Red light enhances the antibacterial properties, biofabrication, and stability of Fagonia indica callus-based silver nanoparticles.

Plant-based nanoparticles can be tuned through the frequency of light for efficient synthesis, structural properties, and antibacterial applications. This research assessed the effect of material type (callus and whole-plant extract) and the interaction with a specific range of light wavelength on AgNP synthesis. All types of AgNPs were characterized by their size, shape, associated functional groups, and surface charge. Interestingly, the size of red light and callus-based AgNPs (RC-AgNPs) was smaller (6.32 nm) compared to 14.59 nm for Ultraviolet light and callus-based AgNPs (UV-C-AgNPs). Zeta potential analysis showed that RC-AgNPs had higher stability (-29.2 mV) compared to UV-C-AgNPs (-16.7 mV). Similarly, red light-based AgNPs had higher Oxidation reduction potential in both whole-plant-based and callus-based AgNPs, indicating a more oxidizing nature compared to those synthesized under UV light. This was confirmed by the lower total phenolic and flavonoid content associated with them and their lower antioxidant activity. The higher antibacterial activities and lower minimum inhibitory concentrations of red light-based AgNPs against highly resistant pathogenic bacteria demonstrated the role of red light in enhancing antibacterial activity. These results indicate that AgNPs synthesized in red light and callus extract are more active compared to those synthesized under other wavelengths and/or in whole-plant extracts.

Assessing the genetic integrity of sugarcane germplasm in the USDA-ARS National Plant Germplasm System collection using single-dose SNP markers.

The World Collection of Sugarcane and Related Grasses, maintained at the USDA-ARS in Miami, FL, is one of the largest sugarcane germplasm repositories in the world. However, the genetic integrity of the Saccharum spp. germplasm in this collection has not been fully analyzed. In this study, we employed a single-dose SNP panel to genotype 901 sugarcane accessions, representing six Saccharum species and various hybrids. Our analysis uncovered a high rate of clone mislabeling in the collection. Specifically, we identified 86 groups of duplicates, characterized by identical SNP genotypes, which encompassed 211 accessions (23% of the total clones), while 135 groups, constituting 471 clones (52% of the total), exhibited near-identical genotypes. In addition, twenty-seven homonymous groups were detected, which shared the same clone name but differed in SNP genotypes. Hierarchical analysis of population structure partitioned the Saccharum germplasm into five clusters, corresponding to S. barberi, S. sinense, S. officinarum, S. spontaneum and S. robustum/S. edule. An assignment test, based on the five Saccharum species, enabled correcting 141 instances of mislabeled species memberships and inaccuracies. Moreover, we clarified the species membership and parentage of 298 clones that had ambiguous passport records (e.g., 'Saccharum spp', 'unknown', and 'hybrid'). Population structure and genetic diversity in these five species were further supported by Principal Coordinate Analysis and neighbor-joining clustering analysis. Analysis of Molecular Variance revealed that within-species genetic variations accounted for 85% of the total molecular variance, with the remaining 15% attributed to among-species genetic variations. The single-dose SNP markers developed in this study offer a robust tool for characterizing sugarcane germplasm worldwide. These findings have important implications for sugarcane genebank management, germplasm exchange, and crop genetic improvement.

LC-MS/MS-based profiling of bioactive metabolites of endophytic bacteria from Cannabis sativa and their anti-Phytophthora activity.

Protection of crop plants from phytopathogens through endophytic bacteria is a newly emerged area of biocontrol. In this study, endophytic bacteria were isolated from the rhizosphere of Cannabis sativa. Based on initial antimicrobial screening, three (03) bacteria Serratia marcescens MOSEL-w2, Enterobacter cloacae MOSEL-w7, and Paenibacillus MOSEL-w13 were selected. Antimicrobial assays of these selected bacteria against Phytophthora parasitica revealed that E. cloacae MOSEL-w7 and Paenibacillus sp. MOSEL-w13 possessed strong activity against P. parasitica. All these bacterial extracts showed strong inhibition against P. parasitica at different concentrations (4-400 µg mL-1). P. parasitica hyphae treated with ethyl acetate extract of E. cloacae MOSEL-w7 resulted in severe growth abnormalities compared to control. The extracts were further evaluated for in vivo detached-leaf assay against P. parasitica on the wild type tobacco. Application of 1% ethyl acetate bacterial extract of S. marcescens MOSEL-w2, E. cloacae MOSEL-w7, and Paenibacillus sp. MOSEL-w13 reduced P. parasitica induced lesion sizes and lesion frequencies by 60-80%. HPLC based fractions of each extract also showed bioactivity against P. parasitica. A total of 24 compounds were found in the S. marcescens MOSEL-w2, 15 compounds in E. cloacae MOSEL-w7 and 20 compounds found in Paenibacillus sp. MOSEL-w13. LC-MS/MS analyses showed different bioactive compounds in the bacterial extracts such as Cotinine (alkylpyrrolidine), L-tryptophan, L-lysine, L-Dopa, and L-ornithine. These results suggest that S. marcescens MOSEL-w2, E. cloacae MOSEL-w7, and Paenibacillus MOSEL-w13 are a source of bioactive metabolites and could be used in combination with other biocontrol agents, with other modes of action for controlling diseases caused by Phytophthora in crops. They could be a clue for the broad-spectrum biopesticides for agriculturally significant crops.

Exploration of microbiome of medicinally important plants as biocontrol agents against Phytophthora parasitica.

In a preliminary plant-based microbiome study, diverse bacterial taxa were identified from different medicinal plants using 16S rRNA gene sequencing. Based on initial antimicrobial screening, eight (8) bacterial endophytes in six (6) different genera, Streptomyces, Pseudomonas, Enterobacter, Bacillus, Arthrobacter, and Delftia, from four important medicinal plants Dodonaea viscosa, Fagonia indica, Caralluma tuberculata, and Calendula arvensis were selected for further analyses. Antimicrobial assays revealed that Pseudomonas taiwanensis MOSEL-RD23 has strong anti-Phytophthora activity. Volatiles produced by P. taiwanensis MOSEL-RD23and Bacillus flexus MOSEL-MIC5 inhibited the growth of Phytophthora parasitica by more than 80%. Ethyl acetate extracts of Streptomyces alboniger MOSEL-RD3, P. taiwanensis MOSEL-RD23, Enterobacter hormaechei MOSEL-FLS1, and Bacillus tequilensis MOSEL-FLS3, and Delftia lacustris MB322 displayed high potency against P. parasitica. All these bacterial extracts showed strong inhibition of more than 80% inhibition in vitro against P. parasitica at different concentrations (4-400 µg/mL). Bacterial extracts showing strong antimicrobial activity were selected for bioactivity-driven fractionation and showed anti-Phytophthoral activity in multiple fractions and different peaks observed in UV-Vis spectroscopy. In the detached-leaf assay against P. parasitica on tobacco, 1% ethyl acetate bacterial extract of S. alboniger MOSEL-RD3, P. taiwanensis MOSEL-RD23, E. hormaechei MOSEL-FLS1, B. tequilensis MOSEL-FLS3, and D. lacustris MB322 reduced lesion sizes and lesion frequencies caused by P. parasitica by 68 to 81%. Overall, P. taiwanensis MOSEL-RD23 showed positive activities for all the assays. Analyzing the potential of bacterial endophytes as biological control agents can potentially lead to the formulation of broad-spectrum biopesticides for the sustainable production of crops.

Production and bioprocess optimization of antitumor Epothilone B analogue from Aspergillus fumigatus, endophyte of Catharanthus roseus, with response surface methodology.

Epothilones are secondary metabolites produced by Sorangium cellulosum with powerful antiproliferative activity against tumor cells by stabilizing their microtubule arrays, arresting their cellular division at G2-M phase. Unfortunately, the lower yield of epothilone is the challenge for its higher accessibility, thus, searching for alternative sources with promising epothilone producing potency is the prospective. Endophytic fungi are the potential repertoire for bioactive metabolites, thus exploring the epothilone producing potency of endophytic fungi of medicinal plants was objective. Thirty-two fungal isolates were recovered from the tested medicinal plants and their potency to produced epothilone have been assessed using the TLC, HPLC and molecular markers epoA, epoC and epoK. Aspergillus fumigatus EFBL, an endophyte of Catharanthus roseus, was the potent epothilone producer (21.5 µg/g biomass) as revealed from the chromatographic analyses and PCR of molecular markers. The chemical identity of extracted epothilone was verified from the HPLC, NMR, FTIR and LC-MS analyses as epothilone B analogue. The putative epoA gene from A. fumigatus was amplified using RT-PCR with the conservative corresponding primers to the active-sites of S. cellulosum. The amplicons of epoA was 517 bp displayed 98 % similarity with A. fumigatus PKS-NRPS domains, and 40 % similarity with epoA of S. cellulosum. From the in silico analyses, Val506, Ala605 and Ser630 are the conservative amino acids of epoA protein of A. fumigatus and S. cellulosum. Epothilone B from A. fumigatus displayed a strong antiproliferative activity against HepG-2, MCF-7 and LS174 T as revealed from the IC50 values 6.4, 8.7 and 10.21 µM, respectively. The productivity of epothilone B from A. fumigatus was optimized by surface response methodology with Plackett-Burman and Faced Centered Central Composite. With the Plackett-Burman design, the yield of epothilone (54.4-60.1 µg/g biomass) by A. fumigatus was increased by about 2.8-3.0 folds comparing to non-optimized cultures (21.5 µg/ g biomass). From the FCCD design, sucrose, tryptone and incubation time being the highest significant variables medium components affecting the epothilone yield of A. fumigatus. This is the first report exploring the feasibility of endophytic fungi for epothilone producing potency, that could be a novel platform for industrial production of epothilone.

Introduction of Arabidopsis's heat shock factor HsfA1d mitigates adverse effects of heat stress on potato (Solanum tuberosum L.) plant.

Thermal stress induces a wide array of morphological and physiological changes in potato affecting its development and economic yield. Response to thermal stress in plants is mostly regulated by heat shock factors (hsfs). The current study aimed at improving heat tolerance by transforming potato plant with heat shock factor, HsfA1d, using Agrobacterium. Gateway cloning strategy was adopted for isolation of HsfA1d from Arabidopsis thaliana and cloning into plant expression vector. The target gene was introduced into potato by infecting internodal explants with Agrobacterium strain GV3101 carrying pGWB402Ω-HsfA1d construct. Upon exposure to heat stress, the wild-type plants turned yellowish, whereas no phenotypic effect on transgenic plants was observed. Expression of HsfA1d in transgenic plants was increased by 5.8-fold under thermal stress compared to room temperature. Transgenic plants exhibited 6-fold increase in the expression of downstream HSP70 under thermal stress compared to wild-type plants. Both chlorophyll a and b were significantly decreased in wild-type plants while no such decrease was recorded in transgenic plants under thermal stress. Heat stress was found to have no significant effect on carotenoid pigments of both wild-type and transgenic plants. Significantly lower electrolyte leakage from transgenic plants was witnessed compared to wild type upon exposure to thermal stress. Transgenic plants accumulated significantly higher proline content compared to wild-type plants under heat stress. It is concluded that HsfA1d plays a vital role in plant thermotolerance and hence can be effectively used to enhance the resistance of crop plants against heat stress.

Variation in surface properties, metabolic capping, and antibacterial activity of biosynthesized silver nanoparticles: comparison of bio-fabrication potential in phytohormone-regulated cell cultures and naturally grown plants.

We compared surface properties, metabolic capping and antibacterial activity of silver nanoparticles, synthesized through extracts of cell cultures of Fagonia indica and its naturally grown form. Extracts from cell cultures (produced with thidiazuron (TDZ) or melatonin (MLN)) were compared to the naturally grown whole plant extracts (WPEs) for their reducing potential, and their effects on physical and biochemical properties of the biosynthesized silver nanoparticles. UV-Vis spectroscopy revealed that the surface plasmon resonance peaked at λ = 415 nm for MLN-AgNPs, λ = 430 nm for TDZ-AgNPs and λ = 460-465 nm for WPE-AgNPs. Transmission electron microscopy and energy dispersive X-rays of AgNPs showed that compared to WPE-AgNPs (mean diameter = 22 nm), extracts from MLN- and TDZ-induced cell cultures produced particles with spherical shapes and smaller diameters (i.e. mean diameter = 15 nm and 19 nm, respectively). Size distribution analysis also showed that TDZ-AgNPs were nearer to a symmetric distribution in terms of diameter (skewness = 0.80) as compared to WPE-AgNPs (skewness = 0.9) and MLN-AgNPs (skewness = 1.4). Furthermore, MLN-induced cell culture extracts produced AgNPs in higher concentration (210 µg mL-1) compared to AgNPs from TDZ-induced cell culture extracts (160 µg mL-1) and WPE (138 µg mL-1). Two-way comparisons of LC-MS/MS profiles of TDZ-AgNPs, MLN-AgNPs, and WPE-AgNPs revealed differences in their secondary metabolite profiles, which might account for differences in their differential response in bio-fabrication, and size distribution. Activity against different pathogenic bacterial strains, Escherichia coli, Bacillus cereus, Xanthomonas citri, Agrobacterium tumefaciens, Streptomyces griseus, and Erwinia carotovora suggested that MLN-AgNPs were more effective compared to TDZ- and WPE-AgNPs. These results indicated that phytohormones induced cell cultures can enhance the production, physical and biochemical properties of AgNPs.

Correction: Variation in surface properties, metabolic capping, and antibacterial activity of biosynthesized silver nanoparticles: comparison of bio-fabrication potential in phytohormone-regulated cell cultures and naturally grown plants.

[This corrects the article DOI: 10.1039/D0RA08419K.].

The PTI to ETI Continuum in Phytophthora-Plant Interactions.

Phytophthora species are notorious pathogens of several economically important crop plants. Several general elicitors, commonly referred to as Pathogen-Associated Molecular Patterns (PAMPs), from Phytophthora spp. have been identified that are recognized by the plant receptors to trigger induced defense responses in a process termed PAMP-triggered Immunity (PTI). Adapted Phytophthora pathogens have evolved multiple strategies to evade PTI. They can either modify or suppress their elicitors to avoid recognition by host and modulate host defense responses by deploying hundreds of effectors, which suppress host defense and physiological processes by modulating components involved in calcium and MAPK signaling, alternative splicing, RNA interference, vesicle trafficking, cell-to-cell trafficking, proteolysis and phytohormone signaling pathways. In incompatible interactions, resistant host plants perceive effector-induced modulations through resistance proteins and activate downstream components of defense responses in a quicker and more robust manner called effector-triggered-immunity (ETI). When pathogens overcome PTI-usually through effectors in the absence of R proteins-effectors-triggered susceptibility (ETS) ensues. Qualitatively, many of the downstream defense responses overlap between PTI and ETI. In general, these multiple phases of Phytophthora-plant interactions follow the PTI-ETS-ETI paradigm, initially proposed in the zigzag model of plant immunity. However, based on several examples, in Phytophthora-plant interactions, boundaries between these phases are not distinct but are rather blended pointing to a PTI-ETI continuum.

The Phytophthora RXLR Effector Avrblb2 Modulates Plant Immunity by Interfering With Ca2+ Signaling Pathway.

In plants, subcellular fluctuations in Ca2+ ion concentration are among the earliest responses to biotic and abiotic stresses. Calmodulin, which is a ubiquitous Ca2+ ion sensor in eukaryotes, plays a major role in translating these Ca2+ signatures to cellular responses by interacting with numerous proteins located in plasma membranes, cytoplasm, organelles and nuclei. In this report, we show that one of the Phytophthora RXLR effector, Avrblb2, interacts with calmodulin at the plasma membrane of the plant cells. Using deletion and single amino acid mutagenesis, we found that calmodulin binds to the effector domain of Avrblb2. In addition, we show that most known homologs of Avrblb2 in three different Phytophthora species interact with different isoforms of calmodulin. Type of amino acids at position 69 in Avrblb2, which determines Rbi-blb2 resistance protein-mediated defense responses, is not involved in the Avrblb2-calmodulin interaction. Using in planta functional analyses, we show that calmodulin binding to Avrblb2 is required for its recognition by Rpi-blb2 to incite hypersensitive response. These findings suggest that Avrblb2 by interacting with calmodulin interfere with plant defense associated Ca2+ signaling in plants.

Biochemical characterization of peptidylarginine deiminase-like orthologs from thermotolerant Emericella dentata and Aspergillus nidulans.

Peptidylarginine deiminases (PADs) are a group of hydrolases, mediating the deimination of peptidylarginine residues into peptidyl-citrulline. Equivocal protein citrullination by PADs of fungal pathogens has a strong relation to the progression of multiple human diseases, however, the biochemical properties of fungal PADs remain ambiguous. Thus, this is the first report exploring the molecular properties of PAD from thermotolerant fungi, to imitate the human temperature. The teleomorph Emericella dentata and anamorph Aspergillus nidulans have been morphologically and molecularly identified, with observed robust growth at 37-40 °C, and strong PAD productivity. The physiological profiles of E. dentata and A. nidulans for PADs production in response to carbon, nitrogen sources, initial medium pH and incubation temperature were relatively identical, emphasizing the taxonomical proximity of these fungal isolates. PADs were purified from E. dentata and A. nidulans with apparent molecular masses 41 and 48 kDa, respectively. The peptide fingerprints of PADs from E. dentata and A. nidulans have been analyzed by MALDI-TOF/MS, displaying a higher sequence similarity to human PAD4 by 18% and 31%, respectively. The conserved peptide sequences of E. dentata and A. nidulans PADs displayed a higher similarity to human PAD than A. fumigatus PADs clade. PADs from both fungal isolates have an optimum pH and pH stability at 7.0-8.0, with putative pI 5.0-5.5, higher structural denaturation at pH 4.0-5.5 and 9.5-12 as revealed from absorbance at λ280nm. E. dentata PAD had a higher conformationally thermal stability than A. nidulans PAD as revealed from its lower Kr value. From the proteolytic mapping, the orientation of trypsinolytic recognition sites on the PADs surface from both fungal isolates was very similar. PADs from both isolates are calcium dependent, with participation of serine and cysteine residues on their catalytic sites. PADs displayed a higher affinity to deiminate the peptidylarginine residues with a feeble affinity to work as ADI. So, PADs from E. dentata and A. nidulans had a relatively similar conformational and kinetic properties. Further molecular modeling analysis are ongoing to explore the role of PADs in citrullination of human proteins in Aspergillosis, that will open a new avenue for unraveling the vague of protein-protein interaction of human A. nidulans pathogen.

Comparative Transcriptome Analysis between a Resistant and a Susceptible Wild Tomato Accession in Response to Phytophthora parasitica.

Phytophthora parasitica is one of the most widespread Phytophthora species, which is known to cause multiple diseases in tomato and is capable of infecting almost all plant parts. Our current understanding of tomato-Phytophthora parasitica interaction is very limited and currently nothing is known at the whole genome or transcriptome level. In this study, we have analyzed and compared the transcriptome of a resistant and a susceptible wild tomato accession in response to P. parasitica infection using the RNA-seq technology. We have identified 2657 and 3079 differentially expressed genes (DEGs) in treatment vs control comparison of resistant (Sp-R) and susceptible (Sp-S) samples respectively. Functional annotation of DEGs revealed substantial transcriptional reprogramming of diverse physiological and cellular processes, particularly the biotic stress responses in both Sp-R and Sp-S upon P. parasitica treatment. However, subtle expression differences among some core plant defense related genes were identified and their possible role in resistance development against P. parasitica is discussed. Our results revealed 1173 genes that were differentially expressed only in Sp-R accession upon P. parasitica inoculation. These exclusively found DEGs in Sp-R accession included some core plant defense genes, for example, several protease inhibitors, chitinases, defensin, PR-1, a downy mildew susceptibility factor, and so on, were all highly induced. Whereas, several R genes, WRKY transcriptions factors and a powdery mildew susceptibility gene (Mlo) were highly repressed during the resistance outcome. Analysis reported here lays out a strong foundation for future studies aimed at improving genetic resistance of tomato cultivars against to Phytopphthora species.

Detection, virulence and genetic diversity of Fusarium species infecting tomato in Northern Pakistan.

In addition to the well-known Fusarium oxysporum f.sp. lycopersici, several other Fusarium species are known to cause extensive worldwide crop losses in tomatoes. Prevalence and identities of Fusarium species infecting tomatoes in Northwest Pakistan is currently not known. In this study, we surveyed and characterized Fusarium species associated with symptomatic tomatoes in Northwest Pakistan using morphological and molecular analyses. Pathogenicity tests revealed varying degrees of virulence with some Fusarium sp. causing severe disease symptoms whereas others displaying mild symptoms. Molecular identification based on Internal Transcribed Spacer (ITS) region and TEF-1α gene sequencing classified all isolates into four major species with a majority (68.9%) belonging to Fusarium incarnatum-equiseti species complex (FIESC), followed by F. graminearum (20.7%), F. acuminatum (6.8%), and F. solani (6.8%). ISSR analyses revealed substantial genetic variability among all the Fusarium population infecting tomatoes. Genetic distance between populations from the central region and the type strain F.o. f.sp. lycopersici from Florida was the highest (0.3662), whereas between the south and central region was the lowest (0.0298), which showed that genetic exchange is negatively effected by distance. High genetic variability suggests that these Fusarium species have the potential to become a major production constraint for tomato growers. Findings in this report would greatly facilitate identification of Fusarium species in developing countries and would provide groundwork for devising and implementing disease management measures for minimizing losses caused by Fusarium species in tomatoes.

A glucanolytic Pseudomonas sp. associated with Smilax bona-nox L. displays strong activity against Phytophthora parasitica.

Biological control is an eco-friendly strategy for mitigating and controlling plant diseases with negligible effects on human health and environment. Biocontrol agents are mostly isolated from field crops, and microbiomes associated with wild native plants is underexplored. The main objective of this study was to characterize the bacterial isolates associated with Smilax bona-nox L, a successful wild plant with invasive growth habits. Forty morphologically distinct bacterial isolates were recovered from S. bona-nox. Based on 16S rRNA gene sequencing, these isolates belonged to 12 different genera namely Burkholderia, Pseudomonas, Xenophilus, Stenotrophomonas, Pantoea, Enterobactriaceae, Kosakonia, Microbacterium, Curtobacterium, Caulobacter, Lysinibacillus and Bacillus. Among them, Pseudomonas sp. EA6 and Pseudomonas sp. EA14 displayed the highest potential for inhibition of Phytophthora. Based on sequence analysis of rpoD gene, these isolates revealed a 97% identity with a Pseudomonas fluorescence strain. Bioactivity-driven assays for finding bioactive compounds revealed that crude proteins of Pseudomonas sp. EA6 inhibited mycelial growth of P. parasitica, whereas crude proteins of Pseudomonas sp. EA14 displayed negligible activity. Fractionation and enzymatic analyses revealed that the bioactivity of Pseudomonas sp. EA6 was mostly due to glucanolytic enzymes. Comparison of chromatographic profile and bioactivity assays indicated that the secreted glucanolytic enzymes consisted of ß-1,3 and ß-1,4 glucanases, which acted together in hydrolyzing Phytophthora cell walls. Since the biological activity of the crude glucanolytic extract was >60-fold higher than the purified ß-1,3 glucanase, the glucanolytic enzyme system of Pseudomonas sp. EA6 likely acts synergistically in cell wall hydrolysis of P. parasitica.

Comparative antileishmanial efficacy of the biosynthesised ZnO NPs from genus Verbena.

This study describes ZnO NPs biosynthesis using leaf extracts of Verbena officinalis and Verbena tenuisecta. The extracts serve as natural reducing, capping and stabilization facilitators. Plant extracts phytochemical analysis, revealed that V. officinalis showed higher total phenolic and flavonoid content (22.12 and 6.38 mg g -1 DW) as compared to V. tennuisecta (12.18 and 2.7 mg g -1 DW). ZnO NPs were characterised by ultraviolet-visible spectroscopy, Fourier transform infrared, X-ray diffraction, scanning electron microscope, transmission electron microscopy (TEM) and energy dispersive X-ray. TEM analysis of ZnO NPs reveals rod and flower shapes and were in the range of 65-75 and 14-31 nm, for V. tenuisecta and V. officinalis, respectively. Bio-potential of ZnO NPs was examined through their leishmanicidal potential against Leishmania tropica. ZnO NPs showed potent leishmanicidal activity with 250 µg ml-1 being the most potent concentration. V. officinalis mediated ZnO NPs showed more potent leishmanicidal activity compared to V. tenuisecta mediated ZnO NPs due to their smaller size and increased phenolics doped onto its surface. These results can be a step forward towards the development of novel compounds that can efficiently replace the current medication schemes for leishmaniasis treatment.

A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants.

BACKGROUND: Most current methods for constructing guide RNAs (gRNA) for the CRISPR/Cas9 genome editing system, depend on traditional cloning using specific type IIS restriction enzymes and DNA ligation. These methods consist of multiple steps of cloning, and are time consuming, resource intensive and not flexible. These issues are particularly exacerbated when multiple guide RNAs need to be assembled in one plasmid such as for multiplexing or for the paired nickases approach. Furthermore, identification of functional gRNA clones usually requires expensive in vitro screening. Addressing these issues will greatly facilitate usage and accessibility of CRISPR/Cas9 genome editing system to resource-limited laboratories. RESULTS: To improve efficiency of cloning multiple guide RNAs for the CRISPR/Cas9 system, we developed a restriction enzyme- and ligation-independent strategy for cloning gRNAs directly in plant expression vectors in one step. Our method relies on a negative selection marker and seamless cloning for combining multiple gRNAs directly in a plant expression vector in one reaction. In addition, using the Agrobacterium-mediated transient assays, this method provides a simple in planta procedure for assaying the effectiveness of multiple gRNAs very rapidly. CONCLUSIONS: For a fraction of resources used in the type IIS restriction enzyme-based cloning method and in vitro screening assays, the system reported here allows efficient construction and testing several ready-to-transfect gRNA constructs in < 3 days. In addition, this system is highly versatile and flexible, and by designing only two additional target-specific primers, multiple gRNAs can be easily assembled in any plasmid in a single reaction.

Genome editing approaches: manipulating of lovastatin and taxol synthesis of filamentous fungi by CRISPR/Cas9 system.

Filamentous fungi are prolific repertoire of structurally diverse secondary metabolites of remarkable biological activities such as lovastatin and paclitaxel that have been approved by FDA as drugs for hypercholesterolemia and cancer treatment. The clusters of genes encoding lovastatin and paclitaxel are cryptic at standard laboratory cultural conditions (Kennedy et al. Science 284:1368-1372, 1999; Bergmann et al. Nature Chem Biol 3:213-217, 2007). The expression of these genes might be triggered in response to nutritional and physical conditions; nevertheless, the overall yield of these metabolites does not match the global need. Consequently, overexpression of the downstream limiting enzymes and/or blocking the competing metabolic pathways of these metabolites could be the most successful technologies to enhance their yield. This is the first review summarizing the different strategies implemented for fungal genome editing, molecular regulatory mechanisms, and prospective of clustered regulatory interspaced short palindromic repeat/Cas9 system in metabolic engineering of fungi to improve their yield of lovastatin and taxol to industrial scale. Thus, elucidating the putative metabolic pathways in fungi for overproduction of lovastatin and taxol was the ultimate objective of this review.

Strategies to enhance biologically active-secondary metabolites in cell cultures of Artemisia - current trends.

The genus Artemisia has been utilized worldwide due to its immense potential for protection against various diseases, especially malaria. Artemisia absinthium, previously renowned for its utilization in the popular beverage absinthe, is gaining resurgence due to its extensive pharmacological activities. Like A. annua, this species exhibits strong biological activities like antimalarial, anticancer and antioxidant. Although artemisinin was found to be the major metabolite for its antimalarial effects, several flavonoids and terpenoids are considered to possess biological activities when used alone and also to synergistically boost the bioavailability of artemisinin. However, due to the limited quantities of these metabolites in wild plants, in vitro cultures were established and strategies have been adopted to enhance medicinally important secondary metabolites in these cultures. This review elaborates on the traditional medicinal uses of Artemisia species and explains current trends to establish cell cultures of A. annua and A. absinthium for enhanced production of medicinally important secondary metabolites.

Molecular and Spectroscopic Characterization of Aspergillus flavipes and Pseudomonas putida L-Methionine γ-Lyase in Vitro.

Pseudomonas putida L-methionine γ-lyase (PpMGL) has been recognized as an efficient anticancer agent, however, its antigenicity and stability remain as critical challenges for its clinical use. From our studies, Aspergillus flavipes L-methionine γ-lyase (AfMGL) displayed more affordable biochemical properties than PpMGL. Thus, the objective of this work was to comparatively assess the functional properties of AfMGL and PpMGL via stability of their internal aldimine linkage, tautomerism of pyridoxal 5'-phosphate (PLP) and structural stability responsive to physicochemical factors. The internal Schiff base of AfMGL and PpMGL have the same stability to hydroxylamine and human serum albumin. Acidic pHs resulted in strong cleavage of the internal Schiff base, inducing the unfolding of MGLs, compared to neutral-alkaline pHs. At λ 280 nm excitation, both AfMGL and PpMGL have identical fluorescence emission spectra at λ 335 nm for the intrinsic tryptophan and λ 560 nm for the internal Schiff base. The maximum PLP tautomeric shift of ketoenamine to enolimine was detected at acidic pH causing complete enzyme unfolding, subunits dissociation and tautomeric shift of intrinsic PLP, rather than neutral-alkaline ones. The T m of AfMGL and PpMGL in presence of thermal stabilizer/ destabilizer was assayed by DSF. The T m of AfMGL and PpMGL was 73.1 °C and 74.4 °C, respectively, suggesting the higher proximity to the tertiary structure of both enzymes. The T m of AfMGL and PpMGL was slightly increased by trehalose and EDTA in contrast to guanidine HCl and urea. The active site and PLP-binding domains are identically conserved in both AfMGL and PpMGL.

Sucrose-enhanced biosynthesis of medicinally important antioxidant secondary metabolites in cell suspension cultures of Artemisia absinthium L.

Natural products are gaining tremendous importance in pharmaceutical industry and attention has been focused on the applications of in vitro technologies to enhance yield and productivity of such products. In this study, we investigated the accumulation of biomass and antioxidant secondary metabolites in response to different carbohydrate sources (sucrose, maltose, fructose and glucose) and sucrose concentrations (1, 3, 5, 7 and 9 %). Moreover, the effects of 3 % repeated sucrose feeding (day-12, -18 and -24) were also investigated. The results showed the superiority of disaccharides over monosaccharides for maximum biomass and secondary metabolites accumulation. Comparable profiles for maximum biomass were observed in response to sucrose and maltose and initial sucrose concentrations of 3 and 5 %. Maximum total phenolic and total flavonoid contents were displayed by cultures treated with sucrose and maltose; however, initial sucrose concentrations of 5 and 7 % were optimum for both classes of metabolites, respectively. Following 3 % extra sucrose feeding, cultures fed on day-24 (late-log phase) showed higher biomass, total phenolic and total flavonoid contents as compared to control cultures. Highest antioxidant activity was exhibited by maltose-treated cultures. Moreover, sucrose-treated cultures displayed positive correlation of antioxidant activity with total phenolics and total flavonoids production. This work describes the stimulatory role of disaccharides and sucrose feeding strategy for higher accumulation of phenolics and flavonoids, which could be potentially scaled up to bioreactor level for the bulk production of these metabolites in suspension cultures of A. absinthium.