Taken together, these data indicate that MsLEC1 and MsLEC2 genes, as well as the orthologous gene MaLEC, are expressed in the youngest cells of indeterminate nodules of both alfalfa and white sweetclover. Moreover, the genes that encode these soluble lectins are also expressed in root tips. MsENOD40 gene expression. We hypothesized that expressing the lectin transgenes would not only have an effect on overall nodule phenotype, but also on downstream nodulin gene expression. We analyzed the expression of MsENOD40, which is expressed within a few hours after rhizobial inoculation . Similar to MsLEC1, MsENOD40 is expressed in the youngest cells of indeterminate nodules and also in root meristems . Accumulation of MsENOD40 RNA was significantly higher in RNA isolated from LEC1AS and LEC2AS nodules than in RNA derived from vector controls, in spite of high variability . The highest mean MsENOD40 RNA accumulation was found for LEC1AS plants, which exhibited the severest symbiotic abnormalities.Lectin concentration is very low in the roots of Phaesolus vulgaris in the absence of Rhizobium phaseoli, but infection causes an increase in the quantity of lectin in the roots . However, lectin levels remain relatively constant in pea roots upon inoculation with R. leguminosarum bv. viciae . Thus,flood and drain table the significance of changes or lack thereof in the regulation of lectin expression levels during nodulation of legume roots is unclear.
Earlier, we found that MsLEC2 mRNA accumulated in uninoculated alfalfa roots , and here, we report that its accumulation increased in the roots of nodulated plants as well, possibly due to the initiation of nodule primordia or, in the case of sweet clover, more lateral roots . Nevertheless, it is difficult to assess the importance of MsLEC2 function in the alfalfa-S. meliloti symbiosis. Most LEC2AS plants did not differ from vector controls in the symbiotic parameters examined, nor did they show any obvious alterations in nodule development or morphology. However, symbiotic parameters measured in some of the LEC2AS plants were at a level intermediate between that of the vector controls and LEC1AS plants. These data collectively suggest that MsLEC2 may have a subtle role in the alfalfa-S. meliloti symbiosis. In contrast, all of the symbiotic parameters that were examined in LEC1AS plants were clearly abnormal. In addition, MsLEC2 mRNA accumulation was up-regulated in many of the LEC1AS nodules and roots of nodulated plants. Moreover, the most abnormal LEC1AS nodule used for Northern blot analysis had the highest level of MsLEC2 mRNA accumulation. These results suggest that the MsLEC1 gene product may repress MsLEC2 expression during symbiosis and that there may be a relationship between the level of MsLEC2 expression and normal symbiotic development. The Mtlec2 gene, which is 93% homologous to MsLEC2, may be inessential for nodulation in M. truncatula because it apparently is a pseudogene.
Although the Mtlec2 promoter was active in mature Medicago varia nodules, it was not active in uninoculated or nodulated M. varia roots . In alfalfa, MsLEC3 is a pseudogene whereas MsLEC2 is not, a finding that is consistent with our results showing MsLEC2 mRNA accumulation in nodules, as well as in uninoculated and nodulated alfalfa roots. . This lectin has been proposed to function as a storage protein. We did not detect MsLEC1 mRNA accumulation in nodules using Northern blots containing total RNA, but we were able to detect it in both root and nodule meristems using in situ hybridization methods. These data correlate with those whereby a promoter-GUS fusion of the Mtlec1 gene was found to be active in mature nodules of transgenic M. varia plants but not with its localization in the nodule peripheral tissue instead of the nodule meristem . However, blue color indicating GUS expression was observed in developing nodule primordia of the M. varia Mtlec1–gusA transgenic plants . Our in situ data also demonstrated MsLEC1 mRNA accumulation in root tips, but the Mtlec1–gusA fusion was not expressed in the root meristems of transgenic M. varia. To help resolve these differences, we examined the expression of MaLEC, which codes for a putative soluble lectin, in white sweet clover. We detected MaLEC mRNA in white sweet clover nodule and root meristematic regions, suggesting that these are the main sites of expression for soluble lectin genes in these organs. The symbiotic abnormalities of LEC1AS plants are consistent with the in situ hybridization data, demonstrating that MsLEC1 is expressed in alfalfa nodules. It appears that MsLEC1 expression at the correct level at nodule initiation and in cells in zones I and II of the nodule may be important for regulation of nodule number , as well as for the regulation of nodule size and persistence . Interestingly, the antisense-MsLEC1 mRNA also accumulated in nodules at almost undetectable levels , in spite of its transcription being driven by the strong cauliflower mosaic virus 35S promoter.
These results suggest that the accumulation of the antisense-MsLEC1 mRNA is regulated in some unknown manner. The abnormally large number and size of nodules seen on LEC1AS plants were unexpected. Based on studies in which lectins promoted nodulation and nodulation-related responses , we predicted that smaller, uninfected nodules would have developed. Indeed, the majority of nodules produced by LEC1AS plants were small, undeveloped, and senesced prematurely . However, although infection thread formation appeared normal, at least based on the organized arrangement of rhizobia in the curled root hairs , it is not known whether or not the premature senescence exhibited by the LEC1AS nodules is due to a lack of MsLEC1 transcript accumulation in nodule meristematic tissues or to a defect in persistence of the infection threads. The latter seems less likely because the rhizobia were not affected by their course through the nodules; large numbers of bacteria were recovered from both LEC1AS and LEC1ST nodules . Based on our previous studies, when the MsLEC1 gene is disturbed, a disorganized proliferation of embryonic and vegetative tissues results . In this report, we have shown that following inoculation with Rm1021, the LEC1AS transgenic root nodules that result are also highly aberrant. In contrast, no abnormal vegetative or reproductive development was detected in the LEC1ST plants , although some abnormal nodulation was observed. This result is compatible with the finding that sensesuppression-induced symbiotic abnormalities are usually milder than those from antisense suppression and further suggests that symbiotic processes may be more sensitive to alterations in MsLEC1 expression. In addition to finding that MsLEC2 gene expression was upregulated in the LEC1AS transgene-containing tissues, we also found that MsENOD40, an early nodulin gene, was expressed at relatively high levels in LEC1AS nodules. The MsENOD40 gene has been shown to be up-regulated in response to cytokinin application, and it has been proposed that nodule development may be influenced by changes in the endogenous cytokinin to auxin ratio . LEC1AS plants exhibit excessive nodule formation, a result that is consistent with an increase in the level or responsiveness to cytokinin. Moreover, LEC1AS plantlets frequently formed severe teratomas with minimal root development , and mature LEC1AS roots were poorly developed , further suggesting an excessive cytokinin response. A mechanism whereby lectin could mediate phytohormone levels and interactions is hypothetical at this time, but hydrophobic ligands, including auxins and cytokinins,rolling bench are known to bind to some soluble legume lectins, albeit to sites independent of the sugar-binding site . Taken together, our findings indicate that the expression of the MsLEC1 and MsLEC2 genes, especially the MsLEC1 gene, is important in the compatible symbiotic interaction between alfalfa and S. meliloti. This hypothesis is consistent with both lectin gain-of-function and loss-of-function experiments. How lectins promote compatible symbiotic interactions is unclear, particularly because lectins with similarity to legume lectins have been found in plant families in addition to the Fabaceae . In Arabidopsis moreover, numerous genes encoding receptor kinases with legume lectin domains have been uncovered , and similar proteins have now been identified in Medicago truncatula . The finding that other plant families have genes that encode proteins with legume lectin domains implies that legume lectins are derived from a lectin gene that was already present in an ancestral flowering plant . Indeed, many proteins that appear to be specific to the legumeRhizobium interaction seem to be recruited from proteins that are common to both legumes and nonlegumes, e.g., NORKand HAR1/ NARK . NORK extracellular sequencelike genes are found not only in nonlegumes, including several grasses and Arabidopsis, but also in a gymnosperm . Similarly, HAR1/NARK genes are very similar to CLAVATA1, a serinethreonine kinase that is important for restricting the floral meristem in Arabidopsis.
It is clear that duplication of NSL, CLAVATA1, and other genes found in nonlegumes has taken place, along with a specialization of their respective proteins for the Rhizobium-legume symbiosis. Gene duplication events often result in the development of new functions for the new proteins. Thus, what makes the rhizobialegume interaction specific may rely more on the details of the interactions between various legume proteins including lectinsand their ligands. Finding a lectinless mutant in an indeterminate nodule-forming legume such as white sweetclover, which appears to have only one gene coding for a soluble lectin, might be one strategy for testing this hypothesis. Alternatively, introducing a gene for a legume lectin, e.g., SBL or PSL into a nonlegume such as Arabidopsis, may also help elucidate whether or not legume lectins can promote colonization of a nonlegume root by rhizobia. Construction of transgenic plants was described previously . Briefly, the MsLEC1 transgene contained 420 bp of DNA encompassing the 3 portion of the open reading frame plus 76 bp of predicted 3 untranslated region. The MsLEC2 transgene contained 400 bp from the 5 portion of the open reading frame beginning 74 bp downstream of the predicted initiator codon . Sense or antisense orientations of transgenes were confirmed using DNA sequence analysis. The CaMV 35S promoter drove transcription of all the transgenes. One plant line of alfalfa cv. Regen SY was used for transformation and regeneration of dozens of independent transformant lines of LEC1AS, LEC1ST, LEC2AS and LEC2ST plants, some of which were grown to maturity for use in nodulation assays. Further control lines containing only the vector used for lectintransgene plants but lacking inserted genes following the promoter were also constructed and were used in nodulation assays. Stable transgene integration and activity, as well as transgene-specific phenotypic effects, have been clearly demonstrated . For nodulation tests, stem cuttings of the transgenic alfalfa plants were placed in sterile 11-liter pans that contained 6 liters of a 1:1 mix of perlite/vermiculite saturated with 2.5 liters of complete Hoagland’s 1 /4-strength nutrient solution and were allowed to root. Stem cuttings were from independent, primary transformant lines because they demonstrated developmental abnormalities that were very similar to progeny resulting from selfing . However, because alfalfa is an outcrossing tetraploid and shows inbreeding depression, it was difficult to obtain progeny plants that survived to maturity. The cuttings were transferred to sterilized Magenta jars containing a similar mix of perlite and vermiculite watered with Hoagland’s 1 /4-strength nutrient solution minus nitrogen. A 5-ml suspension of Sinorhizobium meliloti wild-type strain 1021 cells at an optical density of 600 nm equal to approximately 0.1 to 0.2, labeled either with GUS or with GFP , was added to the Magenta jars after the bacteria were rinsed and diluted in sterile water. One and two weeks after inoculation, the roots were carefully removed from the Magenta jars, were rinsed, and were prepared either for GUS-staining or for viewing under a Zeiss Axiophot fluorescent microscope. Stem cuttings of the transgenic alfalfa plants were made as described above and allowed to root. Cuttings were placed in pots with approximately 400 cm3 of potting soil in a greenhouse. One week before inoculation, nitrogen nutrition was withdrawn from the plants, but other macronutrients were supplied. The potting soil was leached with large quantities of tap water four and one days before inoculation. Rm1021 cells were grown in RDM medium , containing 100 mg of streptomycin per liter to an OD600 of 0.11 or 0.13, depending on the experiment. Rhizobia were pelleted in a clinical centrifuge and were suspended in sterile milli-Q water to an OD600 of 0.1 . Rm1021 suspension was placed on the surface of the potting soil of each plant.