Satoko Yoshida

Inhibition of shoot branching by new terpenoid plant hormones

Shoot branching is a major determinant of plant architecture and is highly regulated by endogenous and environmental cues. Two classes of hormones, auxin and cytokinin, have long been known to have an important involvement in controlling shoot branching. Previous studies using a series of mutants with enhanced shoot branching suggested the existence of a third class of hormone(s) that is derived from carotenoids, but its chemical identity has been unknown. Here we show that levels of strigolactones, a group of terpenoid lactones, are significantly reduced in some of the branching mutants. Furthermore, application of strigolactones inhibits shoot branching in these mutants. Strigolactones were previously found in root exudates acting as communication chemicals with parasitic weeds and symbiotic arbuscular mycorrhizal fungi. Thus, we propose that strigolactones act as a new hormone class—or their biosynthetic precursors—in regulating above-ground plant architecture, and also have a function in underground communication with other neighbouring organisms.

A plant receptor-like kinase required for both bacterial and fungal symbiosis

Most higher plant species can enter a root symbiosis with arbuscular mycorrhizal fungi, in which plant carbon is traded for fungal phosphate. This is an ancient symbiosis, which has been detected in fossils of early land plants. In contrast, the nitrogen-fixing root nodule symbioses of plants with bacteria evolved more recently, and are phylogenetically restricted to the rosid I clade of plants. Both symbioses rely on partially overlapping genetic programmes. We have identified the molecular basis for this convergence by cloning orthologous SYMRK (‘symbiosis receptor-like kinase’) genes from Lotus and pea, which are required for both fungal and bacterial recognition. SYMRK is predicted to have a signal peptide, an extracellular domain comprising leucine-rich repeats, a transmembrane and an intracellular protein kinase domain. Lotus SYMRK is required for a symbiotic signal transduction pathway leading from the perception of microbial signal molecules to rapid symbiosis-related gene activation. The perception of symbiotic fungi and bacteria is mediated by at least one common signalling component, which could have been recruited during the evolution of root nodule symbioses from the already existing arbuscular mycorrhiza symbiosis.

Deregulation of a Ca2+/calmodulin-dependent kinase leads to spontaneous nodule development.

Induced development of a new plant organ in response to rhizobia is the most prominent manifestation of legume root-nodule symbiosis with nitrogen-fixing bacteria. Here we show that the complex root-nodule organogenic programme can be genetically deregulated to trigger de novo nodule formation in the absence of rhizobia or exogenous rhizobial signals. In an ethylmethane sulphonate-induced snf1 (spontaneous nodule formation) mutant of Lotus japonicus, a single amino-acid replacement in a Ca2+/calmodulin-dependent protein kinase (CCaMK) is sufficient to turn fully differentiated root cortical cells into meristematic founder cells of root nodule primordia. These spontaneous nodules are genuine nodules with an ontogeny similar to that of rhizobial-induced root nodules, corroborating previous physiological studies. Using two receptor-deficient genetic backgrounds we provide evidence for a developmentally integrated spontaneous nodulation process that is independent of lipochitin–oligosaccharide signal perception and oscillations in Ca2+ second messenger levels. Our results reveal a key regulatory position of CCaMK upstream of all components required for cell-cycle activation, and a phenotypically divergent series of mutant alleles demonstrates positive and negative regulation of the process.

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.

Molecular regulation of leaf senescence

Leaf senescence is a process of programmed cell death, which is induced in an age-dependent manner and by various environmental cues. The mechanisms that regulate the induction and progression of leaf senescence remain unclear because of their complexity. However, recent genetic and reverse-genetic approaches have identified key components of the regulation of leaf senescence and have revealed glimpses of the underlying molecular mechanisms.

Horizontal Gene Transfer by the Parasitic Plant Striga hermonthica

Genes have transferred from a crop plant to an evolutionarily divergent parasitic weed. Horizontal gene transfer has been postulated to occur between crops to co-occurring parasitic plants, but empirical evidence has been lacking. We present evidence that an HGT event moved a nuclear monocot gene into the genome of the eudicot parasite witchweed (Striga hermonthica), which infects many grass species in Africa. Analysis of expressed sequence tags revealed that the genome of S. hermonthica contains a nuclear gene that is widely conserved among grass species but is not found in other eudicots. Phylogenetically, this gene clusters with sorghum genes, the monocot host of the parasitic weed, suggesting that nuclear genes can be captured by parasitic weeds in nature.

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.

Convergent evolution of strigolactone perception enabled host detection in parasitic plants

How plant parasites evolved to find hosts The seeds of parasitic plants need to be able to sense their hosts presence to germinate at the correct time and in the correct place. This is done through the detection of plant hormones, strigolactones. However, the origin of this sensory system is unknown. Conn et al. investigated the diversity of strigolactone receptors in multiple lineages of parasitic plants and their close relatives. They found a greater copy number and accelerated evolution in parasitic plants as compared with nonparasitic relatives. Functional analyses of parasitic plant strigolactone receptors in transgenic Arabidopsis suggested that convergent evolution has occurred to allow the parasitic plants to detect their hosts. Science, this issue p. 540 Obligate parasitic Orobanchaceae plants germinate after sensing strigolactones exuded from host roots. Obligate parasitic plants in the Orobanchaceae germinate after sensing plant hormones, strigolactones, exuded from host roots. In Arabidopsis thaliana, the α/β-hydrolase D14 acts as a strigolactone receptor that controls shoot branching, whereas its ancestral paralog, KAI2, mediates karrikin-specific germination responses. We observed that KAI2, but not D14, is present at higher copy numbers in parasitic species than in nonparasitic relatives. KAI2 paralogs in parasites are distributed into three phylogenetic clades. The fastest-evolving clade, KAI2d, contains the majority of KAI2 paralogs. Homology models predict that the ligand-binding pockets of KAI2d resemble D14. KAI2d transgenes confer strigolactone-specific germination responses to Arabidopsis thaliana. Thus, the KAI2 paralogs D14 and KAI2d underwent convergent evolution of strigolactone recognition, respectively enabling developmental responses to strigolactones in angiosperms and host detection in parasites.

The D3 F‐box protein is a key component in host strigolactone responses essential for arbuscular mycorrhizal symbiosis

Arbuscular mycorrhiza (AM) represents an ancient endosymbiosis between plant roots and Glomeromycota fungi. Strigolactones (SLs), plant-derived terpenoid lactones, activate hyphal branching of AM fungi before physical contact. Lack of SL biosynthesis results in lower colonization of AM fungi. The F-box protein, DWARF3 (D3), and the hydrolase family protein DWARF14 (D14) are crucial for SL responses in rice. Here we conducted AM fungal colonization assays with the SL-insensitive d3 and d14 mutants. The d3 mutant exhibited strong defects in AM fungal colonization, whereas the d14 mutant showed higher AM fungal colonization. As D14 has a homologous protein, D14-LIKE, we generated D14-LIKE knockdown lines by RNA interference in the wildtype and d14 background. D14 and D14-LIKE double knockdown lines exhibited similar colonization rates as those of the d14-1 mutant. D3 is crucial for establishing AM symbiosis in rice, whereas D14 and D14-LIKE are not. Our results suggest distinct roles for these SL-related components in AM symbiosis.