Erik Limpens

Medicago LYK3, an Entry Receptor in Rhizobial Nodulation Factor Signaling

Rhizobia secrete nodulation (Nod) factors, which set in motion the formation of nitrogen-fixing root nodules on legume host plants. Nod factors induce several cellular responses in root hair cells within minutes, but also are essential for the formation of infection threads by which rhizobia enter the root. Based on studies using bacterial mutants, a two-receptor model was proposed, a signaling receptor that induces early responses with low requirements toward Nod factor structure and an entry receptor that controls infection with more stringent demands. Recently, putative Nod factor receptors were shown to be LysM domain receptor kinases. However, mutants in these receptors, in both Lotus japonicus (nfr1 and nfr5) and Medicago truncatula (Medicago; nfp), do not support the two-receptor model because they lack all Nod factor-induced responses. LYK3, the putative Medicago ortholog of NFR1, has only been studied by RNA interference, showing a role in infection thread formation. Medicago hair curling (hcl) mutants are unable to form curled root hairs, a step preceding infection thread formation. We identified the weak hcl-4 allele that is blocked during infection thread growth. We show that HCL encodes LYK3 and, thus, that this receptor, besides infection, also controls root hair curling. By using rhizobial mutants, we also show that HCL controls infection thread formation in a Nod factor structure-dependent manner. Therefore, LYK3 functions as the proposed entry receptor, specifically controlling infection. Finally, we show that LYK3, which regulates a subset of Nod factor-induced genes, is not required for the induction of NODULE INCEPTION.

Strigolactone Biosynthesis in Medicago truncatula and Rice Requires the Symbiotic GRAS-Type Transcription Factors NSP1 and NSP2

This work examines the functions of the Medicago truncatula and rice GRAS-type transcription factors NSP1 and NSP2. They were found to be essential for strigolactone synthesis, possibly through direct regulation of DWARF27. Legume GRAS (GAI, RGA, SCR)-type transcription factors NODULATION SIGNALING PATHWAY1 (NSP1) and NSP2 are essential for rhizobium Nod factor-induced nodulation. Both proteins are considered to be Nod factor response factors regulating gene expression after symbiotic signaling. However, legume NSP1 and NSP2 can be functionally replaced by nonlegume orthologs, including rice (Oryza sativa) NSP1 and NSP2, indicating that both proteins are functionally conserved in higher plants. Here, we show that NSP1 and NSP2 are indispensable for strigolactone (SL) biosynthesis in the legume Medicago truncatula and in rice. Mutant nsp1 plants do not produce SLs, whereas in M. truncatula, NSP2 is essential for conversion of orobanchol into didehydro-orobanchol, which is the main SL produced by this species. The disturbed SL biosynthesis in nsp1 nsp2 mutant backgrounds correlates with reduced expression of DWARF27, a gene essential for SL biosynthesis. Rice and M. truncatula represent distinct phylogenetic lineages that split approximately 150 million years ago. Therefore, we conclude that regulation of SL biosynthesis by NSP1 and NSP2 is an ancestral function conserved in higher plants. NSP1 and NSP2 are single-copy genes in legumes, which implies that both proteins fulfill dual regulatory functions to control downstream targets after rhizobium-induced signaling as well as SL biosynthesis in nonsymbiotic conditions.

A Nodule-Specific Protein Secretory Pathway Required for Nitrogen-Fixing Symbiosis

Legume Symbiosome Leguminous plants (peas and beans) are major players in global nitrogen cycling by virtue of their symbioses with nitrogen-fixing bacteria that are harbored in specialized structures, called nodules, on the plants roots. Van de Velde et al. (p. 1122) show that the host plant, Medicago truncatula produces nodule-specific cysteine-rich peptides, resembling natural plant defense peptides. The peptides enter the bacterial cells and promote its development into the mature symbiont. In a complementary study, D. Wang et al. (p. 1126), have identified the signal peptidase, also encoded by the plant, that is required for processing these specialized peptides into their active form. Products encoded by the leguminous plant Medicago direct the differentiation of the bacterial partner in symbiosis. The nitrogen-fixing symbiosis between Sinorhizobium meliloti and its leguminous host plant Medicago truncatula occurs in a specialized root organ called the nodule. Bacteria that are released into plant cells are surrounded by a unique plant membrane compartment termed a symbiosome. We found that in the symbiosis-defective dnf1 mutant of M. truncatula, bacteroid and symbiosome development are blocked. We identified the DNF1 gene as encoding a subunit of a signal peptidase complex that is highly expressed in nodules. By analyzing data from whole-genome expression analysis, we propose that correct symbiosome development in M. truncatula requires the orderly secretion of protein constituents through coordinated up-regulation of a nodule-specific pathway exemplified by DNF1.

Single nucleus genome sequencing reveals high similarity among nuclei of an endomycorrhizal fungus

Nuclei of arbuscular endomycorrhizal fungi have been described as highly diverse due to their asexual nature and absence of a single cell stage with only one nucleus. This has raised fundamental questions concerning speciation, selection and transmission of the genetic make-up to next generations. Although this concept has become textbook knowledge, it is only based on studying a few loci, including 45S rDNA. To provide a more comprehensive insight into the genetic makeup of arbuscular endomycorrhizal fungi, we applied de novo genome sequencing of individual nuclei of Rhizophagus irregularis. This revealed a surprisingly low level of polymorphism between nuclei. In contrast, within a nucleus, the 45S rDNA repeat unit turned out to be highly diverged. This finding demystifies a long-lasting hypothesis on the complex genetic makeup of arbuscular endomycorrhizal fungi. Subsequent genome assembly resulted in the first draft reference genome sequence of an arbuscular endomycorrhizal fungus. Its length is 141 Mbps, representing over 27,000 protein-coding gene models. We used the genomic sequence to reinvestigate the phylogenetic relationships of Rhizophagus irregularis with other fungal phyla. This unambiguously demonstrated that Glomeromycota are more closely related to Mucoromycotina than to its postulated sister Dikarya.

Signaling in symbiosis

In recent years, the major focus in nodulation research has been on the genetic dissection of Nod-factor signaling. Components of this pathway appear to be shared with signaling processes that are induced during the formation of mycorrhiza. With the cloning of orthologs of the NIN and DMI2 genes from several legumes, the molecular characteristics of components of the Nod-factor-signaling pathway are now starting to be revealed. Orthologs of HAR1, a key player in the systemic autoregulatory mechanism controlling nodule numbers, have also been cloned recently. The mechanism by which nodulation is autoregulated is related to that by which fixed nitrogen inhibits nodulation. Genes that are involved in Nod-factor signaling may be targets for mechanisms that suppress nodulation. If this is the case, it would bring two fascinating areas of symbiosis together.

Rhizobium–legume symbiosis shares an exocytotic pathway required for arbuscule formation

Endosymbiotic interactions are characterized by the formation of specialized membrane compartments, by the host in which the microbes are hosted, in an intracellular manner. Two well-studied examples, which are of major agricultural and ecological importance, are the widespread arbuscular mycorrhizal symbiosis and the Rhizobium–legume symbiosis. In both symbioses, the specialized host membrane that surrounds the microbes forms a symbiotic interface, which facilitates the exchange of, for example, nutrients in a controlled manner and, therefore, forms the heart of endosymbiosis. Despite their key importance, the molecular and cellular mechanisms underlying the formation of these membrane interfaces are largely unknown. Recent studies strongly suggest that the Rhizobium–legume symbiosis coopted a signaling pathway, including receptor, from the more ancient arbuscular mycorrhizal symbiosis to form a symbiotic interface. Here, we show that two highly homologous exocytotic vesicle-associated membrane proteins (VAMPs) are required for formation of the symbiotic membrane interface in both interactions. Silencing of these Medicago VAMP72 genes has a minor effect on nonsymbiotic plant development and nodule formation. However, it blocks symbiosome as well as arbuscule formation, whereas root colonization by the microbes is not affected. Identification of these VAMP72s as common symbiotic regulators in exocytotic vesicle trafficking suggests that the ancient exocytotic pathway forming the periarbuscular membrane compartment has also been coopted in the Rhizobium–legume symbiosis.

Medicago N2-Fixing Symbiosomes Acquire the Endocytic Identity Marker Rab7 but Delay the Acquisition of Vacuolar Identity

Rhizobium bacteria form N2-fixing organelles, called symbiosomes, inside the cells of legume root nodules. The bacteria are generally thought to enter the cells via an endocytosis-like process. To examine this, we studied the identity of symbiosomes in relation to the endocytic pathway. We show that in Medicago truncatula, the small GTPases Rab5 and Rab7 are endosomal membrane identity markers, marking different (partly overlapping) endosome populations. Although symbiosome formation is considered to be an endocytosis-like process, symbiosomes do not acquire Rab5 at any stage during their development, nor do they accept the trans-Golgi network identity marker SYP4, presumed to mark early endosomes in plants. By contrast, the endosomal marker Rab7 does occur on symbiosomes from an early stage of development when they have stopped dividing up to the senescence stage. However, the symbiosomes do not acquire vacuolar SNAREs (SYP22 and VTI11) until the onset of their senescence. By contrast, symbiosomes acquire the plasma membrane SNARE SYP132 from the start of symbiosome formation throughout their development. Therefore, symbiosomes appear to be locked in a unique SYP132- and Rab7-positive endosome stage and the delay in acquiring (lytic) vacuolar identity (e.g., vacuolar SNAREs) most likely ensures their survival and maintenance as individual units.

IPD3 Controls the Formation of Nitrogen-Fixing Symbiosomes in Pea and Medicago Spp.

A successful nitrogen-fixing symbiosis requires the accommodation of rhizobial bacteria as new organelle-like structures, called symbiosomes, inside the cells of their legume hosts. Two legume mutants that are most strongly impaired in their ability to form symbiosomes are sym1/TE7 in Medicago truncatula and sym33 in Pisum sativum. We have cloned both MtSYM1 and PsSYM33 and show that both encode the recently identified interacting protein of DMI3 (IPD3), an ortholog of Lotus japonicus (Lotus) CYCLOPS. IPD3 and CYCLOPS were shown to interact with DMI3/CCaMK, which encodes a calcium- and calmodulin-dependent kinase that is an essential component of the common symbiotic signaling pathway for both rhizobial and mycorrhizal symbioses. Our data reveal a novel, key role for IPD3 in symbiosome formation and development. We show that MtIPD3 participates in but is not essential for infection thread formation and that MtIPD3 also affects DMI3-induced spontaneous nodule formation upstream of cytokinin signaling. Further, MtIPD3 appears to be required for the expression of a nodule-specific remorin, which controls proper infection thread growth and is essential for symbiosome formation.

A phylogenetic strategy based on a legume-specific whole genome duplication yields symbiotic cytokinin type-A Response Regulators

Legumes host their Rhizobium spp. symbiont in novel root organs called nodules. Nodules originate from differentiated root cortical cells that dedifferentiate and subsequently form nodule primordia, a process controlled by cytokinin. A whole-genome duplication has occurred at the root of the legume Papilionoideae subfamily. We hypothesize that gene pairs originating from this duplication event and are conserved in distinct Papilionoideae lineages have evolved symbiotic functions. A phylogenetic strategy was applied to search for such gene pairs to identify novel regulators of nodulation, using the cytokinin phosphorelay pathway as a test case. In this way, two paralogous type-A cytokinin response regulators were identified that are involved in root nodule symbiosis. Response Regulator9 (MtRR9) and MtRR11 in medicago (Medicago truncatula) and an ortholog in lotus (Lotus japonicus) are rapidly induced upon Rhizobium spp. Nod factor signaling. Constitutive expression of MtRR9 results in arrested primordia that have emerged from cortical, endodermal, and pericycle cells. In legumes, lateral root primordia are not exclusively formed from pericycle cells but also require the involvement of the root cortical cell layer. Therefore, the MtRR9-induced foci of cell divisions show a strong resemblance to lateral root primordia, suggesting an ancestral function of MtRR9 in this process. Together, these findings provide a proof of principle for the applied phylogenetic strategy to identify genes with a symbiotic function in legumes.

Cell- and Tissue-Specific Transcriptome Analyses of Medicago truncatula Root Nodules

Legumes have the unique ability to host nitrogen-fixing Rhizobium bacteria as symbiosomes inside root nodule cells. To get insight into this key process, which forms the heart of the endosymbiosis, we isolated specific cells/tissues at different stages of symbiosome formation from nodules of the model legume Medicago truncatula using laser-capture microdissection. Next, we determined their associated expression profiles using Affymetrix Medicago GeneChips. Cells were collected from the nodule infection zone divided into a distal (where symbiosome formation and division occur) and proximal region (where symbiosomes are mainly differentiating), as well as infected cells from the fixation zone containing mature nitrogen fixing symbiosomes. As non-infected cells/tissue we included nodule meristem cells and uninfected cells from the fixation zone. Here, we present a comprehensive gene expression map of an indeterminate Medicago nodule and selected genes that show specific enriched expression in the different cells or tissues. Validation of the obtained expression profiles, by comparison to published gene expression profiles and experimental verification, indicates that the data can be used as digital “in situ”. This digital “in situ” offers a genome-wide insight into genes specifically associated with subsequent stages of symbiosome and nodule cell development, and can serve to guide future functional studies.