Jacek Plazinski

Plant factors induce expression of nodulation and host-range genes in Rhizobium trifolii

SummaryIn Rhizobium trifolii ANU843, host specific nodulation capability is encoded within a 14kb HindIII fragment of the symbiosis plasmid. To gain a better understanding of the regulation of the nodulation (Nod) genes, we have isolated lac operon transcriptional fusions to several genes within this fragment, using the mini-Mu-lac bacteriophage transposon MudI1734. Using a broad-host-range vector, fragments containing MudI1734 insertions were introduced into R. trifolii ANU845, a derivative of ANU843 which lacks the symbiosis plasmid. Four distinct regions were identified within the Nod fragment, insertions in which resulted in nodulation phenotypes similar to those found previously for Tn5. Region I mutants were Nod- and defective in root hair curling (Hac-) and corresponded to the nodABC and D genes identified by sequence analysis. Region II mutants showed an exaggerated root hair curling (Hac++) response on clover plants and a greatly reduced nodulation ability. Region III mutants were affected in host-range properties, as they gained the ability to nodulate Pisum sativum (peas), but showed only poor nodulation ability on the normal host plant, Trifolium repens (white clover). Region IV mutants showed a delay in the nodulation of Trifolium repens, but only when plants were grown under high light-regimes. When ANU845 strains carrying the above MudI1734 insertions were grown in standard laboratory media, only insertions in nodD expressed β-galactosidase at high levels. However, when cells were placed in medium in which Trifolium repens was growing, insertions in nodA, nodB, region II, region III, and region IV were all induced from 5–10 times above basal levels. This allowed us to determine the directions of transcription in these regions.

Rhizobium nodulation genes involved in root hair curling (Hac) are functionally conserved

SummaryFive specific transposon-induced nodulation defective (Nod−) mutants from different fast-growing species ofRhizobium were used as the recipients for the transfer of each of several endogenous Sym(biosis) plasmids or for recombinant plasmids that encode early nodulation and host-specificity functions. The Nod− mutants were derived fromR. trifolii, R. meliloti and from a broad-host-rangeRhizobium strain which is able to nodulate both cowpea (tropical) legumes and the non-legumeParasponia. These mutants had several common features (a), they were Nod− on all their known plant hosts, (b), they could not induce root hair curling (Hac−) and (c), the mutations were all located on the endogenous Sym-plasmid of the respective strain. Transfer to these mutants of Sym plasmids (or recombinant plasmids) encoding heterologous information for clover nodulation (pBR1AN, pRt032, pRt038), for pea nodulation (pJB5JI, pRL1JI::Tn1831), for lucerne nodulation (pRmSL26), or for the nodulation of both tropical legumes and non-legumes (pNM4AN), was able to restore root hair curling capacity and in most cases, nodulation capacity of the original plant host(s). This demonstrated a functional conservation of at least some genes involved in root hair curling. Positive hybridization between Nod DNA sequences fromR. trifolii and from a broad-host-rangeRhizobium strain (ANU240) was obtained to other fast-growingRhizobium strains. These results indicate that at least some of the early nodulation functions are common in a broad spectrum ofRhizobium strains.

Analysis of the Pectolytic Activity of Rhizobium and Azospirillum Strains Isolated from Trifolium repens

Summary A new pectin plate assay was developed in order to test pectolytic activity of 3 Rhizobium and 5 Azospirillum strains. Azospirillum strains showed a higher pectolytic activity thanrhizobia in culture. In addition, azospirilla isolated from the surface sterilized clover roots previously inoculated with Rhizobium : Azospirillum mixed cultures showed a higher pectolytic enzyme activity than those Azospirillum cells which were not passaged via plants. The possible relation of the pectolytic activity of the Azospirillum strains to an inhibition of clover nodulation is discussed.

Sym Plasmid and Chromosomal Gene Products of Rhizobium trifolii Elicit Developmental Responses on Various Legume Roots

Summary Rhizobium trifolii strains capable of inducing nodulation on clovers can produce heat-stable, water-soluble substances, which can induce a variety of responses on the roots of both clovers and vetches. These responses are: a significant shortening of the root; an increase in the number of root hairs, and a distortion or curling of the root hairs formed. Our results indicate that the Rhizobium genes responsible for the production of these substances are located on the bacterial chromosome. However, substances in the rhizosphere of the roots of clovers and vetches can affect the expression of particular genes in the nodulation region of the Sym plasmid of R. trifolii to enhance the production of these bacterial compounds which inhibit root growth and development.

Taxonomic status ofAnabaena azollae: An overview

Despite the long-standing and widespread use of the symbiotic association between the aquatic fern Azolla and its cyanobacterial symbiontAnabaena azollae to augment nitrogen supplies in rice paddy soils, very little is known about taxonomic aspects of the symbiosis. The two partners normally remain associated throughout vegetative and reproductive development, limiting the opportunities for interchanges. We have used monoclonal antibodies and DNA/DNA hybridization techniques to show that the cyanobacterial partner is not uniform throughout the genus Azolla, and that substantial diversification has occurred. With these procedures it will be possible to characterize genotypes of the cyanobacterium and to monitor experiments aimed at synthesizing new combinations ofAzolla species andAnabaena azollae strains.

Myosins from angiosperms, ferns, and algae amplification of gene fragments with versatile PCR primers and detection of protein products with a monoclonal antibody to a conserved head epitope

SummaryMyosins providing the motors for the actin-based motility that occurs in diverse plants have proved difficult to study. To facilitate those studies, we describe polymerase chain reaction primers that reliably amplify part of the myosin head from diverse plants, consensus sequences that characterise the amplified product as encoding a class V or class VIII myosin, and a monoclonal antibody that recognises an epitope conserved in the head of most plant, fungal, and animal myosins. A pair of stringent oligonucleotide primers was designed that, when used in the polymerase chain reaction, amplified at least eleven different myosins from five species of angiosperms and one sequence from each of the fernAzolla and the algaeNitella andPhaeodactylum. The amplified products, comprising 126 to 135 nucleotides encoding part of the myosin head domain, can be used as myosin-specific probes to screen genomic and cDNA libraries. To identify the products of plant myosin genes, we raised a monoclonal antibody (anti-CHE) to a nine amino acid peptide matching a conserved head epitope showing not more than single amino acid substitutions in most published myosin genes. This antibody recognises rabbit skeletal myosin and multiple polypeptides of >100 kDa in four angiosperms and in the algaNitella. Relating the Mr values of immunoreactive bands inArabidopsis extracts to the predicted Mr values of the products of five myosin genes supports the view that the antibody recognises both myosins V and VIII together with the products of some as yet unsequenced genes. The previously described MB170 antibodies may, in contrast, be specific for one or more type V myosins. Together, the polymerase chain reaction primers and the antibody represent versatile tools for identifying and categorising myosins in diverse plants.

Effect of Azospirillum Strains on Rhizobium-Legume Symbiosis

The interaction of five Azospirillum strains of the legume nodulation capacity of 14 Rhizobium strains was studied by using the rapid plant screening assay (Rolfe 1980). All Azospirillum strains showed an ability to inhibit or enhance nodulation of Rhizobium trifolii, R. meliloti, R. leguminosarum and Rhizobium “cowpea” strains on their respective plant hosts. An inhibition of nodulation was observed when Azospirillum and Rhizobium strains were mixed at a precise cell ratio and inoculated onto plants. Stimulation of nodule formation occurred when plants were inoculated first with an appropriate Rhizobium strains, and an Azospirillum strain added at least 24h later. In addition, the same phenomenon was observed when plants were inoculated first with an Azospirillum strain and Rhizobium added at least 24h later. Another Azospirillum-Rhizobium phenomenon was observed when a particular combination of an R.trifolii strain and an Azospirillum strain formed no nodules but gave a stimulation of clover plant growth on nitrogen free media. When Azospirillum addition caused an increase in nodule number there was a concomitant decrease in the effectiveness of the R.trifolii strains. All Azospirillum strains (SP7,SP59,SP107,SP242 and SP245) showed variation in their ability to inhibit or stimulate Rhizobium nodulation. Our root-segment-squash and nodule isolation methods showed that Azospirillum cells were present in both root segments and nodules of all investigated test legume plants.

Plant-Secreted Factors Induce The Expression of R.Trifolii Nodulation and Host-Range Genes

A 14kb DNA fragment from the Sym plasmid of R.trifolii strain ANU843 was shown to carry several genes which were functionally conserved between different Rhizobium species, as well as, a complex array of genes which determine host specific nodulation ability and strain competitivenes. Extensive mutagenesis of the 14kb Nod region using Tn5 and the mini Mu-lac transposon MudI 1734, revealed a correlation between the site of insertion and the nodulation defect induced. Four distinct regions (designated I, II, III and IV) were identified. The phenotypic analysis of specific subcloned DNA fragments from the Nod region, resulted in the identification of a fifth region (region V) involved in determining host range ability. DNA sequence and mutant analysis showed that region I contained 4 genes (R.trifolii nodA, B,C and D) which affected root hair curling and nodulation ability. Region II mutants were severely debilitated in nodulation ability and predominantly induced short, truncated infection threads. Region III mutants displayed host range properties which differed from the parent strain. In contrast to the parent strain these mutants were able to induce nodules on Pisum sativum including the recalitrant Afghanistan variety. Region IV mutants were consistently delayed in their ability to nodulate clovers. Transcriptional fusion of the E.coli β-galactosidase (lacZ) gene to genes in regions I, II, III and IV were analysed. Appropriately oriented nodD insertions of MudI 1734 were found to be expressed under normal culture conditions while activity from other genes was low. Expression of nodA and B and region II, III and IV genes could only be detected by exposure of-bacteria to plantsecreted factors.

Tn5 Mutagenesis and Symbiotic Mutants of a Fast-Growing Cowpea Rhizobium

The fast-growing cowpea Rhizobium strain NGR234 is able to nodulate a wide range of tropical legumes as well as the non-legume Parasponia (1). Nodulation and structural nitrogenase genes are located on a large Symplasmid in this strain (2). A cryptic megaplasmid of greater than 450 Mdals also exists in NGR234.

Rhizosphere interactions between rhizobia and nonlegumes

The process of infection and nodule development in the Parasponia symbiosis with rhizobia differs significantly from that found in the legume symbiosis (Lancelle and Torrey, 1984, 1985; Bender et al., 1987). Despite these differences, only minor genetic changes are needed in rhizobia to initiate Parasponia nodule development (Bender et al., 1988). Transfer of the nodDl gene from Rhizobium strain NGR234, which nodulates Parasponia, to the clover-specific R. leguminosarum bv trifolii strain ANU843 and derivatives extends the host range of the recipients to include Parasponia. It is also possible to extend the host range of strain ANU843 to Parasponia by inducing single base pair changes in the resident nodD gene in this strain (McIver et al., 1989).