ウンシュウミカン果実の剥皮開始期におけるアルベドに由来するEST の解析

The gene expression and regulation associated with the initial stage of rind peeling of the satsuma mandarin (Citrus unshiu cv. \u27Miyagawa-wase\u27) was characterized through the generation of 626 expressed sequence tags (ESTs) derived from a cDNA library prepared from albedo tissues collected 60 days after anthesis. A Total of 451 ESTs was found to have significant similarity to the known sequences. Using our inhouse database, a catalogue of 222 ESTs annotated with their putative functions was prepared and compared to the previously prepared catalogue of 442 ESTs derived from mature albedo tissues. In consequence, gene homologues associated with cell wall events were preferably found in this library.ウンシュウミカン果実の剥皮開始期アルベドで発現した遺伝子から作成したcDNA ライブラリーについて，ランダムにcDNA を選択してシーケンスし，626 のcDNAクローンのEST を得た．相同性検索によるEST の機能解析の結果，336 クローン（53.7％）で遺伝子機能が推定された．残りのクローンのうち，90 クローン（14.3％）では他の生物のESTと相同性があり，25 クローン（4.0％）についてはシロイヌナズナのゲノム塩基配列とのみ相同性があった．残りの175 クローン（28.0％）については他の生物の塩基配列との相同性は見られなかった．機能が推定されたクローンのうち222 クローンについて，機能推定の結果を一覧表に示した．このcDNA ライブラリーの特徴として，細胞壁にかかわる遺伝子が多くみられた

Arabidopsis subtilases promote defense-related pectin methylesterase activity and robust immune responses to botrytis infection

Plants involve a fine modulation of pectin methylesterase (PME) activity against microbes. PME activity can promote the cell wall stiffening and the production of damage signals able to induce defense responses and plant resistance to pathogens. However, the molecular mechanisms underlying PME activation during disease remain largely unknown. In this study, we explored the role of subtilases (SBTs) as PME activators in Arabidopsis immunity. By using biochemical and reverse genetic approaches, we found that the expression of SBT3.3 and SBT3.5 influences the induction of defense-related PME activity and resistance to the fungus Botrytis cinerea. Arabidopsis sbt3.3 and sbt3.5 knockout mutants showed decreased induction of PME activity and increased susceptibility to the fungus. SBT3.3 expression was stimulated by oligogalacturonides. Overexpression of SBT3.3 overactivated PME activity during fungal infection and enhanced resistance to B. cinerea. A negative correlation was observed between SBT3.3 expression and cell wall methyl ester content in the genotypes analyzed after B. cinerea infection. Increased expression of defense-related genes, including PAD3, CYP81F2 and WAK2, was also revealed in SBT3.3 overexpressing lines. We also demonstrated that SBT3.3 and pro-PME17 are both secreted into the cell wall using distinct protein secretion pathways and different kinetics. Our results propose SBT3.3 and SBT3.5 as modulators of PME activity in Arabidopsis against Botrytis to promptly boost immunity limiting the growth-defense trade-off

An investigation into the role of the ubiquitin-proteasome system in plant defence

Ubiquitin is a highly conserved regulatory protein that is ubiquitously expressed in eukaryotes. The ubiquitin pathway appears to be more diversified in plants compared to the other organisms. In plants, the ubiquitin-26S proteasome pathway appears to be particularly involved in regulating plant growth, development and defence signalling. This project is concerned with establishing potential involvement of UBP12 and UBP13 in plant defence. The Arabidopsis-Pseudomonas interaction was employed to examine the transcriptional response of AtUBP12 and AtUBP13 during pathogen infection. Furthermore, a transient over expression approach was used to investigate possible gain of function phenotypes associated with NtUBP12 (tobacco homologue of AtUBP12 and AtUBP13) activity during Cf-9 triggered HR in tobacco. The second objective of this study was to identify the interactor of UBP12 by employing a yeast two hybrid screen. The final objective was to analyse the functional significance of the mutant forms of ubiquitin during ubiquitination of target protein. Added to the above-mentioned objectives, this study also details the production of plant anti-ubiquitin antibody. The data presented in this study suggests that UBP12 and UBP13 play a critical role in plant defence. The yeast two hybrid assays showed that DIN1 is the interactor of UBP12. The analysis mutant forms of ubiquitin did not show any considerable difference in the pattern of in vitro ubiquitination compared to the wild type ubiquitin; the probable reasons for this were discussed. The anti-ubiquitin antibody generated in this study was shown to have affinity against plant and human ubiquitin

Producing a Subunit Vaccine for Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea virus (PEDv) causes disease and mortality to piglets worldwide. Most vaccines used to combat the disease have been ineffective live attenuated virus vaccines. The goal of this project was to produce a plant-made subunit vaccine based off the membrane protein of the virus. This is the first time this protein has been produced in plants. An elastin-like polypeptide fusion membrane protein accumulated up to 0.8 mg/g of fresh leaf weight when transiently expressed in Nicotiana benthamiana. Virus-like particles were also produced for the first time for PEDv, and were able to form with just the membrane protein, or from co-expression of the membrane and envelope protein. This adds to the limited body evidence that the membrane protein is the only necessary component to make coronavirus-like particles, and represents the first time coronavirus-like particles have been made in plants

The role of the interaction between PiAvr3a and CMPG1 in disease and plant defence

Investigating the individual proteins involved in plant defence and the pathways these proteins are part of is important to gain an understanding of infection and disease resistance processes. It is hoped that this knowledge will help develop solutions to prevent crop infection and increase crop yield. More specifically, CMPG1 was first found to be important in pathogen responses by Kirsch et al. (2001) in parsley. It has further been found to have homology to NbACRE74 and AtPUB20 and AtPUB21 implying it is common to many plant species. CMPG1 is an U-Box E3 Ubiquitin ligase and involved in a number of different defence responses (INF1, Cf-9 & Pto/AvrPto). PiAvr3a is an RxLR cytoplasmic effector protein from potato late blight pathogen Phytophthora infestans. Its virulence function was shown by its ability to suppress INF1 cell death by Bos et al. (2006). This P. infestans protein and the plant protein CMPG1 were found to interact in Yeast-two-hybrid studies performed by Dr M Armstrong (Bos et al., 2010). The aims of this project were to determine the nature of this interaction, functionally and biochemically. Does the interaction found in the Yeast-two-hybrid take place in the plant? What are the biological implications of PiAvr3a and CMPG1 interaction? What is the biochemical nature of the interaction? This project used in planta studies to investigate the CMPG1-PiAvr3a interaction. The biological reason for why this interaction occurred was studied using virus induced gene silencing (VIGS) and hypersensitive response/cell death suppression assays. The biochemical nature of this interaction was investigated using ubiquitination assays, and purified proteins, in vitro. This thesis aimed to provided data to increase the overall understanding of the function of CMPG1 and PiAvr3a during infection. Firstly, this project found evidence for the stabilisation of StCMPG1 by PiAvr3a as well as possible direct interaction inside plant cells. The stronger interaction with, and stabilisation of StCMPG1 by PiAvr3aKI, is in accordance with the stronger suppression of INF1 cell death by this form of the effector (Bos et al., 2006; 2009). Secondly, evidence for the role of CMPG1 in disease resistance to multiple plant pathogens, including oomycetes, bacteria and fungi was found. PiAvr3a suppresses all of these cell death responses. It is likely that CMPG1 is a target for other effectors from other pathogens. Cell death suppression by PiAvr3a is caused by stabilisation, and thus altered function, of CMPG1. Thirdly, promising and surprising data revealed that PiAvr3a may act as an E2 conjugating enzyme. Moreover, it appeared to act via both lysines 48 and 63, perhaps suggesting that it forms a mixed chain on CMPG1 substrates

Functional characterization of the RNA guided DRM2 DNA methyltransferase. Research done in the Faculty of Biology UW and IBB PAS. M.Sc. Thesis Supervisor: prof. dr hab. Ewa Bartnik.

DNA methylation in plants is in part controlled by small RNAs. A key player in RNA directed DNA methylation (RdDM) is Domain Rearranged Methyltransferase 2 (DRM2). In addition to its catalytic domain, which resembles the catalytic domain of animal DNA methyltransferases, the protein has several UBA domains of unknown biochemical significance. Genetic experiments on Arabidopsis thaliana indicate that a DRM2 variant lacking all UBA domains is similarly incapacitated as a catalytic mutant. The aim of this work was to elucidate the role of the UBA domains in DRM2. Towards this end, expression plasmids were generated, which should direct expression of DRM2 variants lacking individual UBA domains only after Agrobacterium mediated transformation. Unfortunately, protein expression of the deletion constructs was not observed, even though a reporter control protein was expressed in parallel experiments