Using laser capture microdissection to study gene expression in early stages of giant cells induced by root‐knot nematodes

Abstract

SUMMARY Root‐knot nematodes (Meloidogyne spp.) are economically important plant parasites that induce specific feeding cells called giant cells in host roots. Study of molecular events involved in induction and differentiation of giant cells has been limited because it is difficult to obtain pure cytoplasm specifically from the highly specialized cells. In this work, laser capture microdissection (LCM) was used to collect cytoplasmic contents from paraffin‐embedded sections of 4 day post‐inoculation giant cells in tomato roots. Total RNA was isolated from the sections, and used in RT‐PCR to investigate expression of cell cycle genes in giant cells. Two D‐type cyclin genes, LeCycD3;2 and LeCycD3;3, were expressed at higher levels in giant cells compared with other cell‐cycle‐related cyclin genes, suggesting that the induction of the G1 phase of the cell cycle may be triggered in response to stimulation by the infecting nematode. LCM provides a powerful new tool to study the molecular basis of host–pathogen interactions at the cellular or subcellular level.

Application of Laser Microdissection to plant pathogenic and symbiotic interactions

“…On the other side, precipitative fixatives, such as Farmer's fixative (ethanol/glacial acetic acid 3:1), are simple to make and provide both sample histological detail of plant tissues and enough RNA recovery for the downstream molecular analyses. RNA extracted from microdissected cells collected from plant tissues fixed with this method has been successfully used for molecular analyses (Kerk et al 2003;Nakazono et al 2003;Ramsay et al 2004;Klink et al 2005Klink et al , 2007aIthal et al 2007). A fixative that leads to a good balance between tissue preservation versus RNA recovery is a solution known as methacarn, which consists of absolute methanolchloroform-glacial acetic acid (6:3:1; Takagi et al 2004;Jiang et al 2006;Balestrini et al 2007;Guether et al 2009).…”

“…The responses in these interactions can in fact be localized in specific cell types or microbial structures. So far only a few studies have been focused on the application of LM in plantmicrobe interactions (Ramsay et al 2004;Klink et al 2005Klink et al , 2007aTang et al 2006;Balestrini et al 2007) and these studies have focused on gene expression. When LM is used, cells associated with particular infection stages can be visualized under the microscope and harvested to verify the response of the plant in different cell types and during the progression of the infection.…”

“…This allows researchers to carry out spatial and temporal gene expression profiling (Day et al 2005). These sorts of projects include investigations of the gene expression that occur during the interaction between plants and nematodes (Ramsay et al 2004;Klink et al 2005Klink et al , 2007aIthal et al 2007), fungal pathogen (Tang et al 2006) and arbuscular mycorrhizal (AM) fungi Guether et al 2009; (Table 1). Each of these studies, which so far have been performed in the field of plant/microbe interactions, employs an embedding step followed by serial sectioning of the tissue since the cells are located deep within the root tissue.…”

“…An area of interest is the development and maintenance of nematode feeding sites. LM methods that faithfully dissect out the feeding sites are important improvements to the study of this significant plant-pathogen interaction (Ramsay et al 2004;Klink et al 2005Klink et al , 2007aIthal et al 2007;Klink and Matthews 2008).…”

“…For example, RNA was extracted 72Á96 h after infection from giant cells induced in tomato roots by root-knot nematodes (Ramsay et al 2004). These cells correspond, in fact, to provascular cells that are induced to re-enter the cell cycle.…”