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The effect of Ascophyllum nodosum extracts on the nodulation and growth of alfalfa was investigated. Plant growth assay revealed that alfalfa treated with 2 g L-1 ANE exhibited a significant increase in leaf area. Under salt stress, alfalfa treated with 0.5 g L-1 ANE exhibited a significant increase in total length compared to controls. A root hair deformation assay indicated that ANE 0.5 g L-1 stimulated the synthesis of Nod factors secreted by rhizobia thus accelerate root hair deformation of alfalfa. Similarly, ANE 0.5 g L-1 caused an increase in nodC gene expression suggesting that ANE may act similarly to flavonoids in the rhizobium-legume symbiosis. Under field conditions, ANE increased the total number of functional nodules, total root length and total leaf area. Taken together, the results suggest that ANE may contain compound(s) that promote specific metabolic pathway both in alfalfa and bacterium thus enhance the symbiotic relationship.

To explore the use of defensins for transgenic plant disease resistance, expression by agroinfiltration of plants, stable transformation of plants and stable transformation of yeast were tested for porcine β-defensin 1 (pbd-1) and Nicotiana tabacum defensin 1 (Ntdef1). Attempts to screen constructs by agroinfiltration of Nicotiana benthamiana leaves revealed that agroinfiltration alone induced localized resistance against Colletotrichum destructivum. A comparison of Agrobacterium tumefaciens strains showed that the induced resistance required the transfer of type IV effectors into plant cells and was independent of salicylic acid or ethylene signaling. Stable expression of pbd-1 in N. tabacum and Pichia pastoris showed that PBD-1 purified from P. pastoris had varying degrees of antimicrobial activity against a broad range of microbes, including P. syringae pv. tabaci, C. destructivum and C. orbiculare, but in transgenic N. tabacum, the protein could not be detected and resistance increased only slightly to P. syringae pv. tabaci but not to C. destructivum or C. orbiculare. Stable expression of Ntdef1 in P. pastoris yielded a protein with no or little antimicrobial activity, and stable expression in N. tabacum did not result in detectable Ntdef1 or increased resistance to those pathogens. Although PBD-1 had strong antimicrobial activity against plant pathogens, plant disease resistance likely did not increase because of the low level of the protein in the plants, whereas resistance did not increase with Ntdef1 likely because of low antimicrobial activity and low levels of the protein in the plant. This research demonstrates that agroinfiltration is not appropriate for testing genes for antimicrobial activity in planta, while the P. pastoris expression system is useful for producing protein for in vitro tests of a gene prior to its transfer to plants.

Microbial succession, composition and ecological distribution within the gastro-intestinal tract are critical areas of study since commensal bacteria have been shown to affect animal health and development. A series of experiments were conducted to determine whether altered microbial succession in neonatal animals would modulate the development and health of pigs later in life. An initial experiment in conventional pigs was conducted to establish the early postnatal microbial succession profile and to identify early colonizing bacterial species. Culture-independent analysis of digesta and mucosal microbiota showed distinct variation between the proximal and distal gastro-intestinal tract (GIT) indicating that fecal or distal gut profiles cannot be used to predict succession in the upper GIT. Temporally, Clostridium spp. were found to be most prevalent in the GIT microbiota of the neonatal pig up to 0.5 d of age, accompanied by a high abundance of Escherichia and Shigella spp. These genera were transiently displaced by Streptococcus spp. followed by a preponderance of Lactobacillus spp. between 3 and 20 d of age. Subsequently, a “snatch-farrow” model was employed to modulate early postnatal microbial succession and investigate the effects on postweaning microbial composition. Pigs were collected into sterile towels directly from the vaginal canal and transferred to a sterile isolator environment for the first 4 days. Pigs were either inoculated with sow feces or not at 1 d of age resulting in significant differences in fecal microbial profile at 4 days of age, prior to removal from isolators. Analysis using terminal restriction fragment length polymorphisms (TRFLP) of intestinal microbiota at 28 d of age did not show significant clustering or variation in diversity indices for either group during the 4-d postnatal isolator phase. However, enumeration of selected taxa using quantitative PCR did indicate significant treatment differences in postweaning microbiota. Despite these results, this approach was rejected for further use as the protocol provided only moderate control of early postnatal colonization and variation and unpredictability of the timing of natural farrowing contributed to significant litter effects. Finally, a gnotobiotic monoassociation model was used investigate the effects of modulating early postnatal microbial succession on postweaning physiology, microbial composition and mucosal gene expression. Twenty-four cesarean-section derived piglets were monoassociated for the first 4 days of life with either L. mucosae (L), S. infantarius (S), C. perfringens (C) or E. coli (E). Pigs from treatments E and L animals showed the highest growth rate during the conventional rearing period (7-28 d of age). Monoassociation with different bacterial species during the first 4 d of life resulted in significant changes in postweaning microbial composition in small intestine and colon as assessed by quantitative PCR, although TRFLP did not identify unique clustering by treatment or variation in diversity. L. mucosae was the only inoculant species with significant variation, with a reduction in the colonic mucosa at 28 days of age. Monoassociation with L. mucosae was also associated with increased nutrition related gene expression in small intestine. Pigs monoassociated with E. coli had low expression of microbial sensing (TLR2 and 4), NFkappaB complex genes and mucins at 28 d of age. This study clearly showed that controlled early microbial succession in neonatal pigs altered post-weaning commensal microbiota composition, postweaning physiology and host gene expression in small and large intestine. The findings suggest the importance of peri-natal management and feeding strategies in promoting postweaning health and performance.

Cytoplasmic incompatibility (CI) is an intriguing, widespread, symbiont-induced reproductive failure that decreases offspring production of arthropods through crossing incompatibility of infected males with uninfected females or with females infected with a distinct symbiont genotype. For years, the molecular mechanism of CI remained unknown. Recent genomic, proteomic, biochemical, and cell biological studies have contributed to understanding of CI in the alphaproteobacterium Wolbachia and implicate genes associated with the WO prophage. Besides a recently discovered additional lineage of alphaproteobacterial symbionts only moderately related to Wolbachia, Cardinium (Bacteroidetes) is the only other symbiont known to cause CI, and genomic evidence suggests that it has very little homology with Wolbachia and evolved this phenotype independently. Here, we present the first transcriptomic study of the CI Cardinium strain cEper1, in its natural host, Encarsia suzannae, to detect important CI candidates and genes involved in the insect-Cardinium symbiosis. Highly expressed transcripts included genes involved in manipulating ubiquitination, apoptosis, and host DNA. Female-biased genes encoding ribosomal proteins suggest an increase in general translational activity of Cardinium in female wasps. The results confirm previous genomic analyses that indicated that Wolbachia and Cardinium utilize different genes to induce CI, and transcriptome patterns further highlight expression of some common pathways that these bacteria use to interact with the host and potentially cause this enigmatic and fundamental manipulation of host reproduction. IMPORTANCE The majority of insects carry maternally inherited intracellular bacteria that are important in their hosts' biology, ecology, and evolution. Some of these bacterial symbionts cause a reproductive failure known as cytoplasmic incompatibility (CI). In CI, the mating of symbiont-infected males and uninfected females produces few or no daughters. The CI symbiont then spreads and can have a significant impact on the insect host population. Cardinium, a bacterial endosymbiont of the parasitoid wasp Encarsia in the Bacteroidetes, is the only bacterial lineage known to cause CI outside the Alphaproteobacteria, where Wolbachia and another recently discovered CI symbiont reside. Here, we sought insight into the gene expression of a CI-inducing Cardinium strain in its natural host, Encarsia suzannae. Our study provides the first insights into the Cardinium transcriptome and provides support for the hypothesis that Wolbachia and Cardinium target similar host pathways with distinct and largely unrelated sets of genes.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20438号 / 農博第2223号 / 新制||農||1049(附属図書館) / 学位論文||H29||N5059(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 矢﨑 一史, 教授 森 直樹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Legumes are unique among plants for their ability to fix atmospheric nitrogen with the help of soil bacteria rhizobia. Medicago truncatula is used as a model legume to study different aspects of symbiotic nitrogen fixation. M. truncatula, in association with its symbiotic partner Sinorhizobium meliloti, fix atmospheric nitrogen into ammonia, which the plant uses for amino acid biosynthesis and the bacteria get reduced photosynthate in return. M. truncatula NPF1.7 previously called MtNIP/LATD is required for symbiotic nitrogen fixing root nodule development and for normal root architecture. Mutations in MtNPF1.7 have defects in these processes. MtNPF1.7 encodes a member of the NPF family of transporters. Experimental results showing that MtNPF1.7 functioning as a high-affinity nitrate transporter are its expression restoring chlorate susceptibility to the Arabidopsis chl1-5 mutant and high nitrate transport in Xenopus laevis oocyte system. However, the weakest Mtnip-3 mutant allele also displays high-affinity nitrate transport in X. laevis oocytes and chlorate susceptibility to the Atchl1-5 mutant, suggesting that MtNPF1.7 might have another biochemical function. Experimental evidence shows that MtNPF1.7 also functions in hormone signaling. Constitutive expression of MtNPF1.7 in several species including M. truncatula results in plants with a robust growth phenotype. Using a synthetic auxin reporter, the presence of higher auxin in both the Mtnip-1 mutant and in M. truncatula plants constitutively expressing MtNPF1.7 was observed. Previous experiments showed MtNPF1.7 expression is hormone regulated and the MtNPF1.7 promoter is active in root and nodule meristems and in the vasculature. Two potential binding sites for an auxin response factors (ARFs) were found in the MtNPF1.7 promoter. Chromatin immunoprecipitation-qRT-PCR confirmed MtARF1 binding these sites. Mutating the MtARF1 binding sites increases MtNPF1.7 expression, suggesting a mechanism for auxin repression of MtNPF1.7. Consistent with these results, constitutive expression of an ARF in wild-type plants partially phenocopies Mtnip-1 mutants’ phenotypes.

Most plant pathogens are specialists where only few plant species are susceptible, while all other plants are resistant. Unraveling the mechanisms behind this can thus provide valuable information for breeding or engineering crops with durable disease resistance. A group of Pseudomonas syringae strains with different host ranges while still closely related were thus chosen for comparative study. We confirmed their close phylogenetic relationship. We found evidence supporting that these strains recombined during evolution. The Arabidopsis thaliana and tomato pathogen P. syringae pv. tomato (Pto) DC3000 was found to be an atypical tomato strain, distinct from the typical Pto strains commonly isolated in the field that do not cause disease in A. thaliana, such as Pto T1. Comparing A. thaliana defense responses to DC3000 and T1, we found that T1 is eliciting stronger responses than DC3000. T1 is likely lacking Type III effector genes necessary to suppress plant defense. To test this, we sequenced the genomes of strains that cause and do not cause disease in A. thaliana. Comparative genomics revealed candidate effector genes responsible for this host range difference. Effector genes conserved in strains pathogenic in A. thaliana were expressed in T1 to test whether they would allow T1 to growth better in A. thaliana. Surprisingly, most of them reduced T1 growth. One of the effectors, HopM1, was of particular interest because it is disrupted in typical Pto strains. Although HopM1 has known virulence function in A. thaliana, HopM1 reduced T1 growth in both A. thaliana and tomato. HopM1 also increased the number of bacterial specks but reduced their average size in tomato. Our data suggest that HopM1 can trigger defenses in these plants. Additionally, transgenic detritivore Pseudomonas fluorescens that can secrete HopM1 shows dramatically increased growth in planta. The importance of genetic background of the pathogen for the functions of individual effectors is discussed. T1 cannot be manipulated to become an A. thaliana pathogen by deleting or adding individual genes. We now have a list of genes that can be studied in the future for the molecular basis of host range determination. / Ph. D.

Orchids are characterized by producing minute endosperm-lacking seeds, which depend on mycorrhizal fungi for germination and embryo development. Some aclorophyllous orchids remain dependent on the mycorrhizal association for carbon acquisition during their whole life history, whereasother orchids develop photosynthesis. Despite the biological significance of orchid mycorrhiza, gene expression studies are lacking. We have used different highthroughput approaches in order to understanding the mechanisms regulating orchid mycorrhiza development and functioning. Firstly, we have used a 2D-LC-MS/MS approach coupled to isobaric tagging for relative and absolute quantification (iTRAQ) to identify proteins with differential accumulation in Oncidium sphacelatum at different stages of mycorrhizal protocorm development (achlorophyllous and green protocorms) after seed inoculation with a Ceratobasidium sp. isolate. Quantitative analysis showed that the expected changes in carbon metabolism in green protocorms were accompanied by enhanced accumulation of proteins involved in the modulation of reactive oxygen species homeostasis, defense related responses, phytoalexins and carotenoid biosynthesis, suggesting that orchid protocorms undergo profound metabolic changes during the switch from the fully mycoheterotrophic to the photosynthethic stage. Secondly, three different proteomic techniques were carried out in independent experiments aiming to identify changes in protein accumulation in mycorrhizal roots of the terrestrial orchid Oeceoclades maculata.Finally, O. maculatamycorrhizal roots were used for transcriptome analyses. The data revealed a strong increase in general stress responses, accompanied by changes in signaling pathways possibly related to fungal recognition and establishment of a compatible interaction. Some of the upregulated genes may be involved in the reorganization of cell structure, likely related to accommodation of the fungal symbiont in the plant roots. We have also observed in mycorrhizal roots up-regulation of genes involved in carbon metabolism, including glycolysis/gluconeogenesis and amino sugars metabolism, as well as genes involved innitrogen assimilation. The down-regulation of genes involved in the jasmonate and ABA transduction pathways, and key genes encoding anti-fungal proteins, such as chitinase and a mannose-specific binding lectin, strongly suggests an alleviation of plant defense responses in O. maculata mycorrhizal roots. In general, our data suggest that the physiology of an orchid mycorrhiza is more similar to a compatible interaction than to an arm-race between plant and fungi. Overall orchid mycorrhiza have proved to be a promising model for investigating plantfungal interactions and further studies should now address the specific roles of the genes showing differential regulation in this study. / As orquídeas são caracterizadas por produzirem sementes diminutas, que não possuem endosperma. Necessitam, portanto, da interação com fungos micorrízicos para germinação e desenvolvimento do embrião. Algumas orquídeas aclorofiladas se mantêm dependentes dos fungos micorrízicos para a aquisição de carbono, enquanto outras desenvolvem a maquinaria fotossintética. Apesar do significado biológico das micorrizas de orquídeas, alterações na expressão gênica e no acúmulo de proteínas foram altamente negligenciads nos últimos anos. Neste trabalho, foram utilizadas diferentes técnicas sequenciamento e identificação de genes e proteínas em larga-escala para acessar as alterações moleculares responsáveis pela regulação das micorrizas de orquídeas. Uma abordagem baseada em 2D-LC MS/MS acoplada a técnica de quantificação absoluta e relativa iTRAQ, foiutilizada para identificar proteínas com acúmulo diferencial em Oncidium sphacelatum em diferentes estágios do desenvolvimento do protocormo (protocormos aclorofilados versus protocormos fotossintetizantes), após inoculação com um fungo do gênero Ceratobasidium. As análises mostraram que, as alterações esperadas no metabolismo do carbono foram acompanhadas de um acúmulo aumentado de proteínas envolvidas na modulação de espécies reativas de oxigênio, respostas de defesa, biossíntese de fitoalexinas e carotenóides, sugerindo que os protocormos de orquídeas passam por profundas alterações metabolicas durante a transição do metabolismo micoheterotrófico para o fotossintético. Posteriormente foram utilizadas três diferentes técnicas de proteômica quantitativa para explorar alterações fisiológicas em raízes micorrizadas e não-micorrizadas de Oeceoclades maculata.Este estudo foi ampliado, pela utilização de uma abordagem transcritômica ao mesmo modelo biológico. Em conjunto, os dados revelaram um forte aumento em respostas relacionadas ao estresse, acompanhadas de alterações em vias de transdução de sinal possivelmente relacionadas ao reconhecimento do simbionte fúngico e estabelecimento de uma interação compatível. Alguns genes com expressão aumentada devem estar envolvidos na reorganização celular, provavelmente ligada a acomodação do simbionte fúngico nas raízes das plantas. Também foi observado o aumento de genes envolvidos no metabolismo do carbono e de açúcares aminados, juntamente a genes relacionados a assimilação de nitrogênio em raízes micorrizadas. A expressão diminuída de genes envolvidas nas vias do jasmonato e ácido abscícico, juntamente a genes-chave que codificam para proteínas anti-fúngicas sugerem fortemente uma atenuação das respostas de defesa da planta em raízes micorrizadas de Oeceoclades maculata. No geral, parece que as micorrizas de orquídeas são fisiológicamente mais próximas de uma simbiose compatível do que de uma interação unilateral em favor da planta. Sobretudo, este sistema biológico provou ser promissor para investigação de interações planta-fungo e, próximas pesquisas devem agora ser focadas em funções específicas dos genes que mostraram regulação diferencial neste estudo.

Orchids are characterized by producing minute endosperm-lacking seeds, which depend on mycorrhizal fungi for germination and embryo development. Some aclorophyllous orchids remain dependent on the mycorrhizal association for carbon acquisition during their whole life history, whereasother orchids develop photosynthesis. Despite the biological significance of orchid mycorrhiza, gene expression studies are lacking. We have used different highthroughput approaches in order to understanding the mechanisms regulating orchid mycorrhiza development and functioning. Firstly, we have used a 2D-LC-MS/MS approach coupled to isobaric tagging for relative and absolute quantification (iTRAQ) to identify proteins with differential accumulation in Oncidium sphacelatum at different stages of mycorrhizal protocorm development (achlorophyllous and green protocorms) after seed inoculation with a Ceratobasidium sp. isolate. Quantitative analysis showed that the expected changes in carbon metabolism in green protocorms were accompanied by enhanced accumulation of proteins involved in the modulation of reactive oxygen species homeostasis, defense related responses, phytoalexins and carotenoid biosynthesis, suggesting that orchid protocorms undergo profound metabolic changes during the switch from the fully mycoheterotrophic to the photosynthethic stage. Secondly, three different proteomic techniques were carried out in independent experiments aiming to identify changes in protein accumulation in mycorrhizal roots of the terrestrial orchid Oeceoclades maculata.Finally, O. maculatamycorrhizal roots were used for transcriptome analyses. The data revealed a strong increase in general stress responses, accompanied by changes in signaling pathways possibly related to fungal recognition and establishment of a compatible interaction. Some of the upregulated genes may be involved in the reorganization of cell structure, likely related to accommodation of the fungal symbiont in the plant roots. We have also observed in mycorrhizal roots up-regulation of genes involved in carbon metabolism, including glycolysis/gluconeogenesis and amino sugars metabolism, as well as genes involved innitrogen assimilation. The down-regulation of genes involved in the jasmonate and ABA transduction pathways, and key genes encoding anti-fungal proteins, such as chitinase and a mannose-specific binding lectin, strongly suggests an alleviation of plant defense responses in O. maculata mycorrhizal roots. In general, our data suggest that the physiology of an orchid mycorrhiza is more similar to a compatible interaction than to an arm-race between plant and fungi. Overall orchid mycorrhiza have proved to be a promising model for investigating plantfungal interactions and further studies should now address the specific roles of the genes showing differential regulation in this study. / As orquídeas são caracterizadas por produzirem sementes diminutas, que não possuem endosperma. Necessitam, portanto, da interação com fungos micorrízicos para germinação e desenvolvimento do embrião. Algumas orquídeas aclorofiladas se mantêm dependentes dos fungos micorrízicos para a aquisição de carbono, enquanto outras desenvolvem a maquinaria fotossintética. Apesar do significado biológico das micorrizas de orquídeas, alterações na expressão gênica e no acúmulo de proteínas foram altamente negligenciads nos últimos anos. Neste trabalho, foram utilizadas diferentes técnicas sequenciamento e identificação de genes e proteínas em larga-escala para acessar as alterações moleculares responsáveis pela regulação das micorrizas de orquídeas. Uma abordagem baseada em 2D-LC MS/MS acoplada a técnica de quantificação absoluta e relativa iTRAQ, foiutilizada para identificar proteínas com acúmulo diferencial em Oncidium sphacelatum em diferentes estágios do desenvolvimento do protocormo (protocormos aclorofilados versus protocormos fotossintetizantes), após inoculação com um fungo do gênero Ceratobasidium. As análises mostraram que, as alterações esperadas no metabolismo do carbono foram acompanhadas de um acúmulo aumentado de proteínas envolvidas na modulação de espécies reativas de oxigênio, respostas de defesa, biossíntese de fitoalexinas e carotenóides, sugerindo que os protocormos de orquídeas passam por profundas alterações metabolicas durante a transição do metabolismo micoheterotrófico para o fotossintético. Posteriormente foram utilizadas três diferentes técnicas de proteômica quantitativa para explorar alterações fisiológicas em raízes micorrizadas e não-micorrizadas de Oeceoclades maculata.Este estudo foi ampliado, pela utilização de uma abordagem transcritômica ao mesmo modelo biológico. Em conjunto, os dados revelaram um forte aumento em respostas relacionadas ao estresse, acompanhadas de alterações em vias de transdução de sinal possivelmente relacionadas ao reconhecimento do simbionte fúngico e estabelecimento de uma interação compatível. Alguns genes com expressão aumentada devem estar envolvidos na reorganização celular, provavelmente ligada a acomodação do simbionte fúngico nas raízes das plantas. Também foi observado o aumento de genes envolvidos no metabolismo do carbono e de açúcares aminados, juntamente a genes relacionados a assimilação de nitrogênio em raízes micorrizadas. A expressão diminuída de genes envolvidas nas vias do jasmonato e ácido abscícico, juntamente a genes-chave que codificam para proteínas anti-fúngicas sugerem fortemente uma atenuação das respostas de defesa da planta em raízes micorrizadas de Oeceoclades maculata. No geral, parece que as micorrizas de orquídeas são fisiológicamente mais próximas de uma simbiose compatível do que de uma interação unilateral em favor da planta. Sobretudo, este sistema biológico provou ser promissor para investigação de interações planta-fungo e, próximas pesquisas devem agora ser focadas em funções específicas dos genes que mostraram regulação diferencial neste estudo.