References

Poole PS etal. (1985) J. Gen. Microbiol. 131, 1441-1448

Quandt J, Hynes MF (1993) Gene 127, 15-21

Walshaw DL, Poole PS (1996) Mol. Microbiol. 21, 1239-1252

The authors thank the DFG for financing this research through the SFB395.

EnodDRl GENE OF WHITE CLOVER RESPONDS TO nod GENES DERIVED FROM RHIZOBIUMLEGUMINOSARUM BV. TRIFOLII STRAIN 4S

A. Suzuki, S. Ishiba, T. Shitaohta, M. Abe, T. Uchiumi, S. Higashi

Dept of Chemistry and Bioscience, Kagoshima University, Kagoshima 890-0065, Japan

The plasmid pC4S8, which possesses the nod genes of Rhizobium leguminosarum bv. trifolii strain 4S (Had+, Hac+, Inf*, Nod+, Nif1", Fix+) as an insert sequence, was constructed and transformed into the pSym-cured strain HI. All the symbiotic features (Had+, Hac+, Inf*, Nod+) except for nitrogen fixation, of strain 4S were restored in the transconjugant Hl(pC4S8).

We attempted to analyze the gene(s) of the host plant (white clover) that responded to Rhizobium nod signal(s) at a very early step of infection. The genes of host plant responsive to the Rhizobium nod genes were differentially screened. As a result, 20 cDNA clones were isolated as up-regulated genes and 44 cDNA clones were isolated as down-regulated genes. After partial sequencing, 27 cDNA clones among the down-regulated genes were represented by A3, designated TrEnodDRl. From the results of full sequence, TrEnodDRl gene is composed of 1755 bp and the number of deduced amino acid residues is 487. There is no similar gene with this nucleotide and amino acid sequences reported in database. A computer search showed that there are two possible transmembrane helices and a pro line-rich domain in the N-terminal region.

The expression level of TrEnodDRl in nodules was reduced to 60% compared with that of the nodule detached root. It was also suppressed in 48 hour-seedlings inoculated with the strain Hl(pC4S8) and strain HI. It was at 20 and 60%, respectively, compared with the signal of un-inoculated seedlings. These results predicted that TrEnodDRl gene may be involved with the nodulation process.

To analyze the function of TrEnodDRl protein, white clover and Lotus japonicus cv. Gifu (a model leguminous plant) were transformed via Agrobacterium tumefaciens using both sense- and antisenss-TrEnodDRl gene driven by CaMV35S promoter. The germination efficiency of the seeds of the T2 generation from transgenic L. japonicus was analyzed. In the case of the seeds from antisense transgenic L. japonicus, about 95% of seeds were normally germinated, whereas only about 45% germinated with the sense seeds. Moreover, the existence of the transgene of T2 germinated plants was confirmed by PCR using specific primers. The germination efficiency of genuine transgenic T2 sense plants is only 13%. These results suggest that TrEnodDRl protein might influence the germination process negatively. We are now using the inoculation test of Rhizobium on the regenerated sense- and wtisense-TrEnodDRl transgenic plants.

ANAEROBIC INDUCTION AND NODULE INFECTED-CELL SPECIFIC EXPRESSION OF LjNOD12 GENE FROM LOTUS JAPONICUS

K. Szczyglowski1, S. Bandyopadhyay2, J. Jorgensen3, L. Amyot1

'Agriculture and Agri-Food Canada, SCPFRC, Ontario, Canada

2DOE-Plant Research Laboratory, Michigan State University, MI, USA

3University of Aarhus, Denmark

Plants respond to hypoxic or anoxic conditions by various anatomical changes and physiological adaptations that include regulation of gene expression. Oxygen concentration in the infected cells of nitrogen-fixing nodules (10 to 50 nM) is among the lowest that has been measured in aerobic cells of any organism. However, the mechanism by which nodule infected cells adapt to microaerobic conditions is poorly understood. Here, we describe a novel late nodulin gene, LjNOD12, from Lotus japonicus and show that its expression is regulated by oxygen concentration. The cDNA corresponding to LJNOD12 gene encodes a polypeptide with a predicted molecular mass of 11.5 kDa (nodulin Nljl2). LjNOD12 mRNA accumulates in the infected cells of L. japonicus nodules concomitantly with the commencement of nitrogen fixation. We show that LjNOD12 mRNA is unstable under atmospheric oxygen concentration and that expression of the LjNOD12 gene can be induced in uninoculated L. japonicus roots within 0.5 h after their exposure to anaerobic conditions. These results suggest that the microaerobic conditions regulate expression of the LjNOD12 gene, and that nodulin Nljl2 may constitute part of a mechanism required for the adaptation of plant cells to the hypoxic milieu of nodules.

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