Central cell-derived peptides regulate early embryo patterning in flowering plants.

Plant embryogenesis initiates with the establishment of an apical-basal axis; however, the molecular mechanisms accompanying this early event remain unclear. Here, we show that a small cysteine-rich peptide family is required for formation of the zygotic basal cell lineage and proembryo patterning in Arabidopsis. EMBRYO SURROUNDING FACTOR 1 (ESF1) peptides accumulate before fertilization in central cell gametes and thereafter in embryo-surrounding endosperm cells. Biochemical and structural analyses revealed cleavage of ESF1 propeptides to form biologically active mature peptides. Further, these peptides act in a non-cell-autonomous manner and synergistically with the receptor-like kinase SHORT SUSPENSOR to promote suspensor elongation through the YODA mitogen-activated protein kinase pathway. Our findings demonstrate that the second female gamete and its sexually derived endosperm regulate early embryonic patterning in flowering plants.

Patterns of divergence of a large family of nodule cysteine-rich peptides in accessions of Medicago truncatula.

The nodule cysteine-rich (NCR) groups of defensin-like (DEFL) genes are one of the largest gene families expressed in the nodules of some legume plants. They have only been observed in the inverted repeat loss clade (IRLC) of legumes, which includes the model legume Medicago truncatula. NCRs are reported to play an important role in plant-microbe interactions. To understand their diversity we analyzed their expression and sequence polymorphisms among four accessions of M. truncatula. A significant expression and nucleotide variation was observed among the genes. We then used 26 accessions to estimate the selection pressures shaping evolution among the accessions by calculating the nucleotide diversity at non-synonymous and synonymous sites in the coding region. The mature peptides of the orthologous NCRs had signatures of both purifying and diversifying selection pressures, unlike the seed DEFLs, which predominantly exhibited purifying selection. The expression, sequence variation and apparent diversifying selection in NCRs within the Medicago species indicates rapid and recent evolution, and suggests that this family of genes is actively evolving to adapt to different environments and is acquiring new functions.

Regulatory patterns of a large family of defensin-like genes expressed in nodules of Medicago truncatula.

Root nodules are the symbiotic organ of legumes that house nitrogen-fixing bacteria. Many genes are specifically induced in nodules during the interactions between the host plant and symbiotic rhizobia. Information regarding the regulation of expression for most of these genes is lacking. One of the largest gene families expressed in the nodules of the model legume Medicago truncatula is the nodule cysteine-rich (NCR) group of defensin-like (DEFL) genes. We used a custom Affymetrix microarray to catalog the expression changes of 566 NCRs at different stages of nodule development. Additionally, bacterial mutants were used to understand the importance of the rhizobial partners in induction of NCRs. Expression of early NCRs was detected during the initial infection of rhizobia in nodules and expression continued as nodules became mature. Late NCRs were induced concomitantly with bacteroid development in the nodules. The induction of early and late NCRs was correlated with the number and morphology of rhizobia in the nodule. Conserved 41 to 50 bp motifs identified in the upstream 1,000 bp promoter regions of NCRs were required for promoter activity. These cis-element motifs were found to be unique to the NCR family among all annotated genes in the M. truncatula genome, although they contain sub-regions with clear similarity to known regulatory motifs involved in nodule-specific expression and temporal gene regulation.

Spatio-temporal expression patterns of Arabidopsis thaliana and Medicago truncatula defensin-like genes.

Plant genomes contain several hundred defensin-like (DEFL) genes that encode short cysteine-rich proteins resembling defensins, which are well known antimicrobial polypeptides. Little is known about the expression patterns or functions of many DEFLs because most were discovered recently and hence are not well represented on standard microarrays. We designed a custom Affymetrix chip consisting of probe sets for 317 and 684 DEFLs from Arabidopsis thaliana and Medicago truncatula, respectively for cataloging DEFL expression in a variety of plant organs at different developmental stages and during symbiotic and pathogenic associations. The microarray analysis provided evidence for the transcription of 71% and 90% of the DEFLs identified in Arabidopsis and Medicago, respectively, including many of the recently annotated DEFL genes that previously lacked expression information. Both model plants contain a subset of DEFLs specifically expressed in seeds or fruits. A few DEFLs, including some plant defensins, were significantly up-regulated in Arabidopsis leaves inoculated with Alternaria brassicicola or Pseudomonas syringae pathogens. Among these, some were dependent on jasmonic acid signaling or were associated with specific types of immune responses. There were notable differences in DEFL gene expression patterns between Arabidopsis and Medicago, as the majority of Arabidopsis DEFLs were expressed in inflorescences, while only a few exhibited root-enhanced expression. By contrast, Medicago DEFLs were most prominently expressed in nitrogen-fixing root nodules. Thus, our data document salient differences in DEFL temporal and spatial expression between Arabidopsis and Medicago, suggesting distinct signaling routes and distinct roles for these proteins in the two plant species.

Expression of coordinately regulated defence response genes and analysis of their role in disease resistance in Medicago truncatula.

Microarray technology was used to identify the genes associated with disease defence responses in the model legume Medicago truncatula. Transcript profiles from M. truncatula cv. Jemalong genotype A17 leaves inoculated with Colletotrichum trifolii and Erysiphe pisi and roots infected with Phytophthora medicaginis were compared to identify the genes expressed in response to all three pathogens and genes unique to an interaction. The A17 genotype is resistant to C. trifolii and E. pisi, exhibiting a hypersensitive response after inoculation, and is moderately susceptible to P. medicaginis. Among the most strongly up-regulated genes in all three interactions were those encoding a hevein-like protein, thaumatin-like protein (TLP) and members of the pathogenesis response (PR)10 family. Transcripts of genes for enzymes in the phenylpropanoid pathway leading to the production of isoflavonoid phytoalexins increased dramatically in response to inoculation with the foliar pathogens. In P. medicaginis-inoculated roots, transcripts of genes in the phenylpropanoid pathway peaked at 5 days post-inoculation, when symptoms became visible. Transcript accumulation of three PR10 family members, a TLP and chalcone synthase (CHS) was assessed in M. truncatula genotype R108 plants. The R108 plants are resistant to C. trifolii and moderately susceptible to E. pisi and P. medicaginis. Transcript accumulation paralleled the stages of pathogen development. To evaluate the role of a TLP, a PR10 family member and CHS in disease resistance, transgenic R108 plants containing interfering RNA (RNAi) constructs were produced. Reduced expression of PR10 and TLP had no effect on the disease phenotype, whereas reduced expression of CHS resulted in increased susceptibility to necrotrophic pathogens.

Transcript profiling of two alfalfa genotypes with contrasting cell wall composition in stems using a cross-species platform: optimizing analysis by masking biased probes.

BACKGROUND: The GeneChip(R) Medicago Genome Array, developed for Medicago truncatula, is a suitable platform for transcript profiling in tetraploid alfalfa [Medicago sativa (L.) subsp. sativa]. However, previous research involving cross-species hybridization (CSH) has shown that sequence variation between two species can bias transcript profiling by decreasing sensitivity (number of expressed genes detected) and the accuracy of measuring fold-differences in gene expression. RESULTS: Transcript profiling using the Medicago GeneChip(R) was conducted with elongating stem (ES) and post-elongation stem (PES) internodes from alfalfa genotypes 252 and 1283 that differ in stem cell wall concentrations of cellulose and lignin. A protocol was developed that masked probes targeting inter-species variable (ISV) regions of alfalfa transcripts. A probe signal intensity threshold was selected that optimized both sensitivity and accuracy. After masking for both ISV regions and previously identified single-feature polymorphisms (SFPs), the number of differentially expressed genes between the two genotypes in both ES and PES internodes was approximately 2-fold greater than the number detected prior to masking. Regulatory genes, including transcription factor and receptor kinase genes that may play a role in development of secondary xylem, were significantly over-represented among genes up-regulated in 252 PES internodes compared to 1283 PES internodes. Several cell wall-related genes were also up-regulated in genotype 252 PES internodes. Real-time quantitative RT-PCR of differentially expressed regulatory and cell wall-related genes demonstrated increased sensitivity and accuracy after masking for both ISV regions and SFPs. Over 1,000 genes that were differentially expressed in ES and PES internodes of genotypes 252 and 1283 were mapped onto putative orthologous loci on M. truncatula chromosomes. Clustering simulation analysis of the differentially expressed genes suggested co-expression of some neighbouring genes on Medicago chromosomes. CONCLUSIONS: The problems associated with transcript profiling in alfalfa stems using the Medicago GeneChip as a CSH platform were mitigated by masking probes targeting ISV regions and SFPs. Using this masking protocol resulted in the identification of numerous candidate genes that may contribute to differences in cell wall concentration and composition of stems of two alfalfa genotypes.

Knockdown of CELL DIVISION CYCLE16 reveals an inverse relationship between lateral root and nodule numbers and a link to auxin in Medicago truncatula.

The postembryonic development of lateral roots and nodules is a highly regulated process. Recent studies suggest the existence of cross talk and interdependency in the growth of these two organs. Although plant hormones, including auxin and cytokinin, appear to be key players in coordinating this cross talk, very few genes that cross-regulate root and nodule development have been uncovered so far. This study reports that a homolog of CELL DIVISION CYCLE16 (CDC16), a core component of the Anaphase Promoting Complex, is one of the key mediators in controlling the overall number of lateral roots and nodules. A partial suppression of this gene in Medicago truncatula leads to a decrease in number of lateral roots and a 4-fold increase in number of nodules. The roots showing lowered expression of MtCDC16 also show reduced sensitivity to phytohormone auxin, thus providing a potential function of CDC16 in auxin signaling.

Physiological and molecular characterization of aluminum resistance in Medicago truncatula.

BACKGROUND: Aluminum (Al) toxicity is an important factor limiting crop production on acid soils. However, little is known about the mechanisms by which legumes respond to and resist Al stress. To explore the mechanisms of Al toxicity and resistance in legumes, we compared the impact of Al stress in Al-resistant and Al-sensitive lines of the model legume, Medicago truncatula Gaertn. RESULTS: A screen for Al resistance in 54 M. truncatula accessions identified eight Al-resistant and eight Al-sensitive lines. Comparisons of hydroponic root growth and root tip hematoxylin staining in an Al-resistant line, T32, and an Al-sensitive line, S70, provided evidence that an inducible Al exclusion mechanism occurs in T32. Transcriptional events associated with the Al resistance response were analyzed in T32 and S70 after 12 and 48 h Al treatment using oligonucleotide microarrays. Fewer genes were differentially regulated in response to Al in T32 compared to S70. Expression patterns of oxidative stress-related genes, stress-response genes and microscopic examination of Al-treated root tips suggested a lower degree of Al-induced oxidative damage to T32 root tips compared to S70. Furthermore, genes associated with cell death, senescence, and cell wall degradation were induced in both lines after 12 h of Al treatment but preferentially in S70 after 48 h of Al treatment. A multidrug and toxin efflux (MATE) transporter, previously shown to exude citrate in Arabidopsis, showed differential expression patterns in T32 and S70. CONCLUSION: Our results identified novel genes induced by Al in Al-resistant and sensitive M. truncatula lines. In T32, transcription levels of genes related to oxidative stress were consistent with reactive oxygen species production, which would be sufficient to initiate cell death of Al-accumulating cells thereby contributing to Al exclusion and root growth recovery. In contrast, transcriptional levels of oxidative stress-related genes were consistent with excessive reactive oxygen species accumulation in S70 potentially resulting in necrosis and irreversible root growth inhibition. In addition, a citrate-exuding MATE transporter could function in Al exclusion and/or internal detoxification in T32 based on Al-induced transcript localization studies. Together, our findings indicate that multiple responses likely contribute to Al resistance in M. truncatula.

Transcriptome profiling identified novel genes associated with aluminum toxicity, resistance and tolerance in Medicago truncatula.

Oligonucleotide microarrays corresponding to over 16,000 genes were used to analyze changes in transcript accumulation in root tips of the Al-sensitive Medicago truncatula cultivar Jemalong genotype A17 in response to Al treatment. Out of 2,782 genes with significant changes in transcript accumulation, 324 genes were up-regulated and 267 genes were down-regulated at least twofold by Al. Up-regulated genes were enriched in transcripts involved in cell-wall modification and abiotic and biotic stress responses while down-regulated genes were enriched in transcripts involved in primary metabolism, secondary metabolism, protein synthesis and processing, and the cell cycle. Known markers of Al-induced gene expression including genes associated with oxidative stress and cell wall stiffening were differentially regulated in this study. Transcript profiling identified novel genes associated with processes involved in Al toxicity including cell wall modification, cell cycle arrest and ethylene production. Novel genes potentially associated with Al resistance and tolerance in M. truncatula including organic acid transporters, cell wall loosening enzymes, Ca(2+) homeostasis maintaining genes, and Al-binding were also identified. In addition, expression analysis of nine genes in the mature regions of the root revealed that Al-induced gene expression in these regions may play a role in Al tolerance. Finally, interfering RNA-induced silencing of two Al-induced genes, pectin acetylesterase and annexin, in A17 hairy roots slightly increased the sensitivity of A17 to Al suggesting that these genes may play a role in Al resistance.

The transcription factor MtSERF1 of the ERF subfamily identified by transcriptional profiling is required for somatic embryogenesis induced by auxin plus cytokinin in Medicago truncatula.

Transcriptional profiling of embryogenic callus produced from Medicago truncatula mesophyll protoplasts indicated up-regulation of ethylene biosynthesis and ethylene response genes. Using inhibitors of ethylene biosynthesis and perception, it was shown that ethylene was necessary for somatic embryogenesis (SE) in this model legume. We chose several genes involved in ethylene biosynthesis and response for subsequent molecular analyses. One of these genes is a gene encoding a transcription factor that belongs to the AP2/ERF superfamily and ERF subfamily of transcription factors. We demonstrate that this gene, designated M. truncatula SOMATIC EMBRYO RELATED FACTOR1 (MtSERF1), is induced by ethylene and is expressed in embryogenic calli. MtSERF1 is strongly expressed in the globular somatic embryo and there is high expression in a small group of cells in the developing shoot meristem of the heart-stage embryo. RNA interference knockdown of this gene causes strong inhibition of SE. We also provide evidence that MtSERF1 is expressed in zygotic embryos. MtSERF1 appears to be essential for SE and may enable a connection between stress and development.

Differential response of the plant Medicago truncatula to its symbiont Sinorhizobium meliloti or an exopolysaccharide-deficient mutant.

Sinorhizobium meliloti forms symbiotic, nitrogen-fixing nodules on the roots of Medicago truncatula. The bacteria invade and colonize the roots through structures called infection threads. S. meliloti unable to produce the exopolysaccharide succinoglycan are unable to establish a symbiosis because they are defective in initiating the production of infection threads and in invading the plant. Here, we use microarrays representing 16,000 M. truncatula genes to compare the differential transcriptional responses of this host plant to wild-type and succinoglycan-deficient S. meliloti at the early time point of 3 days postinoculation. This report describes an early divergence in global plant gene expression responses caused by a rhizobial defect in succinoglycan production, rather than in Nod factor production. The microarray data show that M. truncatula inoculated with wild-type, succinoglycan-producing S. meliloti more strongly express genes encoding translation components, protein degradation machinery, and some nodulins than plants inoculated with succinoglycan-deficient bacteria. This finding is consistent with wild-type-inoculated plants having received a signal, distinct from the well characterized Nod factor, to alter their metabolic activity and prepare for invasion. In contrast, M. truncatula inoculated with succinoglycan-deficient S. meliloti more strongly express an unexpectedly large number of genes in two categories: plant defense responses and unknown functions. One model consistent with our results is that appropriate symbiotically active exopolysaccharides act as signals to plant hosts to initiate infection thread formation and that, in the absence of this signal, plants terminate the infection process, perhaps via a defense response.

Small cysteine-rich peptides resembling antimicrobial peptides have been under-predicted in plants.

Multicellular organisms produce small cysteine-rich antimicrobial peptides as an innate defense against pathogens. While defensins, a well-known class of such peptides, are common among eukaryotes, there are other classes restricted to the plant kingdom. These include thionins, lipid transfer proteins and snakins. In earlier work, we identified several divergent classes of small putatively secreted cysteine-rich peptides (CRPs) in legumes [Graham et al. (2004)Plant Physiol. 135, 1179-97]. Here, we built sequence motif models for each of these classes of peptides, and iteratively searched for related sequences within the comprehensive UniProt protein dataset, the Institute for Genomic Research's 33 plant gene indices, and the entire genomes of the model dicot, Arabidopsis thaliana, and the model monocot and crop species, Oryza sativa (rice). Using this search strategy, we identified approximately 13,000 plant genes encoding peptides with common features: (i) an N-terminal signal peptide, (ii) a small divergent charged or polar mature peptide with conserved cysteines, (iii) a similar intron/exon structure, (iv) spatial clustering in the genomes studied, and (v) overrepresentation in expressed sequences from reproductive structures of specific taxa. The identified genes include classes of defensins, thionins, lipid transfer proteins, and snakins, plus other protease inhibitors, pollen allergens, and uncharacterized gene families. We estimate that these classes of genes account for approximately 2-3% of the gene repertoire of each model species. Although 24% of the genes identified were not annotated in the latest Arabidopsis genome releases (TIGR5, TAIR6), we confirmed expression via RT-PCR for 59% of the sequences attempted. These findings highlight limitations in current annotation procedures for small divergent peptide classes.

A transient decrease in reactive oxygen species in roots leads to root hair deformation in the legume-rhizobia symbiosis.

* A possible role for reactive oxygen species (ROS) in root hair deformation in response to Nod factor (NF) was investigated using Medicago truncatula nodulation mutants, and an inhibitor and precursors of ROS. * In wild-type roots, ROS efflux transiently decreased approximately 1 h after NF treatment. Transcript accumulation of two NADPH oxidase homologs, respiratory burst oxidase homolog 2 (MtRBOH2) and MtRBOH3, also transiently decreased at 1 h. However, in the nonnodulating mutant Nod factor perception (nfp), transcript accumulation did not change. * Exogenous application of ROS prevented root hair swelling and branching induced by NF. When accumulation of ROS was prevented by diphenylene iodonium (DPI), NF did not induce root hair branching. Root treatment with DPI alone reduced ROS efflux and induced root hair tip swelling. Transient treatment of roots with DPI mimicked NF treatment and resulted in root hair branching in the absence of NF. A transient DPI treatment did not induce root hair branching in the nonlegumes Arabidopsis thaliana and tomato (Lycopersicon esculentum). * The results suggest a role for the transient reduction of ROS accumulation in governing NF-induced root hair deformation in legumes.

Molecular and cytological responses of Medicago truncatula to Erysiphe pisi.

SUMMARY Powdery mildew is an economically important disease in a number of crop legumes; however, little is known about resistance to the disease in these species. To gain a better understanding of the genetics of resistance and plant responses to powdery mildew in legumes, we developed a pathosystem with Medicago truncatula and Erysiphe pisi. Screening accessions of M. truncatula identified genotypes that are highly susceptible, moderately resistant and highly resistant to the fungus. In the highly resistant genotype, fungal growth was arrested after appressorium development with no colony formation, while in the moderately resistant genotype a small number of colonies formed. Both resistant and moderately resistant genotypes produced hydrogen peroxide and fluorescent compounds at pathogen penetration sites, consistent with a hypersensitive response (HR), although the response was delayed in the moderately resistant genotype. Very little hydrogen peroxide or fluorescence was detected in the susceptible accession. Microarray analysis of E. pisi-induced early transcriptional changes detected 55 genes associated with the basal defence response that were similarly regulated in all three genotypes. These included pathogenesis-related genes and other genes involved in defence, signal transduction, senescence, cell wall metabolism and abiotic stress. Genes associated with the HR response included flavonoid pathway genes, and others involved in transport, transcription regulation and signal transduction. A total of 34 potentially novel unknown genes, including two legume-specific genes, were identified in both the basal response and the HR categories. Potential binding sites for two defence-related transcription regulators, Myb and Whirly, were identified in promoter regions of induced genes, and four novel motifs were found in promoter regions of genes repressed in the resistant interaction.

Novel paralogous gene families with potential function in legume nodules and seeds.

Within the plant kingdom, legumes are unusual in their ability to form nitrogen-fixing nodules in symbiosis with certain bacteria in the family Rhizobiaceae (rhizhobia). Genes that are required for signaling between plant and symbiont, and for the development and maintenance of the nodule, were either created de novo or adopted from other plant pathways. Only in recent years have genome-scale sequence data from legumes made it possible to identify large, novel families of genes probably evolved to function in nodulation. Members of these novel families are expressed in seeds or nodules, and are homologous to defense-related proteins. Perhaps the most striking example is a large family (of more than 340 members) of cysteine cluster proteins that have homology to plant defensins.

Transcript analysis of early nodulation events in Medicago truncatula.

Within the first 72 h of the interaction between rhizobia and their host plants, nodule primordium induction and infection occur. We predicted that transcription profiling of early stages of the symbiosis between Medicago truncatula roots and Sinorhizobium meliloti would identify regulated plant genes that likely condition key events in nodule initiation. Therefore, using a microarray with about 6,000 cDNAs, we compared transcripts from inoculated and uninoculated roots corresponding to defined stages between 1 and 72 h post inoculation (hpi). Hundreds of genes of both known and unknown function were significantly regulated at these time points. Four stages of the interaction were recognized based on gene expression profiles, and potential marker genes for these stages were identified. Some genes that were regulated differentially during stages I (1 hpi) and II (6-12 hpi) of the interaction belong to families encoding proteins involved in calcium transport and binding, reactive oxygen metabolism, and cytoskeleton and cell wall functions. Genes involved in cell proliferation were found to be up-regulated during stages III (24-48 hpi) and IV (72 hpi). Many genes that are homologs of defense response genes were up-regulated during stage I but down-regulated later, likely facilitating infection thread progression into the root cortex. Additionally, genes putatively involved in signal transduction and transcriptional regulation were found to be differentially regulated in the inoculated roots at each time point. The findings shed light on the complexity of coordinated gene regulation and will be useful for continued dissection of the early steps in symbiosis.

RNA interference identifies a calcium-dependent protein kinase involved in Medicago truncatula root development.

Changes in cellular or subcellular Ca2+ concentrations play essential roles in plant development and in the responses of plants to their environment. However, the mechanisms through which Ca2+ acts, the downstream signaling components, as well as the relationships among the various Ca2+-dependent processes remain largely unknown. Using an RNA interference-based screen for gene function in Medicago truncatula, we identified a gene that is involved in root development. Silencing Ca2+-dependent protein kinase1 (CDPK1), which is predicted to encode a Ca2+-dependent protein kinase, resulted in significantly reduced root hair and root cell lengths. Inactivation of CDPK1 is also associated with significant diminution of both rhizobial and mycorrhizal symbiotic colonization. Additionally, microarray analysis revealed that silencing CDPK1 alters cell wall and defense-related gene expression. We propose that M. truncatula CDPK1 is a key component of one or more signaling pathways that directly or indirectly modulates cell expansion or cell wall synthesis, possibly altering defense gene expression and symbiotic interactions.

Genome organization of more than 300 defensin-like genes in Arabidopsis.

Defensins represent an ancient and diverse set of small, cysteine-rich, antimicrobial peptides in mammals, insects, and plants. According to published accounts, most species' genomes contain 15 to 50 defensins. Starting with a set of largely nodule-specific defensin-like sequences (DEFLs) from the model legume Medicago truncatula, we built motif models to search the near-complete Arabidopsis (Arabidopsis thaliana) genome. We identified 317 DEFLs, yet 80% were unannotated at The Arabidopsis Information Resource and had no prior evidence of expression. We demonstrate that many of these DEFL genes are clustered in the Arabidopsis genome and that individual clusters have evolved from successive rounds of gene duplication and divergent or purifying selection. Sequencing reverse transcription-PCR products from five DEFL clusters confirmed our gene predictions and verified expression. For four of the largest clusters of DEFLs, we present the first evidence of expression, most frequently in floral tissues. To determine the abundance of DEFLs in other plant families, we used our motif models to search The Institute for Genomic Research's gene indices and identified approximately 1,100 DEFLs. These expressed DEFLs were found mostly in reproductive tissues, consistent with our reverse transcription-PCR results. Sequence-based clustering of all identified DEFLs revealed separate tissue- or taxon-specific subgroups. Previously, we and others showed that more than 300 DEFL genes were expressed in M. truncatula nodules, organs not present in most plants. We have used this information to annotate the Arabidopsis genome and now provide evidence of a large DEFL superfamily present in expressed tissues of all sequenced plants.

Grafting between model legumes demonstrates roles for roots and shoots in determining nodule type and host/rhizobia specificity.

Previous grafting experiments have demonstrated that legume shoots play a critical role in symbiotic development of nitrogen-fixing root nodules by regulating nodule number. Here, reciprocal grafting experiments between the model legumes Lotus japonicus and Medicago truncatula were carried out to investigate the role of the shoot in the host-specificity of legume-rhizobia symbiosis and nodule type. Lotus japonicus is nodulated by Mesorhizobium loti and makes determinate nodules, whereas M. truncatula is nodulated by Sinorhizobium meliloti and makes indeterminate nodules. When inoculated with M. loti, L. japonicus roots grafted on M. truncatula shoots produced determinate nodules identical in appearance to those produced on L. japonicus self-grafted roots. Moreover, the hypernodulation phenotype of L. japonicus har1-1 roots grafted on wild-type M. truncatula shoots was restored to wild type when nodulated with M. loti. Thus, L. japonicus shoots appeared to be interchangeable with M. truncatula shoots in the L. japonicus root/M. loti symbiosis. However, M. truncatula roots grafted on L. japonicus shoots failed to induce nodules after inoculation with S. meliloti or a mixture of S. meliloti and M. loti. Instead, only early responses to S. meliloti such as root hair tip swelling and deformation, plus induction of the early nodulation reporter gene MtENOD11:GUS were observed. The results indicate that the L. japonicus shoot does not support normal symbiosis between the M. truncatula root and its microsymbiont S. meliloti, suggesting that an unidentified shoot-derived factor may be required for symbiotic progression in indeterminate nodules.