Viral diseases constitute a major threat to the agricultural production and food security throughout the world. Plants cope with the invading viruses by triggering immune responses and small RNA interference (RNAi) systems. In prokaryotes, CRISPR/Cas systems function as an adaptive immune system to provide bacteria with resistance against invading phages and conjugative plasmids. Interestingly, CRISPR/Cas9 system was shown to interfere with eukaryotic DNA viruses and confer resistance against plant DNA viruses. The majority of the plant viruses have RNA genomes. The aim of this study is to test the ability of the newly discovered CRISPR/Cas13a immune system, that targets and cleaves single stranded RNA (ssRNA) in prokaryotes, to provide resistance against RNA viruses in plants. Here, I employ the CRISPR/Cas13a system for molecular interference against Turnip Mosaic Virus (TuMV), a plant RNA virus. The results of this study established the CRISPR/Cas13a as a molecular interference machinery against RNA viruses in plants. Specifically, my data show that the CRISPR/Cas13a machinery is able to interfere with and degrade the TuMV (TuMV-GFP) RNA genome. In conclusion, these data indicate that the CRISPR/Cas13 systems can be employed for engineering interference and durable resistance against RNA viruses in diverse plant species.

The plant hormone salicylic acid (SA) is an essential activator of plant immune responses directed against biotrophic pathogens. The transcription cofactor NPR1 (Nonexpressor of pathogenesis- related (PR) genes 1) functions to transduce the SA signal into an operational response directed to limited pathogen damage. In the absence of pathogen, NPR1 protein resides in the cytoplasm as a large molecular weight oligomer held together by disulphide bonding. Initiation of defence signalling leads to changes in intracellular redox conditions that promote NPR1 momomer release. Translocation of monomeric NPR1 to the nucleus results in the activation of over 2200 immune-related genes in Arabidopsis. NPR1 lacks a canonical DNA-binding domain but is known to perform part of its regulatory function through engagement of TGA factors (bZIP transcription factor). Induction of SA-dependent signalling is invariably associated with PR-1 gene expression and accumulation of mRNA for this gene serves as a useful marker of defence activation. However, both functional redundancy and stochastic factors limit the effectiveness of standard genetic approaches used in plant research, and thus much of the hierarchal processes surrounding NPR1-dependent gene activation are not fully understood. Using a synthetic biology approach we aim to complete exploratory work and set the foundations for the development of a yeast tool that can be used to manipulate and subsequently understand NPR1 function in relation to interacting partners and gene activation. Accordingly, using this tool we sought to create a conceptual protein circuit based on theoretical plant immunity. In completing this work we have developed a Saccharomyces cerevisiae strain that exhibits a highly oxidising intracellular redox environment. This was achieved by knocking out genes encoding S-nitrosoglutathione reductase (SFA1), flavohemoglobin (YHB1) and YAP1 (bZIP transcription factor), all important components in regulating cellular redox homeostasis and protein S-nitrosylation state in S. cerevisiae. Characterisation of this cell (designated Δsfa1yap1yhb1) reveals a high tolerance to such redox perturbations. Importantly, NPR1 is by default, assembled predominantly in the oligomeric form in this biological chassis. By activating two inducible inputs in the form of Arabidopsis S-nitrosoglutathione reductase (AtGSNOR) and Thioredoxin (AtTRXh5) which both function to promote NPR1 monomerisation, we have created a switch to selectively control NPR1 oligomer-monomer equilibrium. To complete the synthetic circuit, TGA3 was included, along with a modified yeast MEL1 promoter that has been customised to contain the TGA-responsive upstream activation sequence (termed the as-1 element) present in the promoter region of the PR-1 gene. Using FRET tools we were able to confirm nuclear interaction between monomeric NPR1 and TGA3, with this association appearing to induce as-1 element binding. However this process is not sufficient to activate a Luciferase (LUC) reporter gene, even when the GAL4 activation domain (GAL4 AD) is fused to NPR1. Ordinarily, a CUL3-dependent proteolysis-coupled transcription cycle is necessary to maintain efficient NPR1-dependent gene transcription in Arabidopsis. Although S. cerevisiae encodes an evolutionarily related CUL3 ortholog, examination by western blot demonstrates that NPR1 protein is stable in this cell, indicating an endogenous mechanism to degrade NPR1 is either not present or not functional in yeast. As such, this synthetic yeast tool represents a completely novel approach to identify missing components functioning in NPR1-mediated transcriptional regulation. Furthermore, in collaboration with a skilled bioinformatician, and using a rule-based stochastic modeling tool known as Kappa, we have been able to develop, for the first time, a preliminary mathematical simulation representative of NPR1-dependent gene activation that can be used as a foundation for future works.

<p>The activation and maintenance of plant immune responses require a significant amount of energy because they are accompanied by massive transcriptional reprogramming. Spurious activation of plant defense machinery can lead to autoimmune diseases and growth inhibition. So it is important for plants to tightly regulate the immune system to ensure the balance between growth and defense. However, neither the molecular mechanisms nor the design principles of how plants reach this balance are understood. </p><p>In this dissertation work, I showed how transcriptional and translational control of plant immune system can help avoid the constant immune surveillance and elicit a proper level of defense responses when necessary. These fine tunings of the immune system ensure the balance between growth and defense. </p><p>My research on the transcriptional regulation of plant defense responses led to the surprising discovery that even without pathogen, plant can 'anticipate' potential infection according to a circadian schedule under conditions that favor the initiation of infection. Functional analysis of 22 novel immune components unveiled their transient expression at dawn, when the infection is most likely to happen. This pulse expression pattern was shown to be regulated by the central circadian oscillator, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) since these 22 genes are no longer induced in the cca1 mutant. Moreover, the temporal control of the transcription level of these 22 immune genes by CCA1 also fine tunes their expression pattern according to the perceptions of different pathogenic signals. At the basal defense level, the expression of these genes can be transiently induced upon perceptions of critical infection stages of the pathogen. When an elevated level of defense response is needed, the high expression levels of these genes are maintained to confer a stronger immunity against pathogen. Since this stronger form of defense may also cause the suicidal death of the plant cells, the interplay between the circadian clock and defense allows a better decision on the proper level of the immunity to minimize the sacrificial death. The circadian clock is also known to regulate the growth-related cellular functions extensively. So the circadian clock can help to balance the energy used in growth and defense through transcriptional regulation on both sides.</p><p>Besides the integrated control by the circadian clock, the translational control on a key transcription factor involved in the growth-to-defense transition can also maintain the balance between growth and defense.TBF1 is a major transcription factor that can initiate the growth-to-defense transition through transcriptional repression of growth-associated cellular functions and induction of defense-related machinery. Bioinformatics studies identified 2 upstream open reading frames (uORFs) encoding multiple phenylalanine at 5' of the translation initiation codon of TBF1. Under normal conditions, these 2 uORFs can repress the translation of TBF1 to prevent accidental activation. However, pathogen infection may cause rapid and transient depletion of phenylalanine, a well-known precursor for cell wall components and the SAR signal SA. This depletion signal can be reflected by the increase of uncharged tRNAPhe, which subsequently leads to the phosphorylation of eIF2á and the release of uORFs' repression on TBF1. These findings provided the molecular details of how uORF-based translational control can couple transcriptional reprogramming with metabolic status to coordinately trigger the growth-to-defense transition. </p><p>In summary, my dissertation work has identified previously unrecognized regulatory mechanisms by which plant immune responses are balanced with growth. These new findings will further investigations into these novel interfaces between plants and pathogens. Future studies will definitely further improve our understandings of the plant-microbe interactions.</p> / Dissertation

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24675号 / 農博第2558号 / 新制||農||1099(附属図書館) / 学位論文||R5||N5456(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 髙野 義孝, 教授 寺内 良平, 教授 吉田 健太郎 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Plants are surrounded by various threats from the environment such as pathogens, abiotic stresses, and animal attacks. Nutrient content and distribution are essential for plant growth and development as well as plant immunity. Pathogens extract nutrients from host plants to benefit their own growth and reproduction. Sulfate, amino acids, and phosphate are indispensable elements for plant growth, plant nutrition, and plant resistance/susceptibility to disease. However, the role of these nutrients in plant-oomycete interactions is an unexplored area. We developed a hydroponic system to precisely control the nutrients applied to plants. We used Arabidopsis thaliana and Nicotiana benthamiana (N. b) as model plants. Hyaloperonospora arabidopsidis as well as two Phytophthora species, Phytophothora capsici (P. cap) and Phytophothora nicotianae (P. nic) were used as model oomycete pathogens. Hpa is an obligate biotrophic pathogen that obtains nutrients directly from the host plant without causing cell death, while P. cap and P. nic are hemibiotrophic pathogens that display a biotrophic phase followed by a necrotrophic phase where they feed on dead cells. Genomic evidence suggests that these pathogens might obtain nutrients including sulfur in different forms from the host (organic and inorganic respectively). We have optimized the hydroponic system and used Taqman PCR assays and sporangiophore counts to assay the influence of sulfur nutrients on Hpa and P. cap infections. We found that (1) sulfur transporter and metabolism genes play essential roles in plant-oomycete interactions; (2) sulfur is critical components for HR responses against Hpa; (3) low sulfur induces pathogenesis related genes as a systemic acquired response. RNA-seq analysis on Phytophthora-infected Arabidopsis suggested that sulfur transport, assimilation, and metabolism play an important role in plant-oomycete interactions. A second project used RNA-seq analysis on P. nic infected N. b, to identify potential nutrition-related-plant genes that are necessary for full pathogen virulence. RNAi knockdowns of N. b AAP6 (amino acid permease 6) and PHT4 (phosphate transporter 4) genes showed an inhibition of oomycete colonization. These experiments together advance the study on the interplay between nutrient assimilation/metabolism in host plants and oomycete infection which will provide insight into the mechanisms how pathogens intercept nutrients from host. In the long-term, this research could reveal new traits applicable for disease resistance to promote crop and food production. / Doctor of Philosophy / Plants are surrounded by diverse threats from the environment such as pathogens, abiotic stresses, and animal attacks. Oomycetes are the most destructive group of pathogens, triggering severe food security issues. Phytophthora is an oomycete genus causing serious economic loss. Traditional disease control managements including pesticides, crop rotation and culture practices, are not time- or financially- efficient due to the difficulty in managing oomycete spread and oomycete resistance to chemicals. Thus, new plant genes for resistance to oomycete diseases would have a major impact. Plant nutrients are critically important for plant fitness in every aspect of plant growth and plant immunity. Cellular regulatory networks for sulfur, amino acids, and phosphate assimilation and metabolism networks connect to every aspect of plant activity such as functioning enzymes, formation of chlorophyll, synthesis of proteins, and plant immunity. These nutrients are part of the plant defense system but also can be beneficial nutrients fed to the invading pathogens. Studying how nutrients are involved in the responses to oomycete invasions will provide information to introduce resistance strategies into crops. We utilized oomycete pathogens with different lifestyles to study the interactions and found that some sulfate transporter genes, an amino acid transporter and a phosphate transporter might be manipulated by oomycete to obtain nutrients. Sufficient nutrition is a critical factor for successfully triggering plant immunity but also could be reprogrammed by pathogens for successful infection and development. Our studies gave useful information to understand which plant nutrient genes are important during plant–oomycete interactions. These findings could be useful in identifying or engineering new plant genes to control plant diseases.

Diverse pathogens secrete effector proteins into plant cells to manipulate host cellular processes. The genome of Hyaloperonospora arabidopsidis (Hpa), the causative agent of downy mildew of Arabidopsis, contains at least 134 candidate RXLR effector genes. These genes contain an RXLR motif required for effector entry into host cells. Only a small subset of these candidate effectors is conserved in related oomycetes. Here, we describe a comparative functional characterization of the Hpa RXLR effector HaRxL96 and a homologous gene, PsAvh163, from the soybean pathogen Phytophthora sojae. HaRxL96 and PsAvh163 are induced during early stages of infection and carry a functional RXLR motif that is sufficient for protein uptake into plant cells. Both effectors can suppress or activate immune responses in soybean, Nicotiana, and Arabidopsis. Several SA-responsive defense genes are suppressed in Arabidopsis Col:HaRxL96 and Col:PsAvh163 during an incompatible interaction with Hpa Emoy2. Both effectors are localized to the nucleus and cytoplasm of plant cells. Nuclear localization of both effectors is required for proper virulence functions, including suppression of basal resistance and RPP4-mediated immunity to virulent and avirulent Hpa, respectively. In addition, both effectors interact with plant U-box (PUB) proteins that are conserved between diverse plant species. The targeted PUB proteins are negative regulators of plant immunity in Arabidopsis. These experiments demonstrate that evolutionarily-conserved effectors from different oomycete species can suppress immunity in plant species that are divergent from the source pathogen's primary host. / Ph. D.

Unlike mammals, plants do not have specific immune cells. However, plants can still recognize pathogens and defend themselves. They do that by recognizing microbial-associated molecular patterns (MAMPs) and secreted pathogen proteins, called effectors. MAMP-triggered immunity (MTI) relies on recognition of MAMPs by leucine-rich repeats (LRRs) pattern-recognition receptors (PRRs). The best-studied LRR PRR is Flagellin-Sensitive 2 (Fls2), the receptor of a 22-amino acid long epitope of bacterial flagellin, called flg22. In this project, alleles of FLS2 of different tomato cultivars were sequenced and compared to each other to get insight into natural selection acting on FLS2 and to identify residues important for ligand binding. This information may be used in the future to engineer Fls2 for improved ability to recognize flagellin. MTI can be suppressed by effectors secreted by bacteria into plant cells through the type III secretion system. On the other hand, plants are equipped with repertoires of resistance proteins, which can recognize some pathogen effectors. If a pathogen carries an effector that is recognized, effector-triggered immunity (ETI) is activated and the plant is resistant. Here, eggplant breeding lines were screened for their ability to activate ETI upon recognition of effectors of the soil borne pathogen Ralstonia solanacearum, a causative agent of bacterial wilt. Four effectors were found to trigger plant defenses in some of the lines. This is the first step in cloning the genes coding for the responsible resistance proteins. These genes may be used in the future for engineering tomato and potato for resistance to bacterial wilt. / Master of Science in Life Sciences

Post-translational protein modifications (PTM) vastly increase the complexity and functional diversity of the proteome, to precisely regulate crucial cellular processes. The plant immune system is composed of complex signalling networks that are influenced by various PTMs. Activation of plant immunity is associated with a rapid burst of nitric oxide (NO), which can covalently modify cysteine thiols within target proteins by a process termed S-nitrosylation to form S-nitrosothiols (SNOs), constituting a redox-based PTM. Another key PTM involved in plant immunity is SUMOylation, an essential mechanism involving the conjugation of the small ubiquitin-like modifier (SUMO) peptide to lysine residues within target proteins. Although the targets and mechanisms of S-nitrosylation and SUMOylation are becoming evident, how these key PTMs are themselves regulated remains obscure. Work presented in this thesis reveals that during plant immune signalling, the sole Arabidopsis thaliana SUMO conjugating enzyme, SUMO CONJUGATING ENZYME 1 (SCE1), is S-nitrosylated at a highly conserved, but previously uncharacterized cysteine. S-nitrosylation of SCE1 was shown to inhibit its SUMO conjugating activity in vitro and mutational analysis revealed that the site of this modification, Cys139, is not required for enzyme activity but rather constitutes a redox-sensitive inhibitory switch. Generation and characterization of transgenic Arabidopsis plants overexpressing both wild-type and mutant forms of SCE1 revealed that Cys139 is required for efficient immunity against bacterial pathogens. Furthermore, after immune activation, S-nitrosylation of this residue inhibits global SUMOylation of proteins. These results provide evidence of a novel means of crosstalk between S-nitrosylation and SUMOylation in the context of plant immunity. The abundant cellular antioxidant, glutathione (GSH), is S-nitrosylated to form S-nitrosoglutathione (GSNO), which is thought to constitute a stable reservoir of NO bioactivity. In Arabidopsis, GSNO levels are controlled by the enzyme S-NITROSOGLUTATHIONE REDUCTASE 1 (GSNOR1), which indirectly influences the levels of protein SNOs. In this study, transgenic plants overexpressing FLAG-epitope tagged GSNOR1 were generated in various mutant backgrounds, including nitric oxide overproducer 1 (nox1), to further investigate the roles of GSNOR1 and NO in plant immunity. It was shown that ectopic GSNOR1 expression completely recovers developmental and disease susceptibility phenotypes of gsnor1, but not nox1 mutant plants, highlighting in vivo differences between accumulation of GSNO and free NO. Surprisingly, elevated NO levels in nox1 plants promote S-nitrosylation of GSNOR1, inhibiting its enzymatic activity. This suggests a previously unreported means by which NO might regulate its own bioavailability. Further work in this study revealed that recombinant GSNOR1 can be SUMOylated in vitro, which appeared to increase its enzymatic activity. Several potential SUMO modification sites were identified within GSNOR1 and mutational analysis revealed that at least one of these, Lys191, is SUMOylated. Co-immunoprecipitation experiments revealed that transgenic GSNOR1 might be SUMOylated in vivo, although the site(s) and biological function of SUMOylation were not identified. Nonetheless, these results reveal another possible layer of interplay between S-nitrosylation and SUMOylation.

A conspicuous feature of plants responding to pathogen invasion is the synthesis of nitric oxide (NO), a redox signal. NO regulates protein function by S-nitrosylation, the addition of an NO moiety to a cysteine thiol to form an S-nitrosothiol. A key theme of NO function is reprogramming plant immune-related gene expression. However, it is still not clear how the NO signal is translated into transcriptional changes. Here we explored the potential role of a sub-group of SNO Regulated Genes (SRGs) uncovered by global expression profiling. Firstly, transgenic plants containing the SRG1 or SRG3 promoter fused to glucuronidase gene GUS together with qRT-PCR assays confirmed that transcripts of SRGs could be induced by NO and pathogen challenge, suggesting that SRGs may be involved in NO signalling related to plant immunity. More importantly, transient and stable overexpression of SRG genes induced hypersensitive response (HR)-like cell death development, which is often associated with pathogen effector-triggered immunity. Furthermore, transgenic plants constitutively expressing SRG genes exhibited enhanced ROS accumulation, PR1 transcript accumulation, and increased resistance to Pseudomonas syringae (Pst) DC3000 compared with Col-0 wild type plants. In contrast, lines with T-DNA insertions into SRG genes exhibited susceptibility to Pst DC3000. These data suggested SRGs act as the positive regulators in plant immunity. In order to further explore how NO regulates these SRGs in plant immunity, we focused on SRG1 and found SRG1 could be S-nitrosylated in vitro and in vivo. Moreover, electrophoretic mobility shift assays showed SRG1 could bind to an AGT motif and the transcriptional activity was blunted in the presence of NO, suggesting that the DNA binding activity of SRG1 is redox-modulated. Further, a transient repression activity assay showed that SRG1 has repression activity and this activity was impaired in the gsnor1-3 mutant, which has a high S-nitrosothiols level. These data suggested NO could block SRG1 transcriptional activity in vitro and in vivo. Furthermore when the SRG1 overexpression line was crossed with gsnor1-3 the SRG1-mediated resistance related phenotypes were suppressed. These data demonstrated NO negatively regulates SRG1 transcriptional activity during plant immunity. SRG1 may therefore be an important regulator of NO signalling and subsequent regulate transcription during plant immunity. Additionally, NO may negatively feedback to inhibit transcriptional activity of SRG1 to control its repression activity, to enable the activation of plant immunity.

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