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The structure and function of RPW8.1 and RPW8.2, powdery mildew disease resistance proteins from Arabidopsis thaliana (L.) HeyhnJaggard, Daniel Andrew William January 2002 (has links)
No description available.
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The development and characterisation of grapevine virus-based expression vectorsDu Preez, Jacques 03 1900 (has links)
Thesis (PhD (Genetics))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Grapevine (Vitis vinifera L.) is a very important agricultural commodity that needs to be
protected. To achieve this several in vivo tools are needed for the study of this crop and the
pathogens that infect it. Recently the grapevine genome has been sequenced and the next
important step will be gene annotation and function using these in vivo tools. In this study the
use of Grapevine virus A (GVA), genus Vitivirus, family Flexiviridae, as transient expression
and VIGS vector for heterologous protein expression and functional genomics in Nicotiana
benthamiana and V. vinifera were evaluated. Full-length genomic sequences of three South
African variants of the virus (GTR1-1, GTG11-1 and GTR1-2) were generated and used in a
molecular sequence comparison study. Results confirmed the separation of GVA variants into
three groups, with group III (mild variants) being the most distantly related. It showed the
high molecular heterogeneity of the virus and that ORF 2 was the most diverse. The GVA
variants GTG11-1, GTR1-2 and GTR1-1 were placed in molecular groups I, II and III
respectively. A collaboration study investigating the molecular divergence of GVA variants
linked to Shiraz disease (SD), described two interesting GVA variants of group II, namely
GTR1-2 and P163-M5 (Goszczynski et al., 2008). The group II variants were found to be
closely linked to the expression of SD. GTR1-2 was isolated from a susceptible grapevine
plant that never showed SD symptoms (Goszczynski 2007). The P163-M5 variant that
resulted in exceedingly severe symptoms in N. benthamiana and is that used as SD positive
control by the grapevine industry, was found to contain a 119 nt insert within the native
ORF2. Comparative analysis performed on the complete nt and aa sequences of group II GVA
variants suggested that the components in the GVA genome that cause pathogenicity in V.
vinifera are more complex (or different) to those that cause pathogenicity in N. benthamiana.
The three South African variants (GTR1-1, GTG11-1 and GTR1-2) were assembled into fulllength
cDNA clones under control of CaMV 35S promoters. After several strategies were
attempted, including a population cloning strategy for GTR1-2, none of the clones generated
were able to replicate in N. benthamiana plants. A single amino acid substitution at position
13 (Tyr/Y Cys/C) in ORF 5 of the GTR1-2 cDNA clone was shown to abolish or reduce
replication of the virus to below a detectable level. Two infectious clones of Israeli variants of
GVA (T7-GVA-GR5 and T7-GVA118, obtained from M. Mawassi) were brought under
control of a CaMV 35S promoter (35S-GVA-GR5 and 35S-GVA118). Both clones were
infectious, able to replicate, move systemically and induce typical GVA symptoms after
agroinfiltration in N. benthamiana. These Israeli clones served as backbone for further experiments in characterisation of transient expression and VIGS vectors. The use of GVA as
gene insertion vector (35S-GVA118) and gene exchange vector (35S-GVA-GR5-
ORF2+sgMP) in N. benthamiana and V. vinifera was compared. The gene insertion vector,
35S-GVA118 was based on the full-length GVA genome. The gene exchange vector, 35SGVA-
GR5- ORF2+sgMP, was constructed in this study by elimination of ORF 2 and
insertion of a sgMP and unique restriction sites to facilitate transgene insertion. In N.
benthamiana both vectors showed similar GUS expression levels and photobleaching
symptoms upon virus-induced NbPDS silencing. In V. vinifera limited GUS expression levels
and VIGS photobleaching symptoms were observed for the gene insertion vector, 35SGVA118.
No GUS expression was observed for the gene exchange vector 35S-GVA-GR5-
ORF2+sgMP in this host. As for silencing, one plant, agroinfiltrated with 35S-GVA-GR5-
ORF2-VvPDS+sgMP, developed photobleaching symptoms in 3 systemic infected leaves
after 4 months. This study showed that GVA can be used as gene insertion and gene exchange
vector for expression and VIGS in N. benthamiana, but in grapevine its use is limited to
expression and silencing of genes in the phloem tissue. It is also the first report that ORF 2 of
GVA is not needed for long distance movement in grapevine.
To investigate the possible role of the P163-M5 119 nt insertion and the GVA ORF 2 (of
unknown function), in expression of symptoms in plants, ORF 2 of a 35S-GVA-GR5 cDNA
clone was removed and subsequently substituted by the corresponding ORFs of four South
African GVA variants. Upon agro-infiltration into N. benthamiana leaves, all chimaeric GVA
constructs were able to move systemically through the plant. At this stage no correlation
could be found between severity of symptoms, the presence of the P163-M5 insert and the
specific GVA ORF 2 present in the chimaeras, indicating that other factors in the viral
genome or the host plant probably play a crucial role.
This study contributed to the pool of available in vivo tools for study and improvement of the
valuable grapevine crop. It also opened several exciting research avenues to pursue in the near
future. / AFRIKAANSE OPSOMMING: Wingerd (Vitis vinifera L.) is ‘n baie belangrike landboukundige gewas wat beskerm moet
word. Om die rede word verskeie in vivo gereedskap vir die bestudering van die
wingerdplant, en die patogene wat dit infekteer benodig. Die wingerd genoom se volgorde is
bepaal en dus is die volgende logiese stap om die gene te annoteer en funksie daaraan toe te
skryf. In hierdie studie is die gebruik van Grapevine virus A (GVA), genus Vitivirus, familie
Flexiviridae, as tydelike uitdrukking- en virus-geinduseerde geenuitdowingsvektor vir
heteroloë proteïen uitdrukking en funksionele genoomstudies in Nicotiana benthamiana en V.
Vinifera getoets. Vollengte genoomvolgordes van drie Suid-Afrikaanse variante van die virus
(GTR1-1, GTG11-1 en GTR1-2) is gegenereer en in ‘n molekulêre volgorde vergelyking
studie gebruik. Resultate het die verdeling van GVA variante in drie groepe, waar groep III
die verste verwant is, bevestig. Dit het ook gewys dat die virus ‘n baie hoë molekulêre
heterogeniteit het en dat oopleesraam 2 (ORF 2) die mees divers is. ‘n Samewerking studie
waar die molekulêre diversiteit van GVA variante, gekoppel aan Shiraz siekte (SD),
ondersoek is, is twee interessante variante van groep II beskryf, naamlik GTR1-2 en P163-M5
(Goszczynski et al., 2008). Groep II variante is vooraf gevind om nou verwant te wees aan die
ontwikkeling van SD in wingerd. Die GTR1-2 variant is uit ’n vatbare wingerd plant, wat
nooit SD-simptome vertoon het nie, geïsoleer (Goszczynski et al., 2007). In die ORF 2 van
die P163-M5 variant, wat simptome van die ergste graad in N. benthamiana geïnduseer het, en
ook deur die industrie as betroubare SD-positiewe kontrole gebruik word, is ’n 119 nt
invoeging gevind. Die vergelykende analise wat uitgevoer is, het daarop gedui dat die
determinante van patogenisiteit in die GVA genoom moontlik meer kompleks kan wees in V.
vinifera as in N. benthamiana. Die drie Suid-Afrikaanse variante (GTR1-1, GTG11-1 en
GTR1-2) is in afsonderlike vollengte cDNA klone, onder beheer van CaMV 35S promotors,
aanmekaargesit. Nadat verskeie kloneringstrategieë, insluitend ’n populasie kloneringstrategie
vir die GTR1-2 kloon, gebruik is, het geen een van die cDNA klone die vermoë besit om in
N. benthamiana te repliseer nie. ’n Enkele aminosuur substitusie in posisie 13
(Tyr/Y Cys/C) in ORF 5 van die GTR1-2 kloon, het die replisering van die virus tot laer as
’n opspoorbare vlak verlaag. Twee infektiewe klone van Israeliese GVA variante (T7-GVAGR5
en T7-GVA118, verkry van M. Mawassi) is onder beheer van ‘n CaMV 35S promotor
geplaas (35S-GVA-GR5 and 35S-GVA118). Beide klone het na agro-infiltrasie in N.
benthamiana plante gerepliseer, sistemies beweeg en tipiese GVA simptome geinduseer.
Hierdie twee klone het as raamwerk gedien vir verdere eksperimente in karakterisering van tydelike uitdrukkings- en VIGS vektore. Die gebruik van GVA as geen-insvoegingsvektor
(35S-GVA118) en geen-vervangingsvektor (35S-GVA-GR5- ORF2+sgMP) is in N.
benthamiana en V. vinifera vergelyk. Die geen-invoegingsvektor 35S-GVA118, was op die
vollengte GVA genoom gebasseer. Die geen-vervangingsvektor 35S-GVA-GR5-
ORF2+sgMP, was in hierdie studie gekonstrueer. Dit is gemaak eerstens deur eliminasie van
ORF 2 in die 35S-GVA-GR5 kloon, en tweedens deur die invoeging van ’n subgenomiese
promotor van die beweginsproteïen (sgMP) en unieke beperkings-ensiemsetels om klonering
van transgene te fasiliteer. Beide vektore het in N. benthamiana vergelykbare GUS
uitdrukkingsvlakke en fotobleikende simptome getoon na virus-geinduseerde NbPDS
uitdowing. In V. Vinifera is beperkte GUS uitdrukkingsvlakke en VIGS fotobleikende
simptome opgemerk met die geen-invoegingsvektor, 35S-GVA118. Geen GUS uitdrukking is
in hierdie gasheerplant met die geen-vervangingsvektor opgemerk nie. Slegs een wingerdplant
het fotobleikende simptome, na 4 maande in 3 sistemies geïnfekteerde blare gewys, na agroinfiltrasie
van die 35S-GVA-GR5- ORF2-VvPDS+sgMP konstruk. Hierdie studie het
bevestig dat GVA as geen-invoeging en geen-vervangingsvektor, vir heteroloë proteïenuitdrukking
en VIGS, in N. benthamiana gebruik kan word, maar dit blyk of die gebruik
daarvan in wingerd meer tot die floeëm weefsel beperk is. Hierdie studie wys vir die eerste
keer dat ORF 2 nie nodig is vir langafstand beweging van die virus in wingerd nie.
Om die moontlike rol van die P163-M5 119 nt invoeging en die GVA ORF 2 (met onbekende
funksie), in die uitdrukking van simptome in plante te ondersoek, is ORF 2 van die 35SGVA-
GR5 cDNA kloon verwyder en daaropvolgens vervang met die ooreenstemmende
ORFs van vier Suid-Afrikaanse GVA variante. Na agro-infiltrasie in N. benthamiana blare,
het al die chimeras die vermoë gehad om te repliseer, sistemies te beweeg en simptome te
induseer. Geen korrelasie kon gevind word tussen die graad van simptome, die
teenwoordigheid van die P163-M5 insersie en die spesifieke GVA ORF 2 teenwoordig in die
chimeras nie, wat dus daarop dui dat ander faktore in die virusgenoom of die gasheerplant `n
moontlike belangrike rol kan speel.
Hierdie studie het bygedrae tot die beskikbare poel van in vivo gereedskap vir die bestudering
en verbetering van die kosbare wingerdgewas. Dit het ook talle interessante
navorsingsgeleenthede oopgemaak om in die nabye toekoms te betree.
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A functional approach to profiling candidate genes in non model BrassicalesMankowski, Peter J. Unknown Date
No description available.
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EFFECTS OF SILENCING CYC2-LIKE GENES ON FLORAL DEVELOPMENT IN SOLANUM LYCOPERSICUM L. AND NICOTIANA OBTUSIFOLIA M. MARTENS & GALEOTTI (SOLANACEAE)Kim, Joonseog 01 January 2017 (has links)
CYCLOIDEA (CYC) and DICHOTOMA (DICH) of the CYC2 clade of the TCP gene family have been shown to play a significant role in regulating the identity of the dorsal petals and abortion of the single dorsal stamen in Antirrhinum majus. It is believed that CYC2-like genes are responsible for the convergent evolution of floral zygomorphy, but their role in the development of actinomorphic flowers is still unknown. In Solanaceae, previous analysis has identified two paralogs of CYC2-like genes, CYC2A and CYC2B, resulting from a gene duplication that predates the origin the family. Virus-induced gene silencing (VIGS) is a technique to study the gene function by silencing specific target genes of interest, which is shown to be useful in diverse plant species. Here, we report on the role of CYC2-like genes during floral development in Solanaceae based on the results of VIGS using tobacco rattle virus (TRV)-based vector in Solanum lycopersicum having completely actinomorphic flowers and Nicotiana obtusifolia having slightly zygomorphic flowers. Our VIGS experiments in So. lycopersicum show that downregulation of both CYC2A and CYC2B leads to misshaped petals, the unequal growth of the petals, and most frequently increased number of petals, stamens and sepals, while the carpel and ovule morphology remain the same as the wild type. On the contrary, downregulation of CYC2A and CYC2B in N. obtusifolia results in reduced number of flower organs in sepals, stamens, and petals, however carpels remained the same. For both solanaceous species, silencing CYC2A and CYC2B changes the property of cytoplasm and retards the rate of pollen germination. Our findings suggest that the CYC2-like genes are likely involved in the floral development, mainly regulating the number of floral organs and pollen development in Solanaceae.
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Identification of ARGONAUTES Involved in Antiviral RNA Silencing in Nicotiana benthamianaOdokonyero, Denis 1984- 14 March 2013 (has links)
ARGONAUTE proteins (AGOs) are generally accepted as key components of the post transcriptional gene silencing mechanism, also involved in plant antiviral defense. Except for reports on the antiviral roles of AGO1, AGO2 and AGO7 in Arabidopsis, the exact roles played by the individual AGOs in other plant species are largely unknown. This research focused on the identification and characterization of AGOs involved in antiviral RNAi response to various viruses in N. benthamiana. Based on the temporal and spatial distribution of AGO transcripts in 3 and 8-week old plant root, stem and leaf tissues, expressions of NbAGO mRNAs were found to vary with age and tissue specificity. Plant endogenous AGO mRNAs were knocked down through virus induced gene silencing techniques using the Tobacco rattle virus vector system and posteriorly challenged with a GFP-chimeric virus construct deficient of a silencing suppressor. Unlike in control non-silenced plants, the Tomato bushy stunt virus construct deficient of its P19 silencing suppressor was consistently seen to exhibit a strong fluorescence on N. benthamiana plants silenced for NbAGOs 2 and X. Similar results were also obtained upon silencing of NbAGO2 using hairpin vector techniques. Comparable observations were also made when Tobacco mosaic virus GFP constructs were agroinfiltrated on NbAGO2 silenced plants further hinting the antiviral defense roles played by these AGOs. Agroinfiltration of Foxtailmosaic virus, Sunnhemp mosaic virus, and Turnip crinkle virus GFP chimeric constructs on NbAGO2 silenced N. benthamiana plants, however did not result in accumulation of GFP indicating the AGO antiviral defense specificity to TBSV and TMV. The results also hinted at a role for AGO7. Collectively my findings suggest that the expression of AGOs in N. benthamiana is tissue and age dependent, and that unlike in the model plant Arabidopsis where the main antiviral AGO is thought to be AtAGO1; in N. benthamiana, NbAGOs 2 and X seem to be involved in an antiviral defense role against TBSV and TMV with other AGOs perhaps contributing.
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Detection of Acidovorax citrulli, the Causal Agent of Bacterial Fruit Blotch Disease of Cucurbits, Prevention via Seed Treatments and Disease Resistance GenesKiremit, Merve 02 April 2021 (has links)
Melon (Cucumis melo L.) and watermelon (Citrullus lanatus (Thunb.) Matsum and Nakai) belong to the family Cucurbitaceae. Bacterial fruit blotch (BFB) disease of cucurbits is an economically devastating plant disease that has caused an estimated loss of up to $450M on watermelon crops and $75M (worldwide) to the seed and transplant industries since 1996. Disease symptoms include water-soaked cotyledons, leaf necrosis, and internal fruit rot. Current commercial management strategies are very limited and include: seed production field sanitation, greenhouse transplant sanitation, copper-based bactericide sprays, crop rotation, disease-free healthy seeds, isolating diseased plants, and peroxyacetic acid seed treatments. The seedborne disease is usually spread by contaminated seeds, and there is a zero-tolerance policy in the seed industry for infected seeds. No nondestructive assays are commercially available to detect BFB in seeds. This research investigated several different aspects of BFB disease such as non-destructive seed detection, green tea seed treatment, candidate NB-LRR genes for disease resistance, and optimization of virus induced gene silencing for melon and watermelon crops. The potential application of attenuated total reflectance (ATR) Fourier transform infrared spectroscopy (ATR-FTIR) and high-resolution X-ray analysis methods for detection of BFB on seeds were evaluated. It was possible to detect BFB in seeds that were pistil inoculated via x-ray imaging and pericarp inoculated via ATR FT-IR. In vitro and in vivo experiments evaluated the potential of tea (Camellia sinensis) and tea polyphenols as seed treatments to sanitize seeds infected with A. citrulli. Green tea unlike black tea inhibited growth of A. citrulli because of polyphenols. Eighty one melon and forty four watermelon NB-LRR genes were reidentified, and genes that have potential resistance against A. citrulli on melon plants were screened based on host selectivity of the pathogen. Finally, the virus-induced, gene-silencing method was optimized for melon and watermelon for further analysis of potential disease resistance genes. BFB can be nondestructively identified in seeds and green tea may be an effective seed treatment with further development. Promising candidate R genes were identified that might confer stable resistance in the right genetic background. / Doctor of Philosophy / Melon and watermelon crops both belong to the gourd family. Bacterial fruit blotch (BFB) disease of cucurbits is an economically devastating plant disease that has caused an estimated loss of up to $450M on watermelon crops and $75M (worldwide) to the seed and transplant industries since 1996. Disease symptoms include water-soaked cotyledons, leaf necrosis, and internal fruit rot. Current commercial management strategies and detection methods are very limited. The seedborne disease is usually spread by contaminated seeds, and there is a zero-tolerance policy in the seed industry for infected seeds. This research investigated several different aspects of BFB disease such as non-destructive seed detection, green tea seed treatment, candidate disease resistance genes, and optimization of virus induced gene silencing methodology for melon and watermelon crops. There are currently no nondestructive assays available to detect BFB in seeds. We evaluated the potential application of attenuated total reflectance (ATR) Fourier transform infrared spectroscopy (ATR-FTIR) and high-resolution X-ray analysis methods for detection of BFB on seeds. It was possible to detect BFB inside layers of seeds that were naturally inoculated through the flowers via x-ray imaging and seedcoat inoculated via ATR FT-IR. In vitro and in vivo experiments evaluated the potential of tea and tea constituents as seed treatments to sanitize seeds infected with BFB. Green tea unlike black tea inhibited growth of BFB. Eighty one melon and forty four watermelon disease resistance genes were reidentified and genes that have potential resistance against BFB on melon plants were screened based on host selectivity of the pathogen. Finally, the virus induced gene silencing method was optimized for melon and watermelon plants for further analysis of potential disease resistance genes. BFB can be nondestructively identified in seeds and green tea may be an effective seed treatment with further development. Promising candidate resistance genes were identified that might confer stable resistance in the right genetic background.
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Investigating the Molecular Framesworks of Phloem-Cap Fiber Development in Cotton (Gossypium hirsutum)Kaur, Harmanpreet 12 1900 (has links)
The current study focuses on the vascular cambium and the reiterative formation of phloem fiber bundles in cotton stems. The role of the TDIF-PXY-WOX pathway was examined in regulating cambial activity and the differentiation of phloem fibers. A study was conducted to identify and characterize the cotton WOX family genes, focusing on WOX4 and WOX14, aiming to identify and analyze their phylogenetic relationships, tissue-specific expression profiles, functional roles, and metabolic consequences. Through a sequence analysis of the Gossypium hirsutum genome, 42 cotton loci were identified as WOX family members. GhWOX4 exhibited a close homology to 7 loci, while GhWOX14 displayed homology with 8 loci. Tissue-specific expression analysis revealed prominent expression patterns of GhWOX4 and GhWOX14 in cotton internodes and roots, suggesting their involvement in vascular tissue development. Functional studies utilizing VIGS (virus-induced gene silencing) demonstrated that the knockdown of GhWOX4 and GhWOX14 resulted in a significant reduction in stem diameter and bast fiber production. This result suggests that secondary phloem fiber development is regulated by GhWOX4 and GhWOX14 genes in cotton. Additionally, the metabolic profiling of VIGS plants revealed significant alterations in amino acids, organic acids, and sugars, with implications for primary metabolic pathways. These findings suggest that GhWOX4 and GhWOX14 play pivotal roles in cotton plant development, including vascular tissue growth and phloem fiber production, and metabolic regulation.
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Virus-induced gene silencing of putative Diuraphis noxia (Kurdjumov) resistance genes in wheatStarkus, Laura January 1900 (has links)
Master of Science / Department of Entomology / C. Michael Smith / The Russian wheat aphid Diuraphis noxia (Kurdjumov) is a serious pest of world cereal grain crops, primarily barley and wheat. A phenotypic characteristic of D. noxia feeding, leaf rolling, creates a leaf pseudo gall which protects aphids, making it difficult to treat infested plants with insecticides or biological control agents. Therefore, the use of D. noxia-resistant crops is a desirable aphid management tactic. Because of the development of virulent D. noxia biotypes, the identification of new sources of barley and wheat resistance is necessary. Virus-induced gene silencing (VIGS) utilizes the plant defense system to silence viruses in inoculated plants. The accumulation of virus RNA in plants triggers the defense system to silence sequences homologous to the introduced virus and sequences of interest from a plant are inserted into the virus and silenced along with the virus. The VIGS method was tested to determine the ability of barley stripe mosaic virus (BSMV) to serve as a VIGS vector in wheat plants containing the Dnx gene for resistance to D. noxia. Dnx leaves with silenced BSMV virus yielded D. noxia populations that were significantly no different from populations produced on healthy Dnx leaves. Thus, BSMV silencing does not interfere with Dnx resistance. Several different methods were examined to determine how best to confine aphids to the silenced leaf, and a modified plastic straw cage was chosen as the optimum cage type. Microarray and gene expression data were analyzed to select two NBS-LRR type disease resistance protein genes - TaAffx.104814.1.S1_at and TaAffx.28897.1.S1 - (NBS-LRR1 and NBSLRR2), in order to assess their role in Dnx resistance. NBS-LRR1 and NBSLRR2 were silenced by inoculating leaves of Dnx plants with barley stripe mosaic virus (BSMV) containing sequences of each gene. Controls included Dnx and Dn0 plants inoculated with BSMV and non-BSMV inoculated plants. Aphids were allowed to feed on control and treatment plants to assess aphid population and
mean weight of aphids surviving at the end of the experiment. There were no differences among treatments based on aphid population, but there were significant differences the mean weights of aphids reared on several different treatments.
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Elucidation Of The Role Of Gcn2 Gene In Response To Powdery Mildew InfectionOzturk, Ibrahim Kutay 01 August 2012 (has links) (PDF)
Plant immune system is entirely based on the immunities of the individual cells in which systemic signals originate from the infection sites. Powdery mildew disease is one of the agents causing these infection sites, resulting in significant yield losses, if disease develops. Understanding the molecular basis of plant-pathogen interactions is the new trend for fighting against plant pathogens, since classical methods used in selection of resistant plants are becoming less and less efficient nowadays. Thus, finding out the genes which are responsible in plant&rsquo / s resistance is becoming very important.
In this thesis, effect of &lsquo / General Control Nondepressible-2&rsquo / (GCN2) homolog protein in barley defense mechanism was aimed to be studied. The GCN2 of yeast was
v
previously identified in our laboratory as an interacting protein when the yeast cDNA library was screened with a putative yellow rust R gene (Yr10) fragment. There are reports available in the literature for the function of GCN2 protein, which makes it a good candidate for a role in disease resistance. Thus, the barley homologue of GCN2 might have a role in the R protein mediated early disease response of which may be proceeding via Programmed Cell Death (PCD). In order to observe such function of HvGCN2 in barley, silencing of its expression via Virus Induced Gene Silencing (VIGS) was investigated. Therefore, the GCN2 homologue was found to function as dampening the severity of the disease.
The silencing with triple technical replicates was observed in 5 of the 6 samples, at an average of 43.2% by qRT-PCR analysis. The pathogen growth levels at different time points were analyzed under light microscope on the silenced and the control samples by measuring the primary and secondary hyphae lengths. The total of 24 seedlings and 292 individual spores were analyzed, and then the level of disease formation was quantitated with 603 primary hyphae and 106 secondary hyphae measurements. Up to 25% hyphae growth rate differences between the control and silenced groups were observed with a probability value less than 0.05 on t-test.
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Etude du métabolisme des phénylpropanoïdes; analyse de l'interaction de la caféoyl-coenzyme A 3-O-méthyltransférase (CCoAOMT) avec son substrat et caractérisation fonctionnelle d'une nouvelle acyltransférase, l'HydroxyCinnamoyl-CoA : shikimate/quinate hydroxycinnamoyl Transférase (HCT).Hoffmann, Laurent 04 July 2003 (has links) (PDF)
Le métabolisme des phénylpropanoïdes est un métabolisme secondaire spécifique au règne végétal. Il conduit, à partir de la phénylalanine, à la synthèse d'une grande variété de substances telles que les anthocyanes, les isoflavonoïdes, les stilbènes, des esters d'acides hydroxycinnamiques, ou encore à la lignine. Ces métabolites secondaires interviennent dans la pigmentation florale ou encore la protection des tissus végétaux contre divers stress biotiques et abiotiques. Quant à la lignine, elle assure rigidité aux parois cellulaires végétales et imperméabilité aux tissus conducteurs. La lignine est un polymère tridimensionnel constitué de trois unités monomériques qui possèdent le même squelette carboné phénylpropane mais diffèrent par leur degré de méthoxylation et d'hydroxylation. Une partie de mon travail de thèse a consisté à étudier la relation structure/fonction de la caféoyl-coenzyme A O-méthyltransférase (CCoAOMT) de N. tabacum, responsable de l'introduction de la première des deux fonctions méthyles. Des études bioinformatiques couplées à des approches de biochimie et de mutagenèse dirigée, nous ont permis de modéliser l'interaction de la CCoAOMT avec son substrat, le caféoyl-CoA. Trois acides aminés du site actif ont notamment été identifiés comme intervenant dans la reconnaissance spécifique de la chaîne latérale de CoA. J'ai également caractérisé, chez N. tabacum, une nouvelle acyltransférase à activité HydroxyCinnamoyl-CoA : shikimate/quinate hydroxycinnamoyl Transférase (HCT) impliquée dans le métabolisme des phénylpropanoïdes. Nous avons montré que l'enzyme HCT recombinante synthétisait, in vitro, les substrats de l'hydroxylation en position 3 du noyau aromatique. De plus, la répression de l'expression du gène HCT par le «VIGS» conduit à un ralentissement de la croissance des plantes, à une perturbation importante du pool d'acide chlorogénique, ainsi qu'à une diminution de la quantité et à une modification de la composition de la lignine synthétisée.
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