Construction of protoplast genetic transformation system for Mycena--symbiont of Gastrodia elata / 中国中药杂志

Mycena, a symbiont of Gastrodia elata, promotes seed germination of G. elata and plays a crucial role in the sexual reproduction of G. elata. However, the lack of genetic transformation system of Mycena blocks the research on the interaction mechanism of the two. In order to establish the protoplast transformation system of Mycena, this study analyzed the protoplast enzymatic hydrolysis system, screened the resistance markers and regeneration medium, and explored the transient transformation. After hydrolysis of Mycena hyphae with complexes enzymes for 8 h and centrifugation at 4 000 r·min~(-1), high-concentration and quality protoplast was obtained. The optimum regeneration medium for Mycena was RMV, and the optimum resistance marker was 50 mg·mL~(-1) hygromycin. The pLH-HygB-HuSHXG-GFP-HdSHXG was transformed into the protoplast of Mycena which then expressed GFP. The established protoplast transformation system of Mycena laid a foundation for analyzing the functional genes of Mycena and the molecular mechanism of the symbiosis of Mycena and G. elata.

Symbiont modulates expression of specific gene categories in Angomonas deanei

Trypanosomatids are parasites that cause disease in humans, animals, and plants. Most are non-pathogenic and some harbor a symbiotic bacterium. Endosymbiosis is part of the evolutionary process of vital cell functions such as respiration and photosynthesis. Angomonas deanei is an example of a symbiont-containing trypanosomatid. In this paper, we sought to investigate how symbionts influence host cells by characterising and comparing the transcriptomes of the symbiont-containing A. deanei (wild type) and the symbiont-free aposymbiotic strains. The comparison revealed that the presence of the symbiont modulates several differentially expressed genes. Empirical analysis of differential gene expression showed that 216 of the 7625 modulated genes were significantly changed. Finally, gene set enrichment analysis revealed that the largest categories of genes that downregulated in the absence of the symbiont were those involved in oxidation-reduction process, ATP hydrolysis coupled proton transport and glycolysis. In contrast, among the upregulated gene categories were those involved in proteolysis, microtubule-based movement, and cellular metabolic process. Our results provide valuable information for dissecting the mechanism of endosymbiosis in A. deanei.

Multilocus sequence analysis (MLSA) of Bradyrhizobium strains: revealing high diversity of tropical diazotrophic symbiotic bacteria

Symbiotic association of several genera of bacteria collectively called as rhizobia and plants belonging to the family Leguminosae (=Fabaceae) results in the process of biological nitrogen fixation, playing a key role in global N cycling, and also bringing relevant contributions to the agriculture. Bradyrhizobium is considered as the ancestral of all nitrogen-fixing rhizobial species, probably originated in the tropics. The genus encompasses a variety of diverse bacteria, but the diversity captured in the analysis of the 16S rRNA is often low. In this study, we analyzed twelve Bradyrhizobium strains selected from previous studies performed by our group for showing high genetic diversity in relation to the described species. In addition to the 16S rRNA, five housekeeping genes (recA, atpD, glnII, gyrB and rpoB) were analyzed in the MLSA (multilocus sequence analysis) approach. Analysis of each gene and of the concatenated housekeeping genes captured a considerably higher level of genetic diversity, with indication of putative new species. The results highlight the high genetic variability associated with Bradyrhizobium microsymbionts of a variety of legumes. In addition, the MLSA approach has proved to represent a rapid and reliable method to be employed in phylogenetic and taxonomic studies, speeding the identification of the still poorly known diversity of nitrogen-fixing rhizobia in the tropics.

Expression of phenazine biosynthetic genes during the arbuscular mycorrhizal symbiosis of Glomus intraradices

To explore the molecular mechanisms that prevail during the establishment of the arbuscular mycorrhiza symbiosis involving the genus Glomus, we transcriptionally analysed spores of Glomus intraradices BE3 during early hyphal growth. Among 458 transcripts initially identified as being expressed at presymbiotic stages, 20% of sequences had homology to previously characterized eukaryotic genes, 30% were homologous to fungal coding sequences, and 9% showed homology to previously characterized bacterial genes. Among them, GintPbr1a encodes a homolog to Phenazine Biosynthesis Regulator (Pbr) of Burkholderia cenocepacia, an pleiotropic regulatory protein that activates phenazine production through transcriptional activation of the protein D isochorismatase biosynthetic enzyme phzD (Ramos et al., 2010). Whereas GintPbr1a is expressed during the presymbiotic phase, the G. intraradices BE3 homolog of phzD (BGintphzD) is transcriptionally active at the time of the establishment of the arbuscular mycorrhizal symbiosis. DNA from isolated bacterial cultures found in spores of G. intraradices BE3 confirmed that both BGintPbr1a and BGintphzD are present in the genome of its potential endosymbionts. Taken together, our results indicate that spores of G. intraradices BE3 express bacterial phenazine biosynthetic genes at the onset of the fungal-plant symbiotic interaction.

PtSRR1, a putative Pisolithus tinctorius symbiosis related receptor gene is expressed during the first hours of mycorrhizal interaction with Castanea sativa roots / PtSRR1, um possível receptor simbiose-regulado de Pisolithus tinctorius é expresso nas primeiras horas de interação ectomicorrízica com raízes de Castanea sativa

PtSRR1 EST was previously identified in the first hours of Pisolithus tinctorius and Castanea sativa interaction. QRT-PCR confirmed PtSRR1 early expression and in silico preliminary translated peptide analysis indicated a strong probability that PtSRR1 be a transmembrane protein. These data stimulate the PtSRR1 gene research during ectomycorrhiza formation.

PtSRR1 foi isolado preliminarmente de P. tinctorius nas primeiras horas da interação com raízes de C. sativa. Análises de QRT-PCR confirmaram sua expressão positiva (12 h) e seu peptídeo putativo indicou forte possibilidade para proteína transmembranar. Estes dados estimulam o estudo do PtSRR1 durante a formação de ectomicorrizas.

Molecular aspects of soybean cultivar-specific nodulation by Sinorhizobium fredii USDA257.

Sinorhizobium fredii USDA257 forms nitrogen-fixing nodules in association with the primitive soybean cultivar 'Peking' but fails to initiate nodules on many advanced soybean cultivars, including 'McCall'. This distinction is controlled by a set of nodulation genes termed nolXWBTUV. Inactivation of any of these genes enables USDA257 to nodulate McCall and many other improved soybean cultivars. Mutation in the nolXWBTUV locus also alters the Nod factor structure resulting in the production of a novel molecule with glucose incorporated into the chitin backbone. Some of the genes located in the nolXWBTUV locus reveal sequence homologies to known components of the type III secretion system (TTSS) of plant and animal pathogenic bacteria. Recent studies have demonstrated the presence of a complete TTSS in USDA257 and few other symbiotic bacteria. The TTSS cluster of USDA257 contains 27 open reading frames out of which 10 code for the structural components of the TTSS. USDA257, when grown in presence of flavonoids, secrete several proteins called Nops (Nodulation Outer Proteins) into the extracellular environment. Genes located in the TTSS of USDA257 encode some of the extracellular proteins, such as NopX, NopB, and NopL. These type III secreted proteins appear to play an important role in regulating nodulation in a host-dependent manner. Failure to elaborate the Nops results in a drastic phenotypic effect on soybean nodulation, indicating that these proteins may play a pivotal role in soybean cultivar specificity. The secretion of Nops appears to be facilitated by novel filamentous appendages (pili) that are produced by USDA257 upon induction by flavonoids. Biochemical studies have demonstrated the close association of several Nops with the purified pili. However, it remains to be seen if the filamentous appendages can function as conduits for delivery of Nops into the host cell. This review examines the current state of our knowledge on the molecular aspects of soybean cultivar-specific nodulation by USDA257.

Regulation of expression of symbiotic genes in Rhizobium sp. NGR234.

Research in the field of Rhizobium-legume symbiosis faces a new challenge: integrate the wealth of information generated by genomic projects. The goal: apprehend the complexity of the molecular mechanisms involved in symbiotic associations. At the time of writing, the genomes of three micro-symbionts (Bradyrhizobium japonicum, Mesorhizobium loti and Sinorhizobium meliloti) have been sequenced, and two more (those of Rhizobium leguminosarum and Rhizobium etli) will be completed in the near future. Together, completed rhizobial genomes represent already 23,393,822 bp of DNA sequence and 21,797 predicted open reading frames (ORFs). To identify candidate-symbiotic genes in such a broad database, predict their function and dissect the regulatory networks that govern their expression are no simple tasks. One way to confront this problem is to combine different datasets, in particular genetic and transcriptional maps as well as predicted promoters from bioinformatics analyses. Here, we would like to illustrate this type of approach with the analysis of the symbiotic plasmid (pNGR234a) of the broad host-range Rhizobium sp. NGR234.