Investigating the interaction between the nitrogen fixing endophytic bacterium Gluconacetobacter diazotrophicus and rice

Gluconacetobacter diazotrophicus (Gd) is an endophytic, nitrogen-fixing bacterium which has been shown to intracellularly colonise, and improve growth in a variety of crops, including rice. Gd can promote growth in several different ways including nitrogen fixation, hormone production, nutrient solubilisation and stress tolerance, but little is known about the molecular mechanisms of this beneficial bacterial-plant interaction. In this study, the interaction between Gd and rice is explored since rice is a global staple crop. A genome-wide association study (GWAS) was carried out to identify genetic loci associated with the complex trait of improved infection of Gd, and enhanced growth as a result of Gd infection. GWAS was performed using 180 accessions of the Bengal and Assam Aus Panel (BAAP) collection to exploit the natural genetic variation of rice. Seedling root system architecture parameters and shoot length have been quantified. The BAAP accessions showed variation in their growth response to Gd, indicating that some genotypes formed a more successful association than others. GWAS analysis identified significant SNPs, putative QTLs and underlying candidate genes. Candidate genes functions are related to: biotic stress, the ABA pathway, membrane component, trafficking and signalling. Regulation of these pathways could explain the differences in Gd colonisation ability or resulting growth. Long-read sequencing revealed some structural variants that differed in candidate genes. GWAS results, structural variant analysis and initial gene expression hinted that genes MALECTIN/MALECTIN-LIKE RECEPTOR LIKE KINASE 46 and ALDEHYDE OXIDASE 3 are promising candidates. Candidate genes should be validated using transgenic or CRISPR methods in a future longer term study. The GWAS results and use of a nitrogen-fixing null mutant suggested that nitrogen-fixation was not contributing to the resulting variation in response at this seedling stage, hormone regulation is more likely. In addition, bacterial infection was examined visually using different fluorescently-tagged bacterial strains, to pinpoint subcellular localisation and colonisation levels. Overall, this work found QTLs and candidate genes which are potential resources for genetic breeding or genome editing, which works towards generating improved rice varieties, which will provide even greater crop yields.