Doctor of Philosophy / Department of Plant Pathology / Eduard Akhunov / The fungus Puccinia graminis f. sp. tritici (Pgt) is the causal agent of the wheat stem rust disease. Wheat stem rust has attracted a lot of attention after the emergence of the Ug99 race group, which at the time of its origin was virulent on most of the wheat varieties cultivated around the world. The evolution and spread of the Pgt isolates from the Ug99 race group posed a serious threat to worldwide wheat production. To mitigate the potential impact of new rust epidemics in major wheat production areas, it remains critical to identify new strategies for breeding durable resistance traits. A detailed understanding of the plant-pathogen interaction mechanisms in the wheat-Pgt pathosystem should be the foundation of these strategies. The interaction between the matching pair of resistance (R) and avirulence (Avr) genes, an important element of the plant-pathogen interactions, is described by the broadly documented gene-for-gene model. The cloning of the Sr35 gene, which confers near immunity against all isolates from the Ug99 race group provided a unique opportunity to investigate the molecular mechanisms of resistance to stem rust in wheat. The goals of the present study were: (1) to determine whether the Sr35 gene alone is sufficient for conferring resistance against Ug99, (2) to assess the Sr35 transcript levels during the time course of infection, and (3) to identify and validate the corresponding Avr gene interacting with Sr35. The cloning of Avr genes from the biotrophic fungi represents a substantial challenge due to the variability, redundant nature, the lack of similarity to known proteins, and lack of adequate functional tools to validate them. To overcome these limitations, we performed a comparative genomic analysis using multiple Sr35-avirulent and Sr35-virulent races, including 15 chemically mutagenized Pgt strains that acquired virulence on the Sr35 gene. Whole genome shotgun sequencing of the Pgt mutants identified a single candidate gene, which carried strong effect mutations in each mutant strain. The Avr gene candidate (AvrSr35) was expressed at early stages of infection and had a signal peptide indicating that the gene product is secreted. Comparative microscopic analysis of the infected tissues at different time points after infection indicated that AvrSr35 secretion occurs before haustoria formation. The re-sequencing of the AvrSr35 candidate gene in a panel of Sr35-virulent and Sr35-avirulent isolates including isolates from the Ug99 race group, revealed the presence of a mobile DNA element inserted into the coding sequence of virulent isolates. This insertion resulted in a premature termination codon and explains the origin of Pgt field isolates virulent in the presence of the Sr35 gene. Co-expression of AvrSr35 with the Sr35 in N. benthamiana leaves induced a specific hypersensitive response confirming the avirulence function of the candidate effector gene. Subcellular localization, bi-molecular fluorescence complementation, and co-immunoprecipitation assays in N. benthamiana leaves revealed that the AvrSr35 and Sr35 proteins interact and are likely associated with the endoplasmic reticulum and plasma membrane. Thus, this study identified and functionally characterized the first matching pair of Avr/R genes for cereal rusts.

No description available.

Plants have evolved physical barriers and inducible defense responses to combat microbial pathogens. Inducible responses are mediated by R proteins, which recognize invading pathogens. R proteins must be precisely regulated to provide effective resistance, without inhibiting normal plant growth. However, little is known about R gene regulation under defense-inducing conditions. The interaction between the oomycete Hyaloperonospora parasitica and the model plant Arabidopsis thaliana provides an excellent model system to explore R gene regulation. My research focuses on RPP8, a CC-NBS-LRR gene, which provides resistance to the H. parasitica isolate Emco5. Previous work in the McDowell lab suggested that RPP8 is upregulated during defense responses. My research shows that RPP8 alleles from the Columbia and Landsberg erecta ecotypes are upregulated by H. parasitica and the defense signaling molecule salicylic acid, suggesting a potential feedback loop. RPP8-Ler is also systemically upregulated after infection of the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Additionally, RPP8-Ler expression is increased during wounding and heat stress. I also examined the role of regulatory cis elements in the RPP8 promoter. Three W-boxes are essential for basal and inducible RPP8 expression, and are required for resistance to Emco5. The X-box, a unique cis element in the RPP8 promoter, is essential for strong basal expression and wound-induced upregulation, and affects spatial expression of RPP8-Ler. However, the X-box is not required for RPP8-Ler upregulation during pathogen or SA treatment. R genes may be induced as part of global defense responses, which could prime the host for more effective pathogen recognition. / Master of Science

Doctor of Philosophy / Department of Plant Pathology / Bikram S. Gill / H13, a dominant resistance gene transferred from Aegilops tauschii into wheat (Triticum aestivum), confers a high level of antibiosis against a wide range of Hessian fly (HF, Mayetiola destructor) biotypes. Previously, H13 was mapped to the distal arm of chromosome 6DS, where it is flanked by markers Xcfd132 and Xgdm36. A mapping population of 1,368 F2 individuals derived from the cross: PI372129 (h13h13) / PI562619 (Molly, H13H13) was genotyped and H13 was flanked by Xcfd132 at 0.4cM and by Xgdm36 at 1.8cM. Screening of BAC-based physical maps of chromosome 6D of Chinese Spring wheat and Ae. tauschii coupled with high resolution genetic and Radiation Hybrid mapping identified nine candidate genes co-segregating with H13. Candidate gene validation was done on an EMS-mutagenized TILLING population of 2,296 M₃ lines in Molly. Twenty seeds per line were screened for susceptibility to the H13-virulent HF GP biotype. Sequencing of candidate genes from twenty-eight independent susceptible mutants identified three nonsense, and 24 missense mutants for CNL-1 whereas only silent and intronic mutations were found in other candidate genes. 5’ and 3’ RACE was performed to identify gene structure and CDS of CNL-1 from Molly (H13H13) and Newton (h13h13). Increased transcript levels were observed for H13 gene during incompatible interactions at larval feeding stages of GP biotype. The predicted coding sequence of H13 gene is 3,192 bp consisting of two exons with 618 bp 5’UTR and 2,260 bp 3’UTR. It translates into a protein of 1063 amino acids with an N-terminal Coiled-Coil (CC), a central Nucleotide-Binding adapter shared by APAF-1, plant R and CED-4 (NB-ARC) and a C-terminal Leucine-Rich Repeat (LRR) domain. Conserved domain analysis revealed shared domains in Molly and Newton, except for differences in sequence, organization and number of LRR repeat in Newton. Also, the presence of a transposable element towards the C terminal of h13 was indicative of interallelic recombination, recent tandem duplications and gene conversions in the CNL rich region near H13 locus. Comparative analysis of candidate genes in the H13 region indicated that gene duplications in CNL encoding genes during divergence of wheat and barley led to clustering and diversity. This diversity among CNL genes may have a role in defining differences in the recognition specificities of NB-LRR encoding genes. Allele mining for the H13 gene in the core collection of Ae. tauschii and hexaploid wheat cultivars identified different functional haplotypes. Screening of these haplotypes using different HF biotypes would help in the identification of the new sources of resistance to control evolving biotypes of HF. Cloning of H13 will provide perfect markers to breeders for HF resistance breeding programs. It will also provide an opportunity to study R-Avr interactions in the hitherto unexplored field of insect-host interaction.

Plant innate immunity is activated upon pathogen attack by recognizing their avirulent (avr) genes by Resistant (R) genes leading to R-gene resistance or host resistance. Another form of innate immunity is non-host resistance that is exhibited by a given plant species to most strains of a microbial species. R-gene resistance activates salicylic acid (SA) that is synthesized from methyl salicylic acid (MeSA) by Salicylic Acid Binding Protein 2 (SABP2). It was hypothesized that SABP2 plays the similar role in non-host resistance also. Growth experiments and non-host related gene analysis experiments were conducted on tobacco plants using P.s tabaci and P.s. phaseolicola that are host and non-host pathogens on tobacco respectively. Tobacco control plant C3 that expresses SABP2 and 1-2 that is RNAi silenced in SABP2 expression were used in this study. Results suggest that SABP2 may not have any significant role in tobacco non-host resistance.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24680号 / 農博第2563号 / 新制||農||1100(附属図書館) / 学位論文||R5||N5461(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 寺内 良平, 教授 髙野 義孝, 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 / DFAM

Pepper is a popular and important vegetable crop grown and consumed worldwide. However, pepper production is threatened by the gram-negative bacterium Xanthomonas euvesicatoria (Xe) which causes bacterial spot (BS) disease, one of the most common and destructive diseases on pepper. Due to limited genetic resistance resources in host species, a promising strategy for controlling BS disease is to transfer nonhost disease resistance (R) genes from other plant species into pepper plants to confer broad-spectrum and durable resistance. A maize R gene Rxo1 has been functionally transferred to rice plants and confers nonhost resistance to rice pathogen Xanthomonas oryzae pv. oryzicola (Xoc) carrying a type III effector (T3E) AvrRxo1. Most Xe strains carry a T3E Xe4428, a homolog of AvrRxo1. Therefore, Rxo1 could be potentially employed to develop Xe-resistant pepper. In addition, a better understanding of the virulence function of Xe4428 may provide insights into the pathogenesis of Xe and new strategies for crop improvement. In this dissertation, we transformed Rxo1 into the far-related dicot species Nicotiana benthamiana and pepper, and characterized the Rxo1-mediated disease resistance against Xe strains carrying AvrRxo1 or Xe4428. In addition, we explored the virulence function and mechanism of Xe4428. In the Rxo1-transgenic N. benthamiana, we demonstrated that Rxo1 could condition resistance to Xe harboring AvrRxo1 but not Xe4428. We revealed that AvrRxo1 could directly interact with the nucleotide-binding domain of Rxo1 in vivo and in vitro. We further demonstrated that the nucleus localization of AvrRxo1 was required for its avirulence and virulence functions. In addition, the cytosol localization of Rxo1 was also necessary to confer disease resistance. The downstream signaling component NbNDR1 was demonstrated to be involved in Rxo1/AvrRxo1-mediated disease resistance. By RNAseq-based gene expression profiling, we identified six candidate genes of interest up-regulated by the Rxo1-AvrRxo1 recognition. Through virus-induced gene silencing screening, a gene encoding phenylalanine ammonia-lyase 4 was demonstrated to be critical for Rxo1/AvrRxo1-mediated disease resistance in N. benthamiana. Rxo1-transgenic pepper plants were resistant to the Xe strain with the complementary Xoc effector AvrRxo1 but not the wild-type Xe strain that carries Xe4428. A Xe4428 mutant with only one nucleotide substitution could trigger the Rxo1-mediated disease resistance in pepper. Both wild-type and mutant Xe4428 had significant virulence functions that could promote the Xe bacterial proliferation on wild-type pepper plants. In addition, the mutant Xe4428 had a higher expression level than wild-type Xe4428 in Xe bacterial cells, which might explain why the mutant Xe4428 but not wild-type Xe4428, could trigger the Rxo1-mediated disease resistance in pepper. We identified 14 pepper cystatin genes (CaCys), among which two genes (CaCys1 and CaCys13) could be induced, and two genes (CaCys3 and CaCys5) were suppressed by Xe4428. Ectopically expressing one of the induced genes CaCys1 in N. benthamiana increased the stomatal opening and promoted the Xe growth in N. benthamiana plants. Thus, we illuminate one possible mechanism of Xe4428's virulence function is to regulate the stomata apertures in N. benthamiana. Bacterial fruit blotch (BFB) caused by the gram-negative bacterial pathogen Acidovorax citrulli (A. citrulli) is one of the most destructive diseases in cucurbit crops, including melon and watermelon. A better understanding of the virulence and avirulence functions of T3Es in A. citrulli helps breeders engineer crop resistance to BFB. To this end, a clean genetic background of A. citrulli with multiple effector genes deleted is desired. Here, we optimized a marker-exchange-based method for sequential effector deletion and generated an AAC00-1 mutant with five effector genes (Aave2166, Aave3626, Aave1548, Aave2938, Aave2708) deleted (AAC00-15). AAC00-15 was less virulent in watermelon but more virulent in N. benthamiana. Through complementation, we characterized the function of individual effectors and identified a promising R gene, Roq1, that could be used to control BFB disease. / Doctor of Philosophy / As an essential ingredient in almost all cuisines, pepper is grown and consumed worldwide, providing human beings with favorable flavor and nutrients. However, pepper production is threatened by the destructive bacterial spot (BS) disease caused by the bacterial pathogen Xanthomonas euvesicatoria (Xe). Due to limited genetic resistance resources in host species, nonhost resistance (R) genes from other plant species are desired to confer broad-spectrum and durable resistance to the pepper pathogen Xe. Previously, a maize (corn) R gene called Rxo1 was transferred to rice plants. This gene helped these rice plants resist a rice bacterial pathogen that causes leaf streak disease on rice. This rice pathogen has an effector (a virulent protein produced by bacteria to infect plants) that is required for the disease resistance. The pepper pathogen carries a similar effector, so transferring the maize R gene Rxo1 to pepper plants might similarly benefit peppers and help fight against the bacterial spot disease. In this dissertation, we successfully transferred the maize R gene Rxo1 into Nicotiana benthamiana and pepper plants. Our results indicate that this gene can help control disease caused by the pepper pathogen harboring the effector of the rice pathogen but not its native effector. We also illuminate how the disease resistance conferred by this maize gene happens in Nicotiana benthamiana plants. In addition, we explain how the corresponding effector helps infect plants. This research provides insights into the application of R gene transfer between far-related plant species and new tools to improve crop disease resistance.

Plant gene families organized as linked clusters are capable of evolving by a process of unequal crossing-over. This results in the formation of chimeric genes that may impart a novel function. However, the frequency and functional consequences of these unequal cross-over events are poorly characterized. Plant disease resistance genes (R genes) genes are frequently organized as gene clusters. In this study, I constructed an elaborately designed reconfigurable synthetic RPP1 (for resistance to Paranospora parasitica) gene cluster (synthRPP1) to model R gene evolution by meiotic recombination. This experimental design utilizes gain-of-luciferase phenotype (luc+) to identify and isolate recombinant R genes and uses two alternatively marked alleles to distinguish and measure different types of meiotic recombination (intra- vs. inter-chromosomal). Two putative single copy transgenic plants containing the synthRPP1 gene cluster were generated. These synthRPP1 gene clusters were reconfigured in vivo by two kinds of site-specific recombination systems (CRE/Lox, FLP/FRT) to generate two alternative versions of the synthRPP1 gene clusters in vivo. These lines, as well as others being developed, will be used in future genetic crosses to identify and characterize plants expressing chimeric RPP1 genes. My second area of research was to use a previously developed synthetic RBCSB gene cluster (synthRBCSB) gene cluster to investigate the relative frequency of meiotic unequal crossing over between paralogous genes located on either homologous chromosomes (homozygous lines) or sister chromatids (hemizygous lines). In contrast to published somatic recombination frequencies using a different reporter gene system, no statistically significant difference of meiotic unequal crossing over was observed between homo- and hemi-zygous synthRBCSB lines. This result suggests that meiotic unequal crossing-over between paralogs located on homologous chromosomes occurs at about the same frequency as paralogs located on sister chromatids. To investigate the rate of somatic recombination in synthRBCSB lines, a QRT-PCR method was developed to estimate the frequency of somatic recombination. Preliminary results suggest that the somatic recombination frequency was about 10,000 fold higher than meiotic recombination in the same generation. Moreover, two of five cloned chimeric genes that formed by somatic recombination indicated a different distribution of resolution sites than those observed in meiotic recombination. This finding suggests there are significant differences in both the frequency and character of somatic versus meiotic unequal crossing-over between paralogous genes in Arabidopsis. / Ph. D.

To improve understanding of the effectiveness of host resistance against Leptosphaeria spp., three aspects of effectiveness of resistance were investigated. With focus on the major Rlm-mediated resistance against L. maculans, changes in effectiveness of Rlm7-mediated resistance to prevent initiation of disease epidemics at the leaf spot stage were investigated in winter oilseed rape field experiments at five sites in the UK over the period with the cropping seasons 2009/2010 - 2013/2014. L. maculans isolates virulent against Rlm7 were identified in the UK. This may be associated with observed changes in lesion phenotypes on the Rlm7 cultivars in field conditions. However, despite increased severity of phoma leaf spotting on Rlm7 cultivars, there was no associated increase in phoma stem canker severity at the end of the cropping seasons. The effectiveness of winter oilseed rape cultivars for control of phoma stem canker (caused by L. maculans or L. biglobosa) was affected by the coexistence of the two Leptosphaeria species in oilseed rape crops. Weather conditions influenced ascospore release of both species and favoured L. biglobosa ascospore release in 2011, resulting in subsequent increased L. biglobosa phoma leaf spotting and stem canker severity. However, coexistence of Leptosphaeria spp. on oilseed rape crops was affected by the cultivar resistance against L. maculans. CE experiments showed that there were interactions between the two Leptosphaeria spp. in planta. Their coexistence on B. napus was influenced by the different host responses that they trigger during host colonisation. Effects of increased temperature on effectiveness of resistance against L. maculans and on severity of symptoms by Leptosphaeria spp. on B. napus were investigated. Increased temperature affected both Rlm4- and Rlm7-mediated resistance, when assessed by phenotypic and molecular techniques. Increased temperature was associated with increased symptom severity, for both L. maculans and L. biglobosa lesions on plants. Cultivar quantitative resistance background increased effectiveness of resistance against phoma stem canker pathogens at increased temperature and should be deployed in in strategies for adaptation to climate change to avoid increased phoma stem canker epidemics in the future.

Der Endozytoserezeptor Megalin gehört zu einer Gruppe von strukturell und funktionell verwandter Rezeptoren, der LDL R Gen Familie. Es sind zwei Arten von Lipidtransportpartikel beschrieben worden, die durch Megalin in Zellen aufgenommen werden. Zum einen werden Lipoproteine über ihre Apoproteine von Megalin erkannt und endozytiert. Zum anderen nimmt Megalin die hydrophoben Vitamine A und D über ihre Carrierproteine in ihre Zielzellen auf. Es handelt sich um Vitamin D bindendes Protein (DBP) und Retinol bindendes Protein (RBP). Zweck dieser Arbeit war es zu untersuchen, ob die Endozytose von Steroidcarriern durch Megalin ein genereller Mechanismus ist oder ob DBP und RBP Ausnahmen darstellen. Hierzu wurden exemplarisch drei Carrierproteine (24p3, Apo D und CCSP) für Steroide ausgesucht, die in Megalin-exprimierende Gewebe aufgenommen werden. Der (rekombinante) Retinolcarrier 23p3 zeigte bei surface plasmon resonance Analysen keine direkte Bindung an Megalin. Der Progesteron-Carrier Apo D hingegen bindet Megalin, ferner konnte in Zellkulturversuchen Endozytose und lysosomale Degradation von Apo D in Megalin-exprimierende Zellen nachgewiesen werden. Auch der Progesteron-Carrier CCSP wird durch Megalin in Zellen aufgenommen, allerdings ist zur Endozytose von CCSP ein Co-Rezeptor notwendig. Mit dieser Arbeit ist die erste in vivo-Beschreibung eines dualen Rezeptorsystems aus Megalin und einem peripheren Membranprotein namens Cubilin gelungen, welches u.a. im proximalen Tubulus der Niere existiert. Abschließend wurde exemplarisch für ein Steroidhormon-abhängiges Gewebe der murine Uterus hinsichtlich seiner Megalin-Expression untersucht. Es konnte ein bereits bekannter Ligand Megalins, das Glykoprotein Laktoferrin, aus der uterinen, luminalen Flüssigkeit aufgereinigt werden. Ferner konnte gezeigt werden, dass die Expression von Laktoferrin im Uterus strenger hormoneller Kontrolle unterliegt. / The endozytic receptor Megalin belongs to a group of structurally and functionally related receptors called LDL R gene family. Two different types of lipid particles are taken up by Megalin into target cells. The first type, lipoproteins are recognized and internalized by Megalin via their apoproteins. In addition, Megalin mediates the endocytosis of the lipophilic vitamins A and D into target cells by means of their carrier proteins. These proteins are the vitamin D binding protein (DBP) and retinal binding protein (RBP). The aim of the investigations was to determine, if the endocytosis of steroid hormone carriers by Megalin is a common occurrence or restricted only to DBP and RBP. Therefore, three carrier proteins for steroids (24p3, Apo D and CCSP) were chosen as an example. All of them are known to be taken up in Megalin expressing tissues. In surface plasmon resonance analysis recombinant 24p3, a carrier of retinol, showed no affinity to Megalin. Whereas the progesterone carrier Apo D bound to Megalin. Furthermore, it was endozytosed and degraded in lysosomes by Megalin expressing cells. The cellular uptake of the progesterone carrier CCSP is mediated by Megalin as well, however a co-receptor is needed. This work demonstrates for the first time the existence of a dual receptor pathway consisting of Megalin and a peripheral membrane protein named Cubilin in vivo. This systems is functional in addition to other tissues in the proximal tubule of the kidney. Finally, the Megalin expression in the murine uterus as an example of a steroid dependent tissue was investigated. Lactoferrin a known Megalin ligand was purified from the luminal uterine fluid. Furthermore, Lactoferrin expression in the uterus was shown to be under tight hormonal control.