Experimental Evolution of a Plant Pathogen into aLegume SymbiontMarta Marchetti1., Delphine Capela1., Michelle Glew1.¤, Ste´phane Cruveiller2,Be´atriceChane-Woon-Ming2, Carine Gris1, Ton Timmers1,Ve´re´na Poinsot3, Luz B. Gilbert1, Philipp Heeb4,Claudine Me´digue2, Jacques Batut1, Catherine Masson-Boivin1*1 Laboratoire des Interactions Plantes Micro-organismes (LIPM), UMR CNRS-INRA 2594/441, Castanet-Tolosan, France, 2 CNRS-UMR 8030, Evry, France, 3 Laboratoire desIMRCP, UMR UPS/CNRS 5623, Toulouse, France, 4 CNRS, UPS, EDB (Laboratoire e´volution et Diversite´Biologique), UMR5174, Universite´de Toulouse, Toulouse, FranceAbstractRhizobia are phylogenetically disparate a- and b-proteobacteria that have achieved the environmentally essential functionof fixing atmospheric nitrogen in symbiosis with legumes. Ample evidence indicates that horizontal transfer of symbioticplasmids/islands has played a crucial role in rhizobia evolution. However, adaptive mechanisms that allow the recipientgenomes to express symbiotic traits are unknown. Here, we report on the experimental evolution of a pathogenic Ralstoniasolanacearum chimera carrying the symbiotic plasmid of the rhizobium Cupriavidus taiwanensis into Mimosa nodulating andinfecting symbionts. Two types of adaptive mutations in the hrpG-controlled virulence pathway of R. solanacearum wereidentified that are crucial for the transition from pathogenicity towards mutualism. Inactivation of the hrcV structural geneof the type III secretion system allowed nodulation and early infection to take place, whereas inactivation of the mastervirulence regulator hrpG allowed intracellular infection of nodule cells. Our findings predict that natural selection ofadaptive changes in the legume environment following horizontal transfer has been a major driving force in rhizobiaevolution and diversification and show the potential of experimental evolution to decipher the mechanisms leading tosymbiosis.Citation: Marchetti M, Capela D, Glew M, Cruveiller S, Chane-Woon-Ming B, et al. (2010) Experimental Evolution of a Plant Pathogen into a LegumeSymbiont. PLoS Biol 8(1): e1000280. doi:10.1371/journal.pbio.1000280Academic Editor: Graham C. Walker, Massachusetts Institute of Technology, United States of AmericaReceived August 27, 2009; Accepted December 4, 2009; Published January 12, 2010Copyright: ß 2010 Marchetti et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Funding: MG and BG were supporte d by a post-doctoral fellowship from INRA and CNRS, respectively. Work in the CMB and JB laboratory is supported by grantsfrom SPE INRA department, INRA BioRessources, BRG, and ANR-08-BLAN-0295-01. The funders had no role in study design, data collection and analysis, decisionto publish, or preparation of the manuscript.Competing Interests: The authors have declared that no competing interests exist.Abbreviations: HR, hypersensitive response; IT, infection thread; NF, Nod factor; T3SS, type III secretion system* E-mail: [email protected]¤ Current address: Melbourne Dental School, Bio21 Institute of Molecular Science and Biotechnology, The University of Melbourne, Parkville, Victoria, Australia. These authors equally contributed to this work.IntroductionBacteria known as rhizobia have evolved a mutualisticendosymbiosis of major ecological importance with legumes thatcontributes ca. 25% of global nitrogen cycling. Rhizobia inducethe formation on legumes of root nodules that they colonizeintracellularly [1] and in which they fix nitrogen to the benefit ofthe plant. Rhizobia are taxonomically, metabolically, andgenetically diverse soil bacteria [2,3]. They are currentlydistributed in 12 genera of a- and b-proteobacteria intermixedwith saprophytes and pathogens. The occurrence of rhizobia inseveral distant genera is thought to have originated from repeatedand independent events of horizontal transfer of key symbioticfunctions in non symbiotic bacterial genomes [2,4]. Symbioticplasmid/island transfer has been proven both in the field and inthe lab [5,6]. However, horizontal gene transfer cannot solelyaccount for the wide biodiversity of rhizobia, since only a fewrecipient bacteria—phylogenetically close to existing rhizobia [5–8]—turned into nitrogen-fixing legume symbionts. Which phylo-genetic, genetic, or ecological barriers restrict evolution ofsymbiotic properties and how these barriers are overcome havenot been investigated so far.Experimental evolution [9] coupled with genome resequencing[10] is a powerful approach to address the evolution of rhizobia.Ralstonia solanacearum and Cupriavidus taiwanensis are plant-associatedb-proteobacteria with drastically different lifestyles. R. solanacearumis a typical root-infecting pathogen of over 200 host plant species.It intercellularly invades root tissues and heavily colonizes thevascular system, where excessive production of extracellularpolysaccharides blocks water traffic, causing wilting [11,12].Cupriavidus taiwanensis is the major nitrogen-fixing symbiont ofMimosa spp. in Asia [13,14] (see Figure 1A). Due to theirphylogenetic and genomic distance (Figure S1), C. taiwanensis andR. solanacearum are ideally suited to act as symbiotic gene providerand recipient, respectively, in experimental evolution.Here, we report on the experimental evolution of R. solanacearumcarrying the symbiotic plasmid of C. taiwanensis into Mimosa-nodulating and -infecting symbionts. Two types of key adaptivemutations are described that are crucial for the transition frompathogenicity to mutualism. One allows nodulation to occur,PLoS Biology | www.plosbiology.org 1 January 2010 | Volume 8 | Issue 1 | e1000280whereas the other allows intracellular infection of plant cells, avery rare event in plant-associated bacteria.Results/DiscussionEvolution of Symbiotically Proficient R. solanacearumTo generate our starting material, we transferred the 0.55-Mbsymbiotic plasmid pRalta of C. taiwanensis LMG19424 into R.solanacearum strain GMI1000, generating the Ralstonia chimericstrain CBM124. pRalta carries nitrogen-fixation genes and a fullcomplement of nodulation genes required for the synthesis oflipochitooligosaccharide Nod factors (NFs) [15] that trigger theplant developmental program of nodule organogenesis [16].Nevertheless, CBM124