A transient decrease in reactive oxygen species in roots leads to root hair deformation in the legume-rhizobia symbiosis.

* A possible role for reactive oxygen species (ROS) in root hair deformation in response to Nod factor (NF) was investigated using Medicago truncatula nodulation mutants, and an inhibitor and precursors of ROS. * In wild-type roots, ROS efflux transiently decreased approximately 1 h after NF treatment. Transcript accumulation of two NADPH oxidase homologs, respiratory burst oxidase homolog 2 (MtRBOH2) and MtRBOH3, also transiently decreased at 1 h. However, in the nonnodulating mutant Nod factor perception (nfp), transcript accumulation did not change. * Exogenous application of ROS prevented root hair swelling and branching induced by NF. When accumulation of ROS was prevented by diphenylene iodonium (DPI), NF did not induce root hair branching. Root treatment with DPI alone reduced ROS efflux and induced root hair tip swelling. Transient treatment of roots with DPI mimicked NF treatment and resulted in root hair branching in the absence of NF. A transient DPI treatment did not induce root hair branching in the nonlegumes Arabidopsis thaliana and tomato (Lycopersicon esculentum). * The results suggest a role for the transient reduction of ROS accumulation in governing NF-induced root hair deformation in legumes.

Transcript analysis of early nodulation events in Medicago truncatula.

Within the first 72 h of the interaction between rhizobia and their host plants, nodule primordium induction and infection occur. We predicted that transcription profiling of early stages of the symbiosis between Medicago truncatula roots and Sinorhizobium meliloti would identify regulated plant genes that likely condition key events in nodule initiation. Therefore, using a microarray with about 6,000 cDNAs, we compared transcripts from inoculated and uninoculated roots corresponding to defined stages between 1 and 72 h post inoculation (hpi). Hundreds of genes of both known and unknown function were significantly regulated at these time points. Four stages of the interaction were recognized based on gene expression profiles, and potential marker genes for these stages were identified. Some genes that were regulated differentially during stages I (1 hpi) and II (6-12 hpi) of the interaction belong to families encoding proteins involved in calcium transport and binding, reactive oxygen metabolism, and cytoskeleton and cell wall functions. Genes involved in cell proliferation were found to be up-regulated during stages III (24-48 hpi) and IV (72 hpi). Many genes that are homologs of defense response genes were up-regulated during stage I but down-regulated later, likely facilitating infection thread progression into the root cortex. Additionally, genes putatively involved in signal transduction and transcriptional regulation were found to be differentially regulated in the inoculated roots at each time point. The findings shed light on the complexity of coordinated gene regulation and will be useful for continued dissection of the early steps in symbiosis.

Cytokinins play opposite roles in lateral root formation, and nematode and Rhizobial symbioses.

We used the cytokinin-responsive Arabidopsis response regulator (ARR)5 gene promoter fused to a beta-glucuronidase (GUS) reporter gene, and cytokinin oxidase (CKX) genes from Arabidopsis thaliana (AtCKX3) and maize (ZmCKX1) to investigate the roles of cytokinins in lateral root formation and symbiosis in Lotus japonicus. ARR5 expression was undetectable in the dividing initial cells at early stages of lateral root formation, but later we observed high expression in the base of the lateral root primordium. The root tip continues to express ARR5 during subsequent development of the lateral root. These results suggest a dynamic role for cytokinin in lateral root development. We observed ARR5 expression in curled/deformed root hairs, and also in nodule primordia in response to Rhizobial inoculation. This expression declined once the nodule emerged from the parent root. Root penetration and migration of root-knot nematode (RKN) second-stage larvae (L2) did not elevate ARR5 expression, but a high level of expression was induced when L2 reached the differentiating vascular bundle and during early stages of the nematode-plant interaction. ARR5 expression was specifically absent in mature giant cells (GCs), although dividing cells around the GCs continued to express this reporter. The same pattern was observed using a green fluorescent protein (GFP) reporter driven by the ARR5 promoter in tomato. Overexpression of CKX genes rendered the transgenic hairy roots resistant to exogenous application of the cytokinin [N6-(Delta2 isopentenyl) adenine riboside] (iPR). CKX roots have significantly more lateral roots, but fewer nodules and nematode-induced root galls per plant, than control hairy roots.

Lotus japonicus: a new model to study root-parasitic nematodes.

Sedentary plant-parasitic nematodes engage in complex interactions, and induce specialized feeding structures by redirecting plant developmental pathways, and parallels have been observed with rhizobial nodule development on legumes. A model legume would greatly facilitate a better understanding of the differences between parasitic (nematode) and mutualistic (rhizobia and mycorrhizae) symbioses, and we have developed Lotus japonicus as such a model. Conditions for efficient parasitism by root-knot nematode (Meloidogyne spp.) of the widely used Lotus "Gifu" ecotype were established. Features of Lotus biology, such as thin and translucent roots, proved ideal for monitoring the progress of nematode infection both on live specimens and post-staining. We examined L. japonicus mutants with nodulation phenotypes. One, har1, which is a hypernodulated mutant defective in a CLAVATA1-like receptor kinase gene, was found to be hyperinfected by M. incognita. However, another hypernodulated Lotus mutant exhibited the same level of M. incognita infection as wild-type plants. We also established conditions for infection of Lotus by soybean cyst nematode (Heterodera glycines). In contrast to the response to root-knot nematode, the Gifu ecotype is resistant to H. glycines, and elicits a hypersensitive response. This pattern of resistance recapitulates that seen on nematode-resistant soybean plants. We conclude that L. japonicus is a powerful model legume for studying compatible and incompatible plant-nematode interactions.