Proteome analysis of cultivar-specific interactions betweenRhizobium leguminosarum biovartrifolii and subterranean clover cultivar Woogenellup

Abstract

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.

Rooteomics: The Challenge of Discovering Plant Defense-Related Proteins in Roots

“…This study revealed over 250 proteins induced or up-regulated upon infection, which were mainly of bacterial origin present in the nodules as compared with the roots. Other studies followed, such as that reported by Morris and Djordjevic [73] and more recently by Miché et al [74] and van Noorde et al [75], in which proteins involved in rootbacteria interaction were identified. These data reinforce the importance of the study of roots for the discovery of resistance-related genes and proteins in plants.…”

“…The protocols employed to study the nodule proteome are quite diverse, but they have generally recurred to differential centrifugation steps. Recently, this type of procedure has been demonstrated in proteomic studies of M. trunculata root nodules infected by Agrobacterium tumefaciens [28], in soybean root nodules infected by Bradyrhizobium japonicum [29], in pea root and Woogenellup roots infected by Rhizobium leguminosarum [30,31], in Lotus japonicus root nodules infected by Mesorhizobium loti [32]. Furthermore, the characterization of membrane-associated proteins has also been possible with an additional chloroform/methanol extraction step, as demonstrated in the comparative proteomic study of M. truncatula roots inoculated with the fungus Glomus intraradices [33].…”

Comparative Proteomic Analysis of Soybean Nodulation Using a Supernodulation Mutant, SS2-2

“…Through proteomic analysis, several studies have reported identification of symbiosis-related proteins in various plant materials: root and root hairs, [11][12][13][14] nodules, [14][15][16] the peribacteroid membrane, 17,18) nodule mitochondria, 19) the nodule cytosol, 20) xylem, 21) and apoplast. 21) However, most of these studies focused on the protein profile of the specific organelle or tissue at a defined time point, especially at the early stage of nodulation, and development of indeterminate nodules.…”

Proteome Analysis. Novel Proteins Identified at the Peribacteroid Membrane from Lotus japonicus Root Nodules

“…Similar tools were used to identify symbiosis-related proteins in M. truncatula (BestelCorre et al, 2002). Two special studies (Natera et al, 2000;Morris and Djordjevic, 2001) were devoted to the analysis of changes in protein expression in bacteroids compared with free bacteria, and in nodules compared with uninfected roots. Numerous changes were observed representing mostly abundant proteins of known function (like malate dehydrogenase [MDH], DNAK, GroEL, and leghemoglobin).…”

“…Numerous changes were observed representing mostly abundant proteins of known function (like malate dehydrogenase [MDH], DNAK, GroEL, and leghemoglobin). After inoculation of roots with Rhizobium leguminosarum bacteria, 10 developmentally regulated proteins were identified including ␣-fucosidase, ethylene-induced proteins, tubulin, and chaperonin 21 (Morris and Djordjevic, 2001). The results of such studies have demonstrated that proteomics is a powerful tool to gain insight into the expression, abundance, and distribution of proteins in special tissues of model legumes.…”