1. The document describes a strategy to control the plant pathogen Xylella fastidiosa using pathogen confusion by expressing its DSF synthase gene rpfF in transgenic grape plants.
2. Transgenic grape plants expressing rpfF were more resistant to Pierce's disease compared to wild-type grape when challenged with X. fastidiosa.
3. Expression of rpfF and the accessory gene rpfB together in Arabidopsis enhanced DSF production and reduced symptoms caused by a DSF-overproducing strain of Xanthomonas campestris more than rpfF alone.
in this chapter covers the symptoms modulation and diseases severity increases and decreases. and role of SiRNA in diseases severity reduction. and also covers the types of SRNA..
in this chapter covers the symptoms modulation and diseases severity increases and decreases. and role of SiRNA in diseases severity reduction. and also covers the types of SRNA..
RNA silencing refers to related processes of post-transcriptional control of gene expression found in plants, animals and fungi. A unifying feature of RNA silencing is that it mediates sequence-specific degradation of target transcripts, recruiting RNA molecules of 21–23 nucleotides as specificity determinants. In higher plants, RNA silencing serves as an adaptive, antiviral defence system, which is transmitted systemically in response to localized virus challenge. Plant viruses have elaborated a variety of counter-defensive measures to overcome the host silencing response. One of these strategies is to produce proteins that target the cell autonomous or signalling steps of RNA silencing. It is not known whether a similar antiviral mechanism also operates in animal cells
Non-enveloped, flexuous, filamentous,of 720-850 nm long and 12-15 nm in diameter. Symmetry helical. PVY maybe transmitted to potato plants through grafting, plant sap inoculation and through aphid transmission. Presence of characteristic inclusion bodies within infected plant cells.
In potato, causes mild mosaic on leaves,Crinkling and necrosis etc. TGB3 (Triple gene block proteins) is expressed by leaky scanning of the TGB2 subgenomic mRNA. TGBp1 with the presence of TGBp2 and TGBp3 can modify the PD size exclusion limit and move between cells.
RNA silencing refers to related processes of post-transcriptional control of gene expression found in plants, animals and fungi. A unifying feature of RNA silencing is that it mediates sequence-specific degradation of target transcripts, recruiting RNA molecules of 21–23 nucleotides as specificity determinants. In higher plants, RNA silencing serves as an adaptive, antiviral defence system, which is transmitted systemically in response to localized virus challenge. Plant viruses have elaborated a variety of counter-defensive measures to overcome the host silencing response. One of these strategies is to produce proteins that target the cell autonomous or signalling steps of RNA silencing. It is not known whether a similar antiviral mechanism also operates in animal cells
Non-enveloped, flexuous, filamentous,of 720-850 nm long and 12-15 nm in diameter. Symmetry helical. PVY maybe transmitted to potato plants through grafting, plant sap inoculation and through aphid transmission. Presence of characteristic inclusion bodies within infected plant cells.
In potato, causes mild mosaic on leaves,Crinkling and necrosis etc. TGB3 (Triple gene block proteins) is expressed by leaky scanning of the TGB2 subgenomic mRNA. TGBp1 with the presence of TGBp2 and TGBp3 can modify the PD size exclusion limit and move between cells.
Nonhosts of Xylella fastidiosa that sharpshooters would die for. A managemen...huyng
Nonhosts of Xylella fastidiosa that sharpshooters would die for... A management strategy based on trap plants
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II. RNA silencing
RNA silencing/RNAi/ Gene quelling as known in fungi is an endogenous cellular mechanism, that works at the mRNA level and has a sequence dependent mode of action. It is found only in higher eukaryotes and known to be absent in prokaryotes. Initiated by dsRNA or stem loop RNA structures like hairpin RNAs (hpRNAs) which are acted upon by the dicer like enzymes to form 21-25 bp siRNA duplexes, which are further incorporated in the RNA induced silencing complex (RISC) where duplex is unwound and subsequently argonaute 2 protein cleaves the one strand and only the guide strand remains with RISC. Guide strand forms complex with the mRNA through complementary base pairing which is then degraded or silenced through Transcriptional or Post-Transcriptional gene silencing.
III. Transgenic versus non-transgenic RNAi
RNAi in crop protection can be achieved both via transgenic/transformative and non-transgenic/ non-transformative approaches. In transgenic approach, a dsRNA construct has to be genetically engineered into the plant genome by a straightforward strategy of transformation and it provides efficient and long term control. But it also requires generations of crop plants, taking years and delaying practical application also due to the extensive regulatory procedures. As well as during genetic engineering plant transformability and genetic stability needs to be taken into account. Especially, the main issue is that there is very low consumer acceptance of genetically modified crops.
Whereas in case of non-transformative RNAi, dsRNAs are topically applied on the plants either as spray suspension, trunk/stem/petiole injection, root absorption or seed treatment, etc. As this approach silence the genes without introducing any heritable changes in the plant genome, they may not be regulated as a genetically modified crop thus excluding the issue of consumer acceptance. Moreover they are feasible and affordable and incur less cost per tree. The only issue may lie with the continuous supply of dsRNAs which may be overcome using nanoparticles like clay nanosheets, carbon nanotubes, cationic nanoparticles or chemicals like surfactants or peptide based RNA delivery system can be used while not only improves the stability and absorption of dsRNA but also provide for the slow and sustained release of them.
One such study has been done by Mitter et al., wherein they sprayed the dsRNA suspension on the plants either singly or after loading them on clay nanosheets followed by inoculation of the challenge virus. They observed that the clay nanosheets provided enhanced stability and sustained release of dsRNA increasing the protection window from 5 to 20 days in a single spray. This also confirms that the dsRNAs could move or translocate to untreated parts of the plant providing systemic protection to the plants.
IV. Mode of entry and movement of dsRNAs in plants
The dsRNAs sprayed on the plants penetrate the leaves through the wounds o
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Plant immunity towards an integrated view of plant pathogen interaction and i...Pavan R
Plant immunity towards pathogen
Similar to Pathogen confusion as a strategy for controlling diseases caused by Xylella fastidosa - Steve Lindow - Pierce's Disease Conference 2008 (20)
Plant immunity towards an integrated view of plant pathogen interaction and i...
Pathogen confusion as a strategy for controlling diseases caused by Xylella fastidosa - Steve Lindow - Pierce's Disease Conference 2008
1. Pathogen confusion as a strategy for controlling diseases caused by Xylella fastidosa Steven Lindow Department of Plant and Microbial Biology University of California, Berkeley
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4. X. fastidiosa Decreases Virulence by Coordinating Virulence Genes in Cell Density-Dependent Fashion Wild type rpfF mutant
5. Key Virulence Genes Controlled by DSF Downregulated: Type IV pili Polyglacturonase Cellulase Meng et al. 2005 Upregulated: Hemagglutinin adhesins Extracellular polysaccharides ( EPS): gum Type I pili Type IV pili
6. Wild Type - gfp rpfF - gfp 20 µM The RpfF- mutant of Xylella fastidiosa colonizes many more xylem vessels than the wild-type strain
7. Wild type RpfF mutant RpfF- mutant tends to fill xyllem vessels more frequently than wild-type strain of Xylella fastidiosa
8. Over-expression of RpfF in Xylella fastidiosa reduces the movement of the pathogen in the plant and limits disease to site of inoculation
9. Distance of migration (cm) Log cells/g Weeks after inoculation Symptomatic leaves/plant RpfC mutant of X. fastidiosa over-produce DSF and are less virulent since it does not move well within grape
10. Gum Production Plant colonization phase Insect acquisition phase Extensive vessel colonization Low cell numbers in most vessels Disease symptoms may not be present Some vessels have high cell numbers Disease symptoms may be present Further multiplication in crowded vessels slows DSF Abundance Stickiness to Surfaces Expression of adhesins Twitching Motility Type IV pili Pit Membrane Degradation Pgl and Eng expression
11. The goal : Production of DSF in transgenic grape The approach : Expression of rpfF encoding DSF synthase from Xylella fastididosa The strategy : Express RpfF in different cellular locations and with different accessory proteins
12. Strategy 1: Express un-targeted Rpf 35S rpfF pCAMBIA 1390 Results: Very modest production of DSF At threshold of detection in grape, Arabidopsis, and tomato
13. 35S SSU rpfF C-DNA encoding the RUBISCO small subunit N-terminal was isolated from Arabidopsis leaf RNA In-frame fusion with rpfF gene from Xylella fastidiosa Strategy 2: Expression of chloroplast-targeted RpfF Results: Enhanced DSF production in Arabidopsis, tobacco and Tomato Transformation of grape underway MASSMLSSATMVASPAQATMVAPFNGLKSSAAFPATRKANNDITSITSNGGRVNCMQ VWPPIGKKKFETLSYLPDLTDSELAE F MSAVQPFIRTNIGSTLRIIEEPQRDVYWIHMHA DLAINPGRACFSTRLVDDITGYQTNLGQRLNTAGVLAPHVVLASDSDVFNLGGDLALFC QLIREGDRARLLDYAQRCVRGVHAFHVGLGARAHSIALVQGSALGGGFEAALSCHTIIA EEGVMMGLPEVLFXLFPG SSU-rpfF WT-tomato Xf-DSF
14. Strategy 3: C0-expression of chloroplast-targeted RpfF and RpfB Logic: The role of RpfB in DSF synthesis is not clear but it seems to be an “accessory” protein that may help supply needed substrate for RpfF 35S SSU-rpfF nos 35S SSU-rpfB nos 35S gus nos Results: Somewhat higher expression of DSF in Arabidopsis compared to SSU- rpfF construct Transformation of grape is underway
16. Method for challenging with Xylella fastidiosa Stem droplet-needle puncture Vector inoculation may be superior in that it delivers fewer cells to fewer vessels, but it has been unfeasible since transgenic plants have required insecticide sprays to defend against pests in greenhouse
17. Plant Genotype Symptomatic leaves/plant Grape transformed with rpfF gene from Xylella fastidiosa and producing DSF are much more resistant to Pierce’s Disease compared to wild-type grape
18. Disease severity from topical application of Xanthomonas campestris strains varying in DSF production to Arabidopsis transformed with rpfF or with both rpfB and rpfF Arabidopsis genotype Xcc strains Wild type rpfF- Col (WT) ++++ - rpfF transformed ++++ + rpfF & rpfB transformed ++++ ++
19. Distance of migration (cm) Log cells/g Studies of the movement and titer of Xylella fastidiosa in transgenic grape is underway, but we expect both to be reduced based on the observation of disease symptoms that are limited to near the point of inoculation in RpfF- expressing plants and the reduced growth/movement of DSF-overproducing Xylella fastidiosa mutants
20. Susceptible Cabernet sauvignon have been grafted onto transgenic rpfF-expressing rootstocks to test for mobility of DSF within plants
21. 1. Rationale for achieving transgenic protection against Xf 2. Introduced DNA construction, its construction, and its expected affects 3. Observed phenotype of the transformed, uninoculated grapevine 4. Xf challenge method described and compared to vector inoculation 5. Comparison of challenged transgenic and non-transgenic grapevine 6. Xf accumulation (titer) and localization in transgenic vs non-transgenic grapevine 7. Assessment of the value and applicability of the transgenic approach taken Session 4 Panel Discussion Can Transgenic Research Mitigate Pierce’s Disease? Panel:, David Gilchrist, Abhaya Dandekar, John Labavich, Steven Lindow