Howieson, J.

Poole, P.

Terpolilli, J.

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Genetic determinants of suboptimal N2 fixation and  host range in the Medicago-Sinorhizobium symbiosisJason Terpolilli*1, John Howieson2, Philip Poole11Department of Molecular Microbiology, John Innes Centre, Norwich NR4-7UH, United Kingdom2 Centre for Rhizobium Studies, Murdoch University, , Murdoch 6150, Perth, Australia*Author for correspondence: Jason.Terpolilli@bbsrc.ac.ukCosmid Library Constructiono The cosmid pLAFR3, containing the cos sites for packaging into λ-phage and transduction of E. coli, was selected as a starting point.o Although pLAFR3 carries the incC plasmid stability region, work in our lab has suggested the cosmid is not reliably stable in planta.o Therefore, the parDE region was PCR amplified from pJP2 and cloned into pLAFR3 as a HindIII-BamH1 fragment, creating pLMB377 (Figure 2). o parDE encodes an antioxin-toxin protein pair, with demonstrated stability in planta on Pisum sativum3 and M. sativa4.o The stability of pLMB377 in planta is currently being confirmed with Sm1021 (pLMB377) on M. truncatula A17.o pLMB377 will then be used to  construct a cosmid library of S. medicae WSM419 (partial EcoR1 digest of gDNA) and cloned into EcoR1-digested pLMB377. o Based on the known genome size of WSM419 (6.8 Mbp), will require approximately 1,300 colonies for 99% genome coverage.o Mobilise the WSM419 cosmid library into a Sm1021 background.0102030405060708090100M. polymorpha M. murex M. truncatula A17Uninoculated Sm1021 WSM419Screening for effectiveness and host range determinantso Screen Sm1021 carrying the WSM419 cosmid library on M. polymorpha, M. murex and M. truncatula.o Look for large pink nodules, indicative of an effective interaction.o Should be straight-forward for M. polymorpha and M. murex interactions, as wild-type Sm1021 is Fix- and Nod- on these hosts, respectively.o The situation is more complicated for M. truncatula A17; Sm1021 is Fix+ on this host, albeit only partially (Figure 1).o But, nodulation of M. truncatula A17 by Sm1021 compared to WSM419 is very different - Sm1021 yields more numerous nodules which are paler, smaller in size and more widely distributed over the root system than those induced by WSM419 on this host (Figure 3).o Therefore, it will be necessary to screen for a change in nodule morphology on M. truncatula.o Successful putative clones will be isolated from nodules, the symbiotic phenotype will be authenticated and the clone(s) sequenced.Backgroundo The Medicago genus is one of prime importance to sustainable agriculture due to the ability of its members to fix atmospheric N2 in symbiosis with bacteria.o Although two species of bacteria - Sinorhizobium meliloti and S. medicae – can form symbiotic interactions with Medicago spp., the host range exhibited by these microsymbionts varies (Figure 1):• S. meliloti is Nod- on M. murex and Nod+Fix- on M. polymorpha• S. medicae is Nod+Fix+ on both these hostso Recently the complete genome sequence of S. medicae WSM419 was reported1, complementing that of the widely studied ‘lab workhorse’ S. meliloti 1021 (Sm1021), making these two strains key references for study.o While both Sm1021 and WSM419 are able to nodulate and fix N2 on the model legume M. truncatula A17, only WSM419 is able to do so effectively under N-limited conditions2 (Figure 1).o The genetic determinants of these variations in N2 fixation effectiveness and host range are not known.o Therefore, we propose to investigate the basis of the effectiveness and host range differences of these strains using a cosmid library approach.Mean shoot dry weight (% N-fed Control)Figure 1 - Mean shoot dry weight of three Medicago hosts grown in sterile sand for42 days with nutrients in N-free conditions and either uninoculated, or inoculatedseparately with Sm1021 or WSM419. Bars which share a letter for a given plant hostare not significantly different (P<0.05).Figure 2 - Map of the cosmid pLMB377, carrying the cos region for packaging intoλ-phage, the parDE stability region, tetA and tetR encoding tetracycline resistance,as well as transfer functions (tra and trb regions).Figure 3 - Photographs of M. truncatula A17 roots inoculated with (a) Sm1021 and (b)WSM419 and harvested 42 days post-inoculation.1 cm 1 cm(a) (b)References1. Reeve et al. (2010) Stand. Gen. Sci. 2: 77-862. Terpolilli et al. (2008) New Phytol. 179: 62-63. Prell et al. (2009) P.N.A.S. 106: 12477-824. Weinstein et al. (1992) J. Bact. 174: 7486-9a ab baa abcThis research has been supported by a grant from the BBSRC pLMB37721949 bptetAtetRincC regiontra regiontrb regioncos regionparDE stability regionBamHI (1353)EcoRI (1363)HindIII (166)Terpolilli, J., Howieson, J. and Poole, P. (2010) Genetic determinants of suboptimal N2 fixation and host range in the Medicago-Sinorhizobium symbiosis. In: 9th European Nitrogen Fixation Conference, 6 - 10 September, Geneva, Switzerland

Genetic determinants of suboptimal N2 fixation and host range in the Medicago-Sinorhizobium symbiosis

https://researchrepository.murdoch.edu.au/id/eprint/34575/1/Genetic-determinants-of-Medicago-Sinorhizobium-symbiosis.pdf

Genetic determinants of suboptimal N2 fixation and host range in the Medicago-Sinorhizobium symbiosis

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