ハナキカンスウ ノ ガンケンセイ ト バラツキ ヲ ウミダス ハッセイ キバン ノ リロンテキ タンキュウ

本論文第3章の内容はOxford University Press のAnnals of Botany 誌に掲載される予定である（ DOI: 10.1093/aob/mcw034

Co-orthologs of KATANIN1 Impact Plant Morphology and Show Differential Evolution in Maize

Understanding how the size and shape of crop plants and their specific organs are genetically controlled may allow for the development of cultivars with improved plant architecture. A microtubule-severing enzyme called katanin p60 is encoded by KATANIN1 (KTN1) in Arabidopsis or by an ortholog, dwarf and gladius leaf1 (dgl1), in rice. Katanin p60 has been implicated in the control of anisotropic cell growth, which is cell growth directed in a specific direction instead of equally in all directions. Anisotropic cell growth is crucial for proper plant shape and its disruption in ktn1/dgl1 mutants leads to morphological changes such as stunted plant height, shorter leaves and reduced inflorescence size

The interplay between a Phytophthora RXLR effector and an Arabidopsis lectin receptor kinase

Phytophthora infestans – the causal agent of potato late blight – secretes a plethora of effector proteins to facilitate plant infection. The central subject of this thesis is ipiO, one of the first cloned Phytophthora genes with a putative function in pathogenicity as was anticipated based on its in planta induced (ipi) expression, in particular during early stages of host infection. IPI-O contains two striking motifs: RXLR-dEER and RGD. RGD is a cell adhesion motif and was shown to be involved in binding to the extracellular lectin domain of LecRK-I.9, a lectin receptor kinase of Arabidopsis. The RXLR-dEER motif plays a role in effector trafficking into host cells and is shared by several secreted oomycete effector proteins which are known to function as race-specific avirulence (Avr) factors. In a previous study, that was aimed at identifying novel pairs of P. infestans Avr and host plant resistance (R) genes, a high-throughput effector genomics screen identified ipiO as Avr-blb1, the counterpart of the late blight R gene Rpi-blb1 which originates from the nightshade Solanum bulbocastanum. Often R genes exploited in late blight resistance breeding become rapidly ineffective as a result of adaptation of P. infestans. However, unlike most late blight R genes that interact in a gene-for-gene manner with Avr genes, Rpi-blb1 seemed to have the potential to remain its effectiveness. In section 2 we monitored the genetic variation and distribution of the ipiO family in an extensive isolate collection of P. infestans and closely related species. This resulted in the identification of 16 IPI-O variants that could be sub-divided in three distinct classes. Variants from class I and class II were shown to induce cell death when co-infiltrated with Rpi-blb1 in Nicotiana benthamiana. Class III consists solely of the highly divergent variant IPI-O4, that is not able to trigger Rpi-blb1-mediated cell death. Class I is highly diverse and represented in all P. infestans isolates analyzed so far, except in two Mexican P. infestans isolates. The latter two are capable to infect Rpi-blb1 plants, suggesting that the lack of class I variants in the genome of these strains allows them to escape recognition by Rpi-blb1 plants. We propose that profiling of the ipiO variants within P. infestans populations can predict the effectiveness of Rpi-blb1-mediated resistance in potato and, as such, can facilitate integrated disease management. Section 3 of this thesis deals with legume-like lectin receptor kinases (LecRKs), membrane-spanning proteins with potential roles in adaptive responses and cell wall integrity. We present an inventory and a phylogenetic analysis of the Arabidopsis LecRK gene family. The rationale behind this study was to gain better insight into the diversity of LecRKs and their potential roles in plant defense. A comprehensive expression analysis based on exploration of existing databases revealed that several LecRK genes are induced upon treatment with elicitors or during pathogen infection. Based on the phylogenetic analysis we have reclassified the LecRK genes and proposed a new nomenclature. LecRK-I.9, one of the clade I Arabidopsis LecRKs which binds the RGD cell adhesion motif of IPI-O, was shown to mediate adhesion between the cell wall (CW) and plasma membrane (PM). In contrast, IPI-O disrupts these adhesions by virtue of its RGD motif. We analyzed Arabidopsis LecRK-I.9 knock-out lines (lecrk-I.9) for their response to pathogen infection, in particular to Phytophthora brassicae. We also analyzed transgenic Arabidopsis lines expressing ipiO, and observed that both the ipiO-expressing lines and lecrk-I.9 lines are impaired in their resistance to oomycete pathogens. To unravel the mechanisms underlying this phenomenon we analysed callose deposition upon MAMP (i.e. flg22) treatment and investigated the strength of CW-PM adhesions under plasmolysis-inducing conditions. The results indicated that LecRK-I.9 is not only important for the maintenance of the CW-PM continuum, but also in MAMP-triggered immunity. Also here, both the ipiO-expressing lines and the lecrk-I.9 knock-outs displayed a destabilized CW-PM continuum and impaired callose deposition, and hence, they can be regarded as phenocopies. Arabidopsis plants that constitutively express LecRK-I.9 were smaller in size, and displayed increased levels of anthocyanin and lignin. Additionally, these lines were shown to exhibit enhanced resistance to P. brassicae. Furthermore, we studied transgenic potatoes that constitutively Arabidopsis LecRK-I.9. In comparison to the parental control potato line the transgenic lines were less susceptible to mild and moderately aggressive P. infestans isolates, but the increased tolerance was not sufficient to provide resistance to aggressive isolates. These results strongly suggest that LecRK-I.9 is a novel resistance component that plays a role in defense against Phytophthora. In Section 4 we describe a novel method for propagating P. brassicae zoospores on an intermediate host plant. This resulted in the production of high numbers of zoospores thereby facilitating highly reproducible small and large scale inoculation experiments. This thesis is completed with a general discussion (Section 5) addressing the current understanding of effector uptake by host cells, the subsequent recognition by cognate R proteins mediating effector-triggered immunity, and RXLR-dEER effector diversity. We also discuss the role of the RGD motif in effectors of both animal and plant pathogens, and the potential functions of LecRKs. Finally, we high-light the advantages of Arabidopsis-Phytophthora pathosystems as research object. <br/

Exploring the role of cell-wall pectin cross-linking in freezing tolerance and guard cell dynamics in Arabidopsis thaliana

Freezing stress is detrimental to plants, resulting in major crop losses in temperate regions. The plant cell wall is a dynamic network of proteins and polysaccharides including cellulose, hemicellulose and pectins. It is essential for plant survival, providing structural integrity, strength and protection against pathogens. As the cell wall is the site of ice formation, it has also been suggested that the wall could contribute towards protection of the plant against freezing damage. The cell wall undergoes remodelling during cold acclimation, but it is unclear what specific role this restructuring may play in freezing tolerance. The sensitive to freezing8 (sfr8) mutant contains less cell wall fucose due do a mutation in the fucose biosynthetic gene MUR1. This was shown to result in a decrease in dimerisation of the cell wall pectic domain rhamnogalacturonan-II (RG-II), which in wild type plants is predominantly dimerised via a borate-ester cross-link. This decrease in dimerisation likely results in the observed freezing sensitivity of mur1 mutants, as supplementation of plants with boric acid was shown to restore freezing tolerance. Guard cell dynamics were also compromised in the sfr8 mutant, as stomata were found to be more restricted in their movements than wild type in response to ABA, CO2 and changes in humidity. The freezing and guard cell phenotypes of sfr8 may be attributed to a decrease in the tensile modulus of the cell wall with reduced RG-II dimerisation. This makes the wall more vulnerable to deformation during freezing and prevents the guard cells from stiffening to allow an increase in stomatal aperture. RG-II dimerisation also mediates certain structural aspects of the cell wall that may facilitate supercooling by excluding ice nucleation and preventing ice growth. This research reveals the importance of RG-II dimerisation in cell wall dynamics and the impact cell-wall composition has on freezing and desiccation tolerance. These findings could lead to the identification of new targets for crop breeding

Forward genetic analysis of cellulose biosynthesis inhibitor resistance and wall hydrolysis sensitivity.

The functional analysis of components involved in cellulose biosynthesis is central in understanding cell wall assembly and structure in plants. We conducted screens using the herbicides, isoxaben and flupoxam which inhibit cellulose biosynthesis in higher plants. Mutations resulting in a high degree of resistance to isoxaben (ixr) or flupoxam (fxr) were attributed to single amino acid substitutions in primary wall CESAs. Twelve novel resistance alleles were isolated and no cross-resistance was observed. Point mutations were mostly clustered around the C-terminal regions of CESA1 and CESA3, and CESA3 and CESA6 for fxr and ixr respectively. Resistance to isoxaben was also conferred by modification to the putative catalytic regions of CESA3. This resulted in cellulose deficient phenotypes characterized by reduced crystallinity and dwarfism. These results provide genetic evidence supporting CESA1-CESA3, and CESA3-CESA6 association with flupoxam and isoxaben respectively targeting and disrupting these interactions. The ixr and fxr mutants also exhibited enhanced saccharification under enzymatic degradation schemes which is consistent with the observed reduction in cellulose crystallinity. A second forward genetic screen was performed using mild acid hydrolysis to isolate mutants with enhanced saccharification. This screen identified sixty-three responsive to acid hydrolysis (rah) lines. Unconventional strategies to increase sugar yields from plant biomass where highlighted. These included starch hyper-accumulators such as starch excess 4 (sex4) loss-of-function mutants and the perturbation of polar auxin transport. Disruption of the serine/threonine kinase positive regulator of auxin efflux, PINOID (PID) was found to significantly enhance sugar release in Arabidopsis and similar effects were observed in the maize orthologue, BARREN INFLORESENCE 2 (BIF2). Furthermore, the application of N-1-naphthylphthalamic acid (NPA) in Arabidopsis, maize, Miscanthus and switchgrass phenocopied the enhanced wall saccharification effects of PID. This study attempted to elucidate some of the interactions of seemingly unrelated pathways in the context of wall biosynthesis and saccharification enhancement

Plant Development and Organogenesis: From Basic Principles to Applied Research

The way plants grow and develop organs significantly impacts the overall performance and yield of crop plants. The basic knowledge now available in plant development has the potential to help breeders in generating plants with defined architectural features to improve productivity. Plant translational research effort has steadily increased over the last decade due to the huge increase in the availability of crop genomic resources and Arabidopsis-based sequence annotation systems. However, a consistent gap between fundamental and applied science has yet to be filled. One critical point often brought up is the unreadiness of developmental biologists on one side to foresee agricultural applications for their discoveries, and of the breeders to exploit gene function studies to apply to candidate gene approaches when advantageous on the other. In this book, both developmental biologists and breeders make a special effort to reconcile research on the basic principles of plant development and organogenesis with its applications to crop production and genetic improvement. Fundamental and applied science contributions intertwine and chase each other, giving the reader different but complementary perspectives from only apparently distant corners of the same world

Evolution du régulateur floral LEAFY dans la lignée verte

LEAFY (LFY) is a unique transcription factor, highly conserved within land plants. LFY directly regulates a set of genes participating in floral development in angiosperms (flowering plants), but its role in the other groups of land plants is unknown, except in the moss Physcomitrella patens where the LFY ortholog (PpLFY) regulates the first cell division in the zygote. PpLFY does not bind to the same DNA sequences as LFY from Arabidopsis thaliana, in spite of the very high degree of conservation of their DNA binding domains. Thus, it appears that the properties of LFY have changed during evolution ; the goal of my thesis was to find out if such changes had occurred frequently in land plants, and what are their origins and consequences on target genes regulation. I performed SELEX experiments on LFY orthologs from all land plants, which revealed that their DNA binding specificty was highly conserved, except in the case of PpLFY. These results allowed us to build an accurate biophysical model to predict LFY binding on DNA fragments at a genomic level, which we applied on the evolution of the regulation of key target genes by LFY. We were able to predict the regulation of the floral gene AGAMOUS by LFY in various angiosperm species, et we could also show that LFY was very likely regulating gymnosperm orthologs of genes involved in floral organ identity, even before the appearance of the flower. The change in DNA binding specificity observed for PpLFY led us to study more precisely the consequences of this change for the regulation of target genes : for this, I initiated bioinformatic and experimental work in P. patens. Finally, to understand how this change in DNA binding specificity had occurred during evolution, we looked for the ancestor of LFY and found out that LFY already existed in green algae. We are currently investigating the ancestral specificity of LFY in these species.LEAFY (LFY) est un facteur de transcription unique et très conservé chez les plantes terrestres. Il contrôle le développement floral chez les angiospermes (plantes à fleurs), mais son rôle est encore mal connu chez toutes les autres plantes terrestres à l'exception de la mousse Physcomitrella patens où l'orthologue de LFY (PpLFY) est requis pour la première division cellulaire du zygote. PpLFY ne reconnaît pas les mêmes séquences d'ADN que LFY d'Arabidopsis thaliana, malgré la très forte conservation de leurs domaines de liaison à l'ADN. LFY semble donc avoir changé de propriétés au cours de l'évolution ; l'objectif de ma thèse a été de déterminer si de tels changements s'étaient produits fréquemment chez les plantes terrestres, et de comprendre leur origine et leur impact sur la régulation des gènes cibles de LFY. Pour cela, j'ai étudié la spécificité de liaison à l'ADN des orthologues de LFY chez les grands groupes de plantes terrestres par des expériences de SELEX, et cette spécificité s'est révélée très fortement conservée, excepté dans le cas de PpLFY. Ces résultats nous ont permis de construire un modèle biophysique performant pour prédire la liaison de LFY à l'échelle génomique, ce que nous avons appliqué à l'étude de l'évolution de la régulation de quelques gènes clés par LFY. Nous avons ainsi pu prédire la régulation du gène floral AGAMOUS par LFY chez différentes espèces angiospermes, et nous avons pu montrer que LFY régulait très vraisemblablement les orthologues des gènes d'identité florale chez les gymnospermes, c'est-à-dire avant l'apparition de la fleur. La divergence de spécificité de PpLFY nous a poussés à étudier les gènes cibles de PpLFY : pour cela, j'ai initié des approches bioinformatiques et expérimentales chez P. patens. Enfin, pour comprendre comment ce changement de spécificité s'est déroulé au cours de l'évolution, nous nous sommes penchés sur l'ancêtre de LFY et avons découvert que LFY était déjà présent chez les algues vertes. Des études pour déterminer la spécificité ancestrale de LFY chez ces espèces ont été initiées