共查询到20条相似文献,搜索用时 31 毫秒
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Zanek MC Reyes CA Cervera M Peña EJ Velázquez K Costa N Plata MI Grau O Peña L García ML 《Plant cell reports》2008,27(1):57-66
Citrus psorosis is a serious viral disease affecting citrus trees in many countries. Its causal agent is Citrus psorosis virus (CPsV), the type member of genus Ophiovirus. CPsV infects most important citrus varieties, including oranges, mandarins and grapefruits, as well as hybrids and citrus
relatives used as rootstocks. Certification programs have not been sufficient to control the disease and no sources of natural resistance have been found.
Pathogen-derived resistance (PDR) can provide an efficient alternative to control viral diseases in their hosts. For this
purpose, we have produced 21 independent lines of sweet orange expressing the coat protein gene of CPsV and five of them were
challenged with the homologous CPV 4 isolate. Two different viral loads were evaluated to challenge the transgenic plants,
but so far, no resistance or tolerance has been found in any line after 1 year of observations. In contrast, after inoculation
all lines showed characteristic symptoms of psorosis in the greenhouse. The transgenic lines expressed low and variable amounts
of the cp gene and no correlation was found between copy number and transgene expression. One line contained three copies of the cp gene, expressed low amounts of the mRNA and no coat protein. The ORF was cytosine methylated suggesting a PTGS mechanism,
although the transformant failed to protect against the viral load used. Possible causes for the failed protection against
the CPsV are discussed. 相似文献
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Kościańska E Kalantidis K Wypijewski K Sadowski J Tabler M 《Plant molecular biology》2005,59(4):647-661
In this study we analyse several aspects of cytoplasmic RNA silencing by agroinfiltration of DNA constructs encoding single-
and double-stranded RNAs derived from a GFP transgene and from the endogenous Virp1 gene. Both types of inductors resulted after 2–4 days in much higher concentration of siRNAs in the agroinfiltrated zone
than normally seen during systemic silencing. More specifically, infiltration of two transgene hairpin constructs resulted
in elevated levels of siRNAs. However, differences between the two constructs were observed: the antisense–sense arrangement
was more effective than the sense–antisense order. For both double-stranded forms, we observed a relative increase of the
24-mer size class of siRNAs. When a comparable hairpin construct of the endogenous Virp1 gene was assayed, the portion of the 24-mer siRNA class remained low as observed for all kinds of single-stranded inducers.
The lack of increase of Virp1-derived 24-mers was independent of the expression level, as demonstrated by agroinfiltration into a transgenic plant that
overexpressed Virp1 and showed the same pattern. Using transducer constructs, we could detect within a week transitive silencing from GFP to GUS sequences in the infiltrated zone and in either direction 5′–3′ and 3′–5′. Conversely, for the endogenous Virp1 gene neither transitive silencing nor the induction of systemic silencing could be observed. These results are discussed
in view of the current models of RNA silencing. 相似文献
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In plants, transgenes frequently become spontaneously silenced for unknown reasons. Typically, transgene silencing involves
the generation of small interfering RNAs (siRNAs) that directly or indirectly target cognate DNA and mRNA sequences for methylation
and degradation, respectively. In this report, we compared spontaneous silencing of a transgene in Nicotiana benthamiana and Nicotiana tabacum. In both species, abundant siRNAs were produced. In N. benthamiana, the self-silencing process involved mRNA degradation and dense DNA methylation of the homologous coding region. In N.
tabacum, self-silencing occurred without complete mRNA degradation and with low methylation of the cognate coding region. Our data
indicated that in plants, siRNA-mediated spontaneous silencing is, in addition to mRNA degradation, based on translational
inhibition. Differences in the initiation and establishment of self-silencing together with marked differences in the degree
of de novo DNA methylation showed that the mechanistic details of RNA silencing, although largely conserved, may vary also
in genetically close plant species. 相似文献
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Citrus psorosis virus (CPsV) is the causal agent of psorosis, a serious and widespread citrus disease. Two syndromes of psorosis, PsA and PsB, have been described. PsB is the most aggressive and rampant form. Previously, we obtained Pineapple sweet orange plants transformed with a hairpin construct derived from the CPsV coat protein gene (ihpCP). Some of these plants were resistant to CPsV 90-1-1, a PsA isolate homologous to the transgene. In this study, we found that expression of the ihpCP transgene and siRNA production in lines ihpCP-10 and -15 were stable with time and propagation. In particular, line ihpCP-15 has been resistant for more than 2 years, even after re-inoculation. The ihpCP plants were also resistant against a heterologous CPsV isolate that causes severe PsB syndrome. Line ihpCP-15 manifested complete resistance while line ihpCP-10 was tolerant to the virus, although with variable behaviour, showing delay and attenuation in PsB symptoms. These lines are promising for a biotech product aimed at eradicating psorosis. 相似文献
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Aluminum (Al) toxicity is one of the major factors that limit plant growth in acid soils. Al-induced release of organic acids
into rhizosphere from the root apex has been identified as a major Al-tolerance mechanism in many plant species. In this study,
Al tolerance of Yuzu (Citrus Junos Sieb. ex Tanaka) was tested on the basis of root elongation and the results demonstrated that Yuzu was Al tolerant compared
with other plant species. Exposure to Al triggered the exudation of citrate from the Yuzu root. Thus, the mechanism of Al
tolerance in Yuzu involved an Al-inducible increase in citrate release. Aluminum also elicited an increase of citrate content
and increased the expression level of mitochondrial citrate synthase (CjCS) gene and enzyme activity in Yuzu. The CjCS gene was cloned from Yuzu and overexpressed in Nicotiana benthamiana using Agrobacterium tumefaciens-mediated methods. Increased expression level of the CjCS gene and enhanced enzyme activity were observed in transgenic plants compared with the wild-type plants. Root growth experiments
showed that transgenic plants have enhanced levels of Al tolerance. The transgenic Nicotiana plants showed increased levels of citrate in roots compared to wild-type plants. The exudation of citrate from roots of the
transgenic plants significantly increased when exposed to Al. The results with transgenic plants suggest that overexpression
of mitochondrial CS can be a useful tool to achieve Al tolerance. 相似文献
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RNA silencing is a conserved mechanism found ubiquitously in eukaryotic organisms. It has been used to regulate gene expression
and development. In addition, RNA silencing serves as an important mechanism in plants’ defense against invasive nucleic acids,
such as viruses, transposons, and transgenes. As a counter-defense, most plants, and some animal viruses, encode RNA silencing
suppressors to interfere at one or several points of the silencing pathway. In this study, we showed that Pns12 of RGDV (Rice gall dwarf virus) exhibits silencing suppressor activity on the reporter green fluorescent protein in transgenic Nicotiana benthamiana line 16c. Pns12 of RGDV suppressed local silencing induced by sense RNA but had no effect on that induced by dsRNA. Expression
of Pns12 also enhanced Potato virus X pathogenicity in N. benthamiana. Collectively, these results suggested that RGDV Pns12 functions as a virus suppressor of RNA silencing, which might target
an upstream step of dsRNA formation in the RNA silencing pathway. Furthermore, we showed that Pns12 is localized mainly in
the nucleus of N. benthamiana leaf cells. 相似文献
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Shun-Fang Cheng Ying-Ping Huang Zi-Rong Wu Chung-Chi Hu Yau-Heiu Hsu Ching-Hsiu Tsai 《BMC plant biology》2010,10(1):286
Background
The genes of plants can be up- or down-regulated during viral infection to influence the replication of viruses. Identification of these differentially expressed genes could shed light on the defense systems employed by plants and the mechanisms involved in the adaption of viruses to plant cells. Differential gene expression in Nicotiana benthamiana plants in response to infection with Bamboo mosaic virus (BaMV) was revealed using cDNA-amplified fragment length polymorphism (AFLP). 相似文献18.
Yong-Won Kang Jae-Yong Lee Young Jeon Gang-Won Cheong Moonil Kim Hyun-Sook Pai 《Plant molecular biology》2010,72(6):569-583
Sulfite reductase (SiR) performs dual functions, acting as a sulfur assimilation enzyme and as a chloroplast (cp-) nucleoid
binding protein. In this study, we examined the in vivo effects of SiR deficiency on chloroplast development in Nicotiana benthamiana. Virus-induced gene silencing of NbSiR resulted in leaf yellowing and growth retardation phenotypes, which were not rescued by cysteine supplementation. NbSiR:GFP
fusion protein was targeted to chloroplasts and colocalized with cp-nucleoids. Recombinant full-length NbSiR protein and the
C-terminal half of NbSiR possessed cp-DNA compaction activities in vitro, and expression of full-length NbSiR in E. coli caused condensation of genomic DNA. NbSiR silencing differentially affected expression of plastid-encoded genes, inhibiting expression of several genes more severely
than others. In the later stages, depletion of NbSiR resulted in chloroplast ablation. In NbSiR-silenced plants, enlarged cp-nucleoids containing an increased amount of cp-DNA were observed in the middle of the abnormal
chloroplasts, and the cp-DNAs were predominantly of subgenomic sizes based on pulse field gel electrophoresis. The abnormal
chloroplasts developed prolamellar body-like cubic lipid structures in the light without accumulating NADPH:protochlorophyllide
oxidoreductase proteins. Our results suggest that NbSiR plays a role in cp-nucleoid metabolism, plastid gene expression, and
thylakoid membrane development. 相似文献
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The Perilla (Perilla frutescens L. cv. Okdong) oleosin gene, PfOle19, produces a 19-kDa protein that is highly expressed only in seeds. The activity of the −2,015 bp 5′-upstream promoter region
of this gene was investigated in transgenic Arabidopsis plants using the fusion reporter constructs of enhanced green fluorescent protein (EGFP) and β-glucuronidase (GUS). The PfOle19 promoter directs Egfp expression in developing siliques, but not in leaves, stems or roots. In the transgenic Arabidopsis, EGFP fluorescence and histochemical GUS staining were restricted to early seedlings, indehiscent siliques and mature seeds.
Progressive 5′-deletions up to the −963 bp position of the PfOle19 promoter increases the spatial control of the gene expression in seeds, but reduces its quantitative levels of expression.
Moreover, the activity of the PfOle19 promoter in mature seeds is 4- and 5-fold greater than that of the cauliflower mosaic virus 35S promoter in terms of both
EGFP intensity and fluorometric GUS activity, respectively. 相似文献