Jieyu Liu

Nitrogen-fixing nodule symbiosis is an ecologically and economically important trait in legumes and some related species. A critical step in the evolution of nodulation is the recruitment of NODULE INCEPTION (NIN); a homolog of the nitrate-sensing NIN-LIKE PROTEIN (NLP) transcription factors. However, whether adaptations have occurred in the NIN protein upon its recruitment in symbiosis remains elusive. Here we show that non-symbiotic NIN orthologs can function in intracellular infection and even nodule initiation, indicating that these properties of NIN predate the evolution of nodulation. Concurrent with the evolution of nodulation, symbiotic NIN proteins were optimized for their role in symbiosis by acquiring nitrate independent functionality, including constitutive nuclear localization. A single amino acid substitution in the non-symbiotic Arabidopsis AtNLP2 enhances its nuclear localization under low nitrate conditions, making it functionally comparable to the symbiotic Parasponia PanNIN. Our study provides insight in the evolutionary trajectory and molecular adaptation that allowed NIN to function as the central regulator of nitrogen-fixing nodule symbiosis.

The hallmark of the legume lncRNA EARLY NODULIN40 ( ENOD40 ), involved in rhizobium-induced nodulation, is the presence of a highly conserved stretch of 24 nucleotides, designated box2, preceded by a small open-reading-frame (sORF) coding for a peptide of 12 to 13 amino acids. Although there is a well-established link between ENOD40 and nodulation, it is not fully clear by which mechanism ENOD40 functions in this process. Here, we show that a region harboring box2 can complement nodule formation in an ENOD40 knock-out mutant ( enod40-1-2/1 ). The sequence of box2 bears the characteristics of a miR169 target mimic. We show that the artificial target mimic MIM169defg can indeed complement the reduced capability of nodule formation in enod40-1-2/1 , and that box2 exhibits target mimic activity in a transient luciferase assay. In addition, the introduction of a miR169 -resistant form of MtNF-YA1 also elevates the capacity to form nodules in enod40-1-2/1. We conclude that ENOD40 effectuates nodule initiation by posttranscriptional upregulation of the miR169 target NF-YA1 , which encodes an essential transcription factor in this step of the nodulation process.

The importance of auxin in plant organ development, including root nodule formation, is well known. The spatiotemporal distribution pattern of auxin during nodule development has been illustrated using auxin reporter constructs. However, our understanding of how this pattern is established and maintained remains elusive. Here, we studied how the auxin gradient is associated with the spatiotemporal expression patterns of known auxin biosynthesis and transport genes at different stages of nodule development in Medicago (Medicago truncatula). In addition, we examined the Medicago PIN-FORMED10 (MtPIN10) expression pattern and polar positioning on the cell membrane during nodule primordium development to investigate auxin flux. RNA interference and the application of auxin biosynthesis inhibitors were used to demonstrate the importance of auxin biosynthesis and transport at the initial stages of nodulation. Our results show that upon rhizobium inoculation before the first cell divisions, a specific subset of Medicago YUCCA (MtYUC) and MtPIN genes, as well as Medicago LIKE AUXIN RESISTANT2 (MtLAX2), are expressed in the pericycle and contribute to the creation of an auxin maximum. Overall, we demonstrate that the dynamic spatiotemporal expression of both MtYUC and MtPIN genes results in specific auxin outputs during the different stages of nodule primordia and nodule meristem formation.

The importance of auxin in plant organ development including root nodule formation is well established. Using auxin reporter constructs the spatiotemporal auxin distribution pattern during nodule development has previously been illustrated. However, our understanding of how this pattern is built-up and maintained still remains elusive. To this end, we studied how the auxin gradient visualized by DR5 expression patterns at different stages of nodule development in Medicago truncatula (Medicago), is correlated with the spatiotemporal expression patterns of known auxin biosynthesis and auxin transport genes. In addition, we record the MtPIN10-GFP expression pattern and polar positioning on the cell plasma membranes during nodule primordium development to investigate the auxin flux. RNA interference and the application of auxin synthesis blockers were used to demonstrate the relevance of biosynthesis and transport at the initial stages of the nodulation process. Our results show that upon rhizobium inoculation, preceding the first mitotic activity, a specific set of MtYUCs and MtPINs as well as MtLAX2 are expressed in the pericycle contributing to the creation of an auxin maximum. Overall, we demonstrate that dynamic spatiotemporal expression of both, MtYUCs and MtPINs, result in specific auxin outputs in subsequent stages of nodule primordia and nodule meristem formation.

To acquire sufficient mineral nutrients such as phosphate (Pi) from the soil, most plants engage in symbiosis with arbuscular mycorrhizal (AM) fungi. Attracted by plant-secreted strigolactones (SLs), the fungi colonize the roots and form highly branched hyphal structures called arbuscules inside inner cortex cells. The host plant must control the different steps of this interaction to maintain its symbiotic nature. However, how plants sense the amount of Pi obtained from the fungus, and how this determines the arbuscule lifespan, are far from understood. Here, we show that Medicago truncatula SPX-domain containing proteins SPX1 and SPX3 regulate root Pi starvation responses, in part by interacting with PHOSPHATE RESPONSE REGULATOR2, as well as fungal colonization and arbuscule degradation. SPX1 and SPX3 are induced upon Pi starvation but become more restricted to arbuscule-containing cells upon the establishment of symbiosis. This induction in arbuscule-containing cells is associated with the presence of cis-regulatory AW-boxes and transcriptional regulation by the WRINKLED1-like transcription factor WRI5a. Under Pi-limiting conditions, SPX1 and SPX3 facilitate the expression of the SL biosynthesis gene DWARF27, which could help explain the increased fungal branching in response to root exudates. Later, in arbuscule-containing cells, SPX1 and SPX3 redundantly control arbuscule degradation. Thus, SPX proteins play important roles as phosphate sensors to maintain a beneficial AM symbiosis.

To acquire sufficient mineral nutrients such as phosphate (Pi) from the soil, most plants engage in a symbiosis with arbuscular mycorrhizal (AM) fungi. Attracted by plant-secreted strigolactones, the fungi colonize the roots and form highly-branched hyphal structures called arbuscules inside inner cortex cells. It is essential that the host plant controls the different steps of this interaction to maintain its symbiotic nature. However, how plants sense the amount of Pi obtained from the fungus and how this determines the arbuscule lifetime is far from understood. Here, we show that Medicago truncatula SPX-domain containing proteins SPX1 and SPX3 regulate root phosphate starvation responses as well as fungal colonization and arbuscule degradation. SPX1 and SPX3 are induced upon phosphate starvation but become restricted to arbuscule-containing cells upon establishment of the symbiosis. Under Pi-limiting conditions they facilitate the expression of the strigolactone biosynthesis gene DWARF27 , which correlates with increased fungal branching by root exudates and increased root colonization. Later, in the arbuscule-containing cells SPX1 and SPX3 redundantly control the timely degradation of arbuscules. This regulation does not seem to involve direct interactions with known transcriptional regulators of arbuscule degradation. We propose a model where SPX1 and SPX3 control arbuscule degeneration in a Pi-dependent manner via a yet-to-identify negative regulator.

Summary: NIN (NODULE INCEPTION) is a transcription factor that plays a key role during root nodule initiation. However, its role in later nodule developmental stages is unclear. Both NIN mRNA and protein accumulated at the highest level in the proximal part of the infection zone in Medicago truncatula nodules. Two nin weak allele mutants, nin‐13/16, form a rather normal nodule infection zone, whereas a fixation zone is not formed. Instead, a zone with defence responses and premature senescence occurred and symbiosome development gets arrested. Mutations in nin‐13/16 resulted in a truncated NIN lacking the conserved PB1 domain. However, this did not cause the nodule phenotype as nin mutants expressing NIN_ΔPB1 formed wild‐type‐like nodule. The phenotype is likely to be caused by reduced NIN mRNA levels in the cytoplasm. Transcriptome analyses of nin‐16 nodules showed that expression levels of defence/senescence‐related genes are markedly increased, whereas the levels of defence suppressing genes are reduced. Although defence/senescence seems well suppressed in the infection zone, the transcriptome is already markedly changed in the proximal part of infection zone. In addition to its function in infection and nodule organogenesis, NIN also plays a major role at the transition from infection to fixation zone in establishing a functional symbiosis.

Legumes and actinorhizal plants are capable of forming root nodules symbiosis with rhizobia and Frankia bacteria. All these nodulating species belong to the nitrogen fixation clade. Most likely, nodulation evolved once in the last common ancestor of this clade. NIN (NODULE INCEPTION) is a transcription factor that is essential for nodulation in all studied species. Therefore, it seems probable that it was recruited at the start when nodulation evolved. NIN is the founding member of the NIN-like protein (NLP) family. It arose by duplication, and this occurred before nodulation evolved. Therefore, several plant species outside the nitrogen fixation clade have NLP(s), which is orthologous to NIN. In this review, we discuss how NIN has diverged from the ancestral NLP, what minimal changes would have been essential for it to become a key transcription controlling nodulation, and which adaptations might have evolved later.

The legume-rhizobium symbiosis results in nitrogen-fixing root nodules, and their formation involves both intracellular infection initiated in the epidermis and nodule organogenesis initiated in inner root cell layers. NODULE INCEPTION (NIN) is a nodule-specific transcription factor essential for both processes. These NIN-regulated processes occur at different times and locations in the root, demonstrating a complex pattern of spatiotemporal regulation. We show that regulatory sequences sufficient for the epidermal infection process are located within a 5 kb region directly upstream of the NIN start codon in Medicago truncatula. Furthermore, we identify a remote upstream cis-regulatory region required for the expression of NIN in the pericycle, and we show that this region is essential for nodule organogenesis. This region contains putative cytokinin response elements and is conserved in eight more legume species. Both the cytokinin receptor 1, which is essential for nodule primordium formation, and the B-type response regulator RR1 are expressed in the pericycle in the susceptible zone of the uninoculated root. This, together with the identification of the cytokinin-responsive elements in the NIN promoter, strongly suggests that NIN expression is initially triggered by cytokinin signaling in the pericycle to initiate nodule primordium formation.