Differential display analysis of gene expression in the cytoplasm of giant cells induced in tomato roots by Meloidogyne javanica

Abstract

SUMMARY Giant cells induced by root-knot nematodes are highly specialized cells which function as transfer cells and provide nutrients to support the growth and reproduction of the nematode. Changes in the overall pattern of gene expression in giant cells occur during the formation and maintenance of the nematode feeding cells. Differential display analysis has been carried out to detect changes in gene expression in giant cells induced in tomato roots by Meloidogyne javanica, using mRNA isolated directly from mature giant cell cytoplasm, compared to non-infected root tissue. Eighty-one differential displayed bands were generated, and of these, 73 were up-regulated and 8 were down-regulated. Twenty-seven sequences were obtained by direct sequencing of the bands, and 16 fragments were further analysed by real-time quantitative RT-PCR. The most highly up-regulated transcript increased 56-fold in giant cells, and the greatest down-regulation was 11-fold. A time course of expression of the highest and lowest expressed transcripts was also undertaken by quantitative RT-PCR using giant cell enriched tissue. These showed similar changes in expression, but values were dramatically reduced. This result shows the importance of analysing giant cell cytoplasm directly, rather than starting with giant cell enriched tissue, to obtain accurate information on changes in gene expression in nematode feeding cells. Sequenced transcripts showed significant homology to mitogen-activated protein kinase, S-adenosylmethionine decarboxylase, cysteine synthase, cytochrome c reductase subunit, and ribosomal proteins. The expression analysed reflects the high metabolic rate in mature giant cells rather than processes of giant cell induction.

Deciphering genetic diversity and inheritance of tomato fruit weight and composition through a systems biology approach

“…In addition, the interconnections suggested a possible role as a regulator for cysteine synthase whose amount at CE is correlated with several enzymes, glucose, and fruit diameter at OR. This enzyme has been suggested to play a key role in highly metabolically active cells (Wang et al , 2003). Those facts should be taken into account when trying to modify gene expression or protein contents in order to alter metabolite contents.…”

Transcriptional analysis through RNA sequencing of giant cells induced by Meloidogyne graminicola in rice roots

“…Bar-Or et al , 2005; Jammes et al , 2005,). Nevertheless, though technically challenging, giant cell contents can be isolated using microaspiration (Wang et al , 2003) or laser-capture microdissection (LCM; Fosu-Nyarko et al , 2009; Barcala et al , 2010; Portillo et al , 2009, 2013). The research of Barcala et al (2010) and Portillo et al (2013) demonstrated the molecular distinctiveness between the giant cells and the surrounding gall tissue.…”

Early transcriptomic events in microdissected Arabidopsis nematode-induced giant cells

“…Galls are voluminous and complex multicellular structures containing only five to eight GCs. The contribution of GCs to the gall is relatively small, particularly in the early stages of development (Wang et al, 2003). Microcapillar-aspired and hand-dissected tomato GCs contain GC-enriched material (Wilson et al, 1994;Wang et al, 2003 (Ithal et al, 2007b;Klink et al, 2007) and microaspiration in Arabidopsis (Szakasits et al, 2009).…”

“…Microarray technologies have been applied mainly to whole galls containing GCs and the surrounding tissue (Bar-Or et al, 2005;Hammes et al, 2005;Jammes et al, 2005;Fuller et al, 2007). Wang et al (2003) attempted to overcome this limitation through microaspiration of GC contents, but this technique is limited to large GCs at late infection stages, when most of the gene re-programming events leading to cell differentiation are likely to have already occurred. The first microarray analysis from isolated GCs by laser capture microdissection (LCM) has been recently performed in tomato (Portillo et al, 2009), but not in Arabidopsis, which contains smaller root cells.…”