Nitrate uptake by plant cells requires both high- and low-affinity transport activities. nitrate transporter 1/peptide transporter family (NPF) 6.3 is a dual-affinity plasma membrane transport protein that has both high- and low-affinity functions. At-NPF6.3 imports and senses nitrate and is regulated by phosphorylation at Thr-101 (T101). A detailed functional analysis of two maize () homologs of At-NPF6.3 (Zm-NPF6.6 and Zm-NPF6.4) showed that Zm-NPF6.6 was a pH-dependent nonbiphasic high-affinity nitrate-specific transport protein. By contrast, maize NPF6.4 was a low-affinity nitrate transporter with efflux activity. When supplied chloride, NPF6.4 switched to a high-affinity chloride selective transporter, while NPF6.6 had only a low-affinity chloride transport activity. Structural predictions identified a nitrate binding His (H362) in NPF6.6 but not in NPF6.4. Mutation of NPF6.4 Tyr-370 to His (Y370H) resulted in saturable high-affinity nitrate transport activity and nitrate selectivity. Loss of H362 in NPF6.6 (H362Y) eliminated both nitrate and chloride transport. Furthermore, alterations to Thr-104, a conserved phosphorylation site in NPF6.6, resulted in a similar high-affinity nitrate transport activity with increased, whereas equivalent changes in NPF6.4 (T106) disrupted high-affinity chloride transport activity. NPF6 proteins exhibit different substrate specificity in plants and regulate nitrate transport affinity/selectivity using a conserved His residue.

A successful nitrogen-fixing symbiosis requires the accommodation of rhizobial bacteria as new organelle-like structures, called symbiosomes, inside the cells of their legume hosts. Two legume mutants that are most strongly impaired in their ability to form symbiosomes are sym1/TE7 in Medicago truncatula and sym33 in Pisum sativum. We have cloned both MtSYM1 and PsSYM33 and show that both encode the recently identified interacting protein of DMI3 (IPD3), an ortholog of Lotus japonicus (Lotus) CYCLOPS. IPD3 and CYCLOPS were shown to interact with DMI3/CCaMK, which encodes a calcium- and calmodulin-dependent kinase that is an essential component of the common symbiotic signaling pathway for both rhizobial and mycorrhizal symbioses. Our data reveal a novel, key role for IPD3 in symbiosome formation and development. We show that MtIPD3 participates in but is not essential for infection thread formation and that MtIPD3 also affects DMI3-induced spontaneous nodule formation upstream of cytokinin signaling. Further, MtIPD3 appears to be required for the expression of a nodule-specific remorin, which controls proper infection thread growth and is essential for symbiosome formation.

Phenotypic characterization of pea symbiotic mutants has provided a detailed description of the symbiosis with Rhizobium leguminosarum bv. viciae strains. We show here that two allelic non-nodulating pea mutants, RisNod4 and K24, are affected in the PsSym37 gene, encoding a LysM receptor kinase similar to Lotus japonicus NFR1 and Medicago truncatula LYK3. Phenotypic analysis of RisNod4 and K24 suggests a role for the SYM37 in regulation of infection-thread initiation and nodule development from cortical-cell division foci. We show that RisNod4 plants carrying an L to F substitution in the LysM1 domain display a restrictive symbiotic phenotype comparable to the PsSym2(A) lines that distinguish 'European' and 'Middle East' Rhizobium leguminosarum bv. viciae strains. RisNod4 mutants develop nodules only in the presence of a 'Middle East' Rhizobium strain producing O-acetylated Nod factors indicating the SYM37 involvement in Nod-factor recognition. Along with the PsSym37, a homologous LysM receptor kinase gene, PsK1, was isolated and characterized. We show that PsK1 and PsSym37 are genetically linked to each other and to the PsSym2 locus. Allelic complementation analyses and sequencing of the extracellular regions of PsSym37 and PsK1 in several 'European' and 'Afghan' pea cultivars point towards PsK1 as possible candidate for the elusive PsSym2 gene.

SummaryIn root nodules rhizobia enter host cells via infection threads. The release of bacteria to a host cell is possible from cell wall-free regions of the infection thread. We hypothesized that the VAMP721d and VAMP721e exocytotic pathway, identified before in Medicago truncatula, has a role in the local modification of cell wall during the release of rhizobia.To clarify the role of VAMP721d and VAMP721e we used Glycine max, a plant with a determinate type of nodule. The localization of the main polysaccharide compounds of primary cell walls was analysed in control vs nodules with partially silenced GmVAMP721d.The silencing of GmVAMP721d blocked the release of rhizobia. Instead of rhizobiacontaining membrane compartments -symbiosomes -the infected cells contained big clusters of bacteria embedded in a matrix of methyl-esterified and de-methyl-esterified pectin. These clusters were surrounded by a membrane. We found that GmVAMP721d-positive vesicles were not transporting methyl-esterified pectin. We hypothesized that they may deliver the enzymes involved in pectin turnover. Subsequently, we found that GmVAMP721d is partly co-localized with pectate lyase.Therefore, the biological role of VAMP721d may be explained by its action in delivering pectin-modifying enzymes to the site of release.

No abstract