Potential Role and Utilization of Plant Growth Promoting Microbes in Plant Tissue Culture.

Plant growth promoting microbes (PGPMs) play major roles in diverse ecosystems, including atmospheric nitrogen fixation, water uptake, solubilization, and transport of minerals from the soil to the plant. Different PGPMs are proposed as biofertilizers, biostimulants, and/or biocontrol agents to improve plant growth and productivity and thereby to contribute to agricultural sustainability and food security. However, little information exists regarding the use of PGPMs in micropropagation such as the in vitro plant tissue culture. This review presents an overview of the importance of PGPMs and their potential application in plant micropropagation. Our analysis, based on published articles, reveals that the process of in vitro classical tissue culture techniques, under strictly aseptic conditions, deserves to be reviewed to allow vitroplants to benefit from the positive effect of PGPMs. Furthermore, exploiting the potential benefits of PGPMs will lead to lessen the cost production of vitroplants during micropropagation process and will make the technique of plant tissue culture more efficient. The last part of the review will indicate where research is needed in the future.

Establishment of Actinorhizal Symbiosis in Response to Ethylene, Salicylic Acid, and Jasmonate.

Phytohormones play a crucial role in regulating plant developmental processes. Among them, ethylene and jasmonate are known to be involved in plant defense responses to a wide range of biotic stresses as their levels increase with pathogen infection. In addition, these two phytohormones have been shown to inhibit plant nodulation in legumes. Here, exogenous salicylic acid (SA), jasmonate acid (JA), and ethephon (ET) were applied to the root system of Casuarina glauca plants before Frankia inoculation, in order to analyze their effects on the establishment of actinorhizal symbiosis. This protocol further describes how to identify putative ortholog genes involved in ethylene and jasmonate biosynthesis and/or signaling pathways in plant, using the Arabidopsis Information Resource (TAIR), Legume Information System (LIS), and Genevestigator databases. The expression of these genes in response to the bacterium Frankia was analyzed using the gene atlas for Casuarina-Frankia symbiosis (SESAM web site).

Actinorhizal Signaling Molecules: Frankia Root Hair Deforming Factor Shares Properties With NIN Inducing Factor.

Actinorhizal plants are able to establish a symbiotic relationship with Frankia bacteria leading to the formation of root nodules. The symbiotic interaction starts with the exchange of symbiotic signals in the soil between the plant and the bacteria. This molecular dialog involves signaling molecules that are responsible for the specific recognition of the plant host and its endosymbiont. Here we studied two factors potentially involved in signaling between Frankia casuarinae and its actinorhizal host Casuarina glauca: (1) the Root Hair Deforming Factor (CgRHDF) detected using a test based on the characteristic deformation of C. glauca root hairs inoculated with F. casuarinae and (2) a NIN activating factor (CgNINA) which is able to activate the expression of CgNIN, a symbiotic gene expressed during preinfection stages of root hair development. We showed that CgRHDF and CgNINA corresponded to small thermoresistant molecules. Both factors were also hydrophilic and resistant to a chitinase digestion indicating structural differences from rhizobial Nod factors (NFs) or mycorrhizal Myc-LCOs. We also investigated the presence of CgNINA and CgRHDF in 16 Frankia strains representative of Frankia diversity. High levels of root hair deformation (RHD) and activation of ProCgNIN were detected for Casuarina-infective strains from clade Ic and closely related strains from clade Ia unable to nodulate C. glauca. Lower levels were present for distantly related strains belonging to clade III. No CgRHDF or CgNINA could be detected for Frankia coriariae (Clade II) or for uninfective strains from clade IV.

Functional Characterization of the Versatile MYB Gene Family Uncovered Their Important Roles in Plant Development and Responses to Drought and Waterlogging in Sesame.

The MYB gene family constitutes one of the largest transcription factors (TFs) modulating various biological processes in plants. Although genome-wide analysis of this gene family has been carried out in some species, only three MYB members have been functionally characterized heretofore in sesame (Sesamum indicum L.). Here, we identified a relatively high number (287) of sesame MYB genes (SIMYBs) with an uncommon overrepresentation of the 1R-subfamily. A total of 95% of SIMYBs was mapped unevenly onto the 16 linkage groups of the sesame genome with 55 SIMYBs tandemly duplicated. In addition, molecular characterization, gene structure, and evolutionary relationships of SIMYBs were established. Based on the close relationship between sesame and Arabidopsis thaliana, we uncovered that the functions of SIMYBs are highly diverse. A total of 65% of SIMYBs were commonly detected in five tissues, suggesting that they represent key TFs modulating sesame growth and development. Moreover, we found that SIMYBs regulate sesame responses to drought and waterlogging, which highlights the potential of SIMYBs towards improving stress tolerance in sesame. This work presents a comprehensive picture of the MYB gene family in sesame and paves the way for further functional validation of the members of this versatile gene family.

Genomic, transcriptomic, and proteomic approaches towards understanding the molecular mechanisms of salt tolerance in Frankia strains isolated from Casuarina trees.

BACKGROUND: Soil salinization is a worldwide problem that is intensifying because of the effects of climate change. An effective method for the reclamation of salt-affected soils involves initiating plant succession using fast growing, nitrogen fixing actinorhizal trees such as the Casuarina. The salt tolerance of Casuarina is enhanced by the nitrogen-fixing symbiosis that they form with the actinobacterium Frankia. Identification and molecular characterization of salt-tolerant Casuarina species and associated Frankia is imperative for the successful utilization of Casuarina trees in saline soil reclamation efforts. In this study, salt-tolerant and salt-sensitive Casuarina associated Frankia strains were identified and comparative genomics, transcriptome profiling, and proteomics were employed to elucidate the molecular mechanisms of salt and osmotic stress tolerance. RESULTS: Salt-tolerant Frankia strains (CcI6 and Allo2) that could withstand up to 1000 mM NaCl and a salt-sensitive Frankia strain (CcI3) which could withstand only up to 475 mM NaCl were identified. The remaining isolates had intermediate levels of salt tolerance with MIC values ranging from 650 mM to 750 mM. Comparative genomic analysis showed that all of the Frankia isolates from Casuarina belonged to the same species (Frankia casuarinae). Pangenome analysis revealed a high abundance of singletons among all Casuarina isolates. The two salt-tolerant strains contained 153 shared single copy genes (most of which code for hypothetical proteins) that were not found in the salt-sensitive(CcI3) and moderately salt-tolerant (CeD) strains. RNA-seq analysis of one of the two salt-tolerant strains (Frankia sp. strain CcI6) revealed hundreds of genes differentially expressed under salt and/or osmotic stress. Among the 153 genes, 7 and 7 were responsive to salt and osmotic stress, respectively. Proteomic profiling confirmed the transcriptome results and identified 19 and 8 salt and/or osmotic stress-responsive proteins in the salt-tolerant (CcI6) and the salt-sensitive (CcI3) strains, respectively. CONCLUSION: Genetic differences between salt-tolerant and salt-sensitive Frankia strains isolated from Casuarina were identified. Transcriptome and proteome profiling of a salt-tolerant strain was used to determine molecular differences correlated with differential salt-tolerance and several candidate genes were identified. Mechanisms involving transcriptional and translational regulation, cell envelop remodeling, and previously uncharacterized proteins appear to be important for salt tolerance. Physiological and mutational analyses will further shed light on the molecular mechanism of salt tolerance in Casuarina associated Frankia isolates.

Permanent Draft Genome Sequence of Frankia sp. Strain Allo2, a Salt-Tolerant Nitrogen-Fixing Actinobacterium Isolated from the Root Nodules of Allocasuarina.

Frankia sp. strain Allo2 is a member of Frankia lineage Ib, which is able to reinfect plants of the Casuarinaceae family, and exhibits a high level of salt tolerance compared to other isolates. Here, we report the 5.3-Mbp draft genome sequence of Frankia sp. strain Allo2 with a G+C content of 70.0% and 4,224 candidate protein-encoding genes.

Permanent Draft Genome Sequence for Frankia sp. Strain CeD, a Nitrogen-Fixing Actinobacterium Isolated from the Root Nodules of Casuarina equistifolia Grown in Senegal.

Frankiastrain CeD is a member ofFrankialineage Ib that is able to reinfect plants of theCasuarinafamilies. Here, we report a 5.0-Mbp draft genome sequence with a G+C content of 70.1% and 3,847 candidate protein-encoding genes.

Infection-specific activation of the Medicago truncatula Enod11 early nodulin gene promoter during actinorhizal root nodulation.

The MtEnod11 gene from Medicago truncatula is widely used as an early infection-related molecular marker for endosymbiotic associations involving both rhizobia and arbuscular mycorrhizal fungi. In this article, heterologous expression of the MtEnod11 promoter has been studied in two actinorhizal trees, Casuarina glauca and Allocasuarina verticillata. Transgenic C. glauca and A. verticillata expressing a ProMtEnod11::beta-glucuronidase (gus) fusion were generated and the activation of the transgene investigated in the context of the symbiotic associations with the N-fixing actinomycete Frankia and both endo- and ectomycorrhizal fungi (Glomus intraradices and Pisolithus albus, respectively). ProMtEnod11::gus expression was observed in root hairs, prenodules, and nodules and could be correlated with the infection of plant cells by Frankia spp. However, no activation of the gus reporter gene was detected prior to infection or in response to either rhizobial Nod factors or the wasp venom peptide MAS-7. Equally, ProMtEnod11::gus expression was not elicited during the symbiotic associations with either ecto- or endomycorrhizal fungi. These observations suggest that, although there is a conservation of gene regulatory pathways between legumes and actinorhizal plants in cells accommodating endosymbiotic N-fixing bacteria, the events preceding bacterial infection or related to mycorrhization appear to be less conserved.

Comparison of four constitutive promoters for the expression of transgenes in the tropical nitrogen-fixing tree Allocasuarina verticillata.

Allocasuarina verticillata is an actinorhizal tree that lives in symbiotic association with a nitrogen fixing actinomycete called Frankia. In the search for promoters that drive strong constitutive expression in this tropical tree, we studied the organ specificity of four different constitutive promoters (CaMV 35S, e35S, e35S-4ocs and UBQ1 from Arabidopsis thaliana) in stably transformed A. verticillata plants. The ss-glucuronidase (gus) gene was used as a reporter and expression studies were carried out by histochemical analyses on shoots, roots and actinorhizal nodules. While the 35S promoter was poorly expressed in the shoot apex and lateral roots, both the e35S and e35S-4ocs were found to drive high constitutive expression in the transgenic non-nodulated plants. In contrast, the UBQ1 promoter was very poorly expressed and appeared unsuitable for A. verticillata. We also showed that none of the promoters studied were active in the nodule infected cells, whatever the developmental stage studied.