We identified two new members of the GRAS gene family from rice, CIGR1 and CIGR2, which are rapidly induced upon N-acetylchitooligosaccharide elicitor perception. The predicated proteins encoded by CIGR1 and CIGR2 possess significant sequence similarity with previously identified members of the GRAS family, such as Arabidopsis SCARECROW, GAI, RGA, tomato Lateral suppressor, and rice SLR1, all of which have VHIID regions, likely to play a role in cellular signaling. Fusions of CIGR1 and CIGR2 with Green Fluorescent Protein were detected exclusively in the nuclei of onion epidermal cells. The expression of CIGR1 and CIGR2 was dependent on the structure of N-acetylchitooligosaccharides, which parallels the structural specificity for chitin binding to the plasma membrane-localized chitin-binding protein, and independent of de novo protein synthesis. Co-cultivation of rice cells with rice blast fungus strongly induced the expression of CIGR1 and CIGR2, whereas inoculation of suspension cells with phytopathogenic bacteria did not. We hypothesize that CIGR1 and CIGR2 act as transcriptional regulators in the early events of the elicitor-induced defense response in rice.

Various lipo-chitin molecules were tested for their ability to induce the expression of the early nodulin, ENOD2, in Glycine soja roots. When inoculated separately onto G. soja roots, LCO-V (C18:1 delta 11,Mefuc), LCO-V (C18:1 delta 9,Mefuc), LCO-V (C16:0,Mefuc), and LCO-IV (C16:0) were unable to induce ENOD2 expression, even though these compounds had previously been shown to induce root hair curling, the formation of nodule-like primordia, and induction of the early nodulin, ENOD40. ENOD2 expression, however, was induced when any two of these molecules were inoculated in combination. Thus, the lipo-chitin nodulation signals appear to act cooperatively to induce ENOD2 expression. B. japonicum strains USDA110 and USDA135 and B. elkanii strain USDA61, all symbionts of soybean, were found to produce at least two distinct nod signals ([i.e., NodBj-V[C18:1,Mefuc] and NodBj-V[C16:0,Mefuc]). These two compounds were mixed in various ratios and tested for their ability to induce ENOD2 expression. The results indicate that the former compound must be present in equivalent or excess amount in order to obtain maximum ENOD2 expression. Additional nonspecific LCOs (e.g., LCO-IV[C16:2 delta 2,9; SO3]), incapable of inducing root hair curling or cortical cell division, were tested in combination with the four active LCOs listed above. It was found that any combination of one active LCO with a nonspecific LCO was sufficient to induce ENOD2 mRNA expression. The ENOD2 mRNA expression pattern detected by in situ hybridization closely resembled that found in bacterial-induced nodules with expression detected in cortical cells between primary and secondary meristems and around the vascular strands. These data demonstrate that the cooperative action of at least two LCO nodulation signals leads to a greater progression of nodule ontogeny as demonstrated by the expression of ENOD2, a marker gene for the differentiation of nodule parenchyma.

The lipo-chitin (LCO) nodulation signal (nod signal) purified from Bradyrhizobium japonicum induced nodule primordia on soybean (i.e. Glycine soja) roots. These primordia were characterized by a bifurcated vascular connection, cortical cell division, and the accumulation of mRNA of the early nodulin gene, ENOD40. A chemically synthesized LCO identical in structure to the Nod signal purified from B. japonicum cultures showed the same activity when inoculated on to soybean roots. Surprisingly, synthetic LCO or chitin pentamer, inactive in inducing root hair curling (HAD) or cortical cell division (NOI) in G. soja, induced the transient accumulation of ENOD40 mRNA. In roots inoculated with such LCO, ENOD40 mRNA was abundant at 40 h after inoculation but decreased to the background levels 6 days after inoculation. In contrast, nod signals active in inducing HAD and NOI induced high levels of ENOD40 accumulation at 40 h and 6 days after inoculation. In situ hybridization analysis showed that ENOD40 mRNA accumulated in the pericycle of the vascular bundle at 24 h after root inoculation with nod signal. At 6 days post-inoculation with nod signal, ENOD40 expression was seen in dividing subepidermal cortical cells. These results provide morphological and molecular evidence that nodule induction in soybean in response to purified or synthetic nod signal is similar, if not identical, to nodule formation induced by bacterial inoculation. Surprisingly, ENOD40 mRNA accumulation occurs in response to non-specific chitin signals. This suggests that, in the case of ENOD40, nodulation specificity is not determined at the level of initial gene expression.

The soybean response to inoculation with B. japonicum mutants defective in cyclic beta-(1-->3), beta-(1-->6) glucan synthesis was examined by electron microscopy and by monitoring the expression of early and late nodulin genes. Two mutants were examined. Strain AB-14 is an ndvB mutant and is unable to synthesize beta-glucans. Strain AB-1 is an ndvC mutant and produces cyclic glucans containing 95 to 100% beta-(1-->3) glycosidic linkages. Nodules formed by either mutant were defective in nitrogen fixation activity. Soybean plants inoculated with strain AB-14 formed nodules roughly at the same rate as the wild-type strain USDA110, but nodulation by strain AB-1 was significantly delayed. Microscopy of nodules formed by strain AB-14 showed an overall ultrastructure similar to nodules formed by the wild type. However, in some nodules bacteroids were limited to only one part of the infected cells and in others the few bacteroids present showed signs of degradation. Nodulelike structures were formed by strain AB-1 that showed some signs of cellular differentiation. For example, clear parenchyma and sclerenchyma tissue could be seen. However, no infection threads or bacteria were evident in these structures. The expression of early (e.g., ENOD2 and ENOD55) and late (e.g., NOD26 and leghemoglobin) nodulins was examined. Nodules formed by strain AB-14 expressed all of the nodulins tested but at a reduced level. Expression of late nodulins was delayed in strain AB-14-induced nodules. Nodules formed by strain AB-1 were more strongly affected in nodulin expression. Although leghemoglobin synthesis was not detected, infection-specific nodulin transcripts of GmN93 and ENOD55 were detected. However, expression of the early nodulins ENOD2 and ENOD55 was considerably delayed and only apparent when assayed 21 days postinoculation. A low level of expression of intermediate nodulin GmN70 and late nodulin NOD26 could also be detected by 21 days postinoculation. The microscopy data show that tissue differentiation occurs in these nodules even in the absence of active bacterial invasion. These results demonstrate the importance of cyclic beta-(1-->3), beta-(1-->6) glucan synthesis to symbiotic development in soybean.