Bacillus amyloliquefaciens ssp. plantarum FZB42 represents the prototype of Gram-positive plant growth promoting and biocontrol bacteria. In this study, we applied transposon mutagenesis to generate a transposon library, which was screened for genes involved in multicellular behavior and biofilm formation on roots as a prerequisite of plant growth promoting activity. Transposon insertion sites were determined by rescue-cloning followed by DNA sequencing. As in B. subtilis, the global transcriptional regulator DegU was identified as an activator of genes necessary for swarming and biofilm formation, and the DegU-mutant of FZB42 was found impaired in efficient root colonization. Direct screening of 3,000 transposon insertion mutants for plant-growth-promotion revealed the gene products of nfrA and RBAM_017140 to be essential for beneficial effects exerted by FZB42 on plants. We analyzed the performance of GFP-labeled wild-type and transposon mutants in the colonization of lettuce roots using confocal laser scanning microscopy. While the wild-type strain heavily colonized root surfaces, the nfrA mutant did not colonize lettuce roots, although it was not impaired in growth in laboratory cultures, biofilm formation and swarming motility on agar plates. The RBAM17410 gene, occurring in only a few members of the B. subtilis species complex, was directly involved in plant growth promotion. None of the mutant strains were affected in producing the plant growth hormone auxin. We hypothesize that the nfrA gene product is essential for overcoming the stress caused by plant response towards bacterial root colonization. Citation: Budiharjo A, Chowdhury SP, Dietel K, Beator B, Dolgova O, et al. (2014) Transposon Mutagenesis of the Plant-Associated Bacillus amyloliquefaciens ssp. plantarum FZB42 Revealed That the nfrA and RBAM17410 Genes Are Involved in Plant-Microbe-Interactions. PLoS ONE 9(5): e98267. doi:10.1371/journal.pone. 0098267 Copyright: ß 2014 Budiharjo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: German Ministry for Education and Research (PATHCONTROL 0315654A and C) to RB and AH (http://www.genomik-transfer.de/index. php?section = verbund&fkz = 0315654). European Union's Seventh Framework Programme (FP/2007-2013) under Grant agreement n u312117 to RB (http:// www.biofector.info/). ABiTEP GmbH provided support in the form of salaries for authors KD, OD & RB, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the 'author contributions' section.