MtNRLK1, a CLAVATA1-like leucine-rich repeat receptor-like kinase upregulated during nodulation in Medicago truncatula.

Peptides are signaling molecules regulating various aspects of plant development, including the balance between cell division and differentiation in different meristems. Among those, CLAVATA3/Embryo Surrounding Region-related (CLE-ESR) peptide activity depends on leucine-rich-repeat receptor-like-kinases (LRR-RLK) belonging to the subclass XI. In legume plants, such as the Medicago truncatula model, specific CLE peptides were shown to regulate root symbiotic nodulation depending on the LRR-RLK SUNN (Super Numeric Nodules). Amongst the ten M. truncatula LRR-RLK most closely related to SUNN, only one showed a nodule-induced expression, and was so-called MtNRLK1 (Nodule-induced Receptor-Like Kinase 1). MtNRLK1 expression is associated to root and nodule vasculature as well as to the proximal meristem and rhizobial infection zone in the nodule apex. Except for the root vasculature, the MtNRLK1 symbiotic expression pattern is different than the one of MtSUNN. Functional analyses either based on RNA interference, insertional mutagenesis, and overexpression of MtNRLK1 however failed to identify a significant nodulation phenotype, either regarding the number, size, organization or nitrogen fixation capacity of the symbiotic organs formed.

Calcium spiking patterns and the role of the calcium/calmodulin-dependent kinase CCaMK in lateral root base nodulation of Sesbania rostrata.

Nodulation factor (NF) signal transduction in the legume-rhizobium symbiosis involves calcium oscillations that are instrumental in eliciting nodulation. To date, Ca2+ spiking has been studied exclusively in the intracellular bacterial invasion of growing root hairs in zone I. This mechanism is not the only one by which rhizobia gain entry into their hosts; the tropical legume Sesbania rostrata can be invaded intercellularly by rhizobia at cracks caused by lateral root emergence, and this process is associated with cell death for formation of infection pockets. We show that epidermal cells at lateral root bases respond to NFs with Ca2+ oscillations that are faster and more symmetrical than those observed during root hair invasion. Enhanced jasmonic acid or reduced ethylene levels slowed down the Ca2+ spiking frequency and stimulated intracellular root hair invasion by rhizobia, but prevented nodule formation. Hence, intracellular invasion in root hairs is linked with a very specific Ca2+ signature. In parallel experiments, we found that knockdown of the calcium/calmodulin-dependent protein kinase gene of S. rostrata abolished nodule development but not the formation of infection pockets by intercellular invasion at lateral root bases, suggesting that the colonization of the outer cortex is independent of Ca2+ spiking decoding.

Seven in absentia proteins affect plant growth and nodulation in Medicago truncatula.

Protein ubiquitination is a posttranslational regulatory process essential for plant growth and interaction with the environment. E3 ligases, to which the seven in absentia (SINA) proteins belong, determine the specificity by selecting the target proteins for ubiquitination. SINA proteins are found in animals as well as in plants, and a small gene family with highly related members has been identified in the genome of rice (Oryza sativa), Arabidopsis (Arabidopsis thaliana), Medicago truncatula, and poplar (Populus trichocarpa). To acquire insight into the function of SINA proteins in nodulation, a dominant negative form of the Arabidopsis SINAT5 was ectopically expressed in the model legume M. truncatula. After rhizobial inoculation of the 35S:SINAT5DN transgenic plants, fewer nodules were formed than in control plants, and most nodules remained small and white, a sign of impaired symbiosis. Defects in rhizobial infection and symbiosome formation were observed by extensive microscopic analysis. Besides the nodulation phenotype, transgenic plants were affected in shoot growth, leaf size, and lateral root number. This work illustrates a function for SINA E3 ligases in a broad spectrum of plant developmental processes, including nodulation.

Reactive oxygen species and ethylene play a positive role in lateral root base nodulation of a semiaquatic legume.

Lateral root base nodulation on the tropical, semiaquatic legume Sesbania rostrata results from two coordinated, Nod factor-dependent processes: formation of intercellular infection pockets and induction of cell division. Infection pocket formation is associated with cell death and production of hydrogen peroxide. Pharmacological experiments showed that ethylene and reactive oxygen species mediate Nod factor responses and are required for nodule initiation, whereby induction of division and infection could not be uncoupled. Application of purified Nod factors triggered cell division, and both Nod factors and ethylene induced cavities and cell death features in the root cortex. Thus, in S. rostrata, ethylene and reactive oxygen species act downstream from the Nod factors in pathways that lead to formation of infection pockets and initiation of nodule primordia.

pMH11, A tool for gene disruption and expression analysis in Azorhizobium caulinodans.

Tools for mutagenesis and expression analyses are needed to study the role of bacterial genes. Here, we report the construction of pMH11, a small, mobilizable plasmid that replicates in Escherichia coli, but not in Azorhizobium caulinodans, a nodulating microsymbiont of Sesbania rostrata, and that contains a unique BamHI restriction site upstream of a promoterless lacZ gene. pMH11 and two derivatives with the multiple cloning site of pBluescript (KS(II)) are useful for mutagenesis by gene disruption and for expression analyses after selection for cointegration by kanamycin resistance. Weakly constitutive promoter activity from the vector allowed transcription of genes downstream of the integration site, so that no polar effects were caused by gene disruption.