Roots are the primary site of interaction between plants and microorganisms. To meet food demands in changing climates, improved yields and stress resistance are increasingly important, stimulating efforts to identify factors that affect plant productivity. The role of bacterial endophytes that reside inside plants remains largely unexplored, because analysis of their specific functions is impeded by difficulties in cultivating most prokaryotes. Here, we present the first metagenomic approach to analyze an endophytic bacterial community resident inside roots of rice, one of the most important staple foods. Metagenome sequences were obtained from endophyte cells extracted from roots of field-grown plants. Putative functions were deduced from protein domains or similarity analyses of protein-encoding gene fragments, and allowed insights into the capacities of endophyte cells. This allowed us to predict traits and metabolic processes important for the endophytic lifestyle, suggesting that the endorhizosphere is an exclusive microhabitat requiring numerous adaptations. Prominent features included flagella, plant-polymer-degrading enzymes, protein secretion systems, iron acquisition and storage, quorum sensing, and detoxification of reactive oxygen species. Surprisingly, endophytes might be involved in the entire nitrogen cycle, as protein domains involved in N(2)-fixation, denitrification, and nitrification were detected and selected genes expressed. Our data suggest a high potential of the endophyte community for plant-growth promotion, improvement of plant stress resistance, biocontrol against pathogens, and bioremediation, regardless of their culturability.

Sequencing the symbiotic region of Bradyrhizobium japonicum revealed a gene cluster (tts) encoding a type III secretion system (TTSS) that is similar to those found in Mesorhizobium loti MAFF303099 and Rhizobium strain NGR234. In addition to genes that are likely to encode structural core components of the TTSS, the cluster contains several open reading frames that are found exclusively in rhizobia or that are specific to B. japonicum. Depending on the host, mutations within this cluster affected nodulation capacity to different extents. One of the genes likely encodes a transcriptional activator (TtsI) of the two-component regulatory family. Upstream of ttsI, a nod box promoter was identified. Expression of ttsI could be induced by genistein. This induction depended on the transcriptional activator protein NodW as well as the nodD1nodD2nolA gene region. TtsI was found to be involved in transcriptional regulation of the tts gene cluster. Sequence comparison revealed a conserved tts box element within putative promoter regions of several genes. Here, we propose a model of the regulatory cascade leading to the induction of the tts gene cluster.

Azoarcus sp. strain BH72, a mutualistic endophyte of rice and other grasses, is of agrobiotechnological interest because it supplies biologically fixed nitrogen to its host and colonizes plants in remarkably high numbers without eliciting disease symptoms. The complete genome sequence is 4,376,040-bp long and contains 3,992 predicted protein-coding sequences. Genome comparison with the Azoarcus-related soil bacterium strain EbN1 revealed a surprisingly low degree of synteny. Coding sequences involved in the synthesis of surface components potentially important for plant-microbe interactions were more closely related to those of plant-associated bacteria. Strain BH72 appears to be 'disarmed' compared to plant pathogens, having only a few enzymes that degrade plant cell walls; it lacks type III and IV secretion systems, related toxins and an N-acyl homoserine lactones-based communication system. The genome contains remarkably few mobile elements, indicating a low rate of recent gene transfer that is presumably due to adaptation to a stable, low-stress microenvironment.Endophytic bacteria reside within the living tissue of plants without substantively harming them. They are of high interest for agrobiotechnological applications, such as the improvement of plant growth and health, phytoremediation 1 or even as biofertilizer 2 . Supply of nitrogen derived from fixation of atmospheric N 2 by grass endophytes, such as Gluconacetobacter diazotrophicus and Azoarcus sp. strain BH72, which has been shown to occur in sugarcane 3 and Kallar grass 2 , is a process of potential agronomical and ecological importance.Although the lifestyle of these endophytes is relatively well documented, the molecular mechanisms by which they interact beneficially with plants have only been poorly elucidated. A combination of features makes Azoarcus sp. strain BH72 an excellent model grassendophyte 4 . (i) It supplies nitrogen derived from N 2 fixation to its host, Kallar grass (Leptochloa fusca (L.) Kunth); in planta it is usually not culturable, but can be detected by culture-independent methods based on nifH-encoding nitrogenase reductase, the key enzyme for N 2 fixation 2 . (ii) It colonizes nondiseased plants in remarkably high numbers: estimates range from 10 8 cells (culturable cells per gram root dry weight (RDW) of field-grown Kallar grass 5 ) to 10 10 cells (estimated on the basis of abundance of bacterial nifH-mRNA in roots) 2 . (iii) It is the only cultured grass endophyte shown by molecular methods to be the most actively N 2 -fixing bacterium of the natural population in roots 2 . (iv) It also colonizes the roots of rice, a cereal of global importance, in high numbers (10 9 cells per g RDW) in the laboratory, and spreads systemically into shoots 6 . Plant stress response is only very limited in a compatible, that is, well-colonized rice cultivar 7 . Notably, Azoarcus sp. strain BH72 is capable of endophytic N 2 -fixation inside the roots of rice 8 .For a wider application in agriculture, more knowledge is required on mechanisms o...

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