano-sized carriers for conventional antimicrobial agents have fully justified their potential to combat bacterial diseases by reducing cell viability, by attenuating quorum sensing, and by inhibiting/or eradicating biofilms. This communication summarizes emerging nano-antimicrobial therapies in treating bacterial infections, particularly using antibacterial, quorum quenching, and anti-biofilm nanomaterials as new approaches to tackle the current challenges in combating infectious diseases.

Bactericidal, quorum quenching and anti-biofilm nanofactories: a new niche for nanotechnologists

Despite several conventional potent antibacterial therapies, bacterial infections pose a significant threat to human health because they are emerging as the leading cause of death worldwide. Due to the development of antibiotic resistance in bacteria, there is a pressing demand to discover novel approaches for developing more effective therapies to treat multidrug-resistant bacterial strains and biofilm-associated infections. Therefore, attention has been especially devoted to a new and emerging branch of science "nanotechnology" to design non-conventional antimicrobial chemotherapies. A range of nanomaterials and nano-sized carriers for conventional antimicrobial agents have fully justified their potential to combat bacterial diseases by reducing cell viability, by attenuating quorum sensing, and by inhibiting/or eradicating biofilms. This communication summarizes emerging nano-antimicrobial therapies in treating bacterial infections, particularly using antibacterial, quorum quenching, and anti-biofilm nanomaterials as new approaches to tackle the current challenges in combating infectious diseases.

Stress promotes Arabidopsis - Piriformospora indica interaction.

The endophytic fungus Piriformospora indica colonizes Arabidopsis thaliana roots and promotes plant performance, growth and resistance/tolerance against abiotic and biotic stress. Here we demonstrate that the benefits for the plant increase when the two partners are co-cultivated under stress (limited access to nutrient, exposure to heavy metals and salt, light and osmotic stress, pathogen infection). Moreover, physical contact between P. indica and Arabidopsis roots is necessary for optimal growth promotion, and chemical communication cannot replace the physical contact. Lower nutrient availability down-regulates and higher nutrient availability up-regulates the plant defense system including the expression of pathogenesis-related genes in roots. High light, osmotic and salt stresses support the beneficial interaction between the plant and the fungus. P. indica reduces stomata closure and H2O2 production after Alternaria brassicae infection in leaves and suppresses the defense-related accumulation of the phytohormone jasmonic acid. Thus, shifting the growth conditions toward a stress promotes the mutualistic interaction, while optimal supply with nutrients or low stress diminishes the benefits for the plant in the symbiosis.

Microarray analyses during early and later stages of the Arabidopsis/Piriformospora indica interaction.

Colonization of the roots of different plant species by Piriformospora indica results in better plant performance and biotic and abiotic stress tolerance. An increase of the biomass and seed yield is other beneficial effect of P. indica for the host plants. The interaction of P. indica with Arabidopsis thaliana roots is a unique model system to study symbiotic relationships. We describe a co-cultivation system which allows us to investigate the effects of fungal exudates on the root transcriptome before and after the establishment of a physical contact, and during early phases of root colonization. We present a detailed protocol which facilitates easy reproduction of the results (NCBI GEO accession number GSE58771) published by Vahabi et al. (2015) in BMC Plant Biology [1].

The interaction of Arabidopsis with Piriformospora indica shifts from initial transient stress induced by fungus-released chemical mediators to a mutualistic interaction after physical contact of the two symbionts.

BACKGROUND: Piriformospora indica, an endophytic fungus of Sebacinales, colonizes the roots of many plant species including Arabidopsis thaliana. The symbiotic interaction promotes plant performance, growth and resistance/tolerance against abiotic and biotic stress. RESULTS: We demonstrate that exudated compounds from the fungus activate stress and defense responses in the Arabidopsis roots and shoots before the two partners are in physical contact. They induce stomata closure, stimulate reactive oxygen species (ROS) production, stress-related phytohormone accumulation and activate defense and stress genes in the roots and/or shoots. Once a physical contact is established, the stomata re-open, ROS and phytohormone levels decline, and the number and expression level of defense/stress-related genes decreases. CONCLUSIONS: We propose that exudated compounds from P. indica induce stress and defense responses in the host. Root colonization results in the down-regulation of defense responses and the activation of genes involved in promoting plant growth, metabolism and performance.

WRKY6 restricts Piriformospora indica-stimulated and phosphate-induced root development in Arabidopsis.

BACKGROUND: Arabidopsis root growth is stimulated by Piriformospora indica, phosphate limitation and inactivation of the WRKY6 transcription factor. Combinations of these factors induce unexpected alterations in root and shoot growth, root architecture and root gene expression profiles. RESULTS: The results demonstrate that P. indica promotes phosphate uptake and root development under Pi limitation in wrky6 mutant. This is associated with the stimulation of PHOSPHATE1 expression and ethylene production. Expression profiles from the roots of wrky6 seedlings identified genes involved in hormone metabolism, transport, meristem, cell and plastid proliferation, and growth regulation. 25 miRNAs were also up-regulated in these roots. We generated and discuss here a list of common genes which are regulated in growing roots and which are common to all three growth stimuli investigated in this study. CONCLUSION: Since root development of wrky6 plants exposed to P. indica under phosphate limitation is strongly promoted, we propose that common genes which respond to all three growth stimuli are central for the control of root growth and architecture. They can be tested for optimizing root growth in model and agricultural plants.

The beneficial fungus Piriformospora indica protects Arabidopsis from Verticillium dahliae infection by downregulation plant defense responses.

BACKGROUND: Verticillium dahliae (Vd) is a soil-borne vascular pathogen which causes severe wilt symptoms in a wide range of plants. The microsclerotia produced by the pathogen survive in soil for more than 15 years. RESULTS: Here we demonstrate that an exudate preparation induces cytoplasmic calcium elevation in Arabidopsis roots, and the disease development requires the ethylene-activated transcription factor EIN3. Furthermore, the beneficial endophytic fungus Piriformospora indica (Pi) significantly reduced Vd-mediated disease development in Arabidopsis. Pi inhibited the growth of Vd in a dual culture on PDA agar plates and pretreatment of Arabidopsis roots with Pi protected plants from Vd infection. The Pi-pretreated plants grew better after Vd infection and the production of Vd microsclerotia was dramatically reduced, all without activating stress hormones and defense genes in the host. CONCLUSIONS: We conclude that Pi is an efficient biocontrol agent that protects Arabidopsis from Vd infection. Our data demonstrate that Vd growth is restricted in the presence of Pi and the additional signals from Pi must participate in the regulation of the immune response against Vd.

Growth of Arabidopsis seedlings on high fungal doses of Piriformospora indica has little effect on plant performance, stress, and defense gene expression in spite of elevated jasmonic acid and jasmonic acid-isoleucine levels in the roots.

The endophytic fungus Piriformospora indica colonizes the roots of many plant species including Arabidopsis and promotes their performance, biomass, and seed production as well as resistance against biotic and abiotic stress. Imbalances in the symbiotic interaction such as uncontrolled fungal growth result in the loss of benefits for the plants and activation of defense responses against the microbe. We exposed Arabidopsis seedlings to a dense hyphal lawn of P. indica. The seedlings continue to grow, accumulate normal amounts of chlorophyll, and the photosynthetic parameters demonstrate that they perform well. In spite of high fungal doses around the roots, the fungal material inside the roots was not significantly higher when compared with roots that live in a beneficial symbiosis with P. indica. Fifteen defense- and stress-related genes including PR2, PR3, PAL2, and ERF1 are only moderately upregulated in the roots on the fungal lawn, and the seedlings did not accumulate H2O2/radical oxygen species. However, accumulation of anthocyanin in P. indica-exposed seedlings indicates stress symptoms. Furthermore, the jasmonic acid (JA) and jasmonic acid-isoleucine (JA-Ile) levels were increased in the roots, and consequently PDF1.2 and a newly characterized gene for a 2-oxoglurate and Fe2+ -dependent oxygenase were upregulated more than 7-fold on the dense fungal lawn, in a JAR1- and EIN3-dependent manner. We conclude that growth of A. thaliana seedlings on high fungal doses of P. indica has little effect on the overall performance of the plants although elevated JA and JA-Ile levels in the roots induce a mild stress or defense response.