Jillian Perry

Plastid proteins crucial for symbiotic fungal and bacterial entry into plant roots

The roots of most higher plants form arbuscular mycorrhiza, an ancient, phosphate-acquiring symbiosis with fungi, whereas only four related plant orders are able to engage in the evolutionary younger nitrogen-fixing root-nodule symbiosis with bacteria. Plant symbioses with bacteria and fungi require a set of common signal transduction components that redirect root cell development. Here we present two highly homologous genes from Lotus japonicus, CASTOR and POLLUX, that are indispensable for microbial admission into plant cells and act upstream of intracellular calcium spiking, one of the earliest plant responses to symbiotic stimulation. Surprisingly, both twin proteins are localized in the plastids of root cells, indicating a previously unrecognized role of this ancient endosymbiont in controlling intracellular symbioses that evolved more recently.

A TILLING Reverse Genetics Tool and a Web-Accessible Collection of Mutants of the Legume Lotus japonicus

Reverse genetics aims to identify the function of a gene with known sequence by phenotypic analysis of cells or organisms in which the function of this gene is impaired. Commonly used strategies for reverse genetics encompass transposon mutagenesis (Tissier et al., 1999) and RNA-mediated gene silencing or RNA interference (Voinnet, 2002). We adopted a complementary strategy to set up a reverse genetics tool for the legume Lotus japonicus that identifies individuals carrying point mutations in any gene of interest within a large population of ethyl methanesulfonate (EMS)-mutagenized M2 plants. This strategy was first described by McCallum et al. (2000a,b) using the acronym TILLING (Targeted Induced Local Lesions in Genomes). The target sequence is PCR amplified from pooled M2 individuals. DNA with point mutations are detected by melting and reannealing of the PCR products. This results in the formation of heteroduplex DNA in which one strand originates from the mutant and the other from the wild-type PCR product. A mismatch occurs at the site of the point mutation, which can be detected using mismatch-specific endonucleases such as CEL I from celery (Apium graveolens; Yang et al., 2000). This enzyme recognizes mismatches in heteroduplex DNA and cleaves DNA specifically at the mismatched site. The cleavage products can be separated by gel electrophoresis, typically sequencing-type denaturing PAGE. This method of mismatch detection is amenable to pooling strategies. In the Arabidopsis TILLING facility, DNA of eight M2 plants is mixed to form a pool (Colbert et al., 2001). At this pool size, a population of 768 individuals can be screened by PCR in a 96-well microtiter plate, and run on one 96-well gel, each well representing eight individuals. Individuals from pools yielding cleavage products are then PCR amplified individually to identify the mutation bearing plant, progeny of which will segregate the mutation of interest.

CYCLOPS, a mediator of symbiotic intracellular accommodation

The initiation of intracellular infection of legume roots by symbiotic rhizobia bacteria and arbuscular mycorrhiza (AM) fungi is preceded by the induction of calcium signatures in and around the nucleus of root epidermal cells. Although a calcium and calmodulin-dependent kinase (CCaMK) is a key mediator of symbiotic root responses, the decoding of the calcium signal and the molecular events downstream are only poorly understood. Here, we characterize Lotus japonicus cyclops mutants on which microbial infection was severely inhibited. In contrast, nodule organogenesis was initiated in response to rhizobia, but arrested prematurely. This arrest was overcome when a deregulated CCaMK mutant version was introduced into cyclops mutants, conferring the development of full-sized, spontaneous nodules. Because cyclops mutants block symbiotic infection but are competent for nodule development, they reveal a bifurcation of signal transduction downstream of CCaMK. We identified CYCLOPS by positional cloning. CYCLOPS carries a functional nuclear localization signal and a predicted coiled-coil domain. We observed colocalization and physical interaction between CCaMK and CYCLOPS in plant and yeast cell nuclei in the absence of symbiotic stimulation. Importantly, CYCLOPS is a phosphorylation substrate of CCaMK in vitro. Cyclops mutants of rice were impaired in AM, and rice CYCLOPS could restore symbiosis in Lotus cyclops mutants, indicating a functional conservation across angiosperms. Our results suggest that CYCLOPS forms an ancient, preassembled signal transduction complex with CCaMK that is specifically required for infection, whereas organogenesis likely requires additional yet-to-be identified CCaMK interactors or substrates.

Lotus japonicus Nodulation Requires Two GRAS Domain Regulators, One of Which Is Functionally Conserved in a Non-Legume

A new nodulation-defective mutant of Lotus japonicus does not initiate nodule cortical cell division in response to Mesorhizobium loti, but induces root hair deformation, Nod factor-induced calcium spiking, and mycorrhization. This phenotype, together with mapping data, suggested that the mutation could be in the ortholog of the Medicago truncatula NSP1 gene (MtNSP1). The sequence of the orthologous gene (LjNSP1) in the L. japonicus mutant (Ljnsp1-1) revealed a mutation causing a premature stop resulting in loss of the C-terminal 23 amino acids. We also sequenced the NSP2 gene from L. japonicus (LjNSP2). A mutant (Ljnsp2-3) with a premature stop codon was identified by TILLING showing a similar phenotype to Ljnsp1-1. Both LjNSP1 and LjNSP2 are predicted GRAS (GAI, RGA, SCR) domain transcriptional regulators. Transcript steady-state levels of LjNSP1 and LjNSP2 initially decreased and then increased following infection by M. loti. In hairy root transformations, LjNSP1 and MtNSP1 complemented both Mtnsp1-1 and Ljnsp1-1 mutants, demonstrating that these orthologous proteins have a conserved biochemical function. A Nicotiana benthamiana NSP1-like gene (NbNSP1) was shown to restore nodule formation in both Ljnsp1-1 and Mtnsp1-1 mutants, indicating that NSP1 regulators from legumes and non-legumes can propagate the Nod factor-induced signal, activating appropriate downstream targets. The L. japonicus nodules complemented with NbNSP1 contained some cells with abnormal bacteroids and could fix nitrogen. However, the NbNSP1-complemented M. truncatula nodules did not fix nitrogen and contained very few bacteria released from infection threads. These observations suggest that NSP1 is also involved in infection, bacterial release, and normal bacteroid formation in nodule cells.

NENA, a Lotus japonicus Homolog of Sec13, Is Required for Rhizodermal Infection by Arbuscular Mycorrhiza Fungi and Rhizobia but Dispensable for Cortical Endosymbiotic Development

NENA was identified by a genetic screen for Lotus japonicus mutants impaired in arbuscular mycorrhiza and encodes a scaffold nucleoporin. nena mutants are also impaired in rhizobial root hair infection but crack entry leading to fully infected root nodules can overcome the rhizodermal nonresponsiveness of nena-1 to Nod factor. Legumes form symbioses with arbuscular mycorrhiza (AM) fungi and nitrogen fixing root nodule bacteria. Intracellular root infection by either endosymbiont is controlled by the activation of the calcium and calmodulin-dependent kinase (CCaMK), a central regulatory component of the plant’s common symbiosis signaling network. We performed a microscopy screen for Lotus japonicus mutants defective in AM development and isolated a mutant, nena, that aborted fungal infection in the rhizodermis. NENA encodes a WD40 repeat protein related to the nucleoporins Sec13 and Seh1. Localization of NENA to the nuclear rim and yeast two-hybrid experiments indicated a role for NENA in a conserved subcomplex of the nuclear pore scaffold. Although nena mutants were able to form pink nodules in symbiosis with Mesorhizobium loti, root hair infection was not observed. Moreover, Nod factor induction of the symbiotic genes NIN, SbtM4, and SbtS, as well as perinuclear calcium spiking, were impaired. Detailed phenotypic analyses of nena mutants revealed a rhizobial infection mode that overcame the lack of rhizodermal responsiveness and carried the hallmarks of crack entry, including a requirement for ethylene. CCaMK-dependent processes were only abolished in the rhizodermis but not in the cortex of nena mutants. These data support the concept of tissue-specific components for the activation of CCaMK.

Genetics of Symbiosis in Lotus japonicus: Recombinant Inbred Lines, Comparative Genetic Maps, and Map Position of 35 Symbiotic Loci

Development of molecular tools for the analysis of the plant genetic contribution to rhizobial and mycorrhizal symbiosis has provided major advances in our understanding of plant-microbe interactions, and several key symbiotic genes have been identified and characterized. In order to increase the efficiency of genetic analysis in the model legume Lotus japonicus, we present here a selection of improved genetic tools. The two genetic linkage maps previously developed from an interspecific cross between L. japonicus Gifu and L. filicaulis, and an intraspecific cross between the two ecotypes L. japonicus Gifu and L. japonicus MG-20, were aligned through a set of anchor markers. Regions of linkage groups, where genetic resolution is obtained preferentially using one or the other parental combination, are highlighted. Additional genetic resolution and stabilized mapping populations were obtained in recombinant inbred lines derived by a single seed descent from the two populations. For faster mapping of new loci, a selection of reliable markers spread over the chromosome arms provides a common framework for more efficient identification of new alleles and new symbiotic loci among uncharacterized mutant lines. Combining resources from the Lotus community, map positions of a large collection of symbiotic loci are provided together with alleles and closely linked molecular markers. Altogether, this establishes a common genetic resource for Lotus spp. A web-based version will enable this resource to be curated and updated regularly.

TILLING in Lotus japonicus Identified Large Allelic Series for Symbiosis Genes and Revealed a Bias in Functionally Defective Ethyl Methanesulfonate Alleles toward Glycine Replacements

We have established tools for forward and reverse genetic analysis of the legume Lotus (Lotus japonicus). A structured population of M2 progeny of 4,904 ethyl methanesulfonate-mutagenized M1 embryos is available for single nucleotide polymorphism mutation detection, using a TILLING (for Targeting Induced Local Lesions IN Genomes) protocol. Scanning subsets of this population, we identified a mutation load of one per 502 kb of amplified fragment. Moreover, we observed a 1:10 ratio between homozygous and heterozygous mutations in the M2 progeny. This reveals a clear difference in germline genetics between Lotus and Arabidopsis (Arabidopsis thaliana). In addition, we assembled M2 siblings with obvious phenotypes in overall development, starch accumulation, or nitrogen-fixing root nodule symbiosis in three thematic subpopulations. By screening the nodulation-defective population of M2 individuals for mutations in a set of 12 genes known to be essential for nodule development, we identified large allelic series for each gene, generating a unique data set that combines genotypic and phenotypic information facilitating structure-function studies. This analysis revealed a significant bias for replacements of glycine (Gly) residues in functionally defective alleles, which may be explained by the exceptional structural features of Gly. Gly allows the peptide chain to adopt conformations that are no longer possible after amino acid replacement. This previously unrecognized vulnerability of proteins at Gly residues could be used for the improvement of algorithms that are designed to predict the deleterious nature of single nucleotide polymorphism mutations. Our results demonstrate the power, as well as the limitations, of ethyl methanesulfonate mutagenesis for forward and reverse genetic studies. (Original mutant phenotypes can be accessed at http://data.jic.bbsrc.ac.uk/cgi-bin/lotusjaponicus Access to the Lotus TILLING facility can be obtained through http://www.lotusjaponicus.org or http://revgenuk.jic.ac.uk)

A cytosolic invertase is required for normal growth and cell development in the model legume, Lotus japonicus

Neutral/alkaline invertases are a subgroup, confined to plants and cyanobacteria, of a diverse family of enzymes. A family of seven closely-related genes, LjINV1–LjINV7, is described here and their expression in the model legume, Lotus japonicus, is examined. LjINV1 previously identified as encoding a nodule-enhanced isoform is the predominant isoform present in all parts of the plant. Mutants for two isoforms, LjINV1 and LjINV2, were isolated using TILLING. A premature stop codon allele of LjINV2 had no effect on enzyme activity nor did it show a visible phenotype. For LjINV1, premature stop codon and missense mutations were obtained and the phenotype of the mutants examined. Recovery of homozygous mutants was problematic, but their phenotype showed a severe reduction in growth of the root and the shoot, a change in cellular development, and impaired flowering. The cellular organization of both roots and leaves was altered; leaves were smaller and thicker with extra layers of cells and roots showed an extended and broader zone of cell division. Moreover, anthers contained no pollen. Both heterozygotes and homozygous mutants showed decreased amounts of enzyme activity in nodules and shoot tips. Shoot tips also contained up to a 9-fold increased level of sucrose. However, mutants were capable of forming functional root nodules. LjINV1 is therefore crucial to whole plant development, but is clearly not essential for nodule formation or function.

Lotus japonicus Cytokinin Receptors Work Partially Redundantly to Mediate Nodule Formation

In Lotus japonicus, the LHK1 cytokinin receptor performs an essential function but also works partially redundantly with LHK1A and LHK3 to mediate nodule primordium formation within the root cortex. LHK1 is also expressed in the root epidermis, where it likely participates in signaling to restrict rhizobial infection. Previous analysis of the Lotus histidine kinase1 (Lhk1) cytokinin receptor gene has shown that it is required and also sufficient for nodule formation in Lotus japonicus. The L. japonicus mutant carrying the loss-of-function lhk1-1 allele is hyperinfected by its symbiotic partner, Mesorhizobium loti, in the initial absence of nodule organogenesis. At a later time point following bacterial infection, lhk1-1 develops a limited number of nodules, suggesting the presence of an Lhk1-independent mechanism. We have tested a hypothesis that other cytokinin receptors function in at least a partially redundant manner with LHK1 to mediate nodule organogenesis in L. japonicus. We show here that L. japonicus contains a small family of four cytokinin receptor genes, which all respond to M. loti infection. We show that within the root cortex, LHK1 performs an essential role but also works partially redundantly with LHK1A and LHK3 to mediate cell divisions for nodule primordium formation. The LHK1 receptor is also presumed to partake in mediating a feedback mechanism that negatively regulates bacterial infections at the root epidermis. Interestingly, the Arabidopsis thaliana AHK4 receptor gene can functionally replace Lhk1 in mediating nodule organogenesis, indicating that the ability to perform this developmental process is not determined by unique, legume-specific properties of LHK1.

A Suite of Lotus japonicus Starch Mutants Reveals Both Conserved and Novel Features of Starch Metabolism

The metabolism of starch is of central importance for many aspects of plant growth and development. Information on leaf starch metabolism other than in Arabidopsis (Arabidopsis thaliana) is scarce. Furthermore, its importance in several agronomically important traits exemplified by legumes remains to be investigated. To address this issue, we have provided detailed information on the genes involved in starch metabolism in Lotus japonicus and have characterized a comprehensive collection of forward and TILLING (for Targeting Induced Local Lesions IN Genomes) reverse genetics mutants affecting five enzymes of starch synthesis and two enzymes of starch degradation. The mutants provide new insights into the structure-function relationships of ADP-glucose pyrophosphorylase and glucan, water dikinase1 in particular. Analyses of the mutant phenotypes indicate that the pathways of leaf starch metabolism in L. japonicus and Arabidopsis are largely conserved. However, the importance of these pathways for plant growth and development differs substantially between the two species. Whereas essentially starchless Arabidopsis plants lacking plastidial phosphoglucomutase grow slowly relative to wild-type plants, the equivalent mutant of L. japonicus grows normally even in a 12-h photoperiod. In contrast, the loss of GLUCAN, WATER DIKINASE1, required for starch degradation, has a far greater effect on plant growth and fertility in L. japonicus than in Arabidopsis. Moreover, we have also identified several mutants likely to be affected in new components or regulators of the pathways of starch metabolism. This suite of mutants provides a substantial new resource for further investigations of the partitioning of carbon and its importance for symbiotic nitrogen fixation, legume seed development, and perenniality and vegetative regrowth.