The rice endophyte metagenome

Due to its global importance and small genome size, rice was chosen as a model plant to study the biology of grasses. There is world-wide a substantial interest in studying and making best use of beneficial interactions between rice and the associated microflora. Due to the fact that most bacteria cannot be cultivated, cultivation- independent approaches such as metagenomics have to be applied in order to access functional activities of microbes in the environment. Therefore, the rice endophyte metagenome was proposed to the DOE (US Department of Energy) Microbial Genome Program as a sequencing target and was approved for sequencing in 2006. A consortium of four partners (Dr. Sessitsch, ARC; Prof. Reinhold-Hurek, Univ. of Bremen; Prof. van Elsas, Univ. of Groningen and Dr. Brar, IRRI) participated in the isolation of endophytic cells of field-grown rice plants, preparation of DNA for sequencing and complementary analyses. JGI (Joint Genome Institute) constructed libraries and started sequencing. The sequence will be made available to the project partners in late summer 2008. The aim of this proposal is to obtain functional information of yet uncultivated members of the rice endophyte community as well as a better understanding on the endophytic lifestyle by analysing the community sequence. Furthermore, the distribution and abundance of bacteria with interesting functional abilities as well as the expression/activities of these genes will be analysed in different rice cultivars grown in different soils and exposed to different conditions.

Plants are associated with complex microbial communities, which live in the root environment (rhizosphere) or inside the plant. The latter group is called endophytes. They derive mostly from the soil environment and may systematically colonize the plant. Plant-associated microorganisms have been reported to have many plant beneficial characteristics including the out-competition and antagonism of plant pathogens, nutrient mobilization and delivery as well as plant growth and strengthening effects. Most of our understanding on these microbiota is derived from cultivation-based studies and the characterization of isolation, however, it is well known that only a minority of natural microorganisms can be brought into culture. The aim of this project therefore was to better understand the ecology and functioning of bacterial endophyte communities irrespective of their culturability. We chose rice as host plant as rice plays globally a very important role and as plant growth promoting bacteria have been repeatedly reported. Our study was supported by the US Department of Energy through the sequencing and annotation support of the Joint Genome Institute (JGI). We harvested rice plants at the International Rice Research Institute in the Philippines, extracted endophytic, bacterial cells from the roots, isolated their DNA and sent it to the JGI for sequencing. The analysis of the metagenome sequence comprising the genomes of all endophytic bacteria revealed novel information on the characteristics needed for endophytic growth. This understanding was further supported by a comparative genomics analysis of individual endophytic bacteria. Overall, endophytes seem to employ various cellulolytic enzymes for endophytic colonization and are equipped with detoxification mechanisms, which protect them from oxidative stress. Although endophytes have different lifestyle strategies in regard to the interaction with the host as well as with other endophytes, they may use complex communication modes, have a high number of transcriptional regulators or the ability to degrade complex organic compounds. In regard to nutrient cycling we found methanogenic archaea and methane oxidizing bacteria among endophytes as well as bacteria with the capacity to either fix atmospheric nitrogen, denitrify or oxidize ammonium, thereby covering the whole N cycle. We in detail analyzed methanogens and methanotrophs in different cultivars grown in Macedonian fields and found them to be highly diverse and active. The latter study also revealed highly diverse endophytic communities in field grown rice with highly different microbiata associated with roots and stems. In conclusion, we could demonstrate for some functions found in the endophytic microbiome of plants grown in the Philippines global relevance indicating to a certain extent conserved functions of endophytes associated with specific plants. The contribution of endophytic methanogens and methanotrophs to global methane emissions needs to be elucidated.