Genome-wide identification of rhizobial genes associated to the colonization of plant rhizospheres using signature-tagged mutagenesis (STM) and high throughput DNA sequencing

SALAS, M, E.; LOZANO, M. J.; LÓPEZ, J. L.; ALBICORO, F.; NILSSON, J; ALVAREZ, F.; DRAGHI, W. O.; TORRES TEJERIZO, G. A.; PISTORIO, M.; DEL PAPA, M.F.; PARISI, G.; BECKER, A.; LAGARES, A.

Puebla

Congreso; IV Congreso de Bioquímica y Biología Molecular de Bacterias; 2015

Sociedad Mexicana de Bioquímica

Sinorhizobiummeliloti is agram-negative alfa-proteobacterium with the ability to develop nitrogen-fixingroot nodules with legumes of the genera Medicago, Melilotus, and Trigonella.These symbiotic associations are tightly regulated, and rhizobial strainscompete in soil for the colonization of the limited symbiotic niche (i.e. thefinal number of nodules/plant is controlled by the host plant). Thus, differentrhizobial isolates with diverse nitrogen-fixing capacities compete with eachother for root-nodule occupancy. Current evidence indicates that symbiotic competitivenessfor nodule occupancy is a complex phenomenon, and that early processes in therhizosphere are determinant to define which strains will finally associate withthe host plant. Unfortunately, the molecular characterization of early eventsin the rhizosphere by classical transcriptomics and proteomics is technicallychallenging mainly due to the low numbers of colonizing rhizobia. In order tosearch for rhizobial genes that are required for an efficient root colonizationwe used STM technologies coupled to high throughput second-generation DNA sequencing.Classical STM uses different sets of mini-Tn5 mutants where each mutant carriesa specific DNA signature tag (?bar code?). Such a tag can be further used forthe quantitative estimation of the proportion of each mutant in presence of theothers in a given set. Thus, the challenge of a set of mutants against acondition of interest and the subsequent assessment of the proportion of eachtag at the beginning and at the end of the experiment, can be used to determinewhich mutations result in negative/positive effects on the studied phenotype.In this work we present the screening of a library of over 6,000 barcodedmutants of S. meliloti to investigate their early rhizospherecolonization in two different plants: Medicago sativa, a host legume;and Pisum sativum, a non-host legume. The relative analysis of thecolonization behavior of each mutant was performed by a PCR of the mutant tagsfollowed by the massive sequencing of the amplification products from bothinput (initial condition) and output samples (3 and 7 days post-inoculation).The achieved results allowed us to identify more than one hundred S.meliloti genes whose interruption generates negative effects on thecolonization of alfalfa roots. Affected mutants could be grouped according totheir growth in reach medium, minimal medium, and root exudates. Affection inroot colonization strongly correlated with the proportion of nodule occupancylate in symbiosis. Thus, the presence, status, and expression of the identifiedgene set will be a valuable tool at the time of screening for new inoculants rhizobia.Finally, the comparison of the gene sets required for the colonization ofhomologous vs. heterologous plants allowed us to draw out general conclusionsabout the evolutionary origin of the main genetic markers and functionsassociated with the specific colonization of the hostplant root.