MtZR1, a PRAF protein,is involved in the development of roots and symbiotic root nodules in Medicago truncatula

PRAF proteins are present in all plants, but their functions remain unclear. We investigated the role of one member of the PRAF family, MtZR1, on the development of roots and nitrogen‐fixing nodules in Medicago truncatula. We found that MtZR1 was expressed in all M. truncatula organs. Spatiotemporal analysis showed that MtZR1 expression in M. truncatula roots was mostly limited to the root meristem and the vascular bundles of mature nodules. MtZR1 expression in root nodules was down‐regulated in response to various abiotic stresses known to affect nitrogen fixation efficiency. The down‐regulation of MtZR1 expression by RNA interference in transgenic roots decreased root growth and impaired nodule development and function. MtZR1 overexpression resulted in longer roots and significant changes to nodule development. Our data thus indicate that MtZR1 is involved in the development of roots and nodules. To our knowledge, this work provides the first in vivo experimental evidence of a biological role for a typical PRAF protein in plants.     

Peribacteroid space acidification: a marker of mature bacteroid functioning in Medicago truncatula nodules

Legumes form a symbiotic interaction with Rhizobiaceae bacteria, which differentiate into nitrogen‐fixing bacteroids within nodules. Here, we investigated in vivo the pH of the peribacteroid space (PBS) surrounding the bacteroid and pH variation throughout symbiosis. In vivo confocal microscopy investigations, using acidotropic probes, demonstrated the acidic state of the PBS. In planta analysis of nodule senescence induced by distinct biological processes drastically increased PBS pH in the N₂‐fixing zone (zone III). Therefore, the PBS acidification observed in mature bacteroids can be considered as a marker of bacteroid N₂ fixation. Using a pH‐sensitive ratiometric probe, PBS pH was measured in vivo during the whole symbiotic process. We showed a progressive acidification of the PBS from the bacteroid release up to the onset of N₂ fixation. Genetic and pharmacological approaches were conducted and led to disruption of the PBS acidification. Altogether, our findings shed light on the role of PBS pH of mature bacteroids in nodule functioning, providing new tools to monitor in vivo bacteroid physiology.     

Borate promotes the formation of a complex between legume AGP‐extensin and Rhamnogalacturonan II and enhances production of Rhizobium capsular polysaccharide during infection thread development in Pisum sativum symbiotic root nodules

The capacity to bind to biomolecules is considered to be the basis for any physiological role of boron (B). Legume arabinogalactan protein‐extensin (AGPE), a major component of the infection thread matrix of legume nodules is a potential B‐ligand. Therefore, its role in infection threads development was investigated in Pisum sativum grown under B deficiency. Using the AGPE‐specific antibody MAC265, immunochemical analysis revealed that a 175 kDa MAC265 antigen was abundant in +B but much weaker in –B nodule extracts. A B‐dependent complex involving AGPE and rhamnogalacturonan II (RGII) could be co‐purified using anti‐RGII antiserum. Following fractionation of –B nodules, MAC265 antigens were mostly associated with the bacterial pellet. Immunogold staining confirmed that AGPE was closely associated with the surface of rhizobia in the lumen of threads in ?B nodules whereas in +B nodules, AGPE was separated from the bacterial surface by a sheath of capsular polysaccharide. Interestingly, colonies of rhizobia grown in free‐living culture without B developed low capsule production. Therefore, we propose that B could be important for apical growth of infection threads by strengthening thread wall through a B‐dependent AGPE‐RGII interaction and by promoting bacterial advance through a B‐dependent production of a stable rhizobial capsule that prevents AGPE attachment.     

The advantage of laser‐capture microdissection over whole tissue analysis in proteomic profiling studies

Laser‐capture microdissection (LCM) offers a reliable cell population enrichment tool and has been successfully coupled to MS analysis. Despite this, most proteomic studies employ whole tissue lysate (WTL) analysis in the discovery of disease biomarkers and in profiling analyses. Furthermore, the influence of tissue heterogeneity in WTL analysis, nor its impact in biomarker discovery studies have been completely elucidated. In order to address this, we compared previously obtained high resolution MS data from a cohort of 38 breast cancer tissues, of which both LCM enriched tumor epithelial cells and WTL samples were analyzed. Label‐free quantification (LFQ) analysis through MaxQuant software showed a significantly higher number of identified and quantified proteins in LCM enriched samples (3404) compared to WTLs (2837). Furthermore, WTL samples displayed a higher amount of missing data compared to LCM both at peptide and protein levels (p‐value < 0.001). 2D analysis on co‐expressed proteins revealed discrepant expression of immune system and lipid metabolisms related proteins between LCM and WTL samples. We hereby show that LCM better dissected the biology of breast tumor epithelial cells, possibly due to lower interference from surrounding tissues and highly abundant proteins. All data have been deposited in the ProteomeXchange with the dataset identifier PXD002381 ( http://proteomecentral.proteomexchange.org/dataset/PXD002381 ).     

Metabolomic profiling of wild‐type and mutant soybean root nodules using laser‐ablation electrospray ionization mass spectrometry reveals altered metabolism

The establishment of the nitrogen‐fixing symbiosis between soybean and Bradyrhizobium japonicum is a complex process. To document the changes in plant metabolism as a result of symbiosis, we utilized laser ablation electrospray ionization‐mass spectrometry (LAESI‐MS) for in situ metabolic profiling of wild‐type nodules, nodules infected with a B. japonicum nifH mutant unable to fix nitrogen, nodules doubly infected by both strains, and nodules formed on plants mutated in the stearoyl‐acyl carrier protein desaturase (sacpd‐c) gene, which were previously shown to have an altered nodule ultrastructure. The results showed that the relative abundance of fatty acids, purines, and lipids was significantly changed in response to the symbiosis. The nifH mutant nodules had elevated levels of jasmonic acid, correlating with signs of nitrogen deprivation. Nodules resulting from the mixed inoculant displayed similar, overlapping metabolic distributions within the sectors of effective (fix⁺) and ineffective (nifH mutant, fix^?) endosymbionts. These data are inconsistent with the notion that plant sanctioning is cell autonomous. Nodules lacking sacpd‐c displayed an elevation of soyasaponins and organic acids in the central necrotic regions. The present study demonstrates the utility of LAESI‐MS for high‐throughput screening of plant phenotypes. Overall, nodules disrupted in the symbiosis were elevated in metabolites related to plant defense.     

Sinorhizobium meliloti succinylated high‐molecular‐weight succinoglycan and the Medicago truncatula LysM receptor‐like kinase MtLYK10 participate independently in symbiotic infection

The formation of nitrogen‐fixing nodules on legume hosts is a finely tuned process involving many components of both symbiotic partners. Production of the exopolysaccharide succinoglycan by the nitrogen‐fixing bacterium Sinorhizobium meliloti 1021 is needed for an effective symbiosis with Medicago spp., and the succinyl modification to this polysaccharide is critical. However, it is not known when succinoglycan intervenes in the symbiotic process, and it is not known whether the plant lysin‐motif receptor‐like kinase MtLYK10 intervenes in recognition of succinoglycan, as might be inferred from work on the Lotus japonicus MtLYK10 ortholog, LjEPR3. We studied the symbiotic infection phenotypes of S. meliloti mutants deficient in succinoglycan production or producing modified succinoglycan, in wild‐type Medicago truncatula plants and in Mtlyk10 mutant plants. On wild‐type plants, S. meliloti strains producing no succinoglycan or only unsuccinylated succinoglycan still induced nodule primordia and epidermal infections, but further progression of the symbiotic process was blocked. These S. meliloti mutants induced a more severe infection phenotype on Mtlyk10 mutant plants. Nodulation by succinoglycan‐defective strains was achieved by in trans rescue with a Nod factor‐deficient S. meliloti mutant. While the Nod factor‐deficient strain was always more abundant inside nodules, the succinoglycan‐deficient strain was more efficient than the strain producing only unsuccinylated succinoglycan. Together, these data show that succinylated succinoglycan is essential for infection thread formation in M. truncatula, and that MtLYK10 plays an important, but different role in this symbiotic process. These data also suggest that succinoglycan is more important than Nod factors for bacterial survival inside nodules.     

Harnessing the MinION: An example of how to establish long‐read sequencing in a laboratory using challenging plant tissue from Eucalyptus pauciflora

Long‐read sequencing technologies are transforming our ability to assemble highly complex genomes. Realizing their full potential is critically reliant on extracting high‐quality, high‐molecular‐weight (HMW) DNA from the organisms of interest. This is especially the case for the portable MinION sequencer which enables all laboratories to undertake their own genome sequencing projects, due to its low entry cost and minimal spatial footprint. One challenge of the MinION is that each group has to independently establish effective protocols for using the instrument, which can be time‐consuming and costly. Here, we present a workflow and protocols that enabled us to establish MinION sequencing in our own laboratories, based on optimizing DNA extraction from a challenging plant tissue as a case study. Following the workflow illustrated, we were able to reliably and repeatedly obtain >6.5 Gb of long‐read sequencing data with a mean read length of 13 kb and an N50 of 26 kb. Our protocols are open source and can be performed in any laboratory without special equipment. We also illustrate some more elaborate workflows which can increase mean and average read lengths if this is desired. We envision that our workflow for establishing MinION sequencing, including the illustration of potential pitfalls and suggestions of how to adapt it to other tissue types, will be useful to others who plan to establish long‐read sequencing in their own laboratories.     

Phosphorylation of S344 in the calmodulin‐binding domain negatively affects CCaMK function during bacterial and fungal symbioses

Calcium and Ca²⁺/calmodulin‐dependent protein kinase (CCaMK) plays a critical role in the signaling pathway that establishes root nodule symbiosis and arbuscular mycorrhizal symbiosis. Calcium‐dependent autophosphorylation is central to the regulation of CCaMK, and this has been shown to promote calmodulin binding. Here, we report a regulatory mechanism of Medicago truncatula CCaMK (MtCCaMK) through autophosphorylation of S344 in the calmodulin‐binding/autoinhibitory domain. The phospho‐ablative mutation S344A did not have significant effect on its kinase activities, and supports root nodule symbiosis and arbuscular mycorrhizal symbiosis, indicating that phosphorylation at this position is not required for establishment of symbioses. The phospho‐mimic mutation S344D show drastically reduced calmodulin‐stimulated substrate phosphorylation, and this coincides with a compromised interaction with calmodulin and its interacting partner, IPD3. Functional complementation tests revealed that the S344D mutation blocked root nodule symbiosis and reduced the mycorrhizal association. Furthermore, S344D was shown to suppress the spontaneous nodulation associated with a gain‐of‐function mutant of MtCCaMK (T271A), revealing that phosphorylation at S344 of MtCCaMK is adequate for shutting down its activity, and is epistatic over previously identified T271 autophosphorylation. These results reveal a mechanism that enables CCaMK to ‘turn off’ its function through autophosphorylation.     

Deletion of fucose residues in plant N‐glycans by repression of the GDP‐mannose 4,6‐dehydratase gene using virus‐induced gene silencing and RNA interference

Production of pharmaceutical glycoproteins in plants has many advantages in terms of safety and reduced costs. However, plant‐produced glycoproteins have N‐glycans with plant‐specific sugar residues (core β‐1,2‐xylose and α‐1,3‐fucose) and a Lewis a (Le^a) epitope, i.e., Galβ(1‐3)[Fucα(1‐4)]GlcNAc. Because these sugar residues and glycan structures seemed to be immunogenic, several attempts have been made to delete them by repressing their respective glycosyltransferase genes. However, until date, such deletions have not been successful in completely eliminating the fucose residues. In this study, we simultaneously reduced the plant‐specific core α‐1,3‐fucose and α‐1,4‐fucose residues in the Le^a epitopes by repressing the Guanosine 5′‐diphosphate (GDP)‐D‐mannose 4,6‐dehydratase (GMD) gene, which is associated with GDP‐L‐fucose biosynthesis, in Nicotiana benthamiana plants. Repression of GMD was achieved using virus‐induced gene silencing (VIGS) and RNA interference (RNAi). The proportion of fucose‐free N‐glycans found in total soluble protein from GMD gene‐repressed plants increased by 80% and 95% following VIGS and RNAi, respectively, compared to wild‐type plants. A small amount of putative galactose substitution in N‐glycans from the NbGMD gene‐repressed plants was observed, similar to what has been previously reported GMD‐knockout Arabidopsis mutant. On the other hand, the recombinant mouse granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) with fucose‐deleted N‐glycans was successfully produced in NbGMD‐RNAi transgenic N. benthamiana plants. Thus, repression of the GMD gene is thus very useful for deleting immunogenic total fucose residues and facilitating the production of pharmaceutical glycoproteins in plants.