The LysM receptor-like kinase SlLYK10 controls lipochitooligosaccharide signaling in inner cell layers of tomato roots.

Establishment of arbuscular mycorrhiza (AM) relies on a plant signaling pathway that can be activated by fungal chitinic signals such as short chain chitooligosaccharides (CO) and lipo-chitooligosaccharides (LCOs). The tomato LysM receptor-like kinase (LysM RLK) SlLYK10 has high affinity for LCOs and is involved in root colonization by arbuscular mycorrhizal fungi (AMF), however its role in LCO responses has not yet been studied. Here, we show that SlLYK10 proteins produced by the Sllyk10-1 and Sllyk10-2 mutant alleles, which both cause decreases in AMF colonization, and carry mutations in LysM1 and 2 respectively, have similar LCO binding affinities compared to the WT SlLYK10. However, the mutant forms were no longer able to induce cell death in Nicotiana benthamiana when co-expressed with MtLYK3, a Medicago truncatula LCO co-receptor, while they physically interacted with MtLYK3 in co-purification experiments. This suggests that the LysM mutations affect the ability of SlLYK10 to trigger signaling through a potential co-receptor rather than its ability to bind LCOs. Interestingly, tomato lines that contain a calcium (Ca2+) concentration reporter (Genetically Encoded Ca2+ indicators, GECO), showed Ca2+ spiking in response to LCO applications, but this occurred only in inner cell layers of the roots, while short chain COs also induced Ca2+ spiking in the epidermis. Moreover, LCO-induced Ca2+spiking was decreased in Sllyk10-1*GECO plants, suggesting that the decrease in AMF colonization in Sllyk10-1 is due to abnormal LCO signaling.

Analysis of the structure and function of the LYK cluster of Medicago truncatula A17 and R108.

The establishment of the Legume-Rhizobia symbiosis is generally dependent on the production of rhizobial lipochitooligosaccharidic Nod factors (NFs) and their perception by plant Lysin Motif Receptor-Like Kinases (LysM-RLKs). In this study, we characterized a cluster of LysM-RLK genes implicated in strain-specific recognition in two highly divergent and widely-studied Medicago truncatula genotypes, A17 and R108. We then used reverse genetic approaches and biochemical analyses to study the function of selected genes in the clusters and the ability of their encoded proteins to bind NFs. Our study has revealed that the LYK cluster exhibits a high degree of variability among M. truncatula genotypes, which in A17 and R108 includes recent recombination events within the cluster and a transposon insertion in A17. The essential role of LYK3 in nodulation in A17 is not conserved in R108 despite similar sequences and good nodulation expression profiles. Although, LYK2, LYK5 and LYK5bis are not essential for nodulation of the two genotypes, some evidence points to accessory roles in nodulation, but not through high-affinity NF binding. This work shows that recent evolution in the LYK cluster provides a source of variation for nodulation, and potential robustness of signaling through genetic redundancy.

Reprogramming of DNA methylation is critical for nodule development in Medicago truncatula.

The legume-Rhizobium symbiosis leads to the formation of a new organ, the root nodule, involving coordinated and massive induction of specific genes. Several genes controlling DNA methylation are spatially regulated within the Medicago truncatula nodule, notably the demethylase gene, DEMETER (DME), which is mostly expressed in the differentiation zone. Here, we show that MtDME is essential for nodule development and regulates the expression of 1,425 genes, some of which are critical for plant and bacterial cell differentiation. Bisulphite sequencing coupled to genomic capture enabled the identification of 474 regions that are differentially methylated during nodule development, including nodule-specific cysteine-rich peptide genes. Decreasing DME expression by RNA interference led to hypermethylation and concomitant downregulation of 400 genes, most of them associated with nodule differentiation. Massive reprogramming of gene expression through DNA demethylation is a new epigenetic mechanism controlling a key stage of indeterminate nodule organogenesis during symbiotic interactions.

PUB1 Interacts with the Receptor Kinase DMI2 and Negatively Regulates Rhizobial and Arbuscular Mycorrhizal Symbioses through Its Ubiquitination Activity in Medicago truncatula.

PUB1, an E3 ubiquitin ligase, which interacts with and is phosphorylated by the LYK3 symbiotic receptor kinase, negatively regulates rhizobial infection and nodulation during the nitrogen-fixing root nodule symbiosis in Medicago truncatula In this study, we show that PUB1 also interacts with and is phosphorylated by DOES NOT MAKE INFECTIONS 2, the key symbiotic receptor kinase of the common symbiosis signaling pathway, required for both the rhizobial and the arbuscular mycorrhizal (AM) endosymbioses. We also show here that PUB1 expression is activated during successive stages of root colonization by Rhizophagus irregularis that is compatible with its interaction with DOES NOT MAKE INFECTIONS 2. Through characterization of a mutant, pub1-1, affected by the E3 ubiquitin ligase activity of PUB1, we have shown that the ubiquitination activity of PUB1 is required to negatively modulate successive stages of infection and development of rhizobial and AM symbioses. In conclusion, PUB1 represents, to our knowledge, a novel common component of symbiotic signaling integrating signal perception through interaction with and phosphorylation by two key symbiotic receptor kinases, and downstream signaling via its ubiquitination activity to fine-tune both rhizobial and AM root endosymbioses.

Lateral root formation and patterning in Medicago truncatula.

The plant root system is crucial for anchorage and nutrition, and has a major role in plant adaptation, as well as in interactions with soil micro-organisms. Despite the agronomical and ecological importance of legume plants, whose roots can interact symbiotically with soil bacteria called rhizobia that fix atmospheric dinitrogen, and the evidence that lateral root (LR) development programmes are intercepted and influenced by symbiotic organisms, very little is known concerning the cellular and molecular events governing LR development in legumes. To better understand the interconnections between LR formation and symbiotic processes triggered by rhizobia or symbiotic molecules such as lipo-chitooligosaccharides (LCOs), we first need a detailed description of LR development mechanisms in legumes. Using thin sections, we have described the cellular events leading to the formation of a new LR primordium (LRP) in Medicago truncatula, and divided them into seven stages prior to LR emergence. To monitor auxin accumulation we generated transgenic DR5:GUS and DR5:VENUS-N7 reporter lines of M. truncatula, and used them to analyze early stages of LR development. Interesting differences were observed for LR ontogeny compared to Arabidopsis thaliana. Notably, we observed endodermal and cortical contributions to LRP formation, and the associated DR5:GUS expression profile indicated that endodermal and cortical cell divisions were correlated with auxin accumulation. As described for A. thaliana, we observed a preferential zone for LR initiation at 4.45 mm from the root tip. Finally, we studied LR emergence and showed that a significant proportion of new LRP do not emerge straight away and could thus be an additional source of root plasticity. Our results shed new light on the patterning and early development of LRs in M. truncatula.