Advances in plant synthetic biology approaches to control expression of gene circuits.

The applications of synthetic biology range from creating simple circuits to monitor an organism's state to complex circuits capable of reconstructing aspects of life. The latter has the potential to be used in plant synthetic biology to address current societal issues by reforming agriculture and enhancing production of molecules of increased demand. For this reason, development of efficient tools to precisely control gene expression of circuits must be prioritized. In this review, we report the latest efforts towards characterization, standardization and assembly of genetic parts into higher-order constructs, as well as available types of inducible systems to modulate their transcription in plant systems. Subsequently, we discuss recent developments in the orthogonal control of gene expression, Boolean logic gates and synthetic genetic toggle-like switches. Finally, we conclude that by combining different means of controlling gene expression, we can create complex circuits capable of reshaping plant life.

A beneficial fungal root endophyte triggers systemic RNA silencing and DNA methylation of a host reporter gene.

A growing body of evidence suggests that RNA interference (RNAi) plays a pivotal role in the communication between plants and pathogenic fungi, where a bi-directional trans-kingdom RNAi is established to the advantage of either the host or the pathogen. Similar mechanisms acting during plant association with non-pathogenic symbiotic microorganisms have been elusive to this date. To determine whether root endophytes can induce systemic RNAi responses to their host plants, we designed an experimental reporter-based system consisting of the root-restricted, beneficial fungal endophyte, Fusarium solani strain K (FsK) and its host Nicotiana benthamiana. Since not all fungi encode the RNAi machinery, we first needed to validate that FsK does so, by identifying its core RNAi enzymes (2 Dicer-like genes, 2 Argonautes and 4 RNA-dependent RNA polymerases) and by showing its susceptibility to in vitro RNAi upon exogenous application of double stranded RNAs (dsRNAs). Upon establishing this, we transformed FsK with a hairpin RNA (hpRNA) construct designed to target a reporter gene in its host N. benthamiana. The hpRNA was processed by FsK RNAi machinery predominantly into 21-24-nt small RNAs that triggered RNA silencing but not DNA methylation in the fungal hyphae. Importantly, when the hpRNA-expressing FsK was used to inoculate N. benthamiana, systemic RNA silencing and DNA methylation of the host reporter gene was recorded. Our data suggest that RNAi signals can be translocated by root endophytes to their hosts and can modulate gene expression during mutualism, which may be translated to beneficial phenotypes.

Interplay between rhizobial nodulation and arbuscular mycorrhizal fungal colonization in Lotus japonicus roots.

AIMS: This study aims to identify main factors that influence the tripartite association of legumes with arbuscular mycorrhiza fungi (AMF) and nitrogen-fixing rhizobia. METHODS AND RESULTS: Concurrent inoculations with Mesorhizobium loti and four AMF strains were performed on the model legume Lotus japonicus. Nodulation was significantly enhanced by all AMF strains, under normal conditions, and by specific AMF strains under heat-stress conditions. The impact of rhizobia on mycorrhizal colonization was AMF strain dependent. Co-inoculation trials, where either AMF or rhizobia were restricted outside the root, showed that the symbiotic phenotypes are not influenced by microbial interactions at the pre-symbiotic stage. External application of nutrients showed that P enhances nodulation, while N application does not enhance mycorrhizal colonization. CONCLUSIONS: Nodulation and mycorhization affect one another during advanced stages of the symbiosis. AMF strains may enhance nodulation under both normal and high environmental temperatures. Rhizobium-AMF compatibility is critical, as rhizobium may positively affect specific AMF strains, an effect that does not derive from increased N uptake.

Plant terpenoid metabolism co-opts a component of the cell wall biosynthesis machinery.

Glycosylation is one of the most prevalent molecular modifications in nature. Single or multiple sugars can decorate a wide range of acceptors from proteins to lipids, cell wall glycans and small molecules, dramatically affecting their activity. Here, we discovered that by 'hijacking' an enzyme of the cellulose synthesis machinery involved in cell wall assembly, plants evolved cellulose synthase-like enzymes (Csls) and acquired the capacity to glucuronidate specialized metabolites, that is, triterpenoid saponins. Apparently, endoplasmic reticulum-membrane localization of Csls and of other pathway proteins was part of evolving a new glycosyltransferase function, as plant metabolite glycosyltransferases typically act in the cytosol. Discovery of glucuronic acid transferases across several plant orders uncovered the long-pursued enzymatic reaction in the production of a low-calorie sweetener from licorice roots. Our work opens the way for engineering potent saponins through microbial fermentation and plant-based systems.

Unraveling the roles of plant specialized metabolites: using synthetic biology to design molecular biosensors.

Plants are a rich source of specialized metabolites with a broad range of bioactivities and many applications in human daily life. Over the past decades significant progress has been made in identifying many such metabolites in different plant species and in elucidating their biosynthetic pathways. However, the biological roles of plant specialized metabolites remain elusive and proposed functions lack an identified underlying molecular mechanism. Understanding the roles of specialized metabolites frequently is hampered by their dynamic production and their specific spatiotemporal accumulation within plant tissues and organs throughout a plant's life cycle. In this review, we propose the employment of strategies from the field of Synthetic Biology to construct and optimize genetically encoded biosensors that can detect individual specialized metabolites in a standardized and high-throughput manner. This will help determine the precise localization of specialized metabolites at the tissue and single-cell levels. Such information will be useful in developing complete system-level models of specialized plant metabolism, which ultimately will demonstrate how the biosynthesis of specialized metabolites is integrated with the core processes of plant growth and development.

Lotus SHAGGY-like kinase 1 is required to suppress nodulation in Lotus japonicus.

Glycogen synthase kinase/SHAGGY-like kinases (SKs) are a highly conserved family of signaling proteins that participate in many developmental, cell-differentiation, and metabolic signaling pathways in plants and animals. Here, we investigate the involvement of SKs in legume nodulation, a process requiring the integration of multiple signaling pathways. We describe a group of SKs in the model legume Lotus japonicus (LSKs), two of which respond to inoculation with the symbiotic nitrogen-fixing bacterium Mesorhizobium loti. RNAi knock-down plants and an insertion mutant for one of these genes, LSK1, display increased nodulation. Ηairy-root lines overexpressing LSK1 form only marginally fewer mature nodules compared with controls. The expression levels of genes involved in the autoregulation of nodulation (AON) mechanism are affected in LSK1 knock-down plants at low nitrate levels, both at early and late stages of nodulation. At higher levels of nitrate, these same plants show the opposite expression pattern of AON-related genes and lose the hypernodulation phenotype. Our findings reveal an additional role for the versatile SK gene family in integrating the signaling pathways governing legume nodulation, and pave the way for further study of their functions in legumes.

An endophytic Fusarium-legume association is partially dependent on the common symbiotic signalling pathway.

Legumes interact with a wide range of microbes in their root systems, ranging from beneficial symbionts to pathogens. Symbiotic rhizobia and arbuscular mycorrhizal glomeromycetes trigger a so-called common symbiotic signalling pathway (CSSP), including the induction of nuclear calcium spiking in the root epidermis. By combining gene expression analysis, mutant phenotypic screening and analysis of nuclear calcium elevations, we demonstrate that recognition of an endophytic Fusarium solani strain K (FsK) in model legumes is initiated via perception of chitooligosaccharidic molecules and is, at least partially, CSSP-dependent. FsK induced the expression of Lysin-motif receptors for chitin-based molecules, CSSP members and CSSP-dependent genes in Lotus japonicus. In LysM and CSSP mutant/RNAi lines, root penetration and fungal intraradical progression was either stimulated or limited, whereas FsK exudates triggered CSSP-dependent nuclear calcium spiking, in epidermal cells of Medicago truncatula root organ cultures. Our results corroborate CSSP being involved in the perception of signals from other microbes beyond the restricted group of symbiotic interactions sensu stricto.

Colonization of legumes by an endophytic Fusarium solani strain FsK reveals common features to symbionts or pathogens.

Plant cellular responses to endophytic filamentous fungi are scarcely reported, with the majority of described colonization processes in plant-fungal interactions referring to either pathogens or true symbionts. Fusarium solani strain K (FsK) is a root endophyte of Solanum lycopersicum, which protects against root and foliar pathogens. Here, we investigate the association of FsK with two legumes (Lotus japonicus and Medicago truncatula) and report on colonization patterns and plant responses during the establishment of the interaction. L. japonicus plants colonized by FsK complete their life cycle and exhibit no apparent growth defects under normal conditions. We followed the growth of FsK within root-inoculated plants spatiotemporally and showed the capability of the endophyte to migrate to the stem. In a bipartite system comprising of the endophyte and either whole plants or root organ cultures, we studied the plant sub-cellular responses to FsK recognition, using optical, confocal and transmission electron microscopy. A polarized reorganization of the root cell occurs: endoplasmic reticulum/cytoplasm accumulation and nuclear placement at contact sites, occasional development of papillae underneath hyphopodia and membranous material rearrangements towards penetrating hyphae. Fungal hyphae proliferate within the vascular bundle of the plant. Plant cell death is involved in fungal colonization of the root. Our data suggest that the establishment of FsK within legume tissues requires fungal growth adaptations and plant cell-autonomous responses, known to occur during both symbiotic and pathogenic plant-fungal interactions. We highlight the overlooked plasticity of endophytic fungi upon plant colonization, and introduce a novel plant-endophyte association.

Ozone-induced inhibition of kiwifruit ripening is amplified by 1-methylcyclopropene and reversed by exogenous ethylene.

BACKGROUND: Understanding the mechanisms involved in climacteric fruit ripening is key to improve fruit harvest quality and postharvest performance. Kiwifruit (Actinidia deliciosa cv. 'Hayward') ripening involves a series of metabolic changes regulated by ethylene. Although 1-methylcyclopropene (1-MCP, inhibitor of ethylene action) or ozone (O3) exposure suppresses ethylene-related kiwifruit ripening, how these molecules interact during ripening is unknown. RESULTS: Harvested 'Hayward' kiwifruits were treated with 1-MCP and exposed to ethylene-free cold storage (0 °C, RH 95%) with ambient atmosphere (control) or atmosphere enriched with O3 (0.3 µL L- 1) for up to 6 months. Their subsequent ripening performance at 20 °C (90% RH) was characterized. Treatment with either 1-MCP or O3 inhibited endogenous ethylene biosynthesis and delayed fruit ripening at 20 °C. 1-MCP and O3 in combination severely inhibited kiwifruit ripening, significantly extending fruit storage potential. To characterize ethylene sensitivity of kiwifruit following 1-MCP and O3 treatments, fruit were exposed to exogenous ethylene (100 µL L- 1, 24 h) upon transfer to 20 °C following 4 and 6 months of cold storage. Exogenous ethylene treatment restored ethylene biosynthesis in fruit previously exposed in an O3-enriched atmosphere. Comparative proteomics analysis showed separate kiwifruit ripening responses, unraveled common 1-MCP- and O3-dependent metabolic pathways and identified specific proteins associated with these different ripening behaviors. Protein components that were differentially expressed following exogenous ethylene exposure after 1-MCP or O3 treatment were identified and their protein-protein interaction networks were determined. The expression of several kiwifruit ripening related genes, such as 1-aminocyclopropane-1-carboxylic acid oxidase (ACO1), ethylene receptor (ETR1), lipoxygenase (LOX1), geranylgeranyl diphosphate synthase (GGP1), and expansin (EXP2), was strongly affected by O3, 1-MCP, their combination, and exogenously applied ethylene. CONCLUSIONS: Our findings suggest that the combination of 1-MCP and O3 functions as a robust repressive modulator of kiwifruit ripening and provide new insight into the metabolic events underlying ethylene-induced and ethylene-independent ripening outcomes.

A Lotus japonicus E3 ligase interacts with the Nod Factor Receptor 5 and positively regulates nodulation.

BACKGROUND: Post-translational modification of receptor proteins is involved in activation and de-activation of signalling systems in plants. Both ubiquitination and deubiquitination have been implicated in plant interactions with pathogens and symbionts. RESULTS: Here we present LjPUB13, a PUB-ARMADILLO repeat E3 ligase that specifically ubiquitinates the kinase domain of the Nod Factor receptor NFR5 and has a direct role in nodule organogenesis events in Lotus japonicus. Phenotypic analyses of three LORE1 retroelement insertion plant lines revealed that pub13 plants display delayed and reduced nodulation capacity and retarded growth. LjPUB13 expression is spatially regulated during symbiosis with Mesorhizobium loti, with increased levels in young developing nodules. CONCLUSION: LjPUB13 is an E3 ligase with a positive regulatory role during the initial stages of nodulation in L. japonicus.

Metabolic and functional diversity of saponins, biosynthetic intermediates and semi-synthetic derivatives.

Saponins are widely distributed plant natural products with vast structural and functional diversity. They are typically composed of a hydrophobic aglycone, which is extensively decorated with functional groups prior to the addition of hydrophilic sugar moieties, to result in surface-active amphipathic compounds. The saponins are broadly classified as triterpenoids, steroids or steroidal glycoalkaloids, based on the aglycone structure from which they are derived. The saponins and their biosynthetic intermediates display a variety of biological activities of interest to the pharmaceutical, cosmetic and food sectors. Although their relevance in industrial applications has long been recognized, their role in plants is underexplored. Recent research on modulating native pathway flux in saponin biosynthesis has demonstrated the roles of saponins and their biosynthetic intermediates in plant growth and development. Here, we review the literature on the effects of these molecules on plant physiology, which collectively implicate them in plant primary processes. The industrial uses and potential of saponins are discussed with respect to structure and activity, highlighting the undoubted value of these molecules as therapeutics.

AtHESPERIN: a novel regulator of circadian rhythms with poly(A)-degrading activity in plants.

We report the identification and characterization of a novel gene, AtHesperin (AtHESP) that codes for a deadenylase in Arabidopsis thaliana. The gene is under circadian clock-gene regulation and has similarity to the mammalian Nocturnin. AtHESP can efficiently degrade poly(A) substrates exhibiting allosteric kinetics. Size exclusion chromatography and native electrophoresis coupled with kinetic analysis support that the native enzyme is oligomeric with at least 3 binding sites. Knockdown and overexpression of AtHESP in plant lines affects the expression and rhythmicity of the clock core oscillator genes TOC1 and CCA1. This study demonstrates an evolutionary conserved poly(A)-degrading activity in plants and suggests deadenylation as a mechanism involved in the regulation of the circadian clock. A role of AtHESP in stress response in plants is also depicted.

The impact of sodium nitroprusside and ozone in kiwifruit ripening physiology: a combined gene and protein expression profiling approach.

BACKGROUND AND AIMS: Despite their importance in many aspects of plant physiology, information about the function of oxidative and, particularly, of nitrosative signalling in fruit biology is limited. This study examined the possible implications of O3 and sodium nitroprusside (SNP) in kiwifruit ripening, and their interacting effects. It also aimed to investigate changes in the kiwifruit proteome in response to SNP and O3 treatments, together with selected transcript analysis, as a way to enhance our understanding of the fruit ripening syndrome. METHODS: Kiwifruits following harvest were pre-treated with 100 µm SNP, then cold-stored (0 °C, relative humidity 95 %) for either 2 or 6 months in the absence or in the presence of O3 (0·3 µL L(-1)), and subsequently were allowed to ripen at 20 °C. The ripening behaviour of fruit was characterized using several approaches: together with ethylene production, several genes, enzymes and metabolites involved in ethylene biosynthesis were analysed. Kiwifruit proteins were identified using 2-D electrophoresis coupled with nanoliquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Expression patterns of kiwifruit ripening-related genes were also analysed using real-time quantitative reverse transcription-PCR (RT-qPCR). KEY RESULTS: O3 treatment markedly delayed fruit softening and depressed the ethylene biosynthetic mechanism. Although SNP alone was relatively ineffective in regulating ripening, SNP treatment prior to O3 exposure attenuated the O3-induced ripening inhibition. Proteomic analysis revealed a considerable overlap between proteins affected by both SNP and O3. Consistent with this, the temporal dynamics in the expression of selected kiwifruit ripening-related genes were noticeably different between individual O3 and combined SNP and O3 treatments. CONCLUSIONS: This study demonstrates that O3-induced ripening inhibition could be reversed by SNP and provides insights into the interaction between oxidative and nitrosative signalling in climacteric fruit ripening.

A metabolic gene cluster in Lotus japonicus discloses novel enzyme functions and products in triterpene biosynthesis.

Genes for triterpene biosynthetic pathways exist as metabolic gene clusters in oat and Arabidopsis thaliana plants. We characterized the presence of an analogous gene cluster in the model legume Lotus japonicus. In the genomic regions flanking the oxidosqualene cyclase AMY2 gene, genes for two different classes of cytochrome P450 and a gene predicted to encode a reductase were identified. Functional characterization of the cluster genes was pursued by heterologous expression in Nicotiana benthamiana. The gene expression pattern was studied under different developmental and environmental conditions. The physiological role of the gene cluster in nodulation and plant development was studied in knockdown experiments. A novel triterpene structure, dihydrolupeol, was produced by AMY2. A new plant cytochrome P450, CYP71D353, which catalyses the formation of 20-hydroxybetulinic acid in a sequential three-step oxidation of 20-hydroxylupeol was characterized. The genes within the cluster are highly co-expressed during root and nodule development, in hormone-treated plants and under various environmental stresses. A transcriptional gene silencing mechanism that appears to be involved in the regulation of the cluster genes was also revealed. A tightly co-regulated cluster of functionally related genes is involved in legume triterpene biosynthesis, with a possible role in plant development.

Plant-Mediated Effects of Beneficial Microbes and a Plant Strengthener against Spider Mites in Tomato.

The two-spotted spider mite Tetranychus urticae is a polyphagous herbivore with a worldwide distribution, and is a serious pest in tomato and other crops. As an alternative to chemical pesticides, biological control with the release of natural enemies such as predatory mites represent an efficient method to control T. urticae in many crops, but not in tomato. Other biological control agents, such as beneficial microbes, as well as chemical compounds, which can act as plant defense elicitors that confer plant resistance against pests and pathogens, may prove promising biological solutions for the suppression of spider mite populations in tomato. Here, we assessed this hypothesis by recording the effects of a series of fungal and bacterial strains and the plant strengthener acibenzolar-s-methyl for their plant-mediated effects on T. urticae performance in two tomato cultivars. We found significant negative effects on the survival, egg production and spider mite feeding damage on plants inoculated with microbes or treated with the plant strengthener as compared to the control plants. Our results highlight the potential of beneficial microbes and plant strengtheners in spider mite suppression in addition to plant disease control.

Above- and below-ground microbiome in the annual developmental cycle of two olive tree varieties.

The olive tree is a hallmark crop in the Mediterranean region. Its cultivation is characterized by an enormous variability in existing genotypes and geographical areas. As regards the associated microbial communities of the olive tree, despite progress, we still lack comprehensive knowledge in the description of these key determinants of plant health and productivity. Here, we determined the prokaryotic, fungal and arbuscular mycorrhizal fungal (AMF) microbiome in below- (rhizospheric soil, roots) and above-ground (phyllosphere and carposphere) plant compartments of two olive varieties 'Koroneiki' and 'Chondrolia Chalkidikis' grown in Southern and Northern Greece respectively, in five developmental stages along a full fruit-bearing season. Distinct microbial communities were supported in above- and below-ground plant parts; while the former tended to be similar between the two varieties/locations, the latter were location specific. In both varieties/locations, a seasonally stable root microbiome was observed over time; in contrast the plant microbiome in the other compartments were prone to changes over time, which may be related to seasonal environmental change and/or to plant developmental stage. We noted that olive roots exhibited an AMF-specific filtering effect (not observed for bacteria and general fungi) onto the rhizosphere AMF communities of the two olive varieties/locations/, leading to the assemblage of homogenous intraradical AMF communities. Finally, shared microbiome members between the two olive varieties/locations include bacterial and fungal taxa with putative functional attributes that may contribute to olive tree tolerance to abiotic and biotic stress.

Role of lupeol synthase in Lotus japonicus nodule formation.

• Triterpenes are plant secondary metabolites, derived from the cyclization of 2,3-oxidosqualene by oxidosqualene cyclases (OSCs). Here, we investigated the role of lupeol synthase, encoded by OSC3, and its product, lupeol, in developing roots and nodules of the model legume Lotus japonicus. • The expression patterns of OSC3 in different developmental stages of uninfected roots and in roots infected with Mesorhizobium loti were determined. The tissue specificity of OSC3 expression was analysed by in situ hybridization. Functional analysis, in which transgenic L. japonicus roots silenced for OSC3 were generated, was performed. The absence of lupeol in the silenced plant lines was determined by GC-MS. • The expression of ENOD40, a marker gene for nodule primordia initiation, was increased significantly in the OSC3-silenced plant lines, suggesting that lupeol influences nodule formation. Silenced plants also showed a more rapid nodulation phenotype, consistent with this. Exogenous application of lupeol to M. loti-infected wild-type plants provided further evidence for a negative regulatory effect of lupeol on the expression of ENOD40. • The synthesis of lupeol in L. japonicus roots and nodules can be solely attributed to OSC3. Taken together, our data suggest a role for lupeol biosynthesis in nodule formation through the regulation of ENOD40 gene expression.

The impact of biofumigation and chemical fumigation methods on the structure and function of the soil microbial community.

Biofumigation (BIOF) is carried out mainly by the incorporation of brassica plant parts into the soil, and this fumigation activity has been linked to their high glucosinolate (GSL) content. GSLs are hydrolyzed by the endogenous enzyme myrosinase to release isothiocyanates (ITCs). A microcosm study was conducted to investigate the effects induced on the soil microbial community by the incorporation of broccoli residues into soil either with (BM) or without (B) added myrosinase and of chemical fumigation, either as soil application of 2-phenylethyl ITC (PITC) or metham sodium (MS). Soil microbial activity was evaluated by measuring fluorescein diacetate hydrolysis and soil respiration. Effects on the structure of the total microbial community were assessed by phospholipid fatty acid analysis, while the impact on important fungal (ascomycetes (ASC)) and bacterial (ammonia-oxidizing bacteria (AOB)) guilds was evaluated by denaturating gradient gel electrophoresis (DGGE). Overall, B, and to a lesser extent BM, stimulated microbial activity and biomass. The diminished effect of BM compared to B was particularly evident in fungi and Gram-negative bacteria and was attributed to rapid ITC release following the myrosinase treatment. PITC did not have a significant effect, whereas an inhibitory effect was observed in the MS-treated soil. DGGE analysis showed that the ASC community was temporarily altered by BIOF treatments and more persistently by the MS treatment, while the structure of the AOB community was not affected by the treatments. Cloning of the ASC community showed that MS application had a deleterious effect on potential plant pathogens like Fusarium, Nectria, and Cladosporium compared to BIOF treatments which did not appear to inhibit them. Our findings indicate that BIOF induces changes on the structure and function of the soil microbial community that are mostly related to microbial substrate availability changes derived from the soil amendment with fresh organic materials.

Multi-species relationships in legume roots: From pairwise legume-symbiont interactions to the plant - microbiome - soil continuum.

Mutualistic relationships of legume plants with, either bacteria (like rhizobia) or fungi (like arbuscular mycorrhizal fungi), have been investigated intensively, usually as bi-partite interactions. However, diverse symbiotic interactions take place simultaneously or sequentially under field conditions. Their collective, but not additive, contribution to plant growth and performance remains hard to predict, and appears to be furthermore affected by crop species and genotype, non-symbiotic microbial interactions and environmental variables. The challenge is: (i) to unravel the complex overlapping mechanisms that operate between the microbial symbionts as well as between them, their hosts and the rhizosphere (ii) to understand the dynamics of the respective mechanisms in evolutionary and ecological terms. The target for agriculture, food security and the environment, is to use this insight as a solid basis for developing new integrated technologies, practices and strategies for the efficient use of beneficial microbes in legumes and other plants. We review recent advances in our understanding of the symbiotic interactions in legumes roots brought about with the aid of molecular and bioinformatics tools. We go through single symbiont-host interactions, proceed to tripartite symbiont-host interactions, appraise interactions of symbiotic and associative microbiomes with plants in the root-rhizoplane-soil continuum of habitats and end up by examining attempts to validate community ecology principles in the legume-microbe-soil biosystem.

Metal nanoparticles: Phytotoxicity on tomato and effect on symbiosis with the Fusarium solani FsK strain.

The effect of copper (Cu-NPs, CuO-NPs), silver (Ag-NPs) and zinc oxide (ZnO-NPs) nanoparticles (NPs) on plant growth, physiological properties of tomato plants and their symbiotic relationships with the endophytic Fusarium solani FsK strain was investigated. Fungitoxicity tests revealed that the FsK strain was significantly more sensitive to Cu-NPs and ZnO-NPs than CuO-NPs and Ag-NPs both in terms of mycelial growth and spore germination. All NPs were more toxic to FsK compared to their bulk counterparts except for AgNO3, which was 8 to 9-fold more toxic than Ag-NPs. Apart from AgNO3, NPs and bulk counterparts did not affect the number of germinated tomato seeds even in higher concentrations, while root length was significantly reduced in a dose dependent way in most cases. Dry weight of tomato plants was also significantly reduced upon treatment with NPs and counterparts with most pronounced effects in the cases of AgNO3, Cu-NPs, ZnO-NPs, and ZnSO4. Root and shoot length of grown tomato plants was also affected by treatments while differences between NPs and bulk counterparts varied. A marked oxidative stress response was recorded in all cases of NPs/bulk counterparts as indicated by increased MDA and H2O2 levels of treated plants. Treated plants had significantly reduced chlorophyl-a and carotenoid levels compared to the untreated control. NPs and counterparts did not affect FsK colonization of roots indicating a possible shielding effect of tomato plants once the endophyte was established inside the roots. Vice versa, a possible alleviation of CuO-NPs, ZnO-NPs, and ZnSO4 toxicity was observed in the presence of FsK inside tomato roots in terms of plant dry weight. The results suggest that phytotoxicity of NPs in tomato treated plants should be considered before application and while both FsK and tomato are sensitive to NPs, their reciprocal benefits may extent to resistance towards these toxic agents.