Virus‐induced gene silencing in tomato fruit

Fu, Daqi; Zhu, Beiwei; Zhu, Hongliang; Jiang, Weibo; Luo, Yunbo

doi:10.1111/j.1365-313x.2005.02441.x

Cited by 306 publications

(247 citation statements)

References 44 publications

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“…The lack of carotenoids destabilizes the chlorophylls, yielding a readily visible white phenotype (Liu et al, 2002). Silencing of phytoene desaturase also served as reporter in such carotenoidrich organs as tomato fruits (Fu et al, 2005;Orzaez et al, 2006). Similarly, silencing of chalcone synthase, a key enzyme in anthocyanin biosynthesis, has been used as an endogenous reporter for gene silencing in such anthocyanin-rich organs as flower petals (Chen et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

Visual Tracking of Plant Virus Infection and Movement Using a Reporter MYB Transcription Factor That Activates Anthocyanin Biosynthesis

et al. 2012

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Insertion of reporter genes into plant virus genomes is a common experimental strategy to research many aspects of the viral infection dynamics. Their numerous advantages make fluorescent proteins the markers of choice in most studies. However, the use of fluorescent proteins still has some limitations, such as the need of specialized material and facilities to detect the fluorescence. Here, we demonstrate a visual reporter marker system to track virus infection and movement through the plant. The reporter system is based on expression of Antirrhinum majus MYB-related Rosea1 (Ros1) transcription factor (220 amino acids; 25.7 kD) that activates a series of biosynthetic genes leading to accumulation of colored anthocyanins. Using two different tobacco etch potyvirus recombinant clones tagged with Ros1, we show that infected tobacco (Nicotiana tabacum) tissues turn bright red, demonstrating that in this context, the sole expression of Ros1 is sufficient to induce pigment accumulation to a level readily detectable to the naked eye. This marker system also reports viral load qualitatively and quantitatively by means of a very simple extraction process. The Ros1 marker remained stable within the potyvirus genome through successive infectious passages from plant to plant. The main limitation of this marker system is that color output will depend on each particular plant host-virus combination and must be previously tested. However, our experiments demonstrate accurate tracking of turnip mosaic potyvirus infecting Arabidopsis (Arabidopsis thaliana) and either tobacco mosaic virus or potato X virus infecting Nicotiana benthamiana, stressing the general applicability of the method.

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Section: Discussionmentioning

confidence: 99%

Visual Tracking of Plant Virus Infection and Movement Using a Reporter MYB Transcription Factor That Activates Anthocyanin Biosynthesis

et al. 2012

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“…The bags were removed from fruits at 10 DAFB, and these fruits (on the tree) were subjected to A. tumefaciens-mediated tobacco rattle virus vector infiltration. The A. tumefaciens-mediated tobacco rattle virus infiltration was performed according to Fu et al (2005) and Bai et al (2011) with some modifications. A. tumefaciens strains containing pTRV1 and pTRV2 or their derivative vectors, pTRV2-69 and pTRV2-1216, were mixed in a 1:1 ratio and infiltrated into 10-DAFB pear fruit flesh near the petiole.…”

Section: Vigs In Pearmentioning

confidence: 99%

The Methylation of the PcMYB10 Promoter Is Associated with Green-Skinned Sport in Max Red Bartlett Pear

et al. 2013

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Varieties of the European pear (Pyrus communis) can produce trees with both red-and green-skinned fruits, such as the Max Red Bartlett (MRB) variety, although little is known about the mechanism behind this differential pigmentation. In this study, we investigated the pigmentation of MRB and its green-skinned sport (MRB-G). The results suggest that a reduction in anthocyanin concentration causes the MRB-G sport. Transcript levels of PcUFGT (for UDP-glucose:flavonoid 3-O-glucosyltransferase), the key structural gene in anthocyanin biosynthesis, paralleled the change of anthocyanin concentration in both MRB and MRB-G fruit. We cloned the PcMYB10 gene, a transcription factor associated with the promoter of PcUFGT. An investigation of the 2-kb region upstream of the ATG translation start site of PcMYB10 showed the regions 2604 to 2911 bp and 21,218 to 21,649 bp to be highly methylated. A comparison of the PcMYB10 promoter methylation level between the MRB and MRB-G forms indicated a correlation between hypermethylation and the green-skin phenotype. An Agrobacterium tumefaciens infiltration assay was conducted on young MRB fruits by using a plasmid constructed to silence endogenous PcMYB10 via DNA methylation. The infiltrated fruits showed blocked anthocyanin biosynthesis, higher methylation of the PcMYB10 promoter, and lower expression of PcMYB10 and PcUFGT. We suggest that the methylation level of PcMYB10 is associated with the formation of the greenskinned sport in the MRB pear. The potential mechanism behind the regulation of anthocyanin biosynthesis is discussed.

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“…Particularly important for tomato is to understand the genetic basis of the metabolic pathways that operate in fruit, especially those active during the ripening process, which influence the nutritional value and organoleptic features of the crop. It has been shown recently that successful "local" silencing effects can be obtained by direct inoculation (agroinjection) of VIGS (or RNA interference) constructs into developing fruit (Fu et al, 2005;Hoffmann et al, 2006;Orzaez et al, 2006). This methodology speeds up the experimental procedure appreciably.…”

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confidence: 99%

A Visual Reporter System for Virus-Induced Gene Silencing in Tomato Fruit Based on Anthocyanin Accumulation

et al. 2009

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Innes Centre, Colney, Norwich NR4 7UH, United Kingdom (E.B., C.M.)Virus-induced gene silencing (VIGS) is a powerful tool for reverse genetics in tomato (Solanum lycopersicum). However, the irregular distribution of the effects of VIGS hampers the identification and quantification of nonvisual phenotypes. To overcome this limitation, a visually traceable VIGS system was developed for fruit, comprising two elements: (1) a transgenic tomato line (Del/Ros1) expressing Antirrhinum majus Delila and Rosea1 transcription factors under the control of the fruitspecific E8 promoter, showing a purple-fruited, anthocyanin-rich phenotype; and (2) a modified tobacco rattle virus VIGS vector incorporating partial Rosea1 and Delila sequences, which was shown to restore the red-fruited phenotype upon agroinjection in Del/Ros1 plants. Dissection of silenced areas for subsequent chemometric analysis successfully identified the relevant metabolites underlying gene function for three tomato genes, phytoene desaturase, TomloxC, and SlODO1, used for proof of concept. The C-6 aldehydes derived from lipid 13-hydroperoxidation were found to be the volatile compounds most severely affected by TomloxC silencing, whereas geranial and 6-methyl-5-hepten-2-one were identified as the volatiles most severely reduced by phytoene desaturase silencing in ripening fruit. In a third example, silencing of SlODO1, a tomato homolog of the ODORANT1 gene encoding a myb transcription factor, which regulates benzenoid metabolism in petunia (Petunia hybrida) flowers, resulted in a sharp accumulation of benzaldehyde in tomato fruit. Together, these results indicate that fruit VIGS, enhanced by anthocyanin monitoring, can be a powerful tool for reverse genetics in the study of the metabolic networks operating during fruit ripening.

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Virus‐induced gene silencing in tomato fruit

Cited by 306 publications

References 44 publications

Visual Tracking of Plant Virus Infection and Movement Using a Reporter MYB Transcription Factor That Activates Anthocyanin Biosynthesis

Visual Tracking of Plant Virus Infection and Movement Using a Reporter MYB Transcription Factor That Activates Anthocyanin Biosynthesis

The Methylation of the PcMYB10 Promoter Is Associated with Green-Skinned Sport in Max Red Bartlett Pear

A Visual Reporter System for Virus-Induced Gene Silencing in Tomato Fruit Based on Anthocyanin Accumulation

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