Chromosomal barcodes for simultaneous tracking of near-isogenic bacterial strains in plant microbiota.

DNA-amplicon-based microbiota profiling can estimate species diversity and abundance but cannot resolve genetic differences within individuals of the same species. Here we report the development of modular bacterial tags (MoBacTags) encoding DNA barcodes that enable tracking of near-isogenic bacterial commensals in an array of complex microbiome communities. Chromosomally integrated DNA barcodes are then co-amplified with endogenous marker genes of the community by integrating corresponding primer binding sites into the barcode. We use this approach to assess the contributions of individual bacterial genes to Arabidopsis thaliana root microbiota establishment with synthetic communities that include MoBacTag-labelled strains of Pseudomonas capeferrum. Results show reduced root colonization for certain mutant strains with defects in gluconic-acid-mediated host immunosuppression, which would not be detected with traditional amplicon sequencing. Our work illustrates how MoBacTags can be applied to assess scaling of individual bacterial genetic determinants in the plant microbiota.

Functional characterization and physiological roles of the single Shaker outward K+ channel in Medicago truncatula.

The model legume Medicago truncatula possesses a single outward Shaker K+ channel, whereas Arabidopsis thaliana possesses two channels of this type, named AtSKOR and AtGORK, with AtSKOR having been shown to play a major role in K+ secretion into the xylem sap in the root vasculature and with AtGORK being shown to mediate the efflux of K+ across the guard cell membrane, leading to stomatal closure. Here we show that the expression pattern of the single M. truncatula outward Shaker channel, which has been named MtGORK, includes the root vasculature, guard cells and root hairs. As shown by patch-clamp experiments on root hair protoplasts, besides the Shaker-type slowly activating outwardly rectifying K+ conductance encoded by MtGORK, a second K+ -permeable conductance, displaying fast activation and weak rectification, can be expressed by M. truncatula. A knock-out (KO) mutation resulting in an absence of MtGORK activity is shown to weakly reduce K+ translocation to shoots, and only in plants engaged in rhizobial symbiosis, but to strongly affect the control of stomatal aperture and transpirational water loss. In legumes, the early electrical signaling pathway triggered by Nod-factor perception is known to comprise a short transient depolarization of the root hair plasma membrane. In the absence of the functional expression of MtGORK, the rate of the membrane repolarization is found to be decreased by a factor of approximately two. This defect was without any consequence on infection thread development and nodule production in plants grown in vitro, but a decrease in nodule production was observed in plants grown in soil.

Nod Factor Effects on Root Hair-Specific Transcriptome of Medicago truncatula: Focus on Plasma Membrane Transport Systems and Reactive Oxygen Species Networks.

Root hairs are involved in water and nutrient uptake, and thereby in plant autotrophy. In legumes, they also play a crucial role in establishment of rhizobial symbiosis. To obtain a holistic view of Medicago truncatula genes expressed in root hairs and of their regulation during the first hours of the engagement in rhizobial symbiotic interaction, a high throughput RNA sequencing on isolated root hairs from roots challenged or not with lipochitooligosaccharides Nod factors (NF) for 4 or 20 h was carried out. This provided a repertoire of genes displaying expression in root hairs, responding or not to NF, and specific or not to legumes. In analyzing the transcriptome dataset, special attention was paid to pumps, transporters, or channels active at the plasma membrane, to other proteins likely to play a role in nutrient ion uptake, NF electrical and calcium signaling, control of the redox status or the dynamic reprogramming of root hair transcriptome induced by NF treatment, and to the identification of papilionoid legume-specific genes expressed in root hairs. About 10% of the root hair expressed genes were significantly up- or down-regulated by NF treatment, suggesting their involvement in remodeling plant functions to allow establishment of the symbiotic relationship. For instance, NF-induced changes in expression of genes encoding plasma membrane transport systems or disease response proteins indicate that root hairs reduce their involvement in nutrient ion absorption and adapt their immune system in order to engage in the symbiotic interaction. It also appears that the redox status of root hair cells is tuned in response to NF perception. In addition, 1176 genes that could be considered as "papilionoid legume-specific" were identified in the M. truncatula root hair transcriptome, from which 141 were found to possess an ortholog in every of the six legume genomes that we considered, suggesting their involvement in essential functions specific to legumes. This transcriptome provides a valuable resource to investigate root hair biology in legumes and the roles that these cells play in rhizobial symbiosis establishment. These results could also contribute to the long-term objective of transferring this symbiotic capacity to non-legume plants.

Nuclear-localized cyclic nucleotide-gated channels mediate symbiotic calcium oscillations.

Nuclear-associated Ca(2+) oscillations mediate plant responses to beneficial microbial partners--namely, nitrogen-fixing rhizobial bacteria that colonize roots of legumes and arbuscular mycorrhizal fungi that colonize roots of the majority of plant species. A potassium-permeable channel is known to be required for symbiotic Ca(2+) oscillations, but the calcium channels themselves have been unknown until now. We show that three cyclic nucleotide-gated channels in Medicago truncatula are required for nuclear Ca(2+) oscillations and subsequent symbiotic responses. These cyclic nucleotide-gated channels are located at the nuclear envelope and are permeable to Ca(2+) We demonstrate that the cyclic nucleotide-gated channels form a complex with the postassium-permeable channel, which modulates nuclear Ca(2+) release. These channels, like their counterparts in animal cells, might regulate multiple nuclear Ca(2+) responses to developmental and environmental conditions.

Rhizobium-legume symbiosis in the absence of Nod factors: two possible scenarios with or without the T3SS.

The occurrence of alternative Nod factor (NF)-independent symbiosis between legumes and rhizobia was first demonstrated in some Aeschynomene species that are nodulated by photosynthetic bradyrhizobia lacking the canonical nodABC genes. In this study, we revealed that a large diversity of non-photosynthetic bradyrhizobia, including B. elkanii, was also able to induce nodules on the NF-independent Aeschynomene species, A. indica. Using cytological analysis of the nodules and the nitrogenase enzyme activity as markers, a gradient in the symbiotic interaction between bradyrhizobial strains and A. indica could be distinguished. This ranged from strains that induced nodules that were only infected intercellularly to rhizobial strains that formed nodules in which the host cells were invaded intracellularly and that displayed a weak nitrogenase activity. In all non-photosynthetic bradyrhizobia, the type III secretion system (T3SS) appears required to trigger nodule organogenesis. In contrast, genome sequence analysis revealed that apart from a few exceptions, like the Bradyrhizobium ORS285 strain, photosynthetic bradyrhizobia strains lack a T3SS. Furthermore, analysis of the symbiotic properties of an ORS285 T3SS mutant revealed that the T3SS could have a positive or negative role for the interaction with NF-dependent Aeschynomene species, but that it is dispensable for the interaction with all NF-independent Aeschynomene species tested. Taken together, these data indicate that two NF-independent symbiotic processes are possible between legumes and rhizobia: one dependent on a T3SS and one using a so far unknown mechanism.