Sigma Factor Engineering in Actinoplanes sp. SE50/110: Expression of the Alternative Sigma Factor Gene ACSP50_0507 (σHAs) Enhances Acarbose Yield and Alters Cell Morphology.

Sigma factors are transcriptional regulators that are part of complex regulatory networks for major cellular processes, as well as for growth phase-dependent regulation and stress response. Actinoplanes sp. SE50/110 is the natural producer of acarbose, an α-glucosidase inhibitor that is used in diabetes type 2 treatment. Acarbose biosynthesis is dependent on growth, making sigma factor engineering a promising tool for metabolic engineering. ACSP50_0507 is a homolog of the developmental and osmotic-stress-regulating Streptomyces coelicolor σHSc. Therefore, the protein encoded by ACSP50_0507 was named σHAs. Here, an Actinoplanes sp. SE50/110 expression strain for the alternative sigma factor gene ACSP50_0507 (sigHAs) achieved a two-fold increased acarbose yield with acarbose production extending into the stationary growth phase. Transcriptome sequencing revealed upregulation of acarbose biosynthesis genes during growth and at the late stationary growth phase. Genes that are transcriptionally activated by σHAs frequently code for secreted or membrane-associated proteins. This is also mirrored by the severely affected cell morphology, with hyperbranching, deformed and compartmentalized hyphae. The dehydrated cell morphology and upregulation of further genes point to a putative involvement in osmotic stress response, similar to its S. coelicolor homolog. The DNA-binding motif of σHAs was determined based on transcriptome sequencing data and shows high motif similarity to that of its homolog. The motif was confirmed by in vitro binding of recombinantly expressed σHAs to the upstream sequence of a strongly upregulated gene. Autoregulation of σHAs was observed, and binding to its own gene promoter region was also confirmed.

Comprehensive Proteome Profiling of a Xanthomonas campestris pv. Campestris B100 Culture Grown in Minimal Medium with a Specific Focus on Nutrient Consumption and Xanthan Biosynthesis.

Xanthan, a bacterial polysaccharide, is widespread in industrial applications, particularly as a food additive. However, little is known about the process of xanthan synthesis on the proteome level, even though Xanthomonas campestris is frequently used for xanthan fermentation. A label-free LC-MS/MS method was employed to study the protein changes during xanthan fermentation in minimal medium. According to the reference database, 2416 proteins were identified, representing 54.75 % of the proteome. The study examined changes in protein abundances concerning the growth phase and xanthan productivity. Throughout the experiment, changes in nitrate concentration appeared to affect the abundance of most proteins involved in nitrogen metabolism, except Gdh and GlnA. Proteins involved in sugar nucleotide metabolism stay unchanged across all growth phases. Apart from GumD, GumB, and GumC, the gum proteins showed no significant changes throughout the experiment. GumD, the first enzyme in the assembly of the xanthan-repeating unit, peaked during the early stationary phase but decreased during the late stationary phase. GumB and GumC, which are involved in exporting xanthan, increased significantly during the stationary phase. This study suggests that a potential bottleneck for xanthan productivity does not reside in the abundance of proteins directly involved in the synthesis pathways.

Genome-Based Analysis of Virulence Factors and Biofilm Formation in Novel P. aeruginosa Strains Isolated from Household Appliances.

In household washing machines, opportunistic pathogens such as Pseudomonas aeruginosa are present, which represent the household as a possible reservoir for clinical pathogens. Here, four novel P. aeruginosa strains, isolated from different sites of household appliances, were investigated regarding their biofilm formation. Only two isolates showed strong surface-adhered biofilm formation. In consequence of these phenotypic differences, we performed whole genome sequencing using Oxford Nanopore Technology together with Illumina MiSeq. Whole genome data were screened for the prevalence of 285 virulence- and biofilm-associated genes as well as for prophages. Linking biofilm phenotypes and parallelly appearing gene compositions, we assume a relevancy of the las quorum sensing system and the phage-encoded bacteriophage control infection gene bci, which was found on integrated phi297 DNA in all biofilm-forming isolates. Additionally, only the isolates revealing strong biofilm formation harbored the ϕCTX-like prophage Dobby, implicating a role of this prophage on biofilm formation. Investigations on clinically relevant pathogens within household appliances emphasize their adaptability to harsh environments, with high concentrations of detergents, providing greater insights into pathogenicity and underlying mechanisms. This in turn opens the possibility to map and characterize potentially relevant strains even before they appear as pathogens in society.

RcgA and RcgR, Two Novel Proteins Involved in the Conjugative Transfer of Rhizobial Plasmids.

Rhizobia are Gram-negative bacteria that are able to establish a nitrogen-fixing symbiotic interaction with leguminous plants. Rhizobia genomes usually harbor several plasmids which can be transferred to other organisms by conjugation. Two main mechanisms of the regulation of rhizobial plasmid transfer have been described: quorum sensing (QS) and the rctA/rctB system. Nevertheless, new genes and molecules that modulate conjugative transfer have recently been described, demonstrating that new actors can tightly regulate the process. In this work, by means of bioinformatics tools and molecular biology approaches, two hypothetical genes are identified as playing key roles in conjugative transfer. These genes are located between conjugative genes of plasmid pRfaLPU83a from Rhizobium favelukesii LPU83, a plasmid that shows a conjugative transfer behavior depending on the genomic background. One of the two mentioned genes, rcgA, is essential for conjugation, while the other, rcgR, acts as an inhibitor of the process. In addition to introducing this new regulatory system, we show evidence of the functions of these genes in different genomic backgrounds and confirm that homologous proteins from non-closely related organisms have the same functions. These findings set up the basis for a new regulatory circuit of the conjugative transfer of plasmids. IMPORTANCE Extrachromosomal DNA elements, such as plasmids, allow for the adaptation of bacteria to new environments by conferring new determinants. Via conjugation, plasmids can be transferred between members of the same bacterial species, different species, or even to organisms belonging to a different kingdom. Knowledge about the regulatory systems of plasmid conjugative transfer is key in understanding the dynamics of their dissemination in the environment. As the increasing availability of genomes raises the number of predicted proteins with unknown functions, deeper experimental procedures help to elucidate the roles of these determinants. In this work, two uncharacterized proteins that constitute a new regulatory circuit with a key role in the conjugative transfer of rhizobial plasmids were discovered.

MetHoS: a platform for large-scale processing, storage and analysis of metabolomics data.

BACKGROUND: Modern mass spectrometry has revolutionized the detection and analysis of metabolites but likewise, let the data skyrocket with repositories for metabolomics data filling up with thousands of datasets. While there are many software tools for the analysis of individual experiments with a few to dozens of chromatograms, we see a demand for a contemporary software solution capable of processing and analyzing hundreds or even thousands of experiments in an integrative manner with standardized workflows. RESULTS: Here, we introduce MetHoS as an automated web-based software platform for the processing, storage and analysis of great amounts of mass spectrometry-based metabolomics data sets originating from different metabolomics studies. MetHoS is based on Big Data frameworks to enable parallel processing, distributed storage and distributed analysis of even larger data sets across clusters of computers in a highly scalable manner. It has been designed to allow the processing and analysis of any amount of experiments and samples in an integrative manner. In order to demonstrate the capabilities of MetHoS, thousands of experiments were downloaded from the MetaboLights database and used to perform a large-scale processing, storage and statistical analysis in a proof-of-concept study. CONCLUSIONS: MetHoS is suitable for large-scale processing, storage and analysis of metabolomics data aiming at untargeted metabolomic analyses. It is freely available at: https://methos.cebitec.uni-bielefeld.de/ . Users interested in analyzing their own data are encouraged to apply for an account.

Impact of left atrial appendage fibrosis on atrial fibrillation in patients following coronary bypass surgery.

OBJECTIVES: We aimed to assess the relationship of left atrial appendage (LAA) fibrosis with atrial fibrillation (AF) and postoperative events in patients receiving coronary artery bypass graft surgery (CABG). BACKGROUND: Increased atrial fibrosis has been associated with AF and worse outcome following catheter ablation. Only limited data exists focusing on the impact of LAA fibrosis on AF after CABG. METHODS: LAA tissue from 164 CABG-patients was stained with Masson-Goldner trichrome. The histological landscape was scanned and segmented into superpixels for software analysis (QuPath). A classification algorithm was extensively trained to detect fibrotic superpixels for quantification. In 43 propensity score matched pairs with AF or sinus rhythm (SR), LAA fibrosis was compared. Moreover, subgroups of mitral valve regurgitation (MR) were analyzed as follows: SR, SR + MR, AF and AF + MR. The predictive value of LAA fibrosis postoperative stroke, postoperative AF and mortality was assessed. RESULTS: Fibrotic remodeling (%) showed no significant difference for the total cohort between the SR and AF group (SR: 30.8 ± 11.4% and AF: 33.8 ± 16.0%, respectively, p = .32). However, significant fibrotic remodeling was observed for SR and AF subgroups (SR: 27.2 ± 12.2% vs. AF: 35.3 ± 13.7%; respectively, p = .049) and between SR and SR + MR subgroups (SR: 27.2 ± 12.2% vs. SR + MR: 34.9 ± 9.1%, respectively, p = .027). LAA fibrosis was not significantly associated with postoperative stroke, postoperative AF or overall mortality (all p > .05). CONCLUSION: LAA fibrosis may contribute to an individual arrhythmia substrate for AF in patients with AF but also in those with SR and coincidence of MR. LAA fibrosis was not found to be predictive for clinical events in patients after CABG.

Analysis of Gum proteins involved in xanthan biosynthesis throughout multiple cell fractions in a "single-tube".

Xanthomonas is a phytopathogenic bacterium and of industrial interest due to its capability to produce xanthan, used as a thickener and emulsifier in the food and non-food industry. Until now, proteome analyses of Xcc lacking a detailed view on the proteins involved in xanthan biosynthesis. The proteins involved in the biosynthesis of this polysaccharide are located near, in or at the cell membrane. This study aims to establish a robust and rapid protocol for a comprehensive proteome analysis of Xcc strains, without the need to isolate different cell fractions. Therefore, a method for the analysis of the whole cell proteome was compared to the isolation of specific fractions regarding the total number of identified proteins, the overlap, and the differences between the approaches. The whole cell proteome analysis with extended peptide separation methods resulted in more than 3254 identified proteins covering 73.1% of the whole proteome. The protocol was used to study xanthan production in a label-free quantification approach. Expression profiles of 8 Gum proteins were compared between the stationary and logarithmic growth phase. Differential expression levels within the operon structure indicate a complex regulatory mechanism for xanthan biosynthesis. Data are available via ProteomeXchange with identifier PXD027261. SIGNIFICANCE: Bacteria are metabolite factories with a wide variety of natural products. Thus, proteome analyses play a crucial role to understand the biological processes within a cell behind the biosynthesis of those metabolites. Proteins involved in the biosynthesis of secreted products are often organised on, in or around the membrane allowing metabolite channelling. Experiments targeting those biosynthesis pathways on protein level often require the analysis of multiple cell fractions like cytosolic, inner, and outer membrane. This is time consuming and demands different protocols. The protocol presented here is a rapid and robust solution to study biosynthetic pathways of biological or biotechnological interest in a single approach on protein level, where gene products are partitioned across multiple cell fractions. The use of a single method also simplifies the comparison of different experiments, for example, production vs. nonproduction conditions.

Human Coxsackie- and adenovirus receptor is a putative target of neutrophil elastase-mediated shedding.

BACKGROUND: During viral-induced myocarditis, immune cells migrate towards the site of infection and secrete proteases, which in turn can act as sheddases by cleaving extracellular domains of transmembrane proteins. We were interested in the shedding of the Coxsackie- and adenovirus receptor (CAR) that acts as an entry receptor for both eponymous viruses, which cause myocarditis. CAR shedding by secreted immune proteases could result in a favourable outcome of myocarditis as CAR's extracellular domain would be removed from the cardiomyocytes' surface leading to decreased susceptibility to ongoing viral infections. METHODS AND RESULTS: In this work, matrix metalloproteinases and serine proteinases were screened for their proteolytic activity towards human CAR. Whereas matrix metalloproteinases, proteinase 3, and cathepsin G did not cleave human recombinant CAR or only within long incubation times, neutrophil elastase showed a distinct cleavage pattern of CAR's extracellular domain that was time- and dose-dependent. Neutrophil elastase cleaves CAR at its membrane-proximal immunoglobulin domain as we determined by nanoLC-MS/MS. Furthermore, neutrophil elastase treatment of cells reduced CAR surface levels as seen by flow cytometry and immunofluorescence microscopy. CONCLUSIONS: With this study, we show that CAR might be a target for shedding by neutrophil elastase.

Two Flagellar mutants of Xanthomonas campestris are characterized by enhanced xanthan production and higher xanthan viscosity.

Xanthomonas campestris strains are used world-wide for the production of the industrially important exopolysaccharide xanthan. The high industrial relevance of xanthan can be explained by its extraordinary qualities as rheological control agent in aqueous systems and by its stabilizing properties in suspensions and emulsions. The phytopathogen Xanthomonas campestris is a motile bacterium with one polar flagellum. The flagellum is a cost intensive structure, in terms of energy and building block consumption. Based on the assumption that inhibition of the flagellar biosynthesis and related proton driven motility might be beneficial for the xanthan production in Xcc, two genes (fliC and fliM) were mutated to inhibit the motility. Both mutants Xcc JBL007 fliC- and Xcc JBL007 fliM- showed an increased xanthan production. Remarkably, the produced xanthan from both mutants showed enhanced rheological properties. While the chemical composition of the produced xanthan of the initial and both mutant strains did not change, notable differences in persistence length could be measured via atomic force microscopy. Results presented in this study demonstrate the possibility to further improve the xanthan production by Xcc through rational strain design.

Evaluation of virulence potential of methicillin-sensitive and methicillin-resistant Staphylococcus aureus isolates from a German refugee cohort.

BACKGROUND: Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) seem to be highly transmissible, often infect otherwise healthy humans and frequently occur in hospital outbreaks. METHODS: Refugees, living in accommodations in Germany were screened for nasal carriage of S. aureus. The isolates were investigated regarding resistance and virulence, phenotypically and by whole genome data analysis. RESULTS: 5.6% (9/161) of the refugees are carriers of S. aureus. 2.5% (4/161) are MRSA carriers. Among the refugees, spa-types t021, t084, t304, t991 and t4983 were detected, as well as the new spa-types t18794 and t18795. t304 and t991 are assumed to be local spa-types from the middle east. The isolates are less resistant and marginal biofilm formers. Each isolate has a remarkable set of virulence genes, although genes, encoding for proteins strongly associated with invasive S. aureus infections, like Panton-Valentine leucocidin, were not detected. CONCLUSION: The detection of strains from the middle east, supports the assumption that strains co-travel with the refugees and persist despite a transition of the host's living conditions. Whole genome data analysis does not permit to finally evaluate a germ's virulence. Nevertheless, an impression of the virulence potential of the strains, regarding skills in colonization, resistance, immune evasion, and host cell damaging can be pictured.

Subtyping non-small cell lung cancer by histology-guided spatial metabolomics.

PURPOSE: Most cancer-related deaths worldwide are associated with lung cancer. Subtyping of non-small cell lung cancer (NSCLC) into adenocarcinoma (AC) and squamous cell carcinoma (SqCC) is of importance, as therapy regimes differ. However, conventional staining and immunohistochemistry have their limitations. Therefore, a spatial metabolomics approach was aimed to detect differences between subtypes and to discriminate tumor and stroma regions in tissues. METHODS: Fresh-frozen NSCLC tissues (n = 35) were analyzed by matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) of small molecules (< m/z 1000). Measured samples were subsequently stained and histopathologically examined. A differentiation of subtypes and a discrimination of tumor and stroma regions was performed by receiver operating characteristic analysis and machine learning algorithms. RESULTS: Histology-guided spatial metabolomics revealed differences between AC and SqCC and between NSCLC tumor and tumor microenvironment. A diagnostic ability of 0.95 was achieved for the discrimination of AC and SqCC. Metabolomic contrast to the tumor microenvironment was revealed with an area under the curve of 0.96 due to differences in phospholipid profile. Furthermore, the detection of NSCLC with rarely arising mutations of the isocitrate dehydrogenase (IDH) gene was demonstrated through 45 times enhanced oncometabolite levels. CONCLUSION: MALDI-MSI of small molecules can contribute to NSCLC subtyping. Measurements can be performed intraoperatively on a single tissue section to support currently available approaches. Moreover, the technique can be beneficial in screening of IDH-mutants for the characterization of these seldom cases promoting the development of treatment strategies.

Metabolic responses of sugar beet to the combined effect of root hypoxia and NaCl-salinity.

The combined occurrence of salt stress and hypoxia leads to increased growth reduction and severe toxic effects compared to salt stress alone. In the present work, we analyzed the metabolic response of sugar beet (Beta vulgaris L.) to salt stress combined with hypoxia in roots as well as in young and mature leaves. B. vulgaris plants were grown in a hydroponic culture under low and high salt concentrations combined with normoxic and hypoxic conditions. A non-targeted metabolic approach was used to identify the biochemical pathways underlying the metabolic and physiological adaptation mechanisms. Young and mature leaves showed a similar metabolic response to salt stress alone and combined stresses, accumulating sugar compounds. Osmoprotectants such as proline and pinitol were accumulated under combined stress. Roots exposed to hypoxic conditions showed increased TCA (tricarboxylic acid cycle) intermediates levels such as succinate, fumarate and malate. During hypoxia, the concentration of free amino acids as well as intermediates of the GABA (gamma-aminobutyric acid) shunt increased in roots as well as in leaves. The combination of salt stress and hypoxia results in a severe stress response in roots and leaves. A partial flux of the TCA cycle linked with the GABA shunt might be activated during hypoxia to regain reduction equivalents.

Plant Transcriptome Reprograming and Bacterial Extracellular Metabolites Underlying Tomato Drought Resistance Triggered by a Beneficial Soil Bacteria.

Water deficit is one of the major constraints to crop production and food security worldwide. Some plant growth-promoting rhizobacteria (PGPR) strains are capable of increasing plant drought resistance. Knowledge about the mechanisms underlying bacteria-induced plant drought resistance is important for PGPR applications in agriculture. In this study, we show the drought stress-mitigating effects on tomato plants by the Bacillus megaterium strain TG1-E1, followed by the profiling of plant transcriptomic responses to TG1-E1 and the profiling of bacterial extracellular metabolites. Comparison between the transcriptomes of drought-stressed plants with and without TG1-E1 inoculation revealed bacteria-induced transcriptome reprograming, with highlights on differentially expressed genes belonging to the functional categories including transcription factors, signal transduction, and cell wall biogenesis and organization. Mass spectrometry-based analysis identified over 40 bacterial extracellular metabolites, including several important regulators or osmoprotectant precursors for increasing plant drought resistance. These results demonstrate the importance of plant transcriptional regulation and bacterial metabolites in PGPR-induced plant drought resistance.

Elevation of gamma-aminobutyric acid (GABA) and essential amino acids in vacuum impregnation mediated germinated rice traced by MALDI imaging.

Rice is known to contain limiting amino acids. Synthesis of GABA in plants is an adaptive response by initiating glutamic acid. A higher rate of GABA production was observed in samples enriched with glutamic acid and vacuum impregnation (VI) with longer germination time. Heat map profiles classified GABA and essential amino acids into 1) small increments consisting of Arg, His and Met, 2) moderate increments consisting of GABA, Trp, Lys, Phe and Thr, and 3) large increments consisting of Ile, Leu and Val. In Jasmine rice, highest essential amino acids were found in samples soaked with water, enriched with glutamic acid, and germinated for 72-96 h. Highest GABA (44.8 mg/100 g) was noticed after VI for 20-40 min and germinated for 72-96 h. In Riceberry, highest GABA (74.2 mg/100 g) and essential amino acids were associated with samples treated with VI for 20-40 min and germinated for 96 h.

A new technological approach in diagnostic pathology: mass spectrometry imaging-based metabolomics for biomarker detection in urachal cancer.

Urachal adenocarcinomas (UrC) are rare but aggressive. Despite being of profound therapeutic relevance, UrC cannot be differentiated by histomorphology alone from other adenocarcinomas of differential diagnostic importance. As no reliable tissue-based diagnostic biomarkers are available, we aimed to detect such by integrating mass-spectrometry imaging-based metabolomics and digital pathology, thus allowing for a multimodal approach on the basis of spatial information. To achieve this, a cohort of UrC (n = 19) and colorectal adenocarcinomas (CRC, n = 27) as the differential diagnosis of highest therapeutic relevance was created, tissue micro-arrays (TMAs) were constructed, and pathological data was recorded. Hematoxylin and eosin (H&E) stained tissue sections were scanned and annotated, enabling an automized discrimination of tumor and non-tumor areas after training of an adequate algorithm. Spectral information within tumor regions, obtained via matrix-assisted laser desorption/ionization (MALDI)-Orbitrap-mass spectrometry imaging (MSI), were subsequently extracted in an automated workflow. On this basis, metabolic differences between UrC and CRC were revealed using machine learning algorithms. As a result, the study demonstrated the feasibility of MALDI-MSI for the evaluation of FFPE tissue in UrC and CRC with the potential to combine spatial metabolomics data with annotated histopathological data from digitalized H&E slides. The detected Area under the curve (AUC) of 0.94 in general and 0.77 for the analyte taurine alone (diagnostic accuracy for taurine: 74%) makes the technology a promising tool in this differential diagnostic dilemma situation. Although the data has to be considered as a proof-of-concept study, it presents a new adoption of this technology that has not been used in this scenario in which reliable diagnostic biomarkers (such as immunohistochemical markers) are currently not available.

Exopolysaccharide Characterization of Rhizobium favelukesii LPU83 and Its Role in the Symbiosis With Alfalfa.

One of the greatest inputs of available nitrogen into the biosphere occurs through the biological N2-fixation to ammonium as result of the symbiosis between rhizobia and leguminous plants. These interactions allow increased crop yields on nitrogen-poor soils. Exopolysaccharides (EPS) are key components for the establishment of an effective symbiosis between alfalfa and Ensifer meliloti, as bacteria that lack EPS are unable to infect the host plants. Rhizobium favelukesii LPU83 is an acid-tolerant rhizobia strain capable of nodulating alfalfa but inefficient to fix nitrogen. Aiming to identify the molecular determinants that allow R. favelukesii to infect plants, we studied its EPS biosynthesis. LPU83 produces an EPS I identical to the one present in E. meliloti, but the organization of the genes involved in its synthesis is different. The main gene cluster needed for the synthesis of EPS I in E. meliloti, is split into three different sections in R. favelukesii, which probably arose by a recent event of horizontal gene transfer. A R. favelukesii strain devoided of all the genes needed for the synthesis of EPS I is still able to infect and nodulate alfalfa, suggesting that attention should be directed to other molecules involved in the development of the symbiosis.

The two-component system ActJK is involved in acid stress tolerance and symbiosis in Sinorhizobium meliloti.

The nitrogen-fixing α-proteobacterium Sinorhizobium meliloti genome codifies at least 50 response regulator (RR) proteins mediating different and, in many cases, unknown processes. RR-mutant library screening allowed us to identify genes potentially implicated in survival to acid conditions. actJ mutation resulted in a strain with reduced growth rate under mildly acidic conditions as well as a lower capacity to tolerate a sudden shift to lethal acidic conditions compared with the parental strain. Mutation of the downstream gene actK, which encodes for a histidine kinase, showed a similar phenotype in acidic environments suggesting a functional two-component system. Interestingly, even though nodulation kinetics, quantity, and macroscopic morphology of Medicago sativa nodules were not affected in actJ and actK mutants, ActK was required to express the wild-type nitrogen fixation phenotype and ActJK was necessary for full bacteroid development and nodule occupancy. The actJK regulatory system presented here provides insights into an evolutionary process in rhizobium adaptation to acidic environments and suggests that actJK-controlled functions are crucial for optimal symbiosis development.

Fast visual exploration of mass spectrometry images with interactive dynamic spectral similarity pseudocoloring.

Mass Spectrometry Imaging (MSI) is an established and still evolving technique for the spatial analysis of molecular co-location in biological samples. Nowadays, MSI is expanding into new domains such as clinical pathology. In order to increase the value of MSI data, software for visual analysis is required that is intuitive and technique independent. Here, we present QUIMBI (QUIck exploration tool for Multivariate BioImages) a new tool for the visual analysis of MSI data. QUIMBI is an interactive visual exploration tool that provides the user with a convenient and straightforward visual exploration of morphological and spectral features of MSI data. To improve the overall quality of MSI data by reducing non-tissue specific signals and to ensure optimal compatibility with QUIMBI, the tool is combined with the new pre-processing tool ProViM (Processing for Visualization and multivariate analysis of MSI Data), presented in this work. The features of the proposed visual analysis approach for MSI data analysis are demonstrated with two use cases. The results show that the use of ProViM and QUIMBI not only provides a new fast and intuitive visual analysis, but also allows the detection of new co-location patterns in MSI data that are difficult to find with other methods.

Overexpression of alfalfa SIMK promotes root hair growth, nodule clustering and shoot biomass production.

Nitrogen-fixing rhizobia and legumes have developed complex mutualistic mechanism that allows to convert atmospheric nitrogen into ammonia. Signalling by mitogen-activated protein kinases (MAPKs) seems to be involved in this symbiotic interaction. Previously, we reported that stress-induced MAPK (SIMK) shows predominantly nuclear localization in alfalfa root epidermal cells. Nevertheless, SIMK is activated and relocalized to the tips of growing root hairs during their development. SIMK kinase (SIMKK) is a well-known upstream activator of SIMK. Here, we characterized production parameters of transgenic alfalfa plants with genetically manipulated SIMK after infection with Sinorhizobium meliloti. SIMKK RNAi lines, causing strong downregulation of both SIMKK and SIMK, showed reduced root hair growth and lower capacity to form infection threads and nodules. In contrast, constitutive overexpression of GFP-tagged SIMK promoted root hair growth as well as infection thread and nodule clustering. Moreover, SIMKK and SIMK downregulation led to decrease, while overexpression of GFP-tagged SIMK led to increase of biomass in above-ground part of plants. These data suggest that genetic manipulations causing downregulation or overexpression of SIMK affect root hair, nodule and shoot formation patterns in alfalfa, and point to the new biotechnological potential of this MAPK.