Transcriptomic Analyses of Roots of Cereals Associated to the Nitrogen-Fixing Bacteria Azospirillum brasilense and Herbaspirillum seropedicae

Herbaspirillum seropedicae and Azospirillum brasilense are diazotrophic plant growth-promoting bacteria (PGPB) that associate with economically important grasses and enhance productivity. The use of nitrogen-fixing bacteria is an alternative to increase the nitrogen use efficiency of the most produced crops such as maize, wheat and rice. A. brasilense has been used as an inoculant in maize and wheat in Latin America showing significant yield increases. Therefore, understanding the molecular basis that guide these interactions, including both plant and bacteria signaling mechanisms, are of great importance. RNA-seq transcriptomic profiling was used to reveal molecular traits involved in interaction of PGPB with wheat, maize and rice roots. The H. seropedicae SmR1-Triticum aestivum interaction was studied by comparing gene expression of bacteria attached (epiphytic) to wheat roots and planktonic cells in hydroponic system. The results indicate that the bacteria rely on adhesins and cell wall components to attach to the plant roots. The metabolism is adapted to microxic environments of the root surface and nitrogen-fixation genes were highly expressed in epiphytic cells. The H. seropedicae transcript profile was also obtained from bacteria colonizing maize (Zea mays cv. 30F53) rhizosphere in hydroponic culture. We observed that H. seropedicae activates nitrogen metabolism and mobilizes polyhydroxyalkanoate storage at an early stage of root colonization. As observed in wheat roots, epiphytic cells showed altered transcription levels of several genes associated with exopolysaccharide biosynthesis, peptidoglycan turnover and outer membrane proteins, suggesting reorganization of cell wall envelope components. Plant transcription regulation during interaction with PGPB was studied by two different approaches. First, 1019 genes were found differentially expressed in rice (Oryza sativa cv. Nipponbare) roots colonized by H. seropedicae. Genes related to synthesis and efflux of phytosiderophores and transport of phytosiderophores-iron complex were induced by the bacteria. Among the down-regulated genes we observed several coding for proteins related to plant defense and enzymes involved in flavonoid biosynthesis. The reads were also mapped to the bacteria genome revealing that genes related to nitrogen fixation, cell motility and cell wall biogenesis were expressed. The interaction of A. brasilense-wheat was investigated by dual RNA-Seq transcriptional profiling of wheat roots colonized by A. brasilense strain FP2. This study revealed 776 differentially expressed wheat ESTs belonging to various functional categories, ranging from transport activity to biological regulation as well as defense mechanism, production of phytohormones and phytochemicals. In addition, 702 A. brasilense transcripts were identified. Genes encoding proteins related to bacterial chemotaxis, biofilm formation and nitrogen fixation were highly expressed in the sub-set of A. brasilense expressed genes. These studies presented a panorama of the diazotrophic bacteria-cereals interaction in which we were able to identify candidate genes and pathways involved in bacteria colonization and plant growth promotion. The presence of the bacteria enhanced the expression of plant genes related to nutrient up-take, nitrogen assimilation and hormone signaling while down-regulated those involved in the plant-defense allowing efficient colonization by the bacteria. In summary our work revealed several molecular adaptations that PGPB utilize to sense the plant environment, attach to and live on the cereals root surface.