The study of root nodule symbiosis between nitrogen-fixing bacteria and leguminous plant species is important because of the ability to supplement fixed nitrogen fertilizers and increase plant growth in poor soils. Our group has isolated a mutant called nip in the model legume Medicago truncatula that is defective in nodule symbiosis. The nip mutant (numerous infections with polyphenolics) becomes infected by Sinorhizobium meliloti but then accumulates polyphenolic defense compounds in the nodule and fails to progress to a stage where nitrogen fixation can occur. Analysis of the transcriptome of nip roots prior to inoculation with rhizobia was undertaken using Affymetric Medicago Genome Array microarrays. The total RNA of 5-day old uninoculated seedlings was analyzed in triplicate to screen for the NIP gene based on downregulated transcript levels in the mutant as compared to wild type. Further microarray data was generated from 10 days post inoculation (dpi) nip and wild type plants. Analysis of the most highly downregulated transcripts revealed that the NIP gene was not identifiable based on transcript level. Putative gene function was assigned to transcripts with altered expression patterns in order to characterize the nip mutation phenotypically as inferred from the transcriptome. Functional analysis revealed a large number of chaperone proteins were highly expressed in the nip mutant, indicating high stress in the mutant prior to infection by rhizobia. Additionally, a database containing the information regarding the nip expression profile at both 0 days post inoculation (dpi) and 10 dpi were created for screening of candidate genes as predicted from sequence in the genomic region containing NIP.

To elaborate on the function(s) of the ENOD8 gene in the nodules of M. truncatula, several different experimental approaches were used. A census of the ENOD8 genes was first completed indicating that only ENOD8.1 (nt10554-12564 of GenBank AF463407) is highly expressed in nodule tissues. A maltose binding protein-ENOD8 fusion protein was made with an E. coli recombinant system. A variety of biochemical assays were undertaken with the MBP-ENOD8 recombinant protein expressed in E. coli, which did not yield the esterase activity observed for ENOD8 protein nodule fractions purified from M. sativa, tested on general esterase substrates, α-naphthyl acetate, and p-nitrophenylacetate. Attempts were also made to express ENOD8 in a Pichia pastoris system; no ENOD8 protein could be detected from Pichia pastoris strains which were transformed with the ENOD8 expression cassette. Additionally, it was shown that the ENOD8 protein can be recombinantly synthesized by Nicotiana benthamiana in a soluble form, which could be tested for activity toward esterase substrates, bearing resemblance to nodule compounds, such as the Nod factor. Transcription localization studies using an ENOD8 promoter gusA fusion indicated that ENOD8 is expressed in the bacteroid-invaded zone of the nodule. The ENOD8 protein was also detected in that same zone by immunolocalization. Confocal immunomicroscopy with an affinity-purified anti-ENOD8 oligopeptide antibody showed that the ENOD8 protein localizes at the interface between the plant and the bacteroid-differentiated rhizobia, in the symbiosome membrane or symbiosome space. This suggests a possible link between ENOD8 protein and bacteroid differentiation, nitrogen fixation, or plant defense. These possible functions for ENOD8 could be tested with an ENOD8-RNAi transgenic line devoid of detectable ENOD8 proteins.

Cloning and molecular characterization of new genes constitutes a useful approach in studying the symbiotic interactions between the model plant Medicago truncatula and Synorhizobium meliloti. Large numbers of expressed sequence tags (ESTs) available for Medicago truncatula, along with numerous cDNA, oligonucleotides, and Affimetrix DNA microarray chips, represent useful tools for gene discovery. In an attempt to identify a new gene that might be involved in the process of nodulation in Medicago truncatula, preliminary data reported by Fedorova et al. (2002), who identified 340 putative gene products or tentative consensus sequences (TCs) expressed only in nodules, was used. This research was focused on TC33166 (TC103185), which has 3 ESTs in the TC, and whose strongest BLASTX hit of TC103185 is the incomplete root hair elongation (IRE) protein kinase-like protein (NP_192429) from Arabidopsis thaliana. The Arabidopsis IRE gene is required for normal root hair growth, and a role in apical growth was suggested (Oyama et al., 2002). Infection thread growth can be looked at as an inward growth of the root hair. Thus, TC103185 was a good candidate for identifying a gene that may be involved in early events of nodulation. MtIRE (GenBank accession AC122727) is organized in 17 exons and 16 introns, similarly to the Arabidopsis IRE gene. MtIRE is a new member of the IRE family and it is a putative Ser/Thr protein kinase. MtIRE is a nodule- and flower-specific gene, suggesting that nodulation may have recruited it from other developmental processes. MtIRE is likely to be involved in the invasion process, or in the maturation of the symbiosome, or of the cells that contain rhizobia, rather than infection thread initiation and elongation or in nitrogen fixation. Nodule invasion precedes the onset of MtIRE expression and the expression pattern changes in time within the nodule. RNA interference results support MtIRE expression data and suggest a possible role in preventing extensive defense responses. Our study demonstrates the existence of an Arabidopsis IRE homolog in Medicago truncatula root nodules with an entirely new function and regulation.

Plants have many nitrate transporters; in the model legume Medicago truncatula, MtNPF1.7 is among them. MtNPF1.7 is important for M. truncatula growth and it has been established that MtNPF1.7 is a high affinity nitrate transporter. M. truncatula plants with mutations in MtNPF1.7 gene show defects during plants growth, with striking abnormalities in nodule development and root architecture. Nitrogen fixation is an energy expensive process; when legumes have sufficient bioavailable nitrogen like nitrate available, it suppresses nodulation and nitrogen fixation. Previous preliminary results in our lab showed that plants constitutively expressing MtNPF1.7 have a growth phenotype in the absence of nitrate, but no data was available on how M. truncatula plants constitutively expressing MtNPF1.7 are affected by the presence of nitrate. For my research, I confirmed the preliminary results on the growth of M. truncatula plants overexpressing NPF1.7 and examined these plants' phenotypes when nitrate was not provided in the growth media and when it was provided at two different concentrations. Compared with wild type A17, plants constitutively expressing MtNPF1.7 gene grow larger, have more lateral roots and more nodules when grown in the absence of nitrate and when 0.2 mM KNO3 was provided. At 1 mM KNO3, there are fewer differences between wild type A17 and plants constitutively expressing the MtNPF1.7 gene. Compared with wild type A17, plants constitutively expressing the MtNPF1.7 gene flower earlier, which indicates MtNPF1.7 gene may have a function in plant flowering.

Leguminous plants are able to fix nitrogen by establishing a symbiotic relationship with soil dwelling bacteria, called rhizobia. The model plant Medicago truncatula forms a partnership with Sinorhizobium meliloti whereby the plant gains bioavailable nitrogen and in exchange the bacteria gains carbohydrates. This process occurs within nodules, which are structures produced on the roots of the plants within which nitrogen is fixed. M. truncatula incomplete root elongation (MtIRE) was localized to the infection zone, which is zone II of indeterminate nodules. It was shown to encode a signaling kinase so it was anticipated to play a role in nodulation. Mutants of MtIRE in the R108 background, mutagenized with the Tnt1 retrotransposon, were obtained from reverse screen, and were assessed to determine if a disrupted MtIRE gene was the cause of nitrogen fixation defective nodules. Mutant line NF1320, having a mutant phenotype, showed typical Mendelian segregation of 3:1 when backcrossed to R108. Experimental results show that MtIRE gene is not the cause of the mutant phenotype, but was linked to the causative locus. MtIRE co-segregated with the mutant phenotype 83%. Southern blot and the first version of the M. truncatula genome (version 3.5) reported a single MtIRE gene and this was shown to be on chromosome 5 but the latest version of the M. truncatula genome (version 4.0) showed a second copy of the gene on chromosome 4. The genome sequence is based on the A17 reference genome. Both genes are 99% identical. Genetic markers that originate from flanking sequence tags (FSTs) on both chromosome 4 and 5 were tested in an attempt to find an FST that co-segregated with the mutant phenotype 100%. An FST derived from a Tnt1 insertion in Medtr4g060930 (24F) co-segregated with the mutant phenotype closely, with 76% co-segregation. Medtr4g060930 (24F) is on chromosome 4, making it likely that the Tnt1 inserted in the MtIRE gene is also on chromosome 4, and thus the defective gene is on chromosome 4.

Legumes are unique plants because they form special structures “nodules”, via symbiotic relationships with rhizobial bacteria present in the soil. Once rhizobia mature inside nodules, they fix atmospheric nitrogen providing a source of bioavailable nitrogen to the plant. To discover novel genetic components involved in the legume-rhizobia symbiosis by using forward genetic screening, we have isolated Medicago truncatula Tnt1 insertion mutants in the R108 ecotype, which are defective in nodule development and symbiotic nitrogen fixation in response to Sinorhizobium meliloti. Out of three mutants NF11044, NF11217 and NF8324, one of the mutants showed brown nodules and Fix- phenotype that is defective in symbiotic nitrogen fixation. The other two mutants showed white nodules and Fix- phenotype, also indicator of defects in symbiotic nitrogen fixation. To identify the underlying mutation causing the phenotype, we have developed molecular genetic markers by obtaining genomic sequences flanking the Tnt1 insertions by TAIL-PCR and Illumina sequencing. To carry out co-segregation analysis, back-crossed BC1F2 segregating populations were obtained. These are being phenotyped, genotyped and analyzed for co-segregation of the phenotype with the Tnt1 genetic markers. Back-crossing also has the effect of reducing the Tnt1 insertions, which are not linked to the nodulation defective phenotypes. Out of the three mutants, NF8324 harbors exactly the same insertion as in the rsd-1 Tnt1 mutant NF11265. The defect in NF11217 is caused by a Tnt1 insertion in the previously described PLC gene; the site of this insertion is close to that found in a different mutant, NF0217. For mutant NF11044, we developed linkage markers that place the defective locus on chromosome 7. To further characterize co-segregation in NF11044, a mapping population has been created by crossing the mutant with other ecotypes: A17 and A20. We tested mutants and wild type plants with linkage marker A20 X NF11044 BC1F2 that segregates 3:1(wild type: mutant). The recombination frequency ratio is similar as compared to back-crosses to ecotype R108. However, we did not observe mutant phenotypes in the A17 X NF11044 BC1F2 population. Future identification of the defective gene and functional characterization of it once it is identified will be carried out to better understand the mechanism of nodule organogenesis and symbiotic nitrogen fixation.