Dry Stamping Coral Powder: An Effective Method for Isolating Coral Symbiotic Actinobacteria.

Actinobacteria are important sources of antibiotics and have been found repeatedly in coral core microbiomes, suggesting this bacterial group plays important functional roles tied to coral survival. However, to unravel coral-actinobacteria ecological interactions and discover new antibiotics, the complex challenges that arise when isolating symbiotic actinobacteria must be overcome. Moreover, by isolating unknown actinobacteria from corals, novel biotechnological applications may be discovered. In this study, we compared actinobacteria recovery from coral samples between two widely known methods for isolating actinobacteria: dry stamping and heat shock. We found that dry stamping was at least three times better than heat shock. The assembly of isolated strains by dry stamping was unique for each species and consistent across same-species samples, highlighting that dry stamping can be reliably used to characterize coral actinobacteria communities. By analyzing the genomes of the closest related type strains, we were able to identify several functions commonly found among symbiotic organisms, such as transport and quorum sensing. This study provides a detailed methodology for isolating coral actinobacteria for ecological and biotechnological purposes.

Habitat-adapted heterologous symbiont Salinispora arenicola promotes growth and alleviates salt stress in tomato crop plants.

To ensure food security given the current scenario of climate change and the accompanying ecological repercussions, it is essential to search for new technologies and tools for agricultural production. Microorganism-based biostimulants are recognized as sustainable alternatives to traditional agrochemicals to enhance and protect agricultural production. Marine actinobacteria are a well-known source of novel compounds for biotechnological uses. In addition, former studies have suggested that coral symbiont actinobacteria may support co-symbiotic photosynthetic growth and tolerance and increase the probability of corals surviving abiotic stress. We have previously shown that this activity may also hold in terrestrial plants, at least for the actinobacteria Salinispora arenicola during induced heterologous symbiosis with a wild Solanaceae plant Nicotiana attenuata under in vitro conditions. Here, we further explore the heterologous symbiotic association, germination, growth promotion, and stress relieving activity of S. arenicola in tomato plants under agricultural conditions and dig into the possible associated mechanisms. Tomato plants were grown under normal and saline conditions, and germination, bacteria-root system interactions, plant growth, photosynthetic performance, and the expression of salt stress response genes were analyzed. We found an endophytic interaction between S. arenicola and tomato plants, which promotes germination and shoot and root growth under saline or non-saline conditions. Accordingly, photosynthetic and respective photoprotective performance was enhanced in line with the induced increase in photosynthetic pigments. This was further supported by the overexpression of thermal energy dissipation, which fine-tunes energy use efficiency and may prevent the formation of reactive oxygen species in the chloroplast. Furthermore, gene expression analyses suggested that a selective transport channel gene, SlHKT1,2, induced by S. arenicola may assist in relieving salt stress in tomato plants. The fine regulation of photosynthetic and photoprotective responses, as well as the inhibition of the formation of ROS molecules, seems to be related to the induced down-regulation of other salt stress response genes, such as SlDR1A-related genes or SlAOX1b. Our results demonstrate that the marine microbial symbiont S. arenicola establishes heterologous symbiosis in crop plants, promotes growth, and confers saline stress tolerance. Thus, these results open opportunities to further explore the vast array of marine microbes to enhance crop tolerance and food production under the current climate change scenario.

Diving Into Reef Ecosystems for Land-Agriculture Solutions: Coral Microbiota Can Alleviate Salt Stress During Germination and Photosynthesis in Terrestrial Plants.

From their chemical nature to their ecological interactions, coral reef ecosystems have a lot in common with highly productive terrestrial ecosystems. While plants are responsible for primary production in the terrestrial sphere, the photosynthetic endosymbionts of corals are the key producers in reef communities. As in plants, coral microbiota have been suggested to stimulate the growth and physiological performance of the photosynthetic endosymbionts that provide energy sources to the coral. Among them, actinobacteria are some of the most probable candidates. To explore the potential of coral actinobacteria as plant biostimulants, we have analyzed the activity of Salinispora strains isolated from the corals Porites lobata and Porites panamensis, which were identified as Salinispora arenicola by 16S rRNA sequencing. We evaluated the effects of this microorganism on the germination, plant growth, and photosynthetic response of wild tobacco (Nicotiana attenuata) under a saline regime. We identified protective activity of this actinobacteria on seed germination and photosynthetic performance under natural light conditions. Further insights into the possible mechanism showed an endophytic-like symbiosis between N. attenuata roots and S. arenicola and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity by S. arenicola. We discuss these findings in the context of relevant ecological and physiological responses and biotechnological potential. Overall, our results will contribute to the development of novel biotechnologies to cope with plant growth under saline stress. Our study highlights the importance of understanding marine ecological interactions for the development of novel, strategic, and sustainable agricultural solutions.

Herbivore perception decreases photosynthetic carbon assimilation and reduces stomatal conductance by engaging 12-oxo-phytodienoic acid, mitogen-activated protein kinase 4 and cytokinin perception.

Herbivory-induced changes in photosynthesis have been documented in many plant species; however, the complexity of photosynthetic regulation and analysis has thwarted progress in understanding the mechanism involved, particularly those elicited by herbivore-specific elicitors. Here, we analysed the early photosynthetic gas exchange responses in Nicotiana attenuata plants after wounding and elicitation with Manduca sexta oral secretions and the pathways regulating these responses. Elicitation with M. sexta oral secretions rapidly decreased photosynthetic carbon assimilation (AC ) in treated and systemic (untreated, vascularly connected) leaves, which were associated with changes in stomatal conductance, rather than with changes in Rubisco activity and 1-5 ribulose-1,5-bisphosphate turnover. Phytohormone profiling and gas exchange analysis of oral secretion-elicited transgenic plants altered in phytohormone regulation, biosynthesis and perception, combined with micrografting techniques, revealed that the local photosynthetic responses were mediated by 12-oxo-phytodienoic acid, while the systemic responses involved interactions among jasmonates, cytokinins and abscisic acid signalling mediated by mitogen-activated protein kinase 4. The analysis also revealed a role for cytokinins interacting with mitogen-activated protein kinase 4 in CO2 -mediated stomatal regulation. Hence, oral secretions, while eliciting jasmonic acid-mediated defence responses, also elicit 12-oxo-phytodienoic acid-mediated changes in stomatal conductance and AC , an observation illustrating the complexity and economy of the signalling that regulates defence and carbon assimilation pathways in response to herbivore attack.

A Nicotiana attenuata cell wall invertase inhibitor (NaCWII) reduces growth and increases secondary metabolite biosynthesis in herbivore-attacked plants.

Plant invertases are sucrolytic enzymes that are essential for the regulation of carbohydrate metabolism and source-sink relationships. While their activity has been well documented during abiotic and biotic stresses, the role of proteinaceous invertase inhibitors in regulating these changes is unknown. Here, we identify a putative Nicotiana attenuata cell wall invertase inhibitor (NaCWII) which is strongly up-regulated in a jasmonate (JA)-dependent manner following simulated attack by the specialist herbivore Manduca sexta. To understand the role of NaCWII in planta, we silenced its expression by RNA interference and measured changes in primary and secondary metabolism and plant growth following simulated herbivory. NaCWII-silenced plants displayed a stronger depletion of carbohydrates and a reduced capacity to increase secondary metabolite pools relative to their empty vector control counterparts. This coincided with the attenuation of herbivore-induced CWI inhibition and growth suppression characteristic of wild-type plants. Together our findings suggest that NaCWII may act as a regulatory switch located downstream of JA accumulation which fine-tunes the plant's balance between growth and defense metabolism under herbivore attack. Although carbohydrates are not typically viewed as key factors in plant growth and defense, our study shows that interfering with their catabolism strongly influences plant responses to herbivory.

Expression of WNT genes in cervical cancer-derived cells: Implication of WNT7A in cell proliferation and migration.

According to the multifactorial model of cervical cancer (CC) causation, it is now recognized that other modifications, in addition to Human papillomavirus (HPV) infection, are necessary for the development of this neoplasia. Among these, it has been proposed that a dysregulation of the WNT pathway might favor malignant progression of HPV-immortalized keratinocytes. The aim of this study was to identify components of the WNT pathway differentially expressed in CC vs. non-tumorigenic, but immortalized human keratinocytes. Interestingly, WNT7A expression was found strongly downregulated in cell lines and biopsies derived from CC. Restoration of WNT7A in CC-derived cell lines using a lentiviral gene delivery system or after adding a recombinant human protein decreases cell proliferation. Likewise, WNT7A silencing in non-tumorigenic cells markedly accelerates proliferation. Decreased WNT7A expression was due to hypermethylation at particular CpG sites. To our knowledge, this is the first study reporting reduced WNT7A levels in CC-derived cells and that ectopic WNT7A restoration negatively affects cell proliferation and migration.

Cytokinin concentrations and CHASE-DOMAIN CONTAINING HIS KINASE 2 (NaCHK2)- and NaCHK3-mediated perception modulate herbivory-induced defense signaling and defenses in Nicotiana attenuata.

Herbivore attack elicits changes in cytokinins (CKs), but how these changes influence defense signaling remains poorly described. We investigated the influence of the CK pathway on the well-described inducible defense pathways of Nicotiana attenuata in response to wounding with and without elicitors from the specialist herbivore Manduca sexta. CK pathway manipulation often suffers from substantial side effects on plant growth and development. We therefore used multiple manipulation tools including spray application of CKs, chemically-inducible expression of the CK biosynthesis enzyme isopentenyltransferase, and transient and constitutive RNAi-mediated gene silencing of CK receptors to resolve the function of CKs in plant defense. The results demonstrated that CK concentrations in leaves and perception through CHASE-DOMAIN CONTAINING HIS KINASE 2 (NaCHK2) and NaCHK3 were important for the accumulation of jasmonic acid (JA) and phenolamides and proteinase inhibitor activity. By contrast, the CK pathway did not promote the accumulation of the active JA-isoleucine conjugate and negatively regulated the release of specific green leaf volatile esters. Interestingly, CK signaling also promotes the systemic phenolamide accumulation. We conclude that the CK pathway is an important regulator of herbivory-inducible defense signaling and chemistry, which expands its reported participation in adjusting a plant's physiology to abiotic and biotic stress responses.

Cytokinin levels and signaling respond to wounding and the perception of herbivore elicitors in Nicotiana attenuata.

Nearly half a century ago insect herbivores were found to induce the formation of green islands by manipulating cytokinin (CK) levels. However, the response of the CK pathway to attack by chewing insect herbivores remains unclear. Here, we characterize the CK pathway of Nicotiana attenuata (Torr. ex S. Wats.) and its response to wounding and perception of herbivore-associated molecular patterns (HAMPs). We identified 44 genes involved in CK biosynthesis, inactivation, degradation, and signaling. Leaf wounding rapidly induced transcriptional changes in multiple genes throughout the pathway, as well as in the levels of CKs, including isopentenyladenosine and cis-zeatin riboside; perception of HAMPs present in the oral secretions (OS) of the specialist herbivore Manduca sexta amplified these responses. The jasmonate pathway, which triggers many herbivore-induced processes, was not required for these HAMP-triggered changes, but rather suppressed the CK responses. Interestingly CK pathway changes were observed also in systemic leaves in response to wounding and OS application indicating a role of CKs in mediating long distance systemic processes in response to herbivory. Since wounding and grasshopper OS elicited similar accumulations of CKs in Arabidopsis thaliana L., we propose that CKs are integral components of wounding and HAMP-triggered responses in many plant species.

Peripheral T-lymphocytes express WNT7A and its restoration in leukemia-derived lymphoblasts inhibits cell proliferation.

BACKGROUND: WNT7a, a member of the Wnt ligand family implicated in several developmental processes, has also been reported to be dysregulated in some types of tumors; however, its function and implication in oncogenesis is poorly understood. Moreover, the expression of this gene and the role that it plays in the biology of blood cells remains unclear. In addition to determining the expression of the WNT7A gene in blood cells, in leukemia-derived cell lines, and in samples of patients with leukemia, the aim of this study was to seek the effect of this gene in proliferation. METHODS: We analyzed peripheral blood mononuclear cells, sorted CD3 and CD19 cells, four leukemia-derived cell lines, and blood samples from 14 patients with Acute lymphoblastic leukemia (ALL), and 19 clinically healthy subjects. Reverse transcription followed by quantitative Real-time Polymerase chain reaction (qRT-PCR) analysis were performed to determine relative WNT7A expression. Restoration of WNT7a was done employing a lentiviral system and by using a recombinant human protein. Cell proliferation was measured by addition of WST-1 to cell cultures. RESULTS: WNT7a is mainly produced by CD3 T-lymphocytes, its expression decreases upon activation, and it is severely reduced in leukemia-derived cell lines, as well as in the blood samples of patients with ALL when compared with healthy controls (p ≤0.001). By restoring WNT7A expression in leukemia-derived cells, we were able to demonstrate that WNT7a inhibits cell growth. A similar effect was observed when a recombinant human WNT7a protein was used. Interestingly, restoration of WNT7A expression in Jurkat cells did not activate the canonical Wnt/ß-catenin pathway. CONCLUSIONS: To our knowledge, this is the first report evidencing quantitatively decreased WNT7A levels in leukemia-derived cells and that WNT7A restoration in T-lymphocytes inhibits cell proliferation. In addition, our results also support the possible function of WNT7A as a tumor suppressor gene as well as a therapeutic tool.