A comparison between the function of Serendipita indica and Sinorhizobium meliloti in modulating the toxicity of zinc oxide nanoparticles in alfalfa (Medicago sativa L.).

Zinc oxide nanoparticles (ZnO-NPs) are among the most commonly used nano-fertilizers (NF). However, elevated levels of ZnO-NPs in soil may affect plant growth and development due to its potential toxicity when accumulated in large amounts in plant tissues. This research was conducted using an in situ rhizobox system with the aims of evaluating zinc uptake from nano-zinc oxide amended rhizosphere soil by alfalfa plant and the effect of plant growth-promoting microorganisms on alleviating the phytotoxicity of ZnO-NPs. Treatments included microbial inoculations (Sinorhizobium meliloti, Serendipita indica) and different ZnO-NP concentrations (0, 400, and 800 mg kg-1) with three replications. The results indicated that S. indica minimized the phytotoxicity of ZnO-NPs to alfalfa by enhancing growth rate and decreasing zinc (Zn) translocation from root to shoot. Compared with plants inoculated with S. meliloti, co-inoculation with S. indica increased the shoot dry weight by 18.33% and 8.05% at 400 and 800 mg kg-1 ZnO-NPs, respectively. However, at the highest level of ZnO-NPs (800 mg kg-1), root inoculation of S. indica and S. indica + S. meliloti decreased Zn translocation factor by 60.2% and 44.3% compared to S. meliloti, respectively. Furthermore, a distinct relation between tolerance of S. indica-colonized plant to ZnO-NPs and the ability of S. indica in inhibiting or retarding degradation of polyunsaturated lipids through prevention of excess reactive oxygen species formation was observed. Malondialdehyde content of inoculated plants with S. indica either alone or in combination with S. meliloti was significantly lower than non-inoculated plants (p< 0.01). Zn-induced oxidative stress was mitigated by S. indica through enhanced activities of catalase and peroxidase enzymes. The findings of the present study indicate the potential use of endophytes fungus S. indica for ensuring food safety and security, and human health in heavy metal-polluted soil by reducing the phytoavailability of heavy metals in the aerial parts of the host plants.

Renewed interests in the discovery of bioactive actinomycete metabolites driven by emerging technologies.

Actinomycetes are prolific sources of bioactive molecules. Traditional workflows including bacterial isolation, fermentation, metabolite identification and structure elucidation have resulted in high rates of natural product rediscovery in recent years. Recent advancements in multi-omics techniques have uncovered cryptic gene clusters within the genomes of actinomycetes, potentially introducing vast resources for the investigation of bioactive molecules. While developments in culture techniques have allowed for the fermentation of difficult-to-culture actinomycetes, high-throughput metabolite screening has offered plenary tools to accelerate hits discovery. A variety of new bioactive molecules have been isolated from actinomycetes of unique environmental origins, such as endophytic and symbiotic actinomycetes. Synthetic biology and genome mining have also emerged as new frontiers for the discovery of bioactive molecules. This review covers the highlights of recent developments in actinomycete-derived natural product drug discovery.

An Efficient Brome mosaic virus-Based Gene Silencing Protocol for Hexaploid Wheat (Triticum aestivum L.).

Virus-induced gene silencing (VIGS) is a rapid and powerful method to evaluate gene function, especially for species like hexaploid wheat that have large, redundant genomes and are difficult and time-consuming to transform. The Brome mosaic virus (BMV)-based VIGS vector is widely used in monocotyledonous species but not wheat. Here we report the establishment of a simple and effective VIGS procedure in bread wheat using BMVCP5, the most recently improved BMV silencing vector, and wheat genes PHYTOENE DESATURASE (TaPDS) and PHOSPHATE2 (TaPHO2) as targets. Time-course experiments revealed that smaller inserts (~100 nucleotides, nt) were more stable in BMVCP5 and conferred higher silencing efficiency and longer silencing duration, compared with larger inserts. When using a 100-nt insert and a novel coleoptile inoculation method, BMVCP5 induced extensive silencing of TaPDS transcript and a visible bleaching phenotype in the 2nd to 5th systemically-infected leaves from nine to at least 28 days post inoculation (dpi). For TaPHO2, the ability of BMVCP5 to simultaneously silence all three homoeologs was demonstrated. To investigate the feasibility of BMV VIGS in wheat roots, ectopically expressed enhanced GREEN FLUORESCENT PROTEIN (eGFP) in a transgenic wheat line was targeted for silencing. Silencing of eGFP fluorescence was observed in both the maturation and elongation zones of roots. BMVCP5 mediated significant silencing of eGFP and TaPHO2 mRNA expression in roots at 14 and 21 dpi, and TaPHO2 silencing led to the doubling of inorganic phosphate concentration in the 2nd through 4th systemic leaves. All 54 wheat cultivars screened were susceptible to BMV infection. BMVCP5-mediated TaPDS silencing resulted in the expected bleaching phenotype in all eight cultivars examined, and decreased TaPDS transcript was detected in all three cultivars examined. This BMVCP5 VIGS technology may serve as a rapid and effective functional genomics tool for high-throughput gene function studies in aerial and root tissues and in many wheat cultivars.

Combination of Siderophore-Producing Bacteria and Piriformospora indica Provides an Efficient Approach to Improve Cadmium Tolerance in Alfalfa.

Application of siderophore-producing microorganisms (SPMs), as an environmentally friendly approach, facilitates plant growth and survival under heavy metals toxicity. This study evaluated the effectiveness of SPMs, belonging to the bacterial genera Rhizobium and Pseudomonas and a root endophytic fungus (Piriformospora indica) to improve the fitness of alfalfa under cadmium (Cd) stress. A greenhouse experiment was performed as a randomized design with factorial arrangement of treatments. Treatments included microbial inoculations (Sinorhizobium meliloti, Pseudomonas fluorescence, and P. indica) and different Cd concentrations (0, 2, 5, 10 mg/kg) with three replications in potting media containing sand and sterile perlite (v/v, 2:1). The effect of Cd on plant growth and development, antioxidant enzymes activities, and accumulation of Cd and nutrients in alfalfa plant was investigated. Alfalfa inoculated with SPMs showed significantly higher biomass and nutrients uptake under both normal and Cd stress conditions than the controls. Under the highest Cd concentration (10 mg/kg), alfalfa plants inoculated with P. fluorescens and P. indica, either alone or in combination, showed the highest shoot dry weights. Cd-induced oxidative stress was mitigated by SPMs through enhanced antioxidant enzyme activities of catalase, ascorbate peroxidase, and guaiacol peroxidase. We showed that P. indica either alone or in combination with the siderophore producing bacteria (SPB) minimized the toxicity of Cd by enhanced growth rate and the lower Cd concentration in the shoots. In conclusion, metal-resistant SPMs could assist alfalfa to survive in Cd-contaminated soil by enhancing plant growth and development. Application of plant-associated microbes is an efficient, environmentally friendly approach to surmount the adverse effects of heavy metals toxicity on plants, animals, and humans. Graphical abstract.

Plant-Microbe Symbiosis: What Has Proteomics Taught Us?

Beneficial microbes have a positive impact on the productivity and fitness of the host plant. A better understanding of the biological impacts and underlying mechanisms by which the host derives these benefits will help to address concerns around global food production and security. The recent development of omics-based technologies has broadened our understanding of the molecular aspects of beneficial plant-microbe symbiosis. Specifically, proteomics has led to the identification and characterization of several novel symbiosis-specific and symbiosis-related proteins and post-translational modifications that play a critical role in mediating symbiotic plant-microbe interactions and have helped assess the underlying molecular aspects of the symbiotic relationship. Integration of proteomic data with other "omics" data can provide valuable information to assess hypotheses regarding the underlying mechanism of symbiosis and help define the factors affecting the outcome of symbiosis. Herein, an update is provided on the current and potential applications of symbiosis-based "omic" approaches to dissect different aspects of symbiotic plant interactions. The application of proteomics, metaproteomics, and secretomics as enabling approaches for the functional analysis of plant-associated microbial communities is also discussed.

Salinity-associated microRNAs and their potential roles in mediating salt tolerance in rice colonized by the endophytic root fungus Piriformospora indica.

Piriformospora indica (P. indica), an endophytic root fungus, supports the growth and enhanced tolerance of plants to biotic and abiotic stresses. Several recent studies showed the significant role of small RNA (sRNA) molecules including microRNAs (miRNAs) in plant adaption to environmental stress, but little is known concerning the symbiosis-mediated salt stress tolerance regulated at miRNAs level. The overarching goal of this research is to elucidate the impact of miRNAs in regulating the P. indica-mediated salt tolerance in rice. Applying sRNA-seq analysis led to identify a set of 547 differentially abundant miRNAs in response to P. indica inoculation and salt stress. These included 206 rice-specific and 341 previously known miRNAs from other plant species. In silico analysis of miRNAs predictions of the differentially abundant miRNAs led to identifying of 193 putatively target genes, most of which were encoded either genes or transcription factors involved in nutrient uptake, sodium ion transporters, growth regulators, and auxin- responsive proteins. The rice-specific miRNAs targeted the transcription factors involved in the import of potassium ions into the root cells, the export of sodium ions, and plant growth and development. Interestingly, P. indica affected the differential abundance of miRNAs regulated genes and transcription factors linked to salt stress tolerance. Our data helps to understand the molecular basis of salt stress tolerance mediated by symbionts in plant and the potential impact of miRNAs for genetic improvement of rice varieties for tolerance to salt stress.

The AC4 Protein of a Cassava Geminivirus Is Required for Virus Infection.

Geminiviruses (family Geminiviridae) are among the most devastating plant viruses worldwide, causing severe damage in crops of economic and subsistence importance. These viruses have very compact genomes and many of the encoded proteins are multifunctional. Here, we investigated the role of the East African cassava mosaic Cameroon virus (EACMCV) AC4 on virus infectivity in Nicotiana benthamiana. Results showed that plants inoculated with EACMCV containing a knockout mutation in an AC4 open reading frame displayed symptoms 2 to 3 days later than plants inoculated with wild-type virus, and these plants recovered from infection, whereas plants inoculated with the wild-type virus did not. Curiously, when an additional mutation was made in the knockout mutant, the resulting double mutant virus completely failed to cause any apparent symptoms. Interestingly, the role of AC4 on virus infectivity appeared to be dependent on an encoded N-myristoylation motif that mediates cell membrane binding. We previously showed that EACMCV containing the AC4T38I mutant produced virus progeny characterized by second-site mutations and reversion to wild-type virus. These results were confirmed in this study using additional mutations. Together, these results show involvement of EACMCV AC4 in virus infectivity; they also suggest a role for the combined action of mutation and selection, under prevailing environmental conditions, on begomovirus genetic variation and diversity.

Complete genome sequence of a new bipartite begomovirus infecting fluted pumpkin (Telfairia occidentalis) plants in Cameroon.

The complete genome sequence was determined and characterized for a previously unreported bipartite begomovirus from fluted pumpkin (Telfairia occidentalis, family Cucurbitaceae) plants displaying mosaic symptoms in Cameroon. The DNA-A and DNA-B components were ~2.7 kb and ~2.6 kb in size, and the arrangement of viral coding regions on the genomic components was like those characteristic of other known bipartite begomoviruses originating in the Old World. While the DNA-A component was more closely related to that of chayote yellow mosaic virus (ChaYMV), at 78 %, the DNA-B component was more closely related to that of soybean chlorotic blotch virus (SbCBV), at 64 %. This newly discovered bipartite Old World virus is herein named telfairia mosaic virus (TelMV).

Complete genome sequence of a new bipartite begomovirus infecting cotton in the Republic of Benin in West Africa.

Here, we report the complete genome sequence of a novel bipartite begomovirus isolated from cotton plants (Gossypium raimondii, Malvaceae) exhibiting light yellow mosaic symptoms. The genome sequence was determined by Illumina DNA sequencing and confirmed by Sanger sequencing of RCA-enriched, cloned circular genomic components. The DNA-A and DNA-B components were each ~2.7 kb in size, and their genome arrangement was characteristic of other Old World bipartite begomoviruses. While the DNA-A component was most closely related to tobacco leaf curl Comoros virus (TbLCKMV) at 80 %, the DNA-B component had as its closet relative soybean chlorotic blotch virus (SbCBV) at 66 %. This previously undescribed begomovirus is herein named "cotton yellow mosaic virus" (CYMV).

Metabolic and transcriptional response of central metabolism affected by root endophytic fungus Piriformospora indica under salinity in barley.

The root endophytic fungus Piriformospora indica enhances plant adaptation to environmental stress based on general and non-specific plant species mechanisms. In the present study, we integrated the ionomics, metabolomics, and transcriptomics data to identify the genes and metabolic regulatory networks conferring salt tolerance in P. indica-colonized barley plants. To this end, leaf samples were harvested at control (0 mM NaCl) and severe salt stress (300 mM NaCl) in P. indica-colonized and non-inoculated barley plants 4 weeks after fungal inoculation. The metabolome analysis resulted in an identification of a signature containing 14 metabolites and ions conferring tolerance to salt stress. Gene expression analysis has led to the identification of 254 differentially expressed genes at 0 mM NaCl and 391 genes at 300 mM NaCl in P. indica-colonized compared to non-inoculated samples. The integration of metabolome and transcriptome analysis indicated that the major and minor carbohydrate metabolism, nitrogen metabolism, and ethylene biosynthesis pathway might play a role in systemic salt-tolerance in leaf tissue induced by the root-colonized fungus.

High-resolution identification and abundance profiling of cassava (Manihot esculenta Crantz) microRNAs.

BACKGROUND: Small RNAs (sRNAs) are endogenous sRNAs that play regulatory roles in plant growth, development, and biotic and abiotic stress responses. In plants, one subset of sRNAs, microRNAs (miRNAs) exhibit tissue-differential expression and regulate gene expression mainly through direct cleavage of mRNA or indirectly via production of secondary phased siRNAs (phasiRNAs) that silence cognate target transcripts in trans. RESULTS: Here, we have identified cassava (Manihot esculenta Crantz) miRNAs using high resolution sequencing of sRNA libraries from leaf, stem, callus, male and female flower tissues. To analyze the data, we built a cassava genome database and, via sequence analysis and secondary structure prediction, 38 miRNAs not previously reported in cassava were identified. These new cassava miRNAs included two miRNAs not previously been reported in any plant species. The miRNAs exhibited tissue-differential accumulation as confirmed by quantitative RT-PCR and Northern blot analysis, largely reflecting levels observed in sequencing data. Some of the miRNAs identified were predicted to trigger production of secondary phased siRNAs (phasiRNAs) from 80 PHAS loci. CONCLUSIONS: Cassava is a woody perennial shrub, grown principally for its starch-rich storage roots, which are rich in calories. In this study, new miRNAs were identified and their expression was validated using qRT-PCR of RNA from five different tissues. The data obtained expand the list of annotated miRNAs and provide additional new resources for cassava improvement research.

Somatic embryogenesis and plant regeneration of cassava (Manihot esculenta Crantz) landraces from Cameroon.

A procedure to regenerate cassava (Manihot esculenta Crantz) cultivars from Cameroon via somatic embryogenesis (SE) was developed. Shoot apical meristems and immature leaf lobes were used as explants on Murashige and Skoog (MS) basal medium containing 33 or 50 µM of the auxins Picloram (Pic), 2,4-Dichlorophenoxyacetic acid (2,4-D), Dicamba (Dic), and α-Naphthalene acetic acid. Cultivar performance was assessed using SE and number of somatic embryos produced. Overall, the frequency of primary somatic embryogenesis (PSE) and the mean number of somatic embryos produced varied considerably with genotype, type of auxin and concentration tested. For example, cultivar (cv.) Ngan Mbada showed the best performance on MS medium supplemented with 50 µM Pic with a SE frequency of 40 % and an average number of somatic embryos of 90. The second best performance was recorded in cv. Local Red on MS medium supplemented with 33 µM 2,4-D, where the SE frequency was 40 % and an average number of somatic embryos of 60.5. Cultivar Ekona Red recorded the best performance on medium supplemented with 50 µM Pic showing a SE frequency of 47 % and an average number of somatic embryos of 45. We further examined secondary and cyclic somatic embryogenesis (SSE, CSE) and both were also observed to vary with genotype, however, both exhibited significantly higher frequencies of SE compared with PSE. SE started to decline at the fourth cycle of embryogenesis. Examination of organogenesis showed that shoot bud induction from green cotyledons varied across cultivars and benzylaminopurine was shown to outperform Thidiazuron in the ability to induce organogenesis. Furthermore, the frequencies of bud induction were identical under light and dark conditions. Finally, regenerated plants grew easily in the greenhouse with 90-100 % survival rate and did not display detectable variation in morphology.

Proteomics study reveals the molecular mechanisms underlying water stress tolerance induced by Piriformospora indica in barley.

Piriformospora indica is a mutualistic root endophytic fungus, which transfers several benefits to hosts including enhance plant growth and increase yield under both normal and stress conditions. It has been shown that P. indica root-colonization enhances water stress tolerance based on general and non-specific plant-species mechanism. To better understand the molecular mechanism of P. indica-mediated drought stress tolerance, we designed a set of comparative experiments to study the impact of P. indica on barely plants cultivar "Golden Promise" grown under different drought levels [Filed capacity (F.C.) and 25% F.C.]. P. indica enhanced root and shoot biomass of colonized plants under both well-watered and water-deficit conditions. Proteome analysis of P. indica-colonized barley leaves under well-treated and water-deficit conditions resulted in detection of 726 reproducibly protein spots. Mass spectrometry analysis resulted in the identification of 45 differentially accumulated proteins involved in photosynthesis, reactive oxygen scavenging, metabolisms, signal transduction, and plant defense responses. Interestingly, P. indica increased the level of proteins involved in photosynthesis, antioxidative defense system and energy transport. We propose that P. indica-mediated drought stress tolerance in barely is through photosynthesis stimulation, energy releasing and enhanced antioxidative capacity in colonized plants. BIOLOGICAL SIGNIFICANCE: Plant mutualistic symbionts offer long-term abiotic stress tolerance through the host adaptation to environmental stress. There have been a few published proteomic studies of plant symbionts to drought, and this is thought to be the first proteomic analysis, demonstrating the impact of endophyte on barley plant under drought stress. For some of identified proteins like TCTP and PCNA, a connection to physiological function in plants is novel, and can be the best candidates for sources of drought tolerance in future studies.

A proteomics approach to study the molecular basis of enhanced salt tolerance in barley (Hordeum vulgare L.) conferred by the root mutualistic fungus Piriformospora indica.

Piriformospora indica is a root-interacting mutualistic fungus capable of enhancing plant growth, increasing plant resistance to a wide variety of pathogens, and improving plant stress tolerance under extreme environmental conditions. Understanding the molecular mechanisms by which P. indica can improve plant tolerance to stresses will pave the way to identifying the major mechanisms underlying plant adaptability to environmental stresses. We conducted greenhouse experiments at three different salt levels (0, 100 and 300 mM NaCl) on barley (Hordeum vulgare L.) cultivar "Pallas" inoculated with P. indica. Based on the analysis of variance, P. indica had a significant impact on the barley growth and shoot biomass under normal and salt stress conditions. P. indica modulated ion accumulation in colonized plants by increasing the foliar potassium (K(+))/sodium (Na(+)) ratio, as it is considered a reliable indicator of salt stress tolerance. P. indica induced calcium (Ca(2+)) accumulation and likely influenced the stress signal transduction. Subsequently, proteomic analysis of the barley leaf sheath using two-dimensional electrophoresis resulted in detection of 968 protein spots. Of these detected spots, the abundance of 72 protein spots changed significantly in response to salt treatment and P. indica-root colonization. Mass spectrometry analysis of responsive proteins led to the identification of 51 proteins. These proteins belonged to different functional categories including photosynthesis, cell antioxidant defense, protein translation and degradation, energy production, signal transduction and cell wall arrangement. Our results showed that P. indica induced a systemic response to salt stress by altering the physiological and proteome responses of the plant host.

Ethylene in mutualistic symbioses.

Ethylene (ET) is a gaseous phytohormone that participates in various plant physiological processes and essentially contributes to plant immunity. ET conducts its functions by regulating the expression of ET-responsive genes or in crosstalk with other hormones. Several recent studies have shown the significance of ET in the establishment and development of plant-microbe interactions. Therefore, it is not surprising that pathogens and mutualistic symbionts target ET synthesis or signaling to colonize plants. This review introduces the significance of ET metabolism in plant-microbe interactions, with an emphasis on its role in mutualistic symbioses.

Ethylene supports colonization of plant roots by the mutualistic fungus Piriformospora indica.

The mutualistic basidiomycete Piriformospora indica colonizes roots of mono- and dicotyledonous plants, and thereby improves plant health and yield. Given the capability of P. indica to colonize a broad range of hosts, it must be anticipated that the fungus has evolved efficient strategies to overcome plant immunity and to establish a proper environment for nutrient acquisition and reproduction. Global gene expression studies in barley identified various ethylene synthesis and signaling components that were differentially regulated in P. indica-colonized roots. Based on these findings we examined the impact of ethylene in the symbiotic association. The data presented here suggest that P. indica induces ethylene synthesis in barley and Arabidopsis roots during colonization. Moreover, impaired ethylene signaling resulted in reduced root colonization, Arabidopsis mutants exhibiting constitutive ethylene signaling, -synthesis or ethylene-related defense were hyper-susceptible to P. indica. Our data suggest that ethylene signaling is required for symbiotic root colonization by P. indica.

Root cell death and systemic effects of Piriformospora indica: a study on mutualism.

The root systems of most terrestrial plants are confronted with a huge variety of invasive microorganisms that either can cause detrimental effects or in case of mutualistic symbiosis provide benefits for the host. In either case, establishment of the parasitic or mutualistic interaction is the result of a highly sophisticated cross-talk between the partners. Despite the ecological importance of mutualistic symbioses, the molecular events accompanied by this phenomenon are far from being understood. Piriformospora indica represents a recently discovered fungus that transfers considerable beneficial impact to its host plants. In this review, the current knowledge on this novel symbiosis is summarized by focusing on its biological effects in hosts and the role of programmed cell death in the establishment of the mutualistic interaction.