Proteome analysis was used to identify proteins that are involved in the early stages of nodulation between the subterranean clover cultivar Woogenellup and the Rhizobium leguminosarum bv. trifolii strains ANU843 and ANU794. Strain ANU843 induces nitrogen-fixing nodules whereas strain ANU794 forms aberrant nodules on the roots of cv. Woogenellup that fail to develop beyond an early stage. Our aim was to identify proteins that might be involved in the early stages of nodulation over a 48 h period and to identify proteins that are differentially displayed during the interactions between the host and the two microbes. Proteome maps from control Woogenellup roots and inoculated roots were generated and compared at 24 and 48 h post inoculation. Over 1500 spots were resolved on all gels. Of the 16 protein spots that were differentally displayed or developmentally regulated, 10 were assigned putative identities. These included an alpha-fucosidase, several ethylene-induced proteins, a Cu/Zn superoxide dismutase, a hypothetical 16.5 kDa protein, tubulin alpha-chain, chaperonin 21 precursor and triosephosphate isomerase. Of the 22 constitutively expressed proteins spots examined, eight spots were assigned putative protein homologies through N-terminal sequencing and included several pathogenesis and stress-related proteins. The result may suggest that ethylene levels are upregulated during the early stages of infection but that this does not result in the induction of common pathogenesis-related proteins. The specific induction of alpha-fucosidase by ANU794 may be important in the nodulation failure phenotype of strain ANU794.

We tested whether proteome reference maps established for one species can be used for cross-species protein identification by comparing two-dimensional protein gel patterns and protein identification data of two closely related bacterial strains and four plant species. First, proteome profiles of two strains of the fully sequenced bacterium Sinorhizobium meliloti were compared as an example of close relatedness, high reproducibility and sequence availability. Secondly, the proteome profiles of three legumes (Medicago truncatula, Melilotus alba and Trifolium subterraneum), and the nonlegume rice (Oryza sativa) were analysed to test cross-species similarities. In general, we found stronger similarities in gel patterns of the arrayed proteins between the two bacterial strains and between the plant species than could be expected from the sequence similarities. However, protein identity could not be concluded from their gel position, not even when comparing strains of the same species. Surprisingly, in the bacterial strains peptide mass fingerprinting was more reliable for species-specific protein identification than N-terminal sequencing. While peptide masses were found to be unreliable for cross-species protein identification, we present useful criteria to determine confident matching against species-specific expressed sequence tag databases. In conclusion, we present evidence that cautions the use of proteome reference maps and peptide mass fingerprinting for cross-species protein identification.

The clover-nodulating Rhizobium leguminosarum bv. trifolii ANU794 initiates normal root-nodule development with abnormally low efficiency on the Trifolium subterraneum cv. Woogenellup. The cellular and developmental responses of Woogenellup roots to the site- and dose-defined inoculation of green fluorescent protein (gfp)-labeled cells of ANU843 (nodulation proficient) and ANU794 was investigated using light, fluorescence, and confocal microscopy. Strain ANU794-gfp induced three primordia types and four developmental responses at the inoculation site: true or aberrant nodules (on 5 and 25% of plants, respectively), hybrid structures (20% of plants), or lateral roots (50% of plants). The novel hybrid structures possessed nodule and lateral root-like features and unusual vascular patterning. Strain ANU794-gfp induces lateral root formation by stimulating pericycle cell divisions at all nearby protoxylem poles. Only true nodules induced by ANU794-gfp contained intracellular bacteria. In contrast, strain ANU843-gfp induced nodules only and lateral root formation was suppressed at spot inoculation sites. Primordium types were distinguishable by the emission spectrum characteristics of phenolic UV-absorbing and fluorescent compounds that accumulate in primordium cells. Hybrid primordia contained (at least) two fluorescent cell populations, suggesting that they are chimeric. The results suggest that ANU794 may produce both nodule- and lateral root-generating signals simultaneously.

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