Investigating The Role of Brachypodium Distachyon Cellulose Synthase 8 in Gluconacetobacter Diazotrophicus Colonization

Nitrogen is an essential nutrient for plant growth. Significant amount of nitrogen fertilizer is applied to crop field to maintain high yield. Alternatives to chemical nitrogen fertilizer are needed to reduce the costs of crop production and offset environmental damage. Gluconacetobacter diazotrophicus is a nitrogen fixing bacterium that was originally isolated from sugarcane and has been proposed as a possible biofertilizer for monocot crop production. However, the colonization of G. diazotrophicus in most monocot crops is limited and deep understanding of the response of the host plants to G. diazotrophicus colonization is still lacking. In this study, research was conducted to establish and optimize methods for introducing G. diazotrophicus into a new monocot model plant, Brachypodium distachyon. Inoculation was tested in liquid, semi-solid, and solid media with or without a nitrogen supply. To gain insight into the role played by the host plant in the G. diazotrophicus colonization establishment, the gene expression profile of B. distachyon root tissues colonized by G. diazotrophicus was generated using next generation RNA sequencing and then investigated through gene ontology and metabolic pathway analysis. The loss of function mutant for Brachypodium cellulose synthase 8 (BdCESA8) was used to investigate the involvement of the secondary cell wall in the G. diazotrophicus colonization and the function of BdCESA8 in regulating the cellulose content in the xylem. The colonization of G. diazotrophicus in B. distachyon was established and a high level of colonization was achieved in nitrogen free liquid medium. Furthermore, the results indicated the colonization of G. diazotrophicus was stabilized in three weeks after inoculation in Brachypodium and G. diazotrophicus colonized the root through the lateral root emergence sites. The RNA sequencing results indicated that Brachypodium may be actively involved in the establishment of G. diazotrophicus colonization via cell wall synthesis, and jasmonic acid, ethylene, and giberrelin biosynthesis. Therefore, the genes in these biosynthesis pathways potentially play important roles in the beneficial association between the plant and G. diazotrophicus. The increased resistance to G. diazotrophicus colonization in a BdCESA8 knockout mutant suggested that the cellulose synthesis of the secondary cell wall is involved in G. diazotrophicus colonization. The phenotype of the BdCESA8 knockout mutant also indicated that BdCESA8 may have a slightly different function than its homolog in Arabidopsis