Construction and pilot screening of a signature-tagged mutant library of Sinorhizobium fredii
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ORIGINAL PAPER
Construction and pilot screening of a signature‑tagged mutant library of Sinorhizobium fredii Dan Wang1,2 · Yuan Chun Wang1,2 · Li Juan Wu1,2 · Jian Xin Liu1,2 · Pan Zhang1,2 · Jian Jiao1,2 · Hui Yan1,2 · Tao Liu1,2 · Chang Fu Tian1,2 · Wen Xin Chen1,2
Received: 7 May 2015 / Revised: 2 August 2015 / Accepted: 3 October 2015 / Published online: 15 October 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract Sinorhizobium fredii is well known for its ability to establish symbiosis with diverse legumes such as Glycine max (soybean, determinate nodules) and Cajanus cajan (pigeon pea, indeterminate nodules). In order to make screening of S. fredii genes related to symbiosis cost-effective, we constructed a large Tn5 insertion mutant library of S. fredii CCBAU45436 using the signaturetagged mutagenesis (STM) technique. This STM library contains a total of 25,500 independent mutants distributed in 17 sublibraries tagged by corresponding distinct DNA bar-code sequences. After the pilot screening of 255 mutants in 15 batches, Tag85-4, Tag4-17, Tag4-11 and Tag10-13 were found to have attenuated competitiveness (0–30 % in nodule occupation) compared to the wild-type strain when inoculated on soybean. Further characterization of these mutants suggests that Tag4-11 (a pyrC mutant) and Tag10-13 (a nrdJ mutant) are defective in establishing symbiosis with soybean. The pyrC mutant induced uninfected pseudonodules while the nrdJ mutant formed significantly Communicated by Shuang-Jiang Liu. Dan Wang and Yuan Chun Wang have contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00203-015-1161-9) contains supplementary material, which is available to authorized users. * Chang Fu Tian [email protected] 1
State Key Laboratory of Agrobiotechnology, and College of Biological Sciences, China Agricultural University, Beijing, China
2
Rhizobium Research Center, China Agricultural University, Beijing, China
more nodules containing bacteroids with poor persistence ability. When these two mutants were tested on pigeon pea, host-specific symbiotic defects were found. These results demonstrated the STM library as a valuable resource for identifying S. fredii genes relevant to symbiosis. Keywords Signature-tagged mutant · Rhizobia · Soybean · Pigeon pea
Introduction Rhizobia are characterized by their ability to form nitrogenfixing nodules with legumes and could play a significant role in sustainable agriculture. However, our understanding of this symbiosis system is still fragmentary due to the large phylogenetic diversity of rhizobia and the complex multi-step interactions of rhizobium–legume symbiosis (Masson-Boivin et al. 2009; Oldroyd 2013). Recent comparative genomics of rhizobia associated with diverse legumes further suggest that the documented genes relevant to symbiosis generally showed a lineage-specific distribution across different rhizobial genomes, though certain mechanisms with wide phyletic distribution such as the nodulation strategy de
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