Modifications in gene expression and phenolic compounds content by methyl jasmonate and fungal elicitors in Ficus carica. Cv. Siah hairy root cultures.

BACKGROUND: One of the most effective strategies to increase phytochemicals production in plant cultures is elicitation. In the present study, we studied the effect of abiotic and biotic elicitors on the growth, key biosynthetic genes expression, antioxidant capacity, and phenolic compounds content in Rhizobium (Agrobacterium) rhizogenes-induced hairy roots cultures of Ficus carica cv. Siah. METHODS: The elicitors included methyl jasmonate (MeJA) as abiotic elicitor, culture filtrate and cell extract of fungus Piriformospora indica as biotic elicitors were prepared to use. The cultures of F. carica hairy roots were exposed to elicitores at different time points. After elicitation treatments, hairy roots were collected, and evaluated for growth index, total phenolic (TPC) and flavonoids (TFC) content, antioxidant activity (2,2-diphenyl-1-picrylhydrazyl, DPPH and ferric ion reducing antioxidant power, FRAP assays), expression level of key phenolic/flavonoid biosynthesis genes, and high-performance liquid chromatography (HPLC) analysis of some main phenolic compounds in comparison to control. RESULTS: Elicitation positively or negatively affected the growth, content of phenolic/flavonoid compounds and DPPH and FRAP antioxidant activities of hairy roots cultures in depending of elicitor concentration and exposure time. The maximum expression level of chalcone synthase (CHS: 55.1), flavonoid 3'-hydroxylase (F3'H: 34.33) genes and transcription factors MYB3 (32.22), Basic helix-loop-helix (bHLH: 45.73) was induced by MeJA elicitation, whereas the maximum expression level of phenylalanine ammonia-lyase (PAL: 26.72) and UDP-glucose flavonoid 3-O-glucosyltransferase (UFGT: 27.57) genes was obtained after P. indica culture filtrate elicitation. The P. indica elicitation also caused greatest increase in the content of gallic acid (5848 µg/g), caffeic acid (508.2 µg/g), rutin (43.5 µg/g), quercetin (341 µg/g), and apigenin (1167 µg/g) phenolic compounds. CONCLUSIONS: This study support that elicitation of F. carica cv. Siah hairy roots can be considered as an effective biotechnological method for improved phenolic/flavonoid compounds production, and of course this approach requires further research.

Multiple paths of plant host toxicity are associated with the fungal toxin cercosporin.

The Cercospora species of fungi are responsible for leaf spot disease affecting many key economic crops. Most of these fungi secrete a toxic photodynamic molecule, cercosporin, that reacts with light and oxygen to produce reactive singlet oxygen (1 O2 ) contributing to fungal virulence. We show similar cellular localization and aetiology of cercosporin in the non-host Arabidopsis and the host Nicotiana benthamiana. Cercosporin accumulates in cell membranes in an oxidized state and in plastids in a mixture of redox states in a manner that is dependent on ongoing photosynthetic processes. We observed that cercosporin rapidly compromised photosynthesis as measured by Fv /Fm , NPQ, and photosystem I (PSI) parameters. Stomatal guard cells in particular demonstrated rapid light-dependent membrane permeabilization that led to changes in leaf conductance. We showed that cercosporin-mediated 1 O2 generation oxidized RNA to form 8-oxoguanosine (8-oxoG), leading to translational attenuation and induction of 1 O2 signature gene transcripts. We also identified a subset of cercosporin-induced transcripts that were independent of the photodynamic effect. Our results point to the multimodal action of cercosporin that includes the inhibition of photosynthesis, the direct oxidation of nucleic acid residues and the elicitation of complex transcriptome responses.

Warhorses in soil bioremediation: Seed biopriming with PGPF secretome to phytostimulate crop health under heavy metal stress.

The fungal symbiosis with the plant root system is importantly recognized as a plant growth promoting fungi (PGPFs), as well as elicitor of plant defence against different biotic and abiotic stress conditions. Thus PGPFs are playing as a key trouper in enhancing agricultural quality and increased crop production and paving a way towards a sustainable agriculture. Due to increased demand of food production, the over and unscientific usage of chemical fertilizers has led to the contamination of soil by organic and inorganic wastes impacting on soil quality, crops quality effecting on export business of agricultural products. The application of microbial based consortium like plant growth promoting fungi is gaining worldwide importance due to their multidimensional activity. These activities are through plant growth promotion, induction of systemic resistance, disease combating and detoxification of organic and inorganic toxic chemicals, a heavy metal tolerance ability. The master key behind these properties exhibited by PGPFs are attributed towards various secretory biomolecules (secondary metabolites or enzymes or metabolites) secreted by the fungi during interaction mechanism. The present review is focused on the multidimensional role PGPFs as elicitors of Induced systemic resistance against phytopathogens as well as heavy metal detoxifier through seed biopriming and biofortification methods. The in-sights on PGPFs and their probable mechanistic nature contributing towards plants to withstand heavy metal stress and stress alleviation by activating of various stress regulatory pathways leading to secretion of low molecular weight compounds like organic compounds, glomalin, hydrophobins, etc,. Thus projecting the importance of PGPFs and further requirement of research in developing PGPFs based molecules and combining with trending Nano technological approaches for enhanced heavy metal stress alleviations in plant and soil as well as establishing a sustainable agriculture.

The Arabidopsis leucine-rich repeat receptor-like kinase MIK2 is a crucial component of early immune responses to a fungal-derived elicitor.

Fusarium spp. cause severe economic damage in many crops, exemplified by Panama disease of banana or Fusarium head blight of wheat. Plants sense immunogenic patterns (termed elicitors) at the cell surface to initiate pattern-triggered immunity (PTI). Knowledge of fungal elicitors and corresponding plant immune-signaling is incomplete but could yield valuable sources of resistance. We characterized Arabidopsis thaliana PTI responses to a peptide elicitor fraction present in several Fusarium spp. and employed a forward-genetic screen using plants containing a cytosolic calcium reporter to isolate fusarium elicitor reduced elicitation (fere) mutants. We mapped the causal mutation in fere1 to the leucine-rich repeat receptor-like kinase MDIS1-INTERACTING RECEPTOR-LIKE KINASE 2 (MIK2) and confirmed a crucial role of MIK2 in fungal elicitor perception. MIK2-dependent elicitor responses depend on known signaling components and transfer of AtMIK2 is sufficient to confer elicitor sensitivity to Nicotiana benthamiana. Arabidopsis senses Fusarium elicitors by a novel receptor complex at the cell surface that feeds into common PTI pathways. These data increase mechanistic understanding of PTI to Fusarium and place MIK2 at a central position in Arabidopsis elicitor responses.

Fungal elicitor-induced transcriptional changes of genes related to branched-chain amino acid metabolism in Streptomyces natalensis HW-2.

Natamycin is a polyene macrolide antibiotic and widely used as a natural food preservative. Fungal elicitor had positive effects on the natamycin biosynthesis in Streptomyces natalensis HW-2. However, the global gene expression in response to fungal elicitor is not still reported. In the study, RNA-Seq was used to check the change of transcriptome by fungal elicitor in S. natalensis HW-2. The results showed that there were 1265 differential expression genes (DEGs) at 40 h and 2196 DEGs at 80 h. Most of the genes involved in natamycin biosynthesis were upregulated. KEGG pathway analysis showed that fungal elicitor had strong effects on the transcriptional levels of genes related to branch-chained amino acid (BCAA) metabolism. There were 23 upregulated or downregulated DEGs involved in BCAA biosynthesis and degradation at 40 h and 80 h. To confirm whether the improvement of BCAA biosynthesis could produce more natamycin, metabolic engineering was used to homologously overexpress the gene ilvH which encoded the regulatory subunit of acetolactate synthase (ALS) in S. natalensis. The results showed that overexpression of ilvH in S. natalensis HW-2 increased natamycin production to 1.25 g/L in the flask, which was a 32% increase compared with that of the parent strain. Real-time quantitative PCR analysis showed that the transcriptional level of ilvH in mutant strain S. natalensis ZS101 was significantly increased. Acetyl-CoA content was also raised. The results suggested that the fungal elicitor enhanced natamycin biosynthesis by improving precursor supply via BCAA metabolism. This study will open a new avenue for enhancing natamycin production by metabolic engineering and adding fungal elicitor. KEY POINTS: • The fungal elicitor had strong effects on the transcriptional levels of genes related to branch-chained amino acid metabolism by RNA-Seq. • The homologous overexpression of gene ilvH increased natamycin production by 32% and acetyl-CoA content was raised in mutant strain S. natalensis ZS101.

[Effects of exogenous IBA and fungal elicitor on growth of in vitro roots culture of Dysosma versipellis and production of podophyllotoxin].

Using the White as basic medium, the effects of the exogenous IBA and endophytic fungal elicitor on the growth of in vitro roots cultures of Dysosma versipellis and production of podophyllotoxin were investigated in this study. The results showed that the IBA and the endophytic fungus Zasmidium syzygii elicitor could increase the content of podophyllotoxin of in vitro roots of D. versipellis after 3 weeks. The White medium added with 3 mg·L~(-1) IBA induced the highest increase of podophyllotoxin(1 830.86 µg·g~(-1)), which was 2.07 folds greater than the control, and followed by 1.5 mg·L~(-1) IBA, fungal elicitor, 1 mg·L~(-1) IBA, 0.5 mg·L~(-1) IBA and 4.5 mg·L~(-1) IBA, which was 1.82, 1.71, 1.63, 1.43 and 1.1 folds greater than the control, respectively. The results also showed that the growth of roots was certain positively correlated with the change of IBA concentration. Therefore, 3 mg·L~(-1) IBA was the most suitable for the production of podophyllotoxin in the in vitro roots of D. versipellis, and the stimulating effect of Z. syzygii fungal elicitor was between 1.5 mg·L~(-1) and 1 mg·L~(-1) IBA, which was a potential natural elicitor to induce the accumulation of podophyllotoxin in future production.

Physicochemical and microbial responses of Streptomyces natalensis HW-2 to fungal elicitor.

The effects of fungal elicitor on the physicochemical and microbial responses of Streptomyces natalensis HW-2 were investigated. The results showed that the elicitor could decrease dry cell weight (DCW) by 17.7% and increase the utilization of glucose, while the curve of pH was not obviously altered. The elicitor enhanced the yield of natamycin from 1.33 to 2.49 g/L. The morphology of the colony and the mycelium treated with elicitor showed significant differences from that of control. The level of intracellular reactive oxygen species (ROS) increased to 333.8 ng/L, which was a twofold increase comparing with the control. The concentration of Ca2+ reached 421.1 nmol/L, which increased by 32.8% after the addition of the elicitor. The activities of pyruvic carboxylase and phosphoenol pyruvate carboxylase were enhanced by 27.8 and 11.9%, respectively, while citrate synthase activity decreased by 23.1% in comparison with the control.

Activation of phenylpropanoid pathway and PR of potato tuber against Fusarium sulphureum by fungal elicitor from Trichothecium roseum.

The induced resistance of potato tuber (Solanum tuberosum cv. Xindaping) tissue against Fusarium sulphureum by a fungal elicitor from the incompatible pathogen Trichothecium roseum and its possible mechanism were studied. The results showed that the lesion development of the wound-inoculated potato tuber was significantly reduced by treatment with the fungal elicitor from T. roseum (P < 0.05). Inoculation with F. sulphureum on the 16th day after treatment with the fungal elicitor80 at 15.0 µg/ml had the best resistant effect in the potato tuber, with the diameter being only reduced by 47 % that of the control. In addition, the results also showed that the potato tuber treated with the fungal elicitor80 could systemically induce lignin deposition, total phenolic content, flavonoid content and defense enzymes, including three keys phenylpropanoid pathway (PAL, 4CL and C4H) and pathogenesis-related (GLU and CHT) enzymes. The fungal elicitor80 also enhanced the up-regulation of the transcription and expression of PAL, C4H, 4CL, GLU and CHT genes. The treatment with the fungal elicitor80 + F. sulphureum caused the marked and/or prompt enhancement of all indexes when compared to treatment with the fungal elicitor80 or inoculation with the pathogen alone. The results suggested that the fungal elicitor of T. roseum could significantly enhance defense responses in potato tuber against dry rot mainly due to the up-regulation of the transcription and expression of resistance-related genes as well as increasing the activity of resistance-related enzymes and antifungal compounds.

A cerato-platanin-like protein HaCPL2 from Heterobasidion annosum sensu stricto induces cell death in Nicotiana tabacum and Pinus sylvestris.

The cerato-platanin family is a group of small secreted cysteine-rich proteins exclusive for filamentous fungi. They have been shown to be involved in the interactions between fungi and plants. Functional characterization of members from this family has been performed mainly in Ascomycota, except Moniliophthora perniciosa. Our previous phylogenetic analysis revealed that recent gene duplication of cerato-platanins has occurred in Basidiomycota but not in Ascomycota, suggesting higher functional diversification of this protein family in Basidiomycota than in Ascomycota. In this study, we identified three cerato-platanin homologues from the basidiomycete conifer pathogen Heterobasidion annosum sensu stricto. Expression of the homologues under various conditions as well as their roles in the H. annosum s.s.-Pinus sylvestris (Scots pine) pathosystem was investigated. Results showed that HaCPL2 (cerato-platanin-like protein 2) had the highest sequence similarity to cerato-platanin from Ceratocystis platani and hacpl2 was significantly induced during nutrient starvation and necrotrophic growth. The treatment with recombinant HaCPL2 induced cell death, phytoalexin production and defense gene expression in Nicotiana tabacum. Eliciting and cell death-inducing ability accompanied by retardation of apical root growth was also demonstrated in Scots pine seedlings. Our results suggest that HaCPL2 might contribute to the virulence of H. annosum s.s. by promoting plant cell death.

Priming with fungal elicitor elicits early signaling defense against leaf spot of broccoli underlying cellular, biochemical and gene expression.

The phytopathogen Alternaria, which causes leaf spot disease in broccoli plants, has developed the ability to adapt to changing climatic conditions with the failure of conventional fungicides. To restrict its infection and further spread, the application of novel molecules that have the ability to trigger innate immune responses of the plants to concurrent pathogenic invasions is essential. In this context, the current study was set out to assess Trichoderma-derived trehalose molecules' beneficial effects from inciting resistance mechanisms in broccoli plants against Alternaria brassicicola at histochemical, biochemical and genetic levels. From the results of the greenhouse experiment, it was evident that trehalose proved to be an exceptional elicitor in combating broccoli leaf spot disease. The induced resistance is positively associated with an early hypersensitive reaction expressed in the primed seedlings. Additionally, the deposition of histochemical such as callose and phenols was found to be accelerated in the tripartite system at 24 and 48 h after post-inoculation (hpi). At the biochemical level, the activities of the enzymes chitinase (CHI), catalase (CAT) and allene oxide synthase (AOS) were significantly enhanced in trehalose-treated broccoli plants inoculated with A. brassicicola. The enzymatic activities were further validated by quantitative real-time polymerase chain reaction (qRT-PCR) analyses wherein the relative expressions of the corresponding gene transcripts were up-regulated in trehalose primed plants. Conclusively, the investigation results have provided vital insights into the histochemical, biochemical and gene expression underlying the enhanced induced systemic resistance against broccoli leaf spot disease.

Production of Nematicidal Pinosylvin Stilbenes in Cell Suspension Cultures of Pinus koraiensis by Fungal Elicitation.

Pinosylvin stilbenes are natural phenolic compounds found in the Pinaceae family and act as phytoalexins. Some pinosylvin stilbenes have strong nematicidal activity against pine wood nematodes (PWNs: Bursaphelenchus xylophilus). Here, we established the efficient production of two pinosylvin stilbenes, dihydropinosylvin monomethylether (DPME) and pinosylvin monomethylether (PME), by cell suspension culture of Pinus koraiensis after fungal elicitation. DPME and PME were found in small amounts (less than 40 µg/g DW) in the stem bark and leaves of P. koraiensis plants. Cell suspension cultures were established from the cultures of calli derived from mature zygotic embryos of P. koraiensis in 1/2 Litvay medium containing 2.2 µM 2,4-D and 2.2 µM BA. Two types of fungal elicitors, fungal cell extract (CE) and fungal medium filtrate (MF), were prepared from three species of fungi (Penicillium chrysogenum, P. pinophilum, and P. roquefortii). CE and MF treatments strongly stimulated the production of PME and DPME in cultured cells. The production of PME in suspension cells of P. chrysogenum, P. pinophilum, and P. roquefortii MF treatments after 3 days was 5734 µg/g DW, 4051 µg/g DW, and 6724 µg/g DW, respectively. Pinosylvin synthase (PkSTS) and pinosylvin O-methyltransferase (PkPMT) are key genes in DPME and PME biosynthesis. qPCR analysis revealed that the expression of the PkSTS and PkPMT in cultured cells was highly enhanced after fungal elicitor treatment. The cell extracts after MF treatment resulted in 92.5 ± 7.8% immobilization of the adult PWNs and 63.7 ± 3.5% immobilization of the juvenile PWNs within 24 h. However, control cell extracts without MF treatment showed 11.3 ± 1.4% nematicidal activity against adult PWNs. Our results suggest that pinosylvin stilbenes can be produced from the cell culture of P. koraiensis after fungal elicitor treatment and can be used as nematicidal compounds against PWNs.

Stimulatory Effects of Oleci Acid and Fungal Elicitor on Betulinic Acid Production by Submerged Cultivation of Medicinal Mushroom Inonotus obliquus.

To evaluate the novel strategy of oleic acid and fungal elicitor (made from Aspergillus niger) to elicit betulinic acid biosynthesis in medicinal mushroom Inonotus obliquus, we conduct the stimulatory effects investigation for synthesizing betulinic acid from betulin. HPLC results indicated oleic acid and fungal elicitor were effective stimulators. The supplementation of 1.0 g/L oleic acid led to the highest increase of betulinic acid either in dry mycelia or fermentation broth by 2-fold of the control. Fungal elicitor at 45 mg/L markedly increases mycelia growth by 146.0% and enhance intracellular betulinic acid accumulation by 429.5% as compared to the controls. Quantification of transcription levels determined that oleic acid, fungal elicitor and their combinations could induce the expressions of key genes involved in betulinic acid biosynthesis, such as HMG-CoA reductase and squalene synthase. These findings indicated that oleic acid and fungal elicitor could enhance betulinic acid metabolism by up-regulating key genes expression.

Primary and secondary metabolites produced in Salvia miltiorrhiza hairy roots by an endophytic fungal elicitor from Mucor fragilis.

Salvia miltiorrhiza is one of the most commonly used medicinal materials in China. In recent years, the quality of S. miltiorrhiza has attracted much attention. Biotic and abiotic elicitors are widely used in cultivation to improve the quality of medicinal plants. We isolated an endophytic fungus, Mucor fragilis, from S. miltiorrhiza. We compared the effects of endophytic fungal elicitors with those of yeast extract together with silver ion, widely used together as effective elicitors, on S. miltiorrhiza hairy roots. Seventeen primary metabolites (amino acids and fatty acids) and five secondary metabolites (diterpenoids and phenolic acids) were analyzed after elicitor treatment. The mycelium extract promoted the accumulation of salvianolic acid B, rosmarinic acid, stearic acid, and oleic acid in S. miltiorrhiza hairy roots. Additionally, qPCR revealed that elicitors affect the accumulation of primary and secondary metabolites by regulating the expression of key genes (SmAACT, SmGGPPS, and SmPAL). This is the first detection of both the primary and secondary metabolites of S. miltiorrhiza hairy roots, and the results of this work should help guide the quality control of S. miltiorrhiza. In addition, the findings confirm that Mucor fragilis functions as an effective endophytic fungal elicitor with excellent application prospect for cultivation of medicinal plants.

Piriformospora indica based elicitation for overproduction of phenolic compounds by hairy root cultures of Ficus carica.

Ficus carica L. is an important source of phenolic and flavonoid compounds with valuable pharmaceutical application across various diseases. The current study was carried out to investigate the influence of Piriformospora indica elicitation on growth, production of phenolic compounds, antioxidant capacity, and expression level of flavonoid biosynthetic pathway genes in hairy root (HR) cultures of F. carica. The maximum improvement in accumulation of phenolic compounds was observed when HR culture of Ficus carica L. was exposed to 2% culture filtrate of P. indica for 72 h: gallic acid (80.5- fold), caffeic acid (26.2-fold), coumaric acid (4.5-fold), and cinnamic acid (60.1-fold), apigenin (27.6-fold) and rutin (5.7-fold). While the highest levels of chlorogenic acid (4.9-fold) and quercetin flavonoid (8.8-fold) were obtained after 48 h elicitation with culture filtrate and cell extract of P. indica at 6% (v/v), respectively. The analysis of biosynthetic genes revealed that the exposure to fungal elicitors resulted in up-regulation of phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), UDP-glucose flavonoid 3-O-glucosyltransferase (UFGT) and MYB3 transcription factor. This study shows the potential of P. indica as an efficacious elicitor for enhancing the secondary metabolites production by F. carica HRs.

Isolation, characterization, and plant growth-promoting activities of endophytic fungi from a wild orchid Vanda cristata.

Endophytism is one of the widely explored phenomena related to orchids and fungi. Endophytic fungi assist plants by supplementing nutrient acquisition, and synthesis of plant growth regulators. Vanda cristata is an epiphytic orchid that has a great diversity of endophytic fungi. Endophytic fungi were isolated from roots, stems, and leaves of V.cristata and identified by both morphological and molecular study. Furthermore, the isolated endophytic fungi were subjected to auxin synthesis, phosphate solubilization, ammonia synthesis, and elicitor growth test for understanding their growth-promoting effect in a qualitative and quantitative manner. Altogether, 12 different endophytic fungi were isolated from roots, stems, and leaves of V. cristata of which most species belonged to Ascomycota. Unidentified II fungi were found to be most effective for auxin synthesis and phosphate solubilization while Agaricus bisporous and Mycolepto discus were most effective for ammonia synthesis. We have tested the plant growth-promoting activity of the twelve isolated endophytic fungi on Cymbidium aloifolium protocorms (12 weeks old). All the endophytic fungi showed growth-promoting activity. Plant growth of Cymbidium aloifolium was found higher on the MS medium supplemented with all fungal elicitors. Fungal elicitor CVS4, however, showed the highest plant growth-promoting activity toward C. aloifolium.

RNAi-mediated silencing of pinoresinol lariciresinol reductase in Linum album hairy roots alters the phenolic accumulation in response to fungal elicitor.

Lignans are diphenolic compounds produced in plants via coupling of two coniferyl alcohol molecules with the aid of a dirigent protein to form pinoresinol (PINO). The latter is reduced via lariciresinol (LARI) to secoisolariciresinol by the bifunctional pinoresinol-lariciresinol reductase (PLR). In this study, we clarified the consequences of altered lignan biosynthesis on amino acids, phenolics compounds and lignin in the hairy roots of Linum album with an ihpRNAi construct to silence PLR gene expression. Down-regulation of PLR-La1 resulted in up to an 8.3 and 3.3-time increased PINO and LARI content respectively, and reduced levels of podophyllotoxin (PTOX) and 6-methoxy podophyllotoxin (6-MPTOX). By Suppression of PLR expression, the metabolites belonging to shikimate and phenylpropanoid pathways are conducted to phenolic compounds and lignin accumulations. Although PINO and LARI were induced in response to fungal elicitor, the accumulation of PTOX and 6-MPTOX did not occur in PLR down-regulated roots. Our result also demonstrated variation in amino acids, phenolic compounds and lignin levels in presence of the fungal elicitation in PLR down regulated-roots. This data assert the accumulation of aryltetralin lignans in interactions with plant pathogens by PLR activity and the importance this enzyme for defense against pathogens in L. album.

Entomopathogenicity and Biological Attributes of Himalayan Treasured Fungus Ophiocordyceps sinensis (Yarsagumba).

Members of the entomophagous fungi are considered very crucial in the fungal domain relative to their natural phenomenon and economic perspectives; however, inadequate knowledge of their mechanisms of interaction keeps them lagging behind in parallel studies of fungi associated with agro-ecology, forest pathology and medical biology. Ophiocordyceps sinensis (syn. Cordyceps sinensis), an intricate fungus-caterpillar complex after it parasitizes the larva of the moth, is a highly prized medicinal fungus known widely for ages due to its peculiar biochemical assets. Recent technological innovations have significantly contributed a great deal to profiling the variable clinical importance of this fungus and other related fungi with similar medicinal potential. However, a detailed mechanism behind fungal pathogenicity and fungal-insect interactions seems rather ambiguous and is poorly justified, demanding special attention. The goal of the present review is to divulge an update on the published data and provides promising insights on different biological events that have remained underemphasized in previous reviews on fungal biology with relation to life-history trade-offs, host specialization and selection pressures. The infection of larvae by a fungus is not a unique event in Cordyceps; hence, other fungal species are also reviewed for effective comparison. Conceivably, the rationale and approaches behind the inheritance of pharmacological abilities acquired and stored within the insect framework at a time when they are completely hijacked and consumed by fungal parasites, and the molecular mechanisms involved therein, are clearly documented.

Fungal elicitors enhance ginsenosides biosynthesis, expression of functional genes as well as signal molecules accumulation in adventitious roots of Panax ginseng C. A. Mey.

In this work, we selected three fungi strains (Aspergillus niger, Aspergillus flavus and Aspergillus oryzae) as elicitors prepared from mycelium or fermentation broth to improve ginsenosides production in adventitious roots culture. The results showed that ginsenosides production (29.90±4.67mgg-1) was significantly enhanced upon elicitation with 200mgL-1A. niger elicitor prepared from mycelium, which was 3.52-fold of untreated group. HPLC-ESI-MSn analysis was performed, showing that ginsenoside Rb3 was present after treatment with the A. niger. Furthermore, we found that A. niger significantly enhanced accumulation of Nitric oxide (NO), salicylic acid (SA) and jasmonic acid (JA) involved in plant defense response, and significantly up-regulated the expression of the geranyl diphosphate synthase (GPS), farnesyl diphosphate synthase (FPS), squalene synthase (SS), squalene epoxidase (SE), dammarenediol synthase (DS), Two cytochrome P450 (CYP) genes (CYP716A47 and CYP716A53v2) and three UDP-glycosyltransferases (UGT) genes (UGTAE2, UGT94Q2 and UGTpg100).

Low-molecular-weight precursors for defense-related cell wall hydroxycinnamoyl esters in elicited parsley suspension cultures.

Cell walls from suspension cultures of parsley (Petroselinum crispum L.) induced with a fungal elicitor contained hydroxycinnamoyl ester groups presumably not bound to pectic polysaccharides. Extracts from these cells were separated into a range of low-molecular-weight compounds containing esterified ferulic and p-coumaric acid as well as glucose and some arabinose. Similar compounds also accumulated extracellularly in elicited cultures but only in the presence of the peroxidase inhibitor ascorbate, suggesting that they may represent the exported precursors for cell wall hydroxycinnamic acids. From cultures elicited in the presence of ascorbate, alkali released from the cell walls more ferulic, p-coumaric and p-hydroxybenzoic acid, as well as p-hydroxybenzaldehyde and vanillin, indicating that the corresponding wall phenolics can all become further cross-linked.

Identification of fungus-responsive cis-acting element in the promoter of Brassica juncea chitinase gene, BjCHI1.

Chitinases are a group of pathogenesis-related proteins. The Brassica juncea chitinase gene BjCHI1 is highly inducible by pathogenic fungal infection, suggesting that the promoter of BjCHI1 might contain specific cis-acting element responsive to fungal attack. To identify the fungus-responsive element in BjCHI1 promoter (BjC-P), a series of binary plant transformation vectors were constructed by fusing the BjC-P or its deletion-derivatives to ß-glucuronidase (GUS) reporter gene. Expression of the GUS reporter gene was systematically assayed by a transient gene expression system in Nicotiana benthamiana leaves treated with fungal elicitor Hexa-N-Acetyl-Chitohexaose, as well as in transgenic Arabidopsis plants inoculated with fungus Botrytis cinerea. The histochemical and quantitative GUS assays showed that the W-box-like element (GTAGTGACTCAT) in the region (-668 to -657) was necessary for the fungus-response, although there were another five W-box-like elements in BjC-P. In addition, gain-of-function analysis demonstrated that the fragment (-409 to -337) coupled to the W-box-like element was needed for full magnitude of the fungal induction. These results revealed the existence of a novel regulation mechanism of W-box-like element involved in plant pathogenic resistance, and will benefit the potential application of BjC-P in engineering crops.