Análisis funcional de una escilo inositol aminotransferasa en la simbiosis Rhizobium-leguminosa

Resumen

Rhizobium-legume symbiosis results from a complex molecular network that governs the transformation of free-living bacteria into specialized bacteroids capable of nitrogen fixation within the plant nodules. This intricate process entails a series of orchestrated molecular events facilitating the establishment of the mutualistic symbiosis between the microbe and the plant host. Comparative proteomics of bacteroids induced by Rhizobium leguminosarum bv. viciae (Rlv) UPM791 strain in pea (Pisum sativum) and lentil (Lens culinaris) nodules unveiled the identification of approximately 100 rhizobial-proteins with a host dependent expression in response to the symbiotic interaction. Among those exhibiting an overexpression in pea, there was identified an scyllo-inositol aminotransferase (SIAT), an enzyme that might participate in the biosynthesis of rhizopines. The biosynthesis/catabolism of these opine-type compounds has been shown to enhance the competitiveness for nodulation of the legume host, an important trait for the selection of rhizobia strains to elaborate legume inoculants. To elucidate the functional role of SI-AT enzyme in the symbiosis, firstly a bioinformatic analysis was conducted, confirming the classification of this enzyme as an aminotransferase, belonging to the superfamily of aspartate aminotransferases (AAT) within the 3-amino-5-hydroxybenzoic acid (AHBA) synthases group, containing conserved domains for binding to the pyridoxal 5´-phosphate (PLP) cofactor. Subsequently, Rlv UPM791 mutant strain in rlv_1940 gene encoding SI-AT and its derivative expressing wild type copies of the gene in a pBBR-based plasmid were generated. The analysis of the symbiotic phenotype and competitiveness for nodulation in pea plants revealed no significant differences between the wild type and mutant strains in any of the studies conducted. Transcriptional fusions to the reporter gene gusA and RT-qPCR have revealed that SI-AT expression is dependent on the transcriptional regulator GntR, previously shown to be overexpressed in pea bacteroids and that might control genes involved in an adaptation of bacteoids to the legume host. This study endeavors to provide a foundation for the investigation of rhizopines in order to accomplish the production of efficient rhizobia-based biofertilizers as an alternative to synthetic nitrogen fertilizers.