Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium.

Two rapid and simple in planta transformation methods have been developed for the model legume Medicago truncatula. The first approach is based on a method developed for transformation of Arabidopsis thaliana and involves infiltration of flowering plants with a suspension of Agrobacterium. The second method involves infiltration of young seedlings with Agrobacterium. In both cases a proportion of the progeny of the infiltrated plants is transformed. The transformation frequency ranges from 4.7 to 76% for the flower infiltration method, and from 2.9 to 27.6% for the seedling infiltration method. Both procedures resulted in a mixture of independent transformants and sibling transformants. The transformants were genetically stable, and analysis of the T2 generation indicates that the transgenes are inherited in a Mendelian fashion. These transformation systems will increase the utility of M. truncatula as a model system and enable large-scale insertional mutagenesis. T-DNA tagging and the many adaptations of this approach provide a wide range of opportunities for the analysis of the unique aspects of legumes.

Immunolocalization of PsNLEC-1, a lectin-like glycoprotein expressed in developing pea nodules.

The pea (Pisum sativum) nodule lectin gene PsNlec1 is a member of the legume lectin gene family that is strongly expressed in infected pea nodule tissue. A full-length cDNA sequence of PsNlec1 was expressed in Escherichia coli and a specific antiserum was generated from the purified protein. Immunoblotting of material from isolated symbiosomes revealed that the glycoprotein was present in two antigenic isoforms, PsNLEC-1A and PsNLEC-1B. The N-terminal sequence of isoform A showed homology to an eight-amino acid propeptide sequence previously identified from the cDNA sequence of isoform B. In nodule homogenates the antiserum recognized an additional fast-migrating band, PsNLEC-1C. Fractionation studies indicated that PsNLEC-1C was associated with a 100,000 g nodule membrane fraction, suggesting an association with cytoplasmic membrane or vesicles. Immunogold localization in pea nodule tissue sections demonstrated that the PsNLEC-1 antigen was present in the symbiosome compartment and also in the vacuole but revealed differences in distribution between infected host cells in different parts of the nodule. These data suggest that PsNLEC-1 is subject to posttranslational modification and that the various antigenic isoforms can be used to monitor membrane and vesicle targeting during symbiosome development.

Expression of cysteine protease genes in pea nodule development and senescence.

Coding sequences for two cysteine proteases were amplified from cDNA derived from pea nodule mRNA using primers based on conserved regions of plant cysteine proteases. One of the amplified cDNA sequences corresponded to a previously described cysteine protease gene, Cyp15a, expressed in pea shoots in response to dehydration (J.T. Jones and J.E. Mullet, Plant Mol. Biol. 28:1055-1065, 1995). Inside the pea root nodule, in situ hybridization revealed that this gene is expressed strongly in the apical region and more weakly in the uninfected cortex and in the central infected tissue where nitrogen fixation takes place. The complete sequence of the cDNA corresponding to the other gene, PsCyp1, was obtained. Expression of this gene, which was studied both on RNA blots and in situ, showed good correlation with the onset of nodule senescence. In situ hybridization studies revealed that PsCyp1 was expressed in senescent infected tissue at the base of the nodule. This signal was just detectable in normal symbiotically wild-type nodules but was much stronger in the early senescing nodules formed by a symbiotically defective mutant of Rhizobium leguminosarum.

Identification of a new pea gene, PsNlec1, encoding a lectin-like glycoprotein isolated from the symbiosomes of root nodules.

A 27-kD glycoprotein antigen recognized by monoclonal antibody MAC266 was purified from isolated symbiosomes derived from pea (Pisum sativum) root nodules containing Rhizobium. The N-terminal amino acid sequence was obtained, and the corresponding cDNA clone was isolated by a polymerase chain reaction-based strategy. The clone contained a single open reading frame, and the gene was termed PsNlec1. Phylogenetic analysis of 31 legume sequences showed that the PsNlec1 protein is related to the legume lectin family but belongs to a subgroup that is very different from pea seed lectin. Expression of the PsNlec1 transcript was much stronger in nodules than in other parts of the plant. It was found in both infected and uninfected cells in the central tissue of the nodule and in the stele of the root near the attachment point of the nodule. When uninfected pea seedlings were grown on medium containing nitrate, weak transcription of PsNlec1 was observed in the root system. The identification of PsNlec1 inside the symbiosome is consistent with the observation that legume lectins are generally vacuolar proteins that may serve as transient storage components.

Bacterial and plant glycoconjugates at the Rhizobium-legume interface.

Many classes of bacterial and plant glycoconjugate have been shown to be involved in establishing the Rhizobium root nodule symbiosis with peas (Pisum sativum). It was demonstrated, using techniques of molecular genetics, that a group of Rhizobium nodulation genes (nod genes) co-operate to synthesize a lipo-oligosaccharide signal molecule that specifically initiates nodule development on legume hosts. An additional gene function, encoded by nodX, has been found to extend the host range of Rhizobium leguminosarum bv. viciae to include nodulation of a pea mutant, cultivar Afghanistan; the nodX gene product specifies the addition of an acetyl group to the terminal N-acetylglucosamine residue at the reducing end of the pentasaccharide core of this signal molecule. Several other classes of bacterial glycoconjugate have also been shown by genetic analysis to be essential for normal nodule development and function: these include a capsular extracellular polysaccharide; lipopolysaccharide in the outer membrane; and cyclic glucans present in the periplasmic space. Potential functions for these glycoconjugates are discussed in the context of tissue and cell invasion by Rhizobium. Some plant components involved in symbiotic interactions have been identified by the analysis of nodule-specific gene expression (early nodulins). Several of the cDNA clones encoding these early nodulins specify proline-rich proteins that presumably correspond to cell wall glycoproteins or membrane arabinogalactan proteins. Other plant glycoconjugates have been identified using monoclonal antibodies as probes. A plant glycoprotein present in intercellular spaces has been identified as a component of the luminal matrix of infection threads. Because it attaches to the surface of bacteria and is itself susceptible to oxidative cross-linking, this glycoprotein may be involved in limiting the progress of microbial infections. Endocytosis of bacteria into the plant cytoplasm is apparently driven by direct interactions between the bacterial surface and the plasma membrane that is exposed within an unwalled infection droplet; glycoprotein and glycolipid components of the plant membrane glycocalyx have been defined using monoclonal antibodies. Differentiation of endosymbiotic bacteroids is preceded by differentiation of the plant-derived peribacteroid membrane which encloses the symbiosome compartment. Using a monoclonal antibody that identifies a group of plant membrane-associated, inositol-containing glycolipids, we have identified a very early marker for the differentiation of peribacteroid membrane from plasma membrane.