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The nodulation failure resulting from the interaction between Rhizobium leguminosarum biovar trifolii strain ANU794 and the Trifolium subterraneum cv. Woogenellup was examined by transposon mutagenesis to resolve whether multiple determinants were involved in cultivar-specificity. Three new transposon-induced mutants of ANU794 (W72, W78 and W710) with significantly enhanced nodulation ability on cv. Woogenellup were identified. The W72 and W78 mutations are chromosomally-located, whereas the W710 mutation isplasmid-located. The ethylene synthesis inhibitor, aminoethoxyvinylglycine, fails to enhance the nodulation ability of ANU794, ANU7943 (csn1::Tn5) and W78 on cv. Woogenellup, but enhances the nodulation ability of W72,W710 and ANU7941 (nodM::Tn5). DNA sequencing of the W78 locus reveals strong homology to an unknown Mycobacterium open reading frame, and to several bacterial non-haem chloroperoxidases. The previously identified csn1 locus showed homology to the 50S ribosomal protein, L9, with the Tn5 insertion being located in the 5′-untranslated region. The results suggest that cultivar-specificity is mediated by at least two independent mechanisms or determinants, and not by a simple gene-for-gene interaction. The role of ethylene in cultivar specificity is discussed. Cultivar-specific interactions may prove useful in identifying pathways involved in efficient nodule formation and plant-microbe interactions.

Microbes have long exploited plants as favourable niches for colonisation. The surfaces and intercellular spaces of leaves, vascular tissue (phloem and xylem), and the ecto- and endorhizospheres (root surface and intercellular spaces between root cortical cells, respectively) are colonised by microbes to varying extents (Agrios, 1988). About 80 species of bacteria are known to interact with plants either beneficially or detrimentally. The consequence of colonisation or penetration of the plant tissue varies from asymptomatic, to disease, to symbiosis and is influenced greatly by the environment. Products of photosynthesis, such as organic acids and sucrose, can provide energy sources for microbial growth. The roots are not only surrounded by a plethora of soil microbes but up to 40% of fixed carbon can be exported from root cells as organic chemicals or as cellular debris sloughed from the root cap (Lynch and Whipps, 1989). The soil, by comparison, offers a relatively nutrient-poor environment (Suslow, 1982). The cellular energy reserves of microbes bound to soil particles will influence the rapidity of their responsiveness to changes in their environment, for example, the passing of a growing root in their vicinity.

Insertion mutagenesis identified two negatively acting gene loci which restrict the ability of Rhizobium leguminosarum bv. trifolii TA1 to infect the homologous host Trifolium subterraneum cv. Woogenellup. One locus was confirmed by DNA sequence analysis as the nodM gene, while the other locus, designated csn-1 (cultivar-specific nodulation), is not located on the symbiosis plasmid. The presence of these cultivar specificity loci could be suppressed by the introduction of the nodT gene from ANU843, a related R. leguminosarum bv. trifolii strain. Other nod genes, present in R. leguminosarum bv. viciae (including nodX) and R. meliloti, were capable of complementing R. leguminosarum bv. trifolii TA1 for nodulation on cultivar Woogenellup. Nodulation studies conducted with F2 seedlings from a cross between cultivar Geraldton and cultivar Woogenellup indicated that a single recessive gene, designated rwt1, is responsible for the Nod- association between strain TA1 and cultivar Woogenellup. Parallels can be drawn between this association and gene-for-gene systems common in interactions between plants and biotrophic pathogens.

Rhizobium leguminosarum biovar trifolii strain TA1 nodulates a range of Trifolium plants including red, white and subterranean clovers. Nitrogen-fixing nodules are promptly initiated on the tap roots of these plants at the site of inoculation. In contrast to these associations, strain TA1 has a 'Nod-' phenotype on a particular cultivar of subterranean clover called Woogenellup (A.H. Gibson, Aust J Agric Sci 19: (1968) 907-918) where it induces rare, poorly developed, slow-to-appear and ineffective lateral root nodules. By comparing the nodulation gene region of strain TA1 with that of another R. leguminosarum bv. trifolii strain ANU843, which is capable of efficiently nodulating cv. Woogenellup, we have shown that the nodT gene (B.P. Surin et al., Mol Microbiol 4: (1990) 245-252) is essential for nodulation on cv. Woogenellup. The nodT gene is naturally absent in strain TA1. A cosmid clone spanning the entire nodulation gene region of strain TA1 was capable of conferring nodulation ability to R.l. bv. trifolii strains deleted for nodulation genes, but only on cultivars of subterranean clovers nodulated by strain TA1. This shows that cultivar recognition events are, in part, determined by genes in the nodulation region of strain TA1. Complementation studies also indicated that strain TA1 contains negatively-acting genes located on the Sym plasmid and elsewhere, which specifically block nodulation of cv. Woogenellup.

We have been developing Trifolium repens and Trifolium subterraneum for the study of both symbiotic and pathogenic interactions between plants and bacteria. Extensive research into the symbiosis formed between both of these species and Rhizobium leguminosarum by. trifolii has now been broadened to examine the molecular events which take place during the induction of a disease response in these important Australian pasture species. T. subterraneum is proving particularly good as a model system for such study due to a number of valuable genetic and biological features.

The nodulation failure resulting from the interaction between Rhizobium leguminosarum biovar trifolii strain ANU794 and the Trifolium subterraneum cv. Woogenellup was examined by transposon mutagenesis to resolve whether multiple determinants were involved in cultivar-specificity. Three new transposon-induced mutants of ANU794 (W72, W78 and W710) with significantly enhanced nodulation ability on cv. Woogenellup were identified. The W72 and W78 mutations are chromosomally-located, whereas the W710 mutation isplasmid-located. The ethylene synthesis inhibitor, aminoethoxyvinylglycine, fails to enhance the nodulation ability of ANU794, ANU7943 (csn1::Tn5) and W78 on cv. Woogenellup, but enhances the nodulation ability of W72,W710 and ANU7941 (nodM::Tn5). DNA sequencing of the W78 locus reveals strong homology to an unknown Mycobacterium open reading frame, and to several bacterial non-haem chloroperoxidases. The previously identified csn1 locus showed homology to the 50S ribosomal protein, L9, with the Tn5 insertion being located in the 5′-untranslated region. The results suggest that cultivar-specificity is mediated by at least two independent mechanisms or determinants, and not by a simple gene-for-gene interaction. The role of ethylene in cultivar specificity is discussed. Cultivar-specific interactions may prove useful in identifying pathways involved in efficient nodule formation and plant-microbe interactions.

A perennial ryegrass (Lolium perenne L.) cDNA library was screened with a PCR-amplified cad DNA fragment generated from ryegrass cDNA template using degenerate oligonucleotide primers. A full-length cDNA (LpeCad1) was isolated and confirmed to encode a cinnamyl alcohol dehydrogenase (CAD) enzyme by expression of activity in Escherichia coli. The recombinant enzyme catalyses conversion of coniferaldehyde and sinapaldehyde with similar efficiency, and apparent K m values below 10 µM were determined for these substrates, whereas weak substrate inhibition occurs above this concentration. The predicted perennial ryegrass CAD was very similar (88–87percnt; amino acid sequence identity) to the only other monocotyledonous plant CAD sequences available, those of maize and sugarcane, respectively. Southern blot hybridization analysis indicated that there may be two or three cad genes, or alleles, in perennial ryegrass. The ryegrass LpeCad1 gene resembles the maize cadgene in showing strong expression in root and stem tissues, but is also expressed at lower levels in shoot, leaf sheath, leaf blade and floral tissues.

Luciferase genes from Vibrio harveyi (luxAB) and firefly (luc) were introduced into E. coli, Agrobacteriurn, Arabidopsis and tobacco. Transformed bacteria and plants were quantitatively assayed for luciferase activity using a range of in vitro and in vivo assay conditions. Both lux and luc proved efficient reporter genes in bacteria, although it is important to be aware that the sensitive assays may detect expression due to readthrough from distant promoters. LUX activity was undetectable by liquid nitrogen-cooled CCD camera assays on intact tissues of plants which showed strong luxAB expression by in vitro assays. The decanal substrate for the lux assay was toxic to many plant tissues, and caused chemiluminescence in untransformed Arabidopsis leaves. These are serious limitations to application of the lux system for sensitive, non-toxic assays of reporter gene expression in plants. In contrast, LUC activity was readily detectable in intact tissues of all plants with luc expression detectable by luminometer assays on cell extracts. Image intensities of luc-expressing leaves were commonly two to four orders of magnitude above controls under the CCD camera. Provided adequate penetration of the substrate luciferin is obtained, luc is suitable for applications requiring sensitive, non-toxic assays of reporter gene expression in plants.