IN INOCULANTS Nodulaid - 17th International Nitrogen Fixation ...

17 th International Congress on Nitrogen Fixation
Fremantle, Western Australia
27 November – 1 December 2011
Session Details: Tuesday 29 November 2011
Plenary Session 4
1330 - 1500
Authors: Michael Udvardi 1 , Catalina Pislariu 1 , Igor Kryvoruchko 1 , Senjuti Sinharoy 1 , Mingyi
Wang 1 , Rajasekhara R. Duvvuru Muni 1 , Ivone Torres-Jerez 1 , Mark Taylor 1 , Shulan
Zhang 1 , Xiaofei Cheng 1 , Jiangqi Wen 1 , Ji He 1 , Xinbin Dai 1 , Patrick X Zhao 1 , Yuhong
Tang 1 , Rujin Chen 1 , Kirankumar Mysore 1 , Pascal Ratet 2 , Vagner A Benedito 3 , Giles
Oldroyd 4 , and Jeremy D Murray 4
1 Samuel Roberts Noble Foundation, Ardmore, OK, USA; 2 ISV-CNRS, Gif sur Yvette,
France; 3 West Virginia University, USA; 4 John Innes Centre, Norwich, UK;
Presentation Title: Functional genomics of symbiotic nitrogen fixation in legumes
Presentation Time: 1400 – 1430
The past few years have seen tremendous development of tools and resources for functional genomics in
legumes, including completion or near-completion of several legume genomes, comprehensive gene expression
databases for model and crop species, and production of near-saturating mutant populations for efficient forward
and reverse-genetic studies of gene function. This presentation will highlight some of these advances in various
legumes species before focusing on resource development in the model species, Medicago truncatula and how
these resources are accelerating research on symbiotic nitrogen fixation.
Phenotypic screening of several mutant populations of Medicago followed by map-based and other cloning
approaches have identified several genes that are required for nodule development, differentiation, and/or
symbiotic nitrogen fixation (SNF). However, it is clear from transcriptomic studies that hundreds, if not thousands
of plant genes are involved in SNF. Anticipating the completion of the Medicago genome sequence, we invested
heavily in the development of tools and resources that will enable us and others to decipher the functions of
many Medicago genes in the coming years. These resources include a gene expression atlas/database that can
be used to obtain developmental and other types of expression data for most Medicago genes, and two large
mutant populations, a fast-neutron-bombardment-deletion and a tobacco retro-transposonTnt1-insertion
population, which can be used for both forward and high-throughput reverse genetics. About 20,000 Tnt1
insertion lines have been generated in the R108 genotype and phenotypic screens of about half of the population
revealed 172 lines defective in SNF. These were sorted into six distinct phenotypic categories. Thermal
Asymmetric InterLaced PCR (TAIL-PCR) was used to generate 2,422 flanking sequence tags (FSTs) from these
SNF mutant lines. FST analysis identified 33 insertion alleles of the following essential symbiotic genes:
DMI1,2,3; NSP1,2; ERN1; NIN; LYK3; FLOT1,2; ENOD40; SYMREM1; and SUNN. High-throughput 454
sequencing using a 2D-pooling strategy is being used to accelerate our current FST sequencing efforts. This will
lead to a comprehensive FST database for all Tnt1 insertion lines. In parallel, a PCR-based reverse-screening
strategy was established to identify Tnt1 insertions in specific genes of interest. As a result, we have identified
additional insertion alleles of known, essential symbiosis genes. The mutant resources and gene expression
atlas have also been used to identify novel symbiotic genes, including VAPYRIN, which is required for
intracellular accommodation of rhizobia in nodules and of arbuscular mycorrhizal fungi in root cells. Reversegenetics,
using the Tnt1-insertion population is currently being used to identify and characterize transcription
factors and transporters with essential roles in SNF.
This work was supported by the National Science Foundation, the USDA CSREES-NRI, and the Samuel Roberts
Noble Foundation.
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