Dataset Information

Assessment of Globodera pallida RNA Extracted from Solanum Roots.

ABSTRACT: The introduction of high-throughput sequencing technologies has made transcriptome analyses of plant-pathogen interactions almost routine. Nevertheless, it is still challenging to obtain RNA from populations made up of two species. An RNA extraction method that worked well on free-living Caenorhabditis elegans failed when applied to isolated Globodera pallida J2 larva. Furthermore, alternative protocols that extracted RNA from free-living J2 larva produced less satisfactory results once the animals entered their hosts' roots. We have compared several extraction procedures to ascertain whether a single protocol was capable of recovering high-quality, high-molecular-weight RNA from newly hatched J2 larva as well as from larva embedded in roots of both potatoes (Solanum tuberosum L. cv. Desiree) and a very distantly related species, Solanum sisymbriifolium. Although it was possible to recover large amounts of RNA from J2 larvae using Proteinase K treatments, this protocol failed to yield high-quality nematode RNA from infected roots. By comparison, mechanical disruption procedures yielded lower amounts of RNA from infected roots, but what was recovered was of higher quality. We conclude that different extraction protocols need to be developed to sample mixed populations of organisms.

SUBMITTER: Casavant NC

PROVIDER: S-EPMC5411248 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Assessment of Globodera pallida RNA Extracted from Solanum Roots.

Casavant N Carol NC Kuhl Joseph C JC Xiao Fangming F Caplan Allan B AB Dandurand Louise-Marie LM

Journal of nematology 20170301 1

The introduction of high-throughput sequencing technologies has made transcriptome analyses of plant-pathogen interactions almost routine. Nevertheless, it is still challenging to obtain RNA from populations made up of two species. An RNA extraction method that worked well on free-living Caenorhabditis elegans failed when applied to isolated Globodera pallida J2 larva. Furthermore, alternative protocols that extracted RNA from free-living J2 larva produced less satisfactory results ...[more]

PMID: 28512373

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Project description:BACKGROUND:Sedentary endoparasitic cyst nematodes form a feeding structure in plant roots, called a syncytium. Syncytium formation involves extensive transcriptional modifications, which leads to cell modifications such as increased cytoplasmic streaming, enlarged nuclei, increased numbers of organelles, and replacement of a central vacuole by many small vacuoles. When whole root RNA is isolated and analyzed, transcript changes manifested in the infected plant cells are overshadowed by gene expression from cells of the entire root system. Use of microaspiration allows isolation of the content of nematode infected cells from a heterogeneous cell population. However, one challenge with this method is identifying the nematode infected cells under the microscope at early stages of infection. This problem was addressed by staining nematode juveniles with a fluorescent dye prior to infection so that the infected cells could be located and microaspirated. RESULTS:In the present study, we used the fluorescent vital stain PKH26 coupled with a micro-rhizosphere chamber to locate the infected nematode Globodera pallida in Solanum tuberosum root cells. This enabled microaspiration of nematode-infected root cells during the early stages of parasitism. To study the transcriptional events occurring in these cells, an RNA isolation method from microaspirated samples was optimized, and subsequently the RNA was purified using magnetic beads. With this method, we obtained an RNA quality number of 7.8. For transcriptome studies, cDNA was synthesized from the isolated RNA and assessed by successfully amplifying several pathogenesis related protein coding genes. CONCLUSION:The use of PKH26 stained nematode juveniles enabled early detection of nematode infected cells for microaspiration. To investigate transcriptional changes in low yielding RNA samples, bead-based RNA extraction procedures minimized RNA degradation and provided high quality RNA. This protocol provides a robust procedure to analyze gene expression in nematode-infected cells.

Dataset Information

Assessment of Globodera pallida RNA Extracted from Solanum Roots.

Publications

Assessment of <i>Globodera pallida</i> RNA Extracted from <i>Solanum</i> Roots.

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