This paper shows that lipo‐oligosaccharides (Nod factors) synthesized by Rhizobium bacteria elicit the induction of infection‐related early nodulin genes (PsENOD5 and PsENOD12) in pea root hairs. R. leguminosarum bv. viciae secretes a mixture of Nod factors containing a C18 fatty acid chain with 4 (C18:4) or 1 double bond (C18:1). Purified Nod factors harbouring either a C18:4 or a C18:1 acyl moiety induce the expression of the pea early nodulin genes, PsENOD5 and PsENOD12, but the kinetics of induction are different. The expression of both early nodulin genes is induced in a transient manner by the purified Nod factors while a mixture of the Nod factors extends the period during which these genes are expressed. In spite of the host‐specific nature of the infection process, heterologous Nod factors of R. meliloti also induce the expression of PsENOD5 and PsENOD12 genes, though with a marked delay compared with the homologous compounds.

The Rhizobium-legume interaction results in the formation of a new plant organ, the root nodule, in which the bacteria are able to reduce atmospheric N2. These root nodules have a very distinctive tissue organization, which allows the N2 fixation process to occur [1]. In short the Rhizobium-legume interaction involves the following steps: It starts with the deformation and curling of root hairs. The rhizobia then invade the plant by means of a newly formed tube, called the infection thread. Meanwhile, cells in the root cortex start to divide and form the nodule primordium. Infection threads enter individual primordium cells, and bacteria are relased form the infection thread into the cytoplasm of the plant cells. The primordium cells then differentiate into the tissues that make up the mature nodule. Rhizobium thus induces three distinct developmental processes in its host (i) root hair deformation, (ii) infection thread formation, and (iii) induction of cell division followed by differentiation into the different nodule tissues.

During the last few years we have isolated early nodulin clones of both pea and soybean. In this chapter we will summarize the characteristics of these clones (Table 1). Furthermore we will describe how we started to use early nodulin clones to isolate Rhizobium signal molecules.

The role of the Rhizobium nod genes in the induction of nodulin gene expression was examined by analyzing nodules formed on vetch roots by bacterial strains containing only the nod region. Introduction of an 11-kb cloned nod region of the R. leguminosarum sym plasmid pRL1JI into sym plasmid-cured rhizobia conferred on the recipient strains the ability to induce nodules in which all nodulin genes were expressed. This proves that from the sym plasmid only the nod region is involved in the induction of nodulin gene expression. A transconjugant of Agrobacterium carrying the same nod region induces nodules in which only early nodulin gene expression is detected. Thus, the nod region is essential for the induction of early nodulin gene expression. In this case, nodule cytology may indicate that a defense response of the plant interferes with the induction of late nodulin gene expression. Indirect evidence is presented that indeed the Rhizobium nod genes are also in some way involved in the induction of the expression of late noduling genes. The combination between histological data and pattern of nodulin gene expression furthermore reveals a correlation between nodule structure and nodulin gene expression. This correlation may aid in speculations about the functions of nodulins.

Gene expression in pea roots grown in a medium with a low oxygen concentration was compared with that in nitrogen-fixing pea root nodules induced by Rhizobium bacteria. The results show that during microaerobiosis the expression of eight genes is increased. None of these belong to the group of genes earlier identified as nodulin genes. On the other hand, no enhanced transcription of microaerobic genes can be detected during nodule development and hybridizations of Northern blots, containing nodule RNA and RNA isolated from oxygen-stressed roots, show that the alcohol dehydrogenase genes are not expressed at a higher level in pea root nodules whereas a higher expression is observed during microaerobiosis. From these observations it can be concluded that it is unlikely that a low concentration of free oxygen induces the expression of nodulin genes. Furthermore, genes that are activated as a result of oxygen deficiency are not expressed in pea root nodules, indicating that if the concentration of free oxygen is low the nodule cells do not suffer under microaerobic conditions. Probably, leghemoglobin functions as an efficient oxygen buffer for the energy-generating process in both the plant cells and the bacteroids.

Rhizobium bacteria can invade the roots of leguminous plants and elicit the formation of root nodules. This process involves the expression of at least 20 nodule-specific genes encoded by the host plant, the so-called nodulin genes, which include the leghaemoglobin genes1–3. During development of the root nodules the nodulin genes are differentially expressed1. In fast-growing Rhizobium species several essential symbiotic genes are located on a large plasmid, and when fragments of the plasmid which contain the genes essential for nodulation (the nod region) are cloned and transferred to Rhizobium strains lacking the symbiotic plasmid (‘cured’ strains), the recipients regain the ability to form nodules. However, these nodules cannot fix nitrogen because essential nitrogen-fixation genes are absent4–6. We show here that transfer of the nod region (10 kilobases; kb) alone from a Rhizobium leguminosarum symbiotic plasmid into cured Rhizobium strains is sufficient to elicit nodulin gene expression in the host Pisum sativum. We also show that pea root nodules induced by an Agrobacterium strain containing the R. leguminosarum symbiotic plasmid express an early nodulin gene but no other nodulin genes. These results show that at least two signals are involved in the induction of expression of nodulin genes and the presence of the Rhizobium nodulation genes seems to be required to elicit the first signal.

The expression of plant genes involved in the pea‐Rhizobium symbiosis was studied by analysing mRNA from root nodules. The RNA was translated in vitro and the translation products were separated by two‐dimensional gel electrophoresis. The results show differential expression of nodulin genes during root nodule development. One gene encoding N‐40′ is expressed at a significant level 5 days before the leghemoglobin genes. Most other nodulin genes are expressed more or less concomitantly with the leghemoglobin genes whereas the N‐21 mRNA is only present late during the development. In the development of ineffective root nodules induced by infection with different nod+fix− mutants of R. leguminosarum all nodulin genes are expressed except for the N‐21 gene. The results suggest that neither bacteroid development, heme excretion nor nitrogen fixation are essential for the induction of nodulin gene expression in the host plant. Further, it appears that the amount of leghemoglobin in ineffective nodules is regulated at a post‐transcriptional level.

A partial amino acid sequence of cowpea mosaic virus (CPMV) VPg radiochemically modified by chloramine‐T and Bolton‐Hunter reagent has been determined. VPg covalently bound to viral RNA chains (VPg‐RNA) was iodinated with chloramine‐T and Bolton‐Hunter reagent to label tyrosine and lysine residues, respectively. [125I]VPg‐RNA was digested with nuclease P1 and the resulting [125I]VPg‐pU was purified by SDS‐polyacrylamide gel electrophoresis and subjected to automated Edman degradation. Control experiments with chemically synthesized poliovirus VPg showed the feasibility of radiochemical microsequence analysis of protein that had been radiochemically modified by chloramine‐T and Bolton‐Hunter reagent. Analysis of CPMV [125I]VPg‐pU revealed the presence of tyrosine residues at position 12 and 14, and of lysine residues at position 3 and 20, respectively. In combination with Edman degradation of unlabeled CPMV VPg, which showed serine and arginine residues to be present at position 1 and 2, respectively, the data obtained allow the precise positioning of VPg within the 200 000 dalton (200 K) polyprotein encoded by CPMV B RNA and the prediction of its entire amino acid sequence. VPg is located at the COOH terminus of its 60 K, membrane‐bound,precursor and proximal to the amino terminus of the protease‐polymerase domain of the polyprotein. A processing scheme for the 200 K polyprotein is discussed in which Gln‐Ser amino acid pairs act as the major signal for proteolytic cleavage.

Translation of middle-component RNA of cowpea mosaic virus in vitro produced two polypeptides of 95 and 105 kilodaltons (95K and 105K, respectively) with overlapping amino acid sequences, which were specifically cleaved by a protease encoded by the bottom-component RNA. The proteolytic cleavage was studied by the addition of antibodies raised against various bottom-component RNA-encoded proteins to extracts prepared from bottom-component RNA-inoculated cowpea protoplasts. Since antiserum to the 32K polypeptide efficiently inhibited the proteolytic activity of such extracts, although antiserum to VPg or to the 170K polypeptide did not, evidence was obtained which indicates that the 32K polypeptide represents the protease involved. Fractionation of proteolytically active extract by glycerol gradient centrifugation demonstrated that 32K polypeptides do not exist as free proteins but are aggregated to the bottom-component RNA-encoded 170K, 84K, 60K, or 58K polypeptides. Maximal proteolytic activity was observed for 32K polypeptides associated with 170K polypeptides, suggesting that the activity was unstable and confined to newly synthesized molecules.

The expression of the middle-component (M) RNA of cowpea mosaic virus was studied by means of in vitro translation. In both the wheat germ extract and the rabbit reticulocyte lysate, M RNA was translated into two overlapping polypeptides of 95 and 105 kilodaltons. Incubation of these polypeptides with 30,000 x g supernatant fractions from cowpea mesophyll protoplasts inoculated with complete virus or with separate bottom (B) components alone resulted in extensive processing, yielding polypeptides of 60, 58, 48, and 47 kilodaltons. Similar proteolytic activity was found associated with the in vitro translation products from the bottom-component RNA, demonstrating that the protease present in infected cells is encoded by B RNA. Using antisera raised against the separate capsid proteins VP23 and VP37, it was shown that the 60-kilodalton cleavage product is the precursor to both capsid proteins. Cleavage of nascent 95- and 105- kilodalton polypeptides by the in vivo protease demonstrated that this capsid protein precursor is located C terminally within both polypeptides and that the synthesis of these two overlapping polypeptides is the result of two initiation sites on middle-component RNA. In addition, a second virus-induced proteolytic activity, capable of releasing VP23 from the 95- and 105-kilodalton polypeptides, was detected in leaves of infected plants, but not in infected mesophyll protoplasts. A model for the expression of the middle-component RNA is presented.