I

Figure 2. Schematic representation of the multicopy nif gene families of four diazotrophs.

4. References

Carrasco CD et al. (1995) Proc. Natl. Acad. Sei. USA 92, 791-795

Dean DR, Jacobson MR (1992) In Stacey G et al. (eds) Biological Nitrogen Fixation, pp. 763-834, Chapman and Hall, Inc., New York Enderlin CS, Meeks JC (1983) Planta 158, 157-165 Fleischmann RD et al. (1995) Science 269, 496-512 Golden JW et al. (1985) Nature 314, 419-423 Golden JW et al. (1988) J. Bacteriol. 170, 5034-5041 Johansson C, Bergman B (1994) New Phytol. 126, 643-652 Meeks JC (1998) Bioscience 48, 266-276 Potts M et al. (1992) Science 256, 1690-1692 Rippka R et al. (1979) J. Gen. Microbiol. 111, 1-61 Summers ML et al. (1995) J. Bacteriol. 177, 6184-6194 Thiel T (1996) J. Bacteriol. 178, 4493-4499 Thiel T et al. (1997) J. Bacteriol. 179, 5222-5225

Thiel T et al. (1998) In Peschek GA et al. (eds), Phototrophic Prokaryotes, pp. 517-521,

Plenum Press, New York Thiel T, Pratte B (2001) J. Bacteriol. 183, 280-286

5. Acknowledgements

The sequencing of Nostoc punctiforme and Rhodopseudomonas palustris was supported by the DOE through a contract with the Joint Genome Institute. Research in the laboratories of TT, JM, JE and MP is supported by grants from the NSF, USDA and DOE. We thank Elsie Campbell, Kari H├Ągen, John Ingraham and Francis Wong for sequence analyses.

Section 3:

Plant Genomics

INTEGRATED FUNCTIONAL GENOMICS TO DEFINE THE PLANT'S FUNCTION IN SYMBIOTIC NODULATION

P.M. Gresshoff1, J. Stiller1, T. Maguire1, D. Lohar5, S. Ayanru1, D. Buzas1, S. Habamunga1, L. Smith1, B. Carroll2,1. Searle2, K. Meksem4, D. Lightfoot4' S. Grimmond3, A.E. Men1

'Dept of Botany

2Dept of Biochemistry

3Institute for Molecular Bioscience

The University of Queensland, Brisbane St. Lucia, QLD 4072, Australia 4Dept of Plant, Soil, and Agronomy, Southern Illinois University, Carbondale, IL, USA 5Center for Legume Research, University of Tennessee, Knoxville, TN, USA

1. Introduction

Recent advances of high throughput DNA sequencing, bioinformatics, robotics, BAC libraries, microarrays, insertional mutagenesis as well as promoter trapping open the opportunity for an integrated function and structure analysis of the genomes of soybean (Glycine max) and the model legume Lotus japonicus. We are specifically interested in the plant's role during the establishment of nodule morphogenesis, and the genes shared during seemingly related developmental programs leading either to nodule or lateral root formation. Plant mutations were induced using EMS, fast neutron deletion as well as insertion mutagenesis. Single recessive loci were mapped using molecular markers, which were used to isolate soybean BAC clones to generate contigs spanning mutant deletions. Special emphasis was given to the Nts-1 locus of soybean that governs autoregulation of nodulation. Expression analysis of nodulation events using 4200 micro-arrayed root ESTs was initiated to detect gene products temporarily expressed during early nodulation. Insertion of a promoter-less gus-reporter gene into Lotus japonicus allowed the isolation of activated plant lines that showed development specific gus-gene expression. Isolation of flanking DNA sequences provided information of potential promoters and gene function as well as providing a link between structural and functional elements of nodulation-related genes. Evidence suggests that many nodule initiation functions evolved or are shared with lateral root related processes. The possibility exists that several non-legumes share such genes.

2. Positional Cloning of the Supernodulation (nts-I) Locus of Soybean

The original supernodulation mutants were isolated by EMS mutagenesis of wild-type cultivar

Bragg (Carroll et al. 1985). Supernodulation in general is associated with: (a) an increase of nodule number and nodule mass, (b) decreased specific nitrogenase activity, (c) decreased lateral root growth in the inoculated state, (d) shoot control of supernodulation, and (e) nitrate tolerance of nodulation (but not nitrogen fixation) (Gresshoff 1993). These results lead to several important conclusions. First, nitrate regulation of nodulation and internal autoregulation share at least one functional step; second, lateral root initiation and growth are inversely coupled to nodule initiation and growth; and third, nodulation control and nitrogen fixation control are separate processes although they are epistatically related.

RFLP marker pUTG132a was mapped close to the nts-1 gene and used to isolate BAC clones from the Clemson soybean BAC library. End clones were isolated and used to generate a contig region spanning a region also defined by a fast neutron induced deletion of soybean, also exhibiting the supernodulation phenotype. The major BAC clone (138 kb in size; nearly 80% AT content) was sequenced and annotated (in collaboration with AGRF). About ten candidate ORFs with similarity to a variety of plant genes was revealed. Significant micro-synteny with the recently sequenced Arabidopsis genome revealed candidate genes. We are presently testing these for allelic variation among different soybean mutants. Several ESTs exist in the region including two loci for neutral amino acid transporters (pA381-l and pA381-2), aldolase (Gm036), thymidylate synthase and a possible transcription factor.

Several conclusions can be taken from this chromosome walk: (a) it is feasible to walk in a complex genome like soybean (1100 Mb); (b) many endclone sequences reveal repeated DNA elements often related to retrotransposons; (c) molecular markers are preferential in the G. max genome supporting a G. soja to G. max genome expansion hypothesis; (d) a deletion of at least 150 kb leads to a minimal phenotype in nodule regulation but no other physiological alteration, suggesting that functional complementation occurs in a duplicated region of the soybean genome; (e) fast neutron induced deletions are valuable in gene isolation by defining breakage points, and could provide a useful tool if coupled with microarray analysis (see Figure 1). For example, 4200 root ESTs from soybean have been arrayed to investigate gene expression during early nodulation steps in both wild-type and mutant soybeans.

Figure 1. Partial exert of a soybean microarray of 4200 root EST clones hybridized with Cy3/Cy5 labeled RNA from soybean shoots and roots. R = red label from shoot; G = green label from root; L = low expression levels. Note: spot uniformity, low background as well as dynamic range.

3. Promoter Trapping in Lotus japonicus: a Gene Machine for Functional Analysis

Lotus japonicus is a model for its symbioses with both rhizobia and mycorrhizae (Handsberg, Stougaard 1992; Jiang, Gresshoff 1997). Both symbioses are not found in Arabidopsis thaliana. What distinguishes Lotus from most other legumes is its high transformation potential, small genome and true diploidy. Using Agrobacterium tumefaciens or A. rhizogenes, over 1000 transformed lines have been produced. Transformed plants were selected by kanamycin (geneticin, G418) or BASTA selection (Lohar et al. 2001).

Promoter trapping was achieved and generated a large range of developmentally tagged lines (Martirani et al. 1999). Expression patterns ranged from those entirely in the root tips, to broad constitutive expression in the entire root, to those in nodule primordia and those entirely within the nodule. The promoter trapping strategy for plant gene discovery has several advantages: (1) tagged gene expression is followed histologically, (2) the physiological regulation of the trapped promoter can be ascertained leading to prediction of possible function, (3) with a single insert line, inverse PCR gives flanking DNA regions defining the possible promoter and ORF (further substantiating functional predictions), (4) selfed progeny should contain 25% homozygous insertions, which may have a mutant phenotype provided physiological analysis is rigorous and the gene is essential. The approach is especially attractive as it allows discovery of genes intimately involved in nodule initiation but also expressed in lateral root development.

The utility of promoter trapping is best illustrated by line 'Cheetah'. This line contains a single promoter-less gus gene insert that is activated in root tips of the main and lateral roots, the basal root-vascular strand junction as well as nodule primordia and nodule basal regions (Figure 2). The left and right flanking regions of Cheetah were sequenced and allowed the detection of putative open reading frames. The putative promoter has been defined.

Figure 2. Cheetah gus gene expression in lateral root (left) and nodule (right) initials. 4. Molecular Physiology

High frequency transformation allows the testing of known plant genes or promoters in a large number of isolates. We transferred the Arabidopsis ethylene receptor mutant gene etrl-1, which confers ethylene insensitivity, to Gifu, resulting in plants with altered triple response when germinated in the dark. Plants also showed altered flower timing, fruit maturation and abscission of petals (see Figure 3). Insensitivity was directly correlated with the level of etrl-1 expression and the degree of nodule initiation. Transgenic plants exhibited Mendelian segregation of the hypernodulation and ethylene insensitivity phenotypes. One concludes that ethylene controls the initiation of nodule primordia by inhibiting cell divisions off the phloem poles (Wopereis et al. 2000).

Figure 3. Altered nodule pattern (left) and flower maturation (right) in a transgenic Lotus japonicus, altered by the insertion of the ethylene insensitivity receptor gene etrl-1.

We constructed a BAC library of genotype 'Gifu' using HindlU digested DNA (Men et al. 2001). The library has 6.5-fold coverage and holds an average insert size of 94 kb. BACs carrying single genes have been detected using either hybridization of probes onto BAC clones arrayed on nylon membranes or by PCR screening of three-dimensionally pooled BAC pools. Discovery of plant genes from a legume is certain to provide extra insights into nitrogen and phosphorous acquisition and related plant-microbe interactions. As a by-product of this utilitarian goal is the recognition that seemingly organ-specific genes involved in legume nodule formation may have arisen from genes found important in basic plant processes such as lateral root proliferation, constraint of microbial invasion and alteration of phytohormone gradients.

5. References

Carroll BJ et al. (1985) Proc. Natl. Acad. Sci. USA 82, 4162-4166 Gresshoff PM (1993) Plant Breeding Rev. 11, 387-411 Handsberg K, Stougaard J (1992) Plant J. 2, 487-492 Jiang Q, Gresshoff PM (1997) Molec. Plant-Microbe Int. 10, 59-68 Lohar D et al. (2001) J. Exp. Bot.

Martirani L et al. (1999) Molec. Plant-Microbe Int. 12, 275-284 Men A et al. (2001) Molec. Plant-Microbe Int. 14, 375-379 Stiller J et al. (1997) J. Exp. Bot. 48, 1357-1365 Wopereis J et al. (2000) Plant J. 23, 97-114

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