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.

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.

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.

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.

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.

Grapevine wood microbiome analysis identifies key fungal pathogens and potential interactions with the bacterial community implicated in grapevine trunk disease appearance.

BACKGROUND: Grapevine trunk diseases (GTDs) is a disease complex caused by wood pathogenic fungi belonging to genera like Phaeomoniella, Phaeoacremonium, Fomitiporia, Eutypa and members of the family Botryosphaeriaceae. However, the co-occurrence of these fungi in symptomatic and asymptomatic vines at equivalent abundances has questioned their role in GTDs. Hence, we still lack a good understanding of the fungi involved in GTDs, their interactions and the factors controlling their assemblage in vines. We determined the fungal and bacterial microbiome in wood tissues of asymptomatic and symptomatic vines of three main Greek cultivars (Agiorgitiko, Xinomavro, Vidiano), each cultivated in geographically distinct viticultural zones, using amplicon sequencing. RESULTS: We noted that cultivar/biogeography (lumped factor) was the strongest determinant of the wood fungal microbiome (p < 0.001, 22.7%), while GTD symptoms condition had a weaker but still significant effect (p < 0.001, 3.5%), being prominent only in the cultivar Xinomavro. Several fungal Amplicon Sequence Variants (ASVs), reported as GTD-associated pathogens like Kalmusia variispora, Fomitiporia spp., and Phaemoniella chlamydosporα (most dominant in our study), were positively correlated with symptomatic vines in a cultivar/viticultural zone dependent manner. Random Forest analysis pointed to P. chlamydosporα, K. variispora, A. alternata and Cladosporium sp., as highly accurate predictors of symptomatic vines (0% error rate). The wood bacterial microbiome showed similar patterns, with biogeography/cultivar being the main determinant (p < 0.001, 25.5%) of its composition, followed by the GTD status of vines (p < 0.001, 5.2%). Differential abundance analysis revealed a universal positive correlation (p < 0.001) of Bacillus and Streptomyces ASVs with asymptomatic vines. Network analysis identified a significant negative co-occurrence network between these bacterial genera and Phaemoniella, Phaeoacrominum and Seimatosporium. These results point to a plant beneficial interaction between Bacillus/Streptomyces and GTD pathogens. CONCLUSIONS: Our study (a) provides evidence that GTD symptomatic plants support a wood fungal microbiome, showing cultivar and biogeography-dependent patterns, that could be used as a proxy to distinguish between healthy and diseased vines, (b) points to strong interactions between the bacterial and fungal wood microbiome in asymptomatic vines that should be further pursued in the quest for discovery of novel biocontrol agents.

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.

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.

Mutagenesis of a Lotus japonicus GSK3ß/Shaggy-like kinase reveals functionally conserved regulatory residues.

The glycogen synthase kinases 3 family (GSK3s/SKs; serine/threonine protein kinases) is conserved throughout eukaryotic evolution from yeast to plants and mammals. We studied a plant SK kinase from Lotus japonicus (LjSK1), previously implicated in nodule development, by enzyme kinetics and mutagenesis studies to compare it to mammalian homologues. Using a phosphorylated peptide as substrate, LjSK1 displays optimum kinase activity at pH 8.0 and 20 °C following Michaelis-Menten kinetics with Km and Vmax values of 48.2 µM and 111.6 nmol/min/mg, respectively, for ATP. Mutation of critical residues, as inferred by sequence comparison to the human homologue GSK3ß and molecular modeling, showed a conserved role for Lys167, while residues conferring substrate specificity in the human enzyme are not as significant in modulating LjSK1 substrate specificity. Mutagenesis studies also indicate a regulation mechanism for LjSK1 via proteolysis since removal of a 98 residue long N-terminal segment increases its catalytic efficiency by almost two-fold. In addition, we evaluated the alteration of LjSK1 kinase activity in planta, by overexpressing the mutant variants in hairy-roots and a phenotype in nodulation and lateral root development was verified.

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.

An Agrobacterium rhizogenes mediated hairy root transformation protocol for fenugreek.

•This work describes a protocol for hairy root transformation of the medicinal crop legume fenugreek (Trigonella foenum-graecum L.). Hairy root plant transformation mediated by Agrobacterium rhizogenes is an established method for the rapid genetic transformation of various dicotyledonous plants. We have adapted a hairy root transformation protocol from the model legume Medicago truncatula for use in this metabolically rich non-model crop legume. Considering the great variety and abundance of phytochemicals in fenugreek and its established use in traditional medicine, we aim for this method to become a resource for metabolic pathway identification and for production of valuable specialised metabolites via metabolic engineering approaches.•Development rapid transformation (2.5-3 weeks) of fenugreek roots via A. rhizogenes.•Marker gene cassette with suitable promoter for visual detection of transformed fenugreek roots.

A hairy-root transformation protocol for Trigonella foenum-graecum L. as a tool for metabolic engineering and specialised metabolite pathway elucidation.

The development of genetic transformation methods is critical for enabling the thorough characterization of an organism and is a key step in exploiting any species as a platform for synthetic biology and metabolic engineering approaches. In this work we describe the development of an Agrobacterium rhizogenes-mediated hairy root transformation protocol for the crop and medicinal legume fenugreek (Trigonella foenum-graecum). Fenugreek has a rich and diverse content in bioactive specialised metabolites, notably diosgenin, which is a common precursor for synthetic human hormone production. This makes fenugreek a prime target for identification and engineering of specific biosynthetic pathways for the production of triterpene and steroidal saponins, phenolics, and galactomanans. Through this transformation protocol, we identified a suitable promoter for robust transgene expression in fenugreek. Finally, we establish the proof of principle for the utility of the fenugreek system for metabolic engineering programs, by heterologous expression of known triterpene saponin biosynthesis regulators from the related legume Medicago truncatula in fenugreek hairy roots.

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.

Epigenetic Modifications: An Unexplored Facet of Exogenous RNA Application in Plants.

Exogenous RNA interference (exo-RNAi) is a powerful transgene-free tool in modern crop improvement and protection platforms. In exo-RNAi approaches, double-stranded RNAs (dsRNAs) or short-interfering RNAs (siRNAs) are externally applied in plants in order to selectively trigger degradation of target mRNAs. Yet, the applied dsRNAs may also trigger unintended epigenetic alterations and result in epigenetically modified plants, an issue that has not been sufficiently addressed and which merits more careful consideration.

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.

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.

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.