Chengwu Liu

The Root Hair “Infectome” of Medicago truncatula Uncovers Changes in Cell Cycle Genes and Reveals a Requirement for Auxin Signaling in Rhizobial Infection

Transcriptome profiling of M. truncatula root hairs during the initial stages of rhizobial infection helps to interpret two decades of research on Medicago and provides a foundation for future studies on host-symbiont interactions in the rhizosphere. Nitrogen-fixing rhizobia colonize legume roots via plant-made intracellular infection threads. Genetics has identified some genes involved but has not provided sufficient detail to understand requirements for infection thread development. Therefore, we transcriptionally profiled Medicago truncatula root hairs prior to and during the initial stages of infection. This revealed changes in the responses to plant hormones, most notably auxin, strigolactone, gibberellic acid, and brassinosteroids. Several auxin responsive genes, including the ortholog of Arabidopsis thaliana Auxin Response Factor 16, were induced at infection sites and in nodule primordia, and mutation of ARF16a reduced rhizobial infection. Associated with the induction of auxin signaling genes, there was increased expression of cell cycle genes including an A-type cyclin and a subunit of the anaphase promoting complex. There was also induction of several chalcone O-methyltransferases involved in the synthesis of an inducer of Sinorhizobium meliloti nod genes, as well as a gene associated with Nod factor degradation, suggesting both positive and negative feedback loops that control Nod factor levels during rhizobial infection. We conclude that the onset of infection is associated with reactivation of the cell cycle as well as increased expression of genes required for hormone and flavonoid biosynthesis and that the regulation of auxin signaling is necessary for initiation of rhizobial infection threads.

Floral Patterning in Lotus japonicus

Floral patterning in Papilionoideae plants, such as pea (Pisum sativum) and Medicago truncatula, is unique in terms of floral organ number, arrangement, and initiation timing as compared to other well-studied eudicots. To investigate the molecular mechanisms involved in the floral patterning in legumes, we have analyzed two mutants, proliferating floral meristem and proliferating floral organ-2 (pfo-2), obtained by ethyl methanesulfonate mutagenesis of Lotus japonicus. These two mutants showed similar phenotypes, with indeterminate floral structures and altered floral organ identities. We have demonstrated that loss of function of LjLFY and LjUFO/Pfo is likely to be responsible for these mutant phenotypes, respectively. To dissect the regulatory network controlling the floral patterning, we cloned homologs of the ABC function genes, which control floral organ identity in Arabidopsis (Arabidopsis thaliana). We found that some of the B and C function genes were duplicated. RNA in situ hybridization showed that the C function genes were expressed transiently in the carpel, continuously in stamens, and showed complementarity with the A function genes in the heterogeneous whorl. In proliferating floral meristem and pfo-2 mutants, all B function genes were down-regulated and the expression patterns of the A and C function genes were drastically altered. We conclude that LjLFY and LjUFO/Pfo are required for the activation of B function genes and function together in the recruitment and determination of petals and stamens. Our findings suggest that gene duplication, change in expression pattern, gain or loss of functional domains, and alteration of key gene functions all contribute to the divergence of floral patterning in L. japonicus.

A H + -ATPase That Energizes Nutrient Uptake during Mycorrhizal Symbioses in Rice and Medicago truncatula

Electrochemical H+ gradients are essential to drive active transport of solutes through plant membranes. This work describes plant mutants defective in a proton pump that is specifically located in arbuscule-containing root cells and shows that this proton pump is required for the function of the arbuscular mycorrhizal symbiosis and symbiosis-driven phosphate acquisition and plant growth. Most plant species form symbioses with arbuscular mycorrhizal (AM) fungi, which facilitate the uptake of mineral nutrients such as phosphate from the soil. Several transporters, particularly proton-coupled phosphate transporters, have been identified on both the plant and fungal membranes and contribute to delivering phosphate from fungi to plants. The mechanism of nutrient exchange has been studied in plants during mycorrhizal colonization, but the source of the electrochemical proton gradient that drives nutrient exchange is not known. Here, we show that plasma membrane H+-ATPases that are specifically induced in arbuscule-containing cells are required for enhanced proton pumping activity in membrane vesicles from AM-colonized roots of rice (Oryza sativa) and Medicago truncatula. Mutation of the H+-ATPases reduced arbuscule size and impaired nutrient uptake by the host plant through the mycorrhizal symbiosis. Overexpression of the H+-ATPase Os-HA1 increased both phosphate uptake and the plasma membrane potential, suggesting that this H+-ATPase plays a key role in energizing the periarbuscular membrane, thereby facilitating nutrient exchange in arbusculated plant cells.

Rhizobial infection is associated with the development of peripheral vasculature in nodules of Medicago truncatula.

Cross talk between infection and morphogenesis is required to develop a normal nodule in the legume-rhizobia symbiosis. Nodulation in legumes involves the coordination of epidermal infection by rhizobia with cell divisions in the underlying cortex. During nodulation, rhizobia are entrapped within curled root hairs to form an infection pocket. Transcellular tubes called infection threads then develop from the pocket and become colonized by rhizobia. The infection thread grows toward the developing nodule primordia and rhizobia are taken up into the nodule cells, where they eventually fix nitrogen. The epidermal and cortical developmental programs are synchronized by a yet-to-be-identified signal that is transmitted from the outer to the inner cell layers of the root. Using a new allele of the Medicago truncatula mutant Lumpy Infections, lin-4, which forms normal infection pockets but cannot initiate infection threads, we show that infection thread initiation is required for normal nodule development. lin-4 forms nodules with centrally located vascular bundles similar to that found in lateral roots rather than the peripheral vasculature characteristic of legume nodules. The same phenomenon was observed in M. truncatula plants inoculated with the Sinorhizobium meliloti exoY mutant, and the M. truncatula vapyrin-2 mutant, all cases where infections arrest. Nodules on lin-4 have reduced expression of the nodule meristem marker MtCRE1 and do not express root-tip markers. In addition, these mutant nodules have altered patterns of gene expression for the cytokinin and auxin markers CRE1 and DR5. Our work highlights the coordinating role that bacterial infection exerts on the developing nodule and allows us to draw comparisons with primitive actinorhizal nodules and rhizobia-induced nodules on the nonlegume Parasponia andersonii.

The Role of Flavonoids in Nodulation Host-Range Specificity: An Update

Flavonoids are crucial signaling molecules in the symbiosis between legumes and their nitrogen-fixing symbionts, the rhizobia. The primary function of flavonoids in the interaction is to induce transcription of the genes for biosynthesis of the rhizobial signaling molecules called Nod factors, which are perceived by the plant to allow symbiotic infection of the root. Many legumes produce specific flavonoids that only induce Nod factor production in homologous rhizobia, and therefore act as important determinants of host range. Despite a wealth of evidence on legume flavonoids, relatively few have proven roles in rhizobial infection. Recent studies suggest that production of key “infection” flavonoids is highly localized at infection sites. Furthermore, some of the flavonoids being produced at infection sites are phytoalexins and may have a role in the selection of compatible symbionts during infection. The molecular details of how flavonoid production in plants is regulated during nodulation have not yet been clarified, but nitrogen availability has been shown to play a role.

Signaling at the Root Surface: The Role of Cutin Monomers in Mycorrhization

Most vascular plants interact with arbuscular mycorrhizal fungi (AMF) and are thereby provided significant advantages in nutrient acquisition, especially phosphate. The widespread retention of this symbiosis in vascular plants is testament to its importance and ancient origins. This highlight focuses on the reports of two of the first genes identified through forward genetic screens for AMF symbiotic mutants: Required for Abuscular Mycorrhization 1 (RAM1) and RAM2, which respectively encode a GRAS transcription factor and a glycerol-3-phosphate acyl-transferase (GPAT) that are required for the formation of fungal entry structures (hyphopodia) on the root surface. This provides the first insights into a mycorrhization specific signaling pathway and reveals cutin monomers as a critical component of signaling in the mycorrhizal symbiosis. We discuss the proposed links between these genes, and the role of cutin and its precursors in interactions with AMF, and oomycete and fungal pathogens. Root colonization by AMF involves the detection of compounds found in host root exudates such as strigolactones. Strigolactones are carotenoid-derived terpenoids that serve as a rhizosphere signal to activate responses in AMF, including hyphal branching, and thereby promote mycorrhizal colonization of the root surface. This involves the fungus making contact with the plant epidermal cell wall and forming a hyphopodium which is a lobed hyphal contact point with the root that serves as the entry point of the fungus into the epidermis. The hyphopodium is a specialized structure similar to, but distinct from, the pathogenic appressorium. The fungus then passes through an epidermal cell and colonizes the root cortex by extensive intercellular hyphal growth and the formation of terminal intracellular structures called arbuscules. The arbuscule, which serves as the nutrient exchange interface in the symbiosis, is highly branched and is surrounded by plant plasma membrane. It has been known for some time that the evolutionary history of the mycorrhizal symbioses is closely intertwined with that of a second root endosymbiosis called nodulation. The signaling required for the initiation of nodulation in legumes involves several genes that are also essential for mycorrhization (Kistner et al., 2005). This common set of genes is usually referred to as the common signaling pathway. For clarity, throughout this manuscript, common signally pathway genes identified in model legumes will be given as Medicago truncatula/Lotus japonicus. These shared components comprise a signaling circuit, which includes the ion channel DMI1 (Does not Make Infections)/POLLUX, DMI2/SYMRK (SYMbiosis Receptor like Kinase) and the calcium calmodulin kinase DMI3/CCaMK (Catoira et al., 2000) and its substrate IPD3 (Interacting Protein of DMI3)/Cyclops. Downstream of this shared signaling pathway lie the genes regulating the transcriptional outputs, including the GRAS transcription factors NSP1 and NSP2, which interact to regulate expression of nodulation genes (Hirsch et al., 2009).

MtLAX2, a functional homologue of the Arabidopsis auxin influx transporter AUX1, is required for nodule organogenesis

Genetic and molecular biological tools were used to show that MtLAX2 is required for not only lateral root formation but also nodulation, in Medicago truncatula. Most legume plants can form nodules, specialized lateral organs that form on roots, and house nitrogen-fixing bacteria collectively called rhizobia. The uptake of the phytohormone auxin into cells is known to be crucial for development of lateral roots. To test the role of auxin influx in nodulation we used the auxin influx inhibitors 1-naphthoxyacetic acid (1-NOA) and 2-NOA, which we found reduced nodulation of Medicago truncatula. This suggested the possible involvement of the AUX/LAX family of auxin influx transporters in nodulation. Gene expression studies identified MtLAX2, a paralogue of Arabidopsis (Arabidopsis thaliana) AUX1, as being induced at early stages of nodule development. MtLAX2 is expressed in nodule primordia, the vasculature of developing nodules, and at the apex of mature nodules. The MtLAX2 promoter contains several auxin response elements, and treatment with indole-acetic acid strongly induces MtLAX2 expression in roots. mtlax2 mutants displayed root phenotypes similar to Arabidopsis aux1 mutants, including altered root gravitropism, fewer lateral roots, shorter root hairs, and auxin resistance. In addition, the activity of the synthetic DR5-GUS auxin reporter was strongly reduced in mtlax2 roots. Following inoculation with rhizobia, mtlax2 roots developed fewer nodules, had decreased DR5-GUS activity associated with infection sites, and had decreased expression of the early auxin responsive gene ARF16a. Our data indicate that MtLAX2 is a functional analog of Arabidopsis AUX1 and is required for the accumulation of auxin during nodule formation in tissues underlying sites of rhizobial infection.

Identification of a core set of rhizobial infection genes using data from single cell-types.

Genome-wide expression studies on nodulation have varied in their scale from entire root systems to dissected nodules or root sections containing nodule primordia (NP). More recently efforts have focused on developing methods for isolation of root hairs from infected plants and the application of laser-capture microdissection technology to nodules. Here we analyze two published data sets to identify a core set of infection genes that are expressed in the nodule and in root hairs during infection. Among the genes identified were those encoding phenylpropanoid biosynthesis enzymes including Chalcone-O-Methyltransferase which is required for the production of the potent Nod gene inducer 4′,4-dihydroxy-2-methoxychalcone. A promoter-GUS analysis in transgenic hairy roots for two genes encoding Chalcone-O-Methyltransferase isoforms revealed their expression in rhizobially infected root hairs and the nodule infection zone but not in the nitrogen fixation zone. We also describe a group of Rhizobially Induced Peroxidases whose expression overlaps with the production of superoxide in rhizobially infected root hairs and in nodules and roots. Finally, we identify a cohort of co-regulated transcription factors as candidate regulators of these processes.

A Medicago truncatula Cystathionine-β-Synthase-like Domain-Containing Protein Is Required for Rhizobial Infection and Symbiotic Nitrogen Fixation

A Cystathionine-β-Synthase like protein, exclusively expressed during Medicago-Rhizobium symbiosis, is required for infection thread propagation and bacterial endocytosis. The symbiosis between leguminous plants and soil rhizobia culminates in the formation of nitrogen-fixing organs called nodules that support plant growth. Two Medicago truncatula Tnt1-insertion mutants were identified that produced small nodules, which were unable to fix nitrogen effectively due to ineffective rhizobial colonization. The gene underlying this phenotype was found to encode a protein containing a putative membrane-localized domain of unknown function (DUF21) and a cystathionine-β-synthase domain. The cbs1 mutants had defective infection threads that were sometimes devoid of rhizobia and formed small nodules with greatly reduced numbers of symbiosomes. We studied the expression of the gene, designated M. truncatula Cystathionine-β-Synthase-like1 (MtCBS1), using a promoter-β-glucuronidase gene fusion, which revealed expression in infected root hair cells, developing nodules, and in the invasion zone of mature nodules. An MtCBS1-GFP fusion protein localized itself to the infection thread and symbiosomes. Nodulation factor-induced Ca2+ responses were observed in the cbs1 mutant, indicating that MtCBS1 acts downstream of nodulation factor signaling. MtCBS1 expression occurred exclusively during Medicago-rhizobium symbiosis. Induction of MtCBS1 expression during symbiosis was found to be dependent on Nodule Inception (NIN), a key transcription factor that controls both rhizobial infection and nodule organogenesis. Interestingly, the closest homolog of MtCBS1, MtCBS2, was specifically induced in mycorrhizal roots, suggesting common infection mechanisms in nodulation and mycorrhization. Related proteins in Arabidopsis have been implicated in cell wall maturation, suggesting a potential role for CBS1 in the formation of the infection thread wall.

Nitrogen sensing in legumes

Legumes fix atmospheric nitrogen (N) in a symbiotic relationship with bacteria. For this reason, although legume crops can be low yielding and less profitable when compared with cereals, they are frequently included in crop rotations. Grain legumes form only a minor part of most human diets, and legume crops are greatly underutilized. Food security and soil fertility could be significantly improved by greater grain legume usage and increased improvement of a range of grain legumes. One limitation for the use of legumes as a source of N input into agricultural systems is the fact that the formation of N-fixing nodules is suppressed when soils are replete with n. In this review, we report what is known about this process and how soil N supply might be sensed and feed back to regulate nodulation.