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

The triterpene echinocystic acid and its 3-O-glucoside derivative are revealed as potent and selective glucocorticoid receptor agonists

Glucocorticoids are steroid hormones widely used to control many inflammatory conditions. These effects are primarily attributed to glucocorticoid receptor transrepressional activities but with concomitant receptor transactivation associated with considerable side effects. Accordingly, there is an immediate need for selective glucocorticoid receptor agonists able to dissociate transactivation from transrepression. Triterpenoids have structural similarities with glucocorticoids and exhibit anti-inflammatory and apoptotic activities via mechanisms that are not well-defined. In this study, we examined whether echinocystic acid and its 3-O-glucoside derivative act, at least in part, through the regulation of glucocorticoid receptor and whether they can constitute selective receptor activators. We showed that echinocystic acid and its glucoside induced glucocorticoid receptor nuclear translocation by 75% and 55%. They suppressed the nuclear factor-kappa beta transcriptional activity by 20% and 70%, respectively, whereas they have no glucocorticoid receptor transactivation capability and stimulatory effect on the expression of the phosphoenolopyruvate carboxykinase target gene in HeLa cells. Interestingly, their suppressive effect is diminished in glucocorticoid receptor low level COS-7 cells, verifying the receptor involvement in this process. Induced fit docking calculations predicted favorable binding in the ligand binding domain and structural characteristics which can be considered consistent with the experimental observations. Further, glucocorticoids exert apoptotic activities; we have demonstrated here that the echinocystic acids in combination with the synthetic glucocorticoid, dexamethasone, induce apoptosis. Taken together, our results indicate that echinocystic acids are potent glucocorticoid receptor regulators with selective transrepressional activities (dissociated from transactivation), highlighting the potential of echinocystic acid derivatives as more promising treatments for inflammatory conditions

sRNAseq-data

<p>We have previously shown that the beneficial soilborne endophytic fungus <i>Fusarium solani</i> strain K (FsK) translocates small RNAs (sRNAs) to its host <i>Nicotiana benthamiana</i> leading to systemic silencing of a reporter gene. Here, we aimed to address the mechanistic details of this sRNA-based interaction. In the systemically silenced plants, we performed sRNA sequencing in root and leaf tissues. Our findings revealed that the translocated fungal sRNAs induced transitivity and secondary sRNA production already in the root tissues. The secondary sRNA pattern did not differ between the root and leaf tissues. Secondary sRNAs were mainly of 22-24 nt in size, with the conspicuous absence of 21-nt sRNAs. Importantly, systemic silencing and RNA-directed DNA methylation (RdDM) could be induced only in an intronless gene, but not in an intron-containing gene or in an intronless gene at RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) knockout background. Overall, our data suggest that FsK-induced silencing in the host requires RDR6-mediated transitivity and amplification of silencing signals and, in this view, not all genes are equally efficient targets for FsK-induced silencing.</p&gt

Different factors are operative in shaping the epiphytic grapevine microbiome across different geographical scales: Biogeography, cultivar or vintage?

Abstract Introduction The composition of the grapevine microbiome is controlled by a range of factors all contributing to the establishment of microbial terroir. Most studies have focused on the grape must microbiome, while less is known about the assemblage mechanisms of the epiphytic grapevine microbiome. We aimed to disentangle the role of geographic location, cultivar and vintage on the composition of the epiphytic fungal and bacterial communities of grapevine: within (regional scale) and across (national scale) viticultural zones in Greece. We hypothesised that: (i) the influence of all three factors varies at different geographical scales; (ii) fungi and bacteria respond differently to the factors shaping the grapevine microbiome. Materials and Methods We analysed leaves and berries collected from Greek cultivars (i) Vidiano and Agiorgitiko located in three geographically distant regions (from 100 to >600 km), (ii) Roditis and Sideritis from different terroir units of the viticultural zone of Aigialeia. Results Amplicon sequencing analysis identified strong regional signatures on the epiphytic microbiome across viticultural zones, whereas cultivar becomes a strong determinant at the viticultural zone scale. Fungal communities were more responsive to all studied factors compared to bacterial communities. Differential abundance (DA) analysis identified dominant fungal (Alternaria, Cladosporium, Nigrospora, Aureobasidium, Vishniacozyma) and bacterial (Sphingomonas, Masillia, Streptococcus, Staphylococcus, Enterobacterales) amplicon sequence variants (ASVs) exhibiting geographical and cultivar‐specific patterns. Conclusion Our data suggest that different factors are operative in shaping the epiphytic grapevine microbiome at different geographical scales with bacterial and fungal communities showing different responses to the tested structural factors

Cytological and Other Aspects of Pathogenesis-related Gene Expression in Tomato Plants Grown on a Suppressive Compost

• Background and Aims Recent studies have shown that certain composts may trigger indirect defence mechanisms by sensitizing the plant to create an increased state of resistance, similar to systemic acquired resistance. In this study, the capacity of a disease-suppressive compost to alter the expression pattern of certain pathogenesis-related (PR) genes in the root system of tomato plants (Solanum lycopersicum) provided the opportunity to study their cellular expression pattern and to investigate putative roles of these genes in the mechanisms of plant defence

THE EFFECT OF RHIZOPHAGUS IRREGULARIS AND MESORHIZOBIUM LOTI CO-INOCULATION ON LOTUS JAPONICUS

Plants establish symbiotic relationships with soil bacteria or fungi, which colonize the plant root and provide the plant with inorganic nutrients, in exchange for photosynthesis products. Legume plants associate with both arbuscular mycorrhizal fungi (AMF) and the nitrogen-fixing soil bacteria called rhizobia. During the legume-rhizobium symbiosis, biological nitrogen fixation takes place in specific plants organs formed on the root, called nodules. Using the model legume Lotus japonicus, we studied the establishment of the legume-rhizobia-AMF tripartite symbiosis. We examined how the AM fungus Rhizophagus irregularis and the rhizobium Mesorhizobium loti affected one another during the colonization of the same legume roots, by performing co-inoculations. Moreover, we monitored the effect of the co-inoculation on the general plant performance. According to our results, the presence of M. loti had no effect on the root colonization by R. irregularis. However, root colonization by R. irregularis had a positive effect on the formation of root nodules. This study aims to enhance our understanding on how the plant selects, combines and controls its symbionts, towards to a more efficient use of legume plants in agroecosystems

Cytological and other aspects of pathogenesis-related gene expression in tomato plants grown on a suppressive compost

center dot Background and Aims Recent studies have shown that certain composts may trigger indirect defence mechanisms by sensitizing the plant to create an increased state of resistance, similar to systemic acquired resistance. In this study, the capacity of a disease-suppressive compost to alter the expression pattern of certain pathogenesis-related (PR) genes in the root system of tomato plants (Solanum lycopersicum) provided the opportunity to study their cellular expression pattern and to investigate putative roles of these genes in the mechanisms of plant defence. center dot Methods Employing the reverse transcription-polymerase chain reaction (RT-PCR) and in situ RNA:RNA hybridization techniques, the accumulation and distribution of the transcripts of the differentially expressed PR genes were examined in plants grown on compost and compared with those of control plants grown on peat. center dot Key Results Elevated levels of expression of the pathogenesis-related genes PR-1, PR-5 and P69/PR-7 were detected in the roots of tomato plants grown on the compost. A clearly distinguished spatial induction pattern was observed for these PR genes: PR-1 transcripts were almost exclusively detected in the pericycle cells surrounding the root stele of the main and lateral roots; PR-5 transcripts were present in the phloem of the root and stem tissues; and the accumulation and distribution of PR-7 transcripts was detected in discrete groups of cells that appeared sporadically in both the parenchyma and vascular system of the root, suggesting that the gene is not expressed in a tissue-specific manner. In addition, a novel cDNA clone was isolated (P69G), which probably encodes a new tomato P69 isoform. center dot Conclusions This study provides evidence that a supressive compost is able to elicit consistent and increased expression of certain PR genes in the roots of tomato plants, even in the absence of any pathogen. The in situ localization studies reveal expression patterns which are in accordance with the presence of protein or with the putative roles of the respective encoded proteins. The expression of the PR genes may be triggered by the microflora of the compost or could be associated with abiotic factors of the compost

THE EFFECT OF RHIZOPHAGUS IRREGULARIS AND MESORHIZOBIUM LOTI CO-INOCULATION ON LOTUS JAPONICUS

Plants establish symbiotic relationships with soil bacteria or fungi, which colonize the plant root and provide the plant with inorganic nutrients, in exchange for photosynthesis products. Legume plants associate with both arbuscular mycorrhizal fungi (AMF) and the nitrogen-fixing soil bacteria called rhizobia. During the legume-rhizobium symbiosis, biological nitrogen fixation takes place in specific plants organs formed on the root, called nodules. Using the model legume Lotus japonicus, we studied the establishment of the legume-rhizobia-AMF tripartite symbiosis. We examined how the AM fungus Rhizophagus irregularis and the rhizobium Mesorhizobium loti affected one another during the colonization of the same legume roots, by performing co-inoculations. Moreover, we monitored the effect of the co-inoculation on the general plant performance. According to our results, the presence of M. loti had no effect on the root colonization by R. irregularis. However, root colonization by R. irregularis had a positive effect on the formation of root nodules. This study aims to enhance our understanding on how the plant selects, combines and controls its symbionts, towards to a more efficient use of legume plants in agroecosystems