Development of a fluorescent in situ hybridization (FISH) technique for visualizing CGMMV in plant tissues

Shargil, Dorit; Zemach, Hanita; Belausov, Eduard; Lachman, Oded; Kamenetsky, Rina; Dombrovsky, Aviv

doi:10.1016/j.jviromet.2015.07.014

Cited by 18 publications

(7 citation statements)

References 32 publications

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“…In addition, honeybees may use their mandibles to bite (Papachristoforou et al ., ), which may cause damage to reproductive tissues and allow the virus to penetrate the plant. The field experiments here clearly demonstrated that the honeybees were capable of spreading CGMMV during pollination, probably due to microscopic wounding events rather than by direct inoculation of the pollen grains (Shargil et al ., ). Primary CGMMV inoculum in proximity to the path of the foraging honeybees contributes to the disease spread.…”

Section: Discussionmentioning

confidence: 97%

“…Two plausible ways of transmission of CGMMV in these experiments are either through the injury of the flower made by the honeybees during pollination or via fertilization made by infected pollen (Shipp et al, 2008;Liu, 2010;Liu et al, 2014). However, a recent study of the male flowers using high resolution of organ visualization by the FISH technique showed virus detection in anther tissue, whereas the pollen grains were virus-free (Shargil et al, 2015). It is therefore suggested that mechanical adherence of the virus to the honeybee occurs.…”

Section: Discussionmentioning

confidence: 98%

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The honeybee Apis mellifera contributes to Cucumber green mottle mosaic virus spread via pollination

et al. 2017

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The tobamovirus Cucumber green mottle mosaic virus (CGMMV) is efficiently transmitted between plants by mechanical contact. So far, no clear evidence has been reported regarding the transmission potential of the virus by beneficial pollinator insects. This study examined the capability of the well-known pollinator honeybee Apis mellifera to transmit CGMMV in cucurbits using melon and cucumber plants as a model. In order to provide a clear answer to that question, five experiments were designed on various scales performed under three environmental conditions. The results show that under protected cropping conditions, CGMMV is transmitted by the honeybees. The location of the beehive in relation to both the CGMMV primary inoculum source and the healthy plants during honeybee foraging plays an important role in the efficiency of CGMMV spread. Furthermore, in the presence of early stage CGMMV-inoculated plants, the efficiency of CGMMV spread to uninoculated plants placed on the honeybees' path to the beehive may increase. To the authors' knowledge, CGMMV transmission by honeybees has not yet been shown, and this study can be adopted for other tobamovirus related research.

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Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 98%

The honeybee Apis mellifera contributes to Cucumber green mottle mosaic virus spread via pollination

et al. 2017

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“…However, the authors could not exclude the possible transmission by mechanical inoculation due to contact while rubbing the infected male flowers [ 37 ]. In a later study, using FISH to follow CGMMV RNA in infected cucumber and melon plants, anther tissue of the male flowers was found to be heavily infected, but pollen grains were found to be virus-free [ 31 ].…”

Section: Discussionmentioning

confidence: 99%

“…FISH was performed following Shargil et al [ 31 ] using a ToBRFV-specific single-stranded DNA primer labeled with the fluorophore cyanine-Cy3 at the 3′ end (TBR-Cy3-6190; 5′-TTTAAGCATCTCGATTATCTCA/3Cy3Sp/-3′) corresponding to ToBRFV nucleotides 6190-6212.…”

Section: Methodsmentioning

confidence: 99%

ToBRFV Infects the Reproductive Tissues of Tomato Plants but Is Not Transmitted to the Progenies by Pollination

Avni

Gelbart

Sufrin-Ringwald

et al. 2022

Cells

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Tomato brown rugose fruit virus (ToBRFV), a newly identified Tobamovirus, has recently emerged as a significant pathogen of tomato plants (Solanum lycopersicum). The virus can evade or overcome the known tobamovirus resistance in tomatoes, i.e., Tm-1, Tm-2, and its allele Tm-22. ToBRFV was identified for the first time only a few years ago, and its interactions with the tomato host are still not clear. We investigated ToBRFV’s presence in the reproductive tissues of tomato using fluorescent in situ hybridization (FISH) and RT-PCR. In infected plants, the virus was detected in the leaves, petals, ovary, stamen, style, stigma, and pollen grains but not inside the ovules. Fruits and seeds harvested from infected plants were contaminated with the virus. To test whether the virus is pollen transmitted, clean mother plants were hand pollinated with pollen from ToBRFV-infected plants and grown to fruit. None of the fruits and seeds harvested from the pollinated clean mother plants contained ToBRFV. Pollen germination assays revealed the germination arrest of ToBRFV-infected pollen. We concluded that ToBRFV might infect reproductive organs and pollen grains of tomato but that it is not pollen transmitted.

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“…To determine RNA localization at the cellular level, fluorescence in situ hybridization (FISH) was developed and has become a vital tool in analyzing animal and yeast gene expression (Levsky and Singer, 2003). In plants, FISH has been used to visualize entire chromosomes or individual genetic loci (Lamb et al, 2007;Tirichine et al, 2009;Jiang, 2019) as well as pathogen DNA (Shargil et al, 2015). However, although there are notable examples using whole-mounted tissues (Bruno et al, 2011(Bruno et al, , 2015Himanen et al, 2012;Rozier et al, 2014;Bleckmann and Dresselhaus, 2016;Woloszynska et al, 2019), the use of FISH to investigate plant RNA localization at high resolution and deep in tissues remains to be fully exploited.…”

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confidence: 99%

Visualization of Protein Coding, Long Noncoding, and Nuclear RNAs by Fluorescence in Situ Hybridization in Sections of Shoot Apical Meristems and Developing Flowers

et al. 2019

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In addition to transcriptional regulation, gene expression is further modulated through mRNA spatiotemporal distribution, by RNA movement between cells, and by RNA localization within cells. Here, we have adapted RNA fluorescence in situ hybridization (FISH) to explore RNA localization in Arabidopsis (Arabidopsis thaliana). We show that RNA FISH on sectioned material can be applied to investigate the tissue and subcellular localization of meristem and flower development genes, cell cycle transcripts, and plant long noncoding RNAs. We also developed double RNA FISH to dissect the coexpression of different mRNAs at the shoot apex and nuclear-cytoplasmic separation of cell cycle gene transcripts in dividing cells. By coupling RNA FISH with fluorescence immunocytochemistry, we further demonstrate that a gene's mRNA and protein may be simultaneously detected, for example revealing uniform distribution of PIN-FORMED1 (PIN1) mRNA and polar localization of PIN1 protein in the same cells. Therefore, our method enables the visualization of gene expression at both transcriptional and translational levels with subcellular spatial resolution, opening up the possibility of systematically tracking the dynamics of RNA molecules and their cognate proteins in plant cells.

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Development of a fluorescent in situ hybridization (FISH) technique for visualizing CGMMV in plant tissues

Cited by 18 publications

References 32 publications

The honeybee Apis mellifera contributes to Cucumber green mottle mosaic virus spread via pollination

The honeybee Apis mellifera contributes to Cucumber green mottle mosaic virus spread via pollination

ToBRFV Infects the Reproductive Tissues of Tomato Plants but Is Not Transmitted to the Progenies by Pollination

Visualization of Protein Coding, Long Noncoding, and Nuclear RNAs by Fluorescence in Situ Hybridization in Sections of Shoot Apical Meristems and Developing Flowers

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