PHYTOCHROME-INTERACTING FACTOR 4/HEMERA-mediated thermosensory growth requires the Mediator subunit MED14.

While moderately elevated ambient temperatures do not trigger stress responses in plants, they do substantially stimulate the growth of specific organs through a process known as thermomorphogenesis. The basic helix-loop-helix transcription factor PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) plays a central role in regulating thermomorphogenetic hypocotyl elongation in various plant species, including Arabidopsis (Arabidopsis thaliana). Although it is well known that PIF4 and its co-activator HEMERA (HMR) promote plant thermosensory growth by activating genes involved in the biosynthesis and signaling of the phytohormone auxin, the detailed molecular mechanism of such transcriptional activation is not clear. In this report, we investigated the role of the Mediator complex in the PIF4/HMR-mediated thermoresponsive gene expression. Through the characterization of various mutants of the Mediator complex, a tail subunit named MED14 was identified as an essential factor for thermomorphogenetic hypocotyl growth. MED14 was required for the thermal induction of PIF4 target genes but had a marginal effect on the levels of PIF4 and HMR. Further transcriptomic analyses confirmed that the expression of numerous PIF4/HMR-dependent, auxin-related genes required MED14 at warm temperatures. Moreover, PIF4 and HMR physically interacted with MED14 and both were indispensable for the association of MED14 with the promoters of these thermoresponsive genes. While PIF4 did not regulate MED14 levels, HMR was required for the transcript abundance of MED14. Taken together, these results unveil an important thermomorphogenetic mechanism, in which PIF4 and HMR recruit the Mediator complex to activate auxin-related growth-promoting genes when plants sense moderate increases in ambient temperature.

Transcription factor OsbZIP49 controls tiller angle and plant architecture through the induction of indole-3-acetic acid-amido synthetases in rice.

Tiller angle is an important determinant of plant architecture in rice (Oryza sativa L.). Auxins play a critical role in determining plant architecture; however, the underlying metabolic and signaling mechanisms are still largely unknown. In this study, we have identified a member of the bZIP family of TGA class transcription factors, OsbZIP49, that participates in the regulation of plant architecture and is specifically expressed in gravity-sensing tissues, including the shoot base, nodes and lamina joints. Transgenic rice plants overexpressing OsbZIP49 displayed a tiller-spreading phenotype with reduced plant height and internode lengths. In contrast, CRISPR/Cas9-mediated knockout of OsbZIP49 resulted in a compact architecture. Follow-up studies indicated that the effects of OsbZIP49 on tiller angles are mediated through changes in shoot gravitropic responses. Additionally, we provide evidence that OsbZIP49 activates the expression of indole-3-acetic acid-amido synthetases OsGH3-2 and OsGH3-13 by directly binding to TGACG motifs located within the promoters of both genes. Increased GH3-catalyzed conjugation of indole-3-acetic acid (IAA) in rice transformants overexpressing OsbZIP49 resulted in the increased accumulation of IAA-Asp and IAA-Glu, and a reduction in local free auxin, tryptamine and IAA-Glc levels. Exogenous IAA or naphthylacetic acid (NAA) partially restored shoot gravitropic responses in OsbZIP49-overexpressing plants. Knockout of OsbZIP49 led to reduced expression of both OsGH3-2 and OsGH3-13 within the shoot base, and increased accumulation of IAA and increased OsIAA20 expression levels were observed in transformants following gravistimulation. Taken together, the present results reveal the role transcription factor OsbZIP49 plays in determining plant architecture, primarily due to its influence on local auxin homeostasis.

In vivo assembly of the sorgoleone biosynthetic pathway and its impact on agroinfiltrated leaves of Nicotiana benthamiana.

Sorgoleone, a hydrophobic compound exuded from root hair cells of Sorghum spp., accounts for much of the allelopathic activity of the genus. The enzymes involved in the biosynthesis of this compound have been identified and functionally characterized. Here, we report the successful assembly of the biosynthetic pathway and the significant impact of in vivo synthesized sorgoleone on the heterologous host Nicotiana benthamiana. A multigene DNA construct was prepared for the expression of genes required for sorgoleone biosynthesis in planta and deployed in N. benthamiana leaf tissues via Agrobacterium-mediated transient expression. RNA-sequencing was conducted to investigate the effects of sorgoleone, via expression of its biosynthesis pathway, on host gene expression. The production of sorgoleone in agroinfiltrated leaves as detected by gas chromatography/mass spectrometry (GC/MS) resulted in the formation of necrotic lesions, indicating that the compound caused severe phytotoxicity to these tissues. RNA-sequencing profiling revealed significant changes in gene expression in the leaf tissues expressing the pathway during the formation of sorgoleone-induced necrotic lesions. Transcriptome analysis suggested that the compound produced in vivo impaired the photosynthetic system as a result of downregulated gene expression for the photosynthesis apparatus and elevated expression of proteasomal genes which may play a major role in the phytotoxicity of sorgoleone.

Puupehenone, a Marine-Sponge-Derived Sesquiterpene Quinone, Potentiates the Antifungal Drug Caspofungin by Disrupting Hsp90 Activity and the Cell Wall Integrity Pathway.

The cell wall-targeting echinocandin antifungals, although potent and well tolerated, are inadequate in treating fungal infections due to their narrow spectrum of activity and their propensity to induce pathogen resistance. A promising strategy to overcome these drawbacks is to combine echinocandins with a molecule that improves their activity and also disrupts drug adaptation pathways. In this study, we show that puupehenone (PUUP), a marine-sponge-derived sesquiterpene quinone, potentiates the echinocandin drug caspofungin (CAS) in CAS-resistant fungal pathogens. We have conducted RNA sequencing (RNA-seq) analysis, followed by genetic and molecular studies, to elucidate PUUP's CAS-potentiating mechanism. We found that the combination of CAS and PUUP blocked the induction of CAS-responding genes required for the adaptation to cell wall stress through the cell wall integrity (CWI) pathway. Further analysis showed that PUUP inhibited the activation of Slt2 (Mpk1), the terminal mitogen-activated protein (MAP) kinase in this pathway. We also found that PUUP induced heat shock response genes and inhibited the activity of heat shock protein 90 (Hsp90). Molecular docking studies predicted that PUUP occupies a binding site on Hsp90 required for the interaction between Hsp90 and its cochaperone Cdc37. Thus, we show that PUUP potentiates CAS activity by a previously undescribed mechanism which involves a disruption of Hsp90 activity and the CWI pathway. Given the requirement of the Hsp90-Cdc37 complex in Slt2 activation, we suggest that inhibitors of this complex would disrupt the CWI pathway and synergize with echinocandins. Therefore, the identification of PUUP's CAS-potentiating mechanism has important implications in the development of new antifungal combination therapies.IMPORTANCE Fungal infections cause more fatalities worldwide each year than malaria or tuberculosis. Currently available antifungal drugs have various limitations, including host toxicity, narrow spectrum of activity, and pathogen resistance. Combining these drugs with small molecules that can overcome these limitations is a useful strategy for extending their clinical use. We have investigated the molecular mechanism by which a marine-derived compound potentiates the activity of the antifungal echinocandin caspofungin. Our findings revealed a mechanism, different from previously reported caspofungin potentiators, in which potentiation is achieved by the disruption of Hsp90 activity and signaling through the cell wall integrity pathway, processes that play important roles in the adaptation to caspofungin in fungal pathogens. Given the importance of stress adaptation in the development of echinocandin resistance, this work will serve as a starting point in the development of new combination therapies that will likely be more effective and less prone to pathogen resistance.

Diterpenoids with herbicidal and antifungal activities from hulls of rice (Oryza sativa).

Diterpenoids are the main secondary metabolites of plants and with a range of biological activities. In the present study, 7 compounds were isolated from the hulls of rice (Oryza sativa L.). Among them, 3 diterpenoids are new namely, 3,20-epoxy-3α-hydroxy- 8,11,13-abietatrie-7-one (1), 4,6-epoxy-3ß-hydroxy-9ß-pimara-7,15-diene (2) and 2-((E)-3- (4-hydroxy-3-methoxyphenyl) allylidene) momilactone A (3). While, 4 terpenoids are known, namely momilactone A (4), momilactone B (5), ent-7-oxo-kaur-15-en-18-oic acid (6) and orizaterpenoid (7). The structures of these diterpenoids were elucidated using 1D and 2D NMR in combination with ESI-MS and HR-EI-MS. Furthermore, all isolated compounds displayed antifungal activities against four crop pathogenic fungi Magnaporthe grisea, Rhizoctonia solani, Blumeria graminearum and Fusarium oxysporum, and phytotoxicity against paddy weed Echinochloa crusgalli. The results suggested that rice could produce plenty of secondary metabolites to defense against weeds and pathogens.

The roles of jasmonate signalling in nitrogen uptake and allocation in rice (Oryza sativa L.).

Herbivore damage by chewing insects activates jasmonate (JA) signalling that can elicit systemic defense responses in rice. Few details are known, however, concerning the mechanism, whereby JA signalling modulates nutrient status in rice in response to herbivory. (15 NH4 )2 SO4 labelling experiments, proteomic surveys, and RT-qPCR analyses were used to identify the roles of JA signalling in nitrogen (N) uptake and allocation in rice plants. Exogenous applications of methyl jasmonate (MeJA) to rice seedlings led to significantly reduced N uptake in roots and reduced translocation of recently-absorbed 15 N from roots to leaves, likely occurring as a result of down-regulation of glutamine synthetase cytosolic isozyme 1-2 and ferredoxin-nitrite reductase. Shoot MeJA treatment resulted in a remobilization of endogenous unlabelled 14 N from leaves to roots, and root MeJA treatment also increased 14 N accumulation in roots but did not affect 14 N accumulation in leaves of rice. Additionally, proteomic and RT-qPCR experiments showed that JA-mediated plastid disassembly and dehydrogenases GDH2 up-regulation contribute to N release in leaves to support production of defensive proteins/compounds under N-limited condition. Collectively, our results indicate that JA signalling mediates large-scale systemic changes in N uptake and allocation in rice plants.

Inferring Roles in Defense from Metabolic Allocation of Rice Diterpenoids.

Among their responses to microbial infection, plants deploy an arsenal of natural antibiotic products. Historically these have been identified on the basis of their antibiotic activity in vitro, which leaves open the question of their relevance to defense in planta. The vast majority of such natural products from the important crop plant rice (Oryza sativa) are diterpenoids whose biosynthesis proceeds via either ent- or syn-copalyl diphosphate (CPP) intermediates, which were isolated on the basis of their antibiotic activity against the fungal blast pathogen Magnaporthe oryzae However, rice plants in which the gene for the syn-CPP synthase Os-CPS4 is knocked out do not exhibit increased susceptibility to M. oryzae Here, we show that knocking out or knocking down Os-CPS4 actually decreases susceptibility to the bacterial leaf blight pathogen Xanthomonas oryzae By contrast, genetic manipulation of the gene for the ent-CPP synthase Os-CPS2 alters susceptibility to both M. oryzae and X. oryzae Despite the secretion of diterpenoids dependent on Os-CPS2 or Os-CPS4 from roots, neither knockout exhibited significant changes in the composition of their rhizosphere bacterial communities. Nevertheless, rice plants allocate substantial metabolic resources toward syn- as well as ent-CPP derived diterpenoids upon infection/induction. Further investigation revealed that Os-CPS4 plays a role in fungal non-host disease resistance. Thus, examination of metabolic allocation provides important clues into physiological function.

Identification and Functional Analysis of a Novel Cytochrome P450 Gene CYP9A105 Associated with Pyrethroid Detoxification in Spodoptera exigua Hübner.

In insects, cytochrome P450 monooxygenases (P450s or CYPs) are known to be involved in the detoxification and metabolism of insecticides, leading to increased resistance in insect populations. Spodoptera exigua is a serious polyphagous insect pest worldwide and has developed resistance to various insecticides. In this study, a novel CYP3 clan P450 gene CYP9A105 was identified and characterized from S. exigua. The cDNAs of CYP9A105 encoded 530 amino acid proteins, respectively. Quantitative real-time PCR analyses showed that CYP9A105 was expressed at all developmental stages, with maximal expression observed in fifth instar stage larvae, and in dissected fifth instar larvae the highest transcript levels were found in midguts and fat bodies. The expression of CYP9A105 in midguts was upregulated by treatments with the insecticides α-cypermethrin, deltamethrin and fenvalerate at both LC15 concentrations (0.10, 0.20 and 5.0 mg/L, respectively) and LC50 concentrations (0.25, 0.40 and 10.00 mg/L, respectively). RNA interference (RNAi) mediated silencing of CYP9A105 led to increased mortalities of insecticide-treated 4th instar S. exigua larvae. Our results suggest that CYP9A105 might play an important role in α-cypermethrin, deltamethrin and fenvalerate detoxification in S. exigua.

A cytochrome P450 CYP71 enzyme expressed in Sorghum bicolor root hair cells participates in the biosynthesis of the benzoquinone allelochemical sorgoleone.

Sorgoleone, a major component of the hydrophobic root exudates of Sorghum spp., is probably responsible for many of the allelopathic properties attributed to members of this genus. Much of the biosynthetic pathway for this compound has been elucidated, with the exception of the enzyme responsible for the catalysis of the addition of two hydroxyl groups to the resorcinol ring. A library prepared from isolated Sorghum bicolor root hair cells was first mined for P450-like sequences, which were then analyzed by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) to identify those preferentially expressed in root hairs. Full-length open reading frames for each candidate were generated, and then analyzed biochemically using both a yeast expression system and transient expression in Nicotiana benthamiana leaves. RNA interference (RNAi)-mediated repression in transgenic S. bicolor was used to confirm the roles of these candidates in the biosynthesis of sorgoleone in planta. A P450 enzyme, designated CYP71AM1, was found to be capable of catalyzing the formation of dihydrosorgoleone using 5-pentadecatrienyl resorcinol-3-methyl ether as substrate, as determined by gas chromatography-mass spectroscopy (GC-MS). RNAi-mediated repression of CYP71AM1 in S. bicolor resulted in decreased sorgoleone contents in multiple independent transformant events. Our results strongly suggest that CYP71AM1 participates in the biosynthetic pathway of the allelochemical sorgoleone.

Two plant-derived aporphinoid alkaloids exert their antifungal activity by disrupting mitochondrial iron-sulfur cluster biosynthesis.

Eupolauridine and liriodenine are plant-derived aporphinoid alkaloids that exhibit potent inhibitory activity against the opportunistic fungal pathogens Candida albicans and Cryptococcus neoformans However, the molecular mechanism of this antifungal activity is unknown. In this study, we show that eupolauridine 9591 (E9591), a synthetic analog of eupolauridine, and liriodenine methiodide (LMT), a methiodide salt of liriodenine, mediate their antifungal activities by disrupting mitochondrial iron-sulfur (Fe-S) cluster synthesis. Several lines of evidence supported this conclusion. First, both E9591 and LMT elicited a transcriptional response indicative of iron imbalance, causing the induction of genes that are required for iron uptake and for the maintenance of cellular iron homeostasis. Second, a genome-wide fitness profile analysis showed that yeast mutants with deletions in iron homeostasis-related genes were hypersensitive to E9591 and LMT. Third, treatment of wild-type yeast cells with E9591 or LMT generated cellular defects that mimicked deficiencies in mitochondrial Fe-S cluster synthesis including an increase in mitochondrial iron levels, a decrease in the activities of Fe-S cluster enzymes, a decrease in respiratory function, and an increase in oxidative stress. Collectively, our results demonstrate that E9591 and LMT perturb mitochondrial Fe-S cluster biosynthesis; thus, these two compounds target a cellular pathway that is distinct from the pathways commonly targeted by clinically used antifungal drugs. Therefore, the identification of this pathway as a target for antifungal compounds has potential applications in the development of new antifungal therapies.

Interactions between Nitrogen and Silicon in Rice and Their Effects on Resistance toward the Brown Planthopper Nilaparvata lugens.

Nitrogen (N) and silicon (Si) are two important nutritional elements required for plant growth, and both impact host plant resistance toward insect herbivores. The interaction between the two elements may therefore play a significant role in determining host plant resistance. We investigated this interaction in rice (Oryza sativa L.) and its effect on resistance to the herbivore brown planthopper Nilaparvata lugens (BPH). Our results indicate that high-level (5.76 mM) N fertilization reduced Si accumulation in rice leaves, and furthermore, this decrease was likely due to decreased expression of Si transporters OsLsi1 and OsLsi2. Conversely, reduced N accumulation was observed at high N fertilization levels when Si was exogenously provided, and this was associated with down-regulation of OsAMT1;1 and OsGS1;1, which are involved in ammonium uptake and assimilation, respectively. Under lower N fertilization levels (0.72 and/or 1.44 mM), Si amendment resulted in increased OsNRT1:1, OsGS2, OsFd-GOGAT, OsNADH-GOGAT2, and OsGDH2 expression. Additionally, bioassays revealed that high N fertilization level significantly decreased rice resistance to BPH, and the opposite effect was observed when Si was provided. These results provide additional insight into the antagonistic interaction between Si and N accumulation in rice, and the effects on plant growth and susceptibility to herbivores.

Ratoon rice generated from primed parent plants exhibit enhanced herbivore resistance.

Rice ratooning is practiced in many rice-growing countries for achieving increased rice production with limited labour input. Here, we report that attack by insect herbivores, or treatment with a defense signaling compound in parent plants, can prime anti-herbivore defense responses in subsequent ratoon plants. We compared the defense responses of rice ratoons generated from parent plants that had been either infested by Cnaphalocrocis medinalis (rice leaffolder, LF) caterpillars or treated with methyl jasmonate (MeJA) during vegetative growth, with ratoons generated from control parent plants. Ratoon plants generated from parents receiving prior LF infestation or MeJA treatment exhibited higher jasmonic acid (JA) levels, as well as elevated levels of transcripts of defense-related genes associated with JA signaling. In addition, elevated activities of peroxidase, polyphenol oxidase and trypsin protease inhibitor were observed, as well as enhanced resistance towards subsequent LF infestation. Pre-priming of ratoon defense responses was significantly reduced in plants where expression of OsAOS (allene oxide synthase, involved in JA biosynthesis) or OsCOI1 (CORONATINE INSENSITIVE1, involved in JA perception) was inhibited by RNA interference. Our results indicate that herbivore exposure or MeJA treatment in rice parent plants enhances anti-herbivore resistance in subsequently generated ratoons through priming of JA-mediated defenses.

Identification of a novel cytochrome P450 CYP321B1 gene from tobacco cutworm (Spodoptera litura) and RNA interference to evaluate its role in commonly used insecticides.

Insect cytochrome P450 monooxygenases (CYPs or P450s) play an important role in detoxifying insecticides leading to resistance in insect populations. A polyphagous pest, Spodoptera litura, has developed resistance to a wide range of insecticides. In the present study, a novel P450 gene, CYP321B1, was cloned from S. litura. The function of CYP321B1 was assessed using RNA interference (RNAi) and monitoring resistance levels for three commonly used insecticides, including chlorpyrifos, ß-cypermethrin and methomyl. The full-length complementary DNA sequence of CYP321B1 is 1814 bp long with an open reading frame of 1 488 bp encoding 495 amino acid residues. Quantitative reverse-transcriptase polymerase chain reaction analyses during larval and pupal development indicated that CYP321B1 expression was highest in the midgut of fifth-instar larvae, followed by fat body and cuticle. The expression of CYP321B1 in the midgut was up-regulated by chlorpyrifos, ß-cypermethrin and methomyl with both lethal concentration at 15% (LC15 ) (50, 100 and 150 µg/mL, respectively) and 50%(LC50 ) dosages (100, 200 and 300 µg/mL, respectively). Addition of piperonyl butoxide (PBO) significantly increased the toxicity of chlorpyrifos, ß-cypermethrin and methomyl to S. litura, suggesting a marked synergism of the three insecticides with PBO and P450-mediated detoxification. RNAi-mediated silencing of CYP321B1 further increased mortality by 25.6% and 38.9% when the fifth-instar larvae were exposed to chlorpyrifos and ß-cypermethrin, respectively, at the LC50 dose levels. The results demonstrate that CYP321B1 might play an important role in chlorpyrifos and ß-cypermethrin detoxification in S. litura.

Investigating sesquiterpene biosynthesis in Ginkgo biloba: molecular cloning and functional characterization of (E,E)-farnesol and α-bisabolene synthases.

Ginkgo biloba is one of the oldest living tree species and has been extensively investigated as a source of bioactive natural compounds, including bioactive flavonoids, diterpene lactones, terpenoids and polysaccharides which accumulate in foliar tissues. Despite this chemical diversity, relatively few enzymes associated with any biosynthetic pathway from ginkgo have been characterized to date. In the present work, predicted transcripts potentially encoding enzymes associated with the biosynthesis of diterpenoid and terpenoid compounds, including putative terpene synthases, were first identified by mining publicly-available G. biloba RNA-seq data sets. Recombinant enzyme studies with two of the TPS-like sequences led to the identification of GbTPS1 and GbTPS2, encoding farnesol and bisabolene synthases, respectively. Additionally, the phylogenetic analysis revealed the two terpene synthase genes as primitive genes that might have evolved from an ancestral diterpene synthase.

A novel cytochrome P450 CYP6AB14 gene in Spodoptera litura (Lepidoptera: Noctuidae) and its potential role in plant allelochemical detoxification.

Cytochrome P450 monooxygenases (P450s) play a prominent role in the adaptation of insects to host plant chemical defenses. To investigate the potential role of P450s in adaptation of the lepidopteran pest Spodoptera litura to host plant allelochemicals, an expressed sequence data set derived from 6th instar midgut tissues was first mined. One sequence identified from the S. litura 6th instar midgut EST database was determined by phylogenetic analysis to belong to the CYP6AB P450 subfamily, and named CYP6AB14. Dietary supplementation of S. litura larvae with either xanthotoxin (XAN), coumarin (COU) and flavone (FLA) led to elevated CYP6AB14 transcript levels in both midgut and fat body tissues. Injection of CYP6AB14-derived double-stranded RNA (dsRNA) into S. litura individuals significantly reduced CYP6AB14 transcript levels, and resulted in increased developmental abnormalities and higher mortality rates among XAN, COU and FLA-fed larvae. Our results strongly suggest a key role for CYP6AB14 in plant allelochemical detoxification in S. litura.

Priming of jasmonate-mediated antiherbivore defense responses in rice by silicon.

Although the function of silicon (Si) in plant physiology has long been debated, its beneficial effects on plant resistance against abiotic and biotic stresses, including insect herbivory, have been well documented. In addition, the jasmonate (JA) signaling pathway plays a crucial role in mediating antiherbivore defense responses in plants. However, potential interactions between JA and Si in response to insect attack have not been examined directly. To explore the role JA may play in Si-enhanced resistance, we silenced the expression of allene oxide synthase (OsAOS; active in JA biosynthesis) and CORONATINE INSENSITIVE1 (OsCOI1; active in JA perception) genes in transgenic rice plants via RNAi and examined resulting changes in Si accumulation and defense responses against caterpillar Cnaphalocrocis medinalis (rice leaffolder, LF) infestation. Si pretreatment increased rice resistance against LF larvae in wild-type plants but not in OsAOS and OsCOI1 RNAi lines. Upon LF attack, wild-type plants subjected to Si pretreatment exhibited enhanced defense responses relative to untreated controls, including higher levels of JA accumulation; increased levels of transcripts encoding defense marker genes; and elevated activities of peroxidase, polyphenol oxidase, and trypsin protease inhibitor. Additionally, reduced Si deposition and Si cell expansion were observed in leaves of OsAOS and OsCOI1 RNAi plants in comparison with wild-type plants, and reduced steady-state transcript levels of the Si transporters OsLsi1, OsLsi2, and OsLsi6 were observed in Si-pretreated plants after LF attack. These results suggest a strong interaction between Si and JA in defense against insect herbivores involving priming of JA-mediated defense responses by Si and the promotion of Si accumulation by JA.

Sorghum allelopathy--from ecosystem to molecule.

Sorghum allelopathy has been reported in a series of field experiments following sorghum establishment. In recent years, sorghum phytotoxicity and allelopathic interference also have been well-described in greenhouse and laboratory settings. Observations of allelopathy have occurred in diverse locations and with various sorghum plant parts. Phytotoxicity has been reported when sorghum was incorporated into the soil as a green manure, when residues remained on the soil surface in reduced tillage settings, or when sorghum was cultivated as a crop in managed fields. Allelochemicals present in sorghum tissues have varied with plant part, age, and cultivar evaluated. A diverse group of sorghum allelochemicals, including numerous phenolics, a cyanogenic glycoside (dhurrin), and a hydrophobic p-benzoquinone (sorgoleone) have been isolated and identified in recent years from sorghum shoots, roots, and root exudates, as our capacity to analyze and identify complex secondary products in trace quantities in the plant and in the soil rhizosphere has improved. These allelochemicals, particularly sorgoleone, have been widely investigated in terms of their mode(s) of action, specific activity and selectivity, release into the rhizosphere, and uptake and translocation into sensitive indicator species. Both genetics and environment have been shown to influence sorgoleone production and expression of genes involved in sorgoleone biosynthesis. In the soil rhizosphere, sorgoleone is released continuously by living root hairs where it accumulates in significant concentrations around its roots. Further experimentation designed to study the regulation of sorgoleone production by living sorghum root hairs may result in increased capacity to utilize sorghum cover crops more effectively for suppression of germinating weed seedlings, in a manner similar to that of soil-applied preemergent herbicides like trifluralin.

A potent plant-derived antifungal acetylenic acid mediates its activity by interfering with fatty acid homeostasis.

6-Nonadecynoic acid (6-NDA), a plant-derived acetylenic acid, exhibits strong inhibitory activity against the human fungal pathogens Candida albicans, Aspergillus fumigatus, and Trichophyton mentagrophytes. In the present study, transcriptional profiling coupled with mutant and biochemical analyses were conducted using the model yeast Saccharomyces cerevisiae to investigate its mechanism of action. 6-NDA elicited a transcriptome response indicative of fatty acid stress, altering the expression of genes that are required for yeast growth in the presence of oleate. Mutants of S. cerevisiae lacking transcription factors that regulate fatty acid ß-oxidation showed increased sensitivity to 6-NDA. Fatty acid profile analysis indicated that 6-NDA inhibited the formation of fatty acids longer than 14 carbons in length. In addition, the growth inhibitory effect of 6-NDA was rescued in the presence of exogenously supplied oleate. To investigate the response of a pathogenic fungal species to 6-NDA, transcriptional profiling and biochemical analyses were also conducted in C. albicans. The transcriptional response and fatty acid profile of C. albicans were comparable to those obtained in S. cerevisiae, and the rescue of growth inhibition with exogenous oleate was also observed in C. albicans. In a fluconazole-resistant clinical isolate of C. albicans, a fungicidal effect was produced when fluconazole was combined with 6-NDA. In hyphal growth assays, 6-NDA inhibited the formation of long hyphal filaments in C. albicans. Collectively, our results indicate that the antifungal activity of 6-NDA is mediated by a disruption in fatty acid homeostasis and that 6-NDA has potential utility in the treatment of superficial Candida infections.

In planta production of the highly potent resveratrol analogue pterostilbene via stilbene synthase and O-methyltransferase co-expression.

Resveratrol and related stilbenes are thought to play important roles in defence responses in several plant species and have also generated considerable interest as nutraceuticals owing to their diverse health-promoting properties. Pterostilbene, a 3,5-dimethylether derivative of resveratrol, possesses properties similar to its parent compound and, additionally, exhibits significantly higher fungicidal activity in vitro and superior pharmacokinetic properties in vivo. Recombinant enzyme studies carried out using a previously characterized O-methyltransferase sequence from Sorghum bicolor (SbOMT3) demonstrated its ability to catalyse the A ring-specific 3,5-bis-O-methylation of resveratrol, yielding pterostilbene. A binary vector was constructed for the constitutive co-expression of SbOMT3 with a stilbene synthase sequence from peanut (AhSTS3) and used for the generation of stably transformed tobacco and Arabidopsis plants, resulting in the accumulation of pterostilbene in both species. A reduced floral pigmentation phenotype observed in multiple tobacco transformants was further investigated by reversed-phase HPLC analysis, revealing substantial decreases in both dihydroquercetin-derived flavonoids and phenylpropanoid-conjugated polyamines in pterostilbene-producing SbOMT3/AhSTS3 events. These results demonstrate the potential utility of this strategy for the generation of pterostilbene-producing crops and also underscore the need for the development of additional approaches for minimizing concomitant reductions in key phenylpropanoid-derived metabolites.

The marine sponge-derived polyketide endoperoxide plakortide F acid mediates its antifungal activity by interfering with calcium homeostasis.

Plakortide F acid (PFA), a marine-derived polyketide endoperoxide, exhibits strong inhibitory activity against the opportunistic fungal pathogens Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus. In the present study, transcriptional profiling coupled with mutant and biochemical analyses were conducted using the model organism Saccharomyces cerevisiae to investigate the mechanism of action of this compound. PFA elicited a transcriptome response indicative of a Ca(2+) imbalance, affecting the expression of genes known to be responsive to altered cellular calcium levels. Several additional lines of evidence obtained supported a role for Ca(2+) in PFA's activity. First, mutants lacking calcineurin and various Ca(2+) transporters, including pumps (Pmr1 and Pmc1) and channels (Cch1 and Mid1), showed increased sensitivity to PFA. In addition, the calcineurin inhibitors FK506 and cyclosporine strongly enhanced PFA activity in wild-type cells. Furthermore, PFA activated the transcription of a lacZ reporter gene driven by the calcineurin-dependent response element. Finally, elemental analysis indicated a significant increase in intracellular calcium levels in PFA-treated cells. Collectively, our results demonstrate that PFA mediates its antifungal activity by perturbing Ca(2+) homeostasis, thus representing a potentially novel mechanism distinct from that of currently used antifungal agents.