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. N.; 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

SMB

Sinorhizobium meliloti is a gram-negative alfa-proteobacterium with the ability to develop nitrogen-fixing root nodules with legumes of the genera Medicago, Melilotus, and Trigonella.These symbiotic associations are tightly regulated, and rhizobial strains compete in soil for the colonization of the limited symbiotic niche (i.e. the final number of nodules/plant is controlled by the host plant). Thus, different rhizobial isolates with diverse nitrogen-fixing capacities compete with each other for root-nodule occupancy. Current evidence indicates that symbiotic competitiveness for nodule occupancy is a complex phenomenon, and that early processes in the rhizosphere 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 technically challenging mainly due to the low numbers of colonizing rhizobia. In order to search for rhizobial genes that are required for an efficient root colonization we 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 the others in a given set. Thus, the challenge of a set of mutants against a condition 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 determine which mutations result in negative/positive effects on the studied phenotype.In this work we present the screening of a library of over 6,000 barcoded mutants of S. meliloti to investigate their early rhizosphere colonization in two different plants: Medicago sativa, a host legume;and Pisum sativum, a non-host legume. The relative analysis of the colonization behavior of each mutant was performed by a PCR of the mutant tags followed 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 the colonization of alfalfa roots. Affected mutants could be grouped according to their 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 identified gene 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 of homologous vs. heterologous plants allowed us to draw out general conclusions about the evolutionary origin of the main genetic markers and functions associated with the specific colonization of the host plant root.