Fast track in vitro mycorrhization of potato plantlets allow studies on gene expression dynamics

Abstract

Root colonization by arbuscular mycorrhizal (AM) fungi is a dynamic process involving major changes in plant gene expression. Here, the expression of a phosphate transporter gene (PT3) and several defense genes, already known to be involved in the various stages of AM establishment, were monitored in the mycelium donor plant (MDP) in vitro culture system associating potato plantlets with an AM fungus. This system allows fast and homogenous mycorrhization of seedlings at their early stage of development by growing the plantlets in active mycelial networks, but has never been validated for gene expression analysis. Here, QRT-PCR analyses were conducted in parallel to pre- (1 day), early (2 and 3 days), and late (6, 9, and 15 days) stages of root colonization. We observed the induction of a plant gene marker of AM root colonization (PT3) at the late stage and the induction of MAPK and PAL genes at the early and late stages of root colonization. We also demonstrated the induction of PR1 and PR2 genes at pre- and late stages and of GST1 and Lox genes at a late stage of root colonization. These results validated the MDP in vitro culture system as an optimal tool to study gene expression analysis during the AM fungi establishment. This system further opened the door to investigate gene networks associated with the plants-AM fungi symbiosis.

Transcriptional regulation of defence genes and involvement of the WRKY transcription factor in arbuscular mycorrhizal potato root colonization

“…As previously described by Gallou et al (2010), three stages of root colonization (i.e. referring to the pre-, early and late stages, respectively) were observed and further confirmed by molecular analysis ( Table 1).…”

“…referring to the pre-, early and late stages, respectively) were observed and further confirmed by molecular analysis ( Table 1). The potato pathogenesis-related 1 (PR1) gene (i. e. a marker gene used to identify the three stages of root colonization, Gallou et al 2010) was up-regulated during the pre-and late stages of root colonization as well as the phosphate transporter gene StPT3 (i.e. a marker gene induced specifically in arbuscule-containing cells, Rausch et al 2001) at the late stage of root colonization.…”

“…βeta-tubulin and Elongation Factor-1-alpha), which showed the most stable expression in potato roots during root colonization by Glomus sp. MUCL 41833, determined by the geNorm software (http:// medgen.ugent.be/∼jvdesomp/geNorm) (see Gallou et al 2010). The data were analysed statistically with the software REST 2009 (relative expression software tool; Pfaffl et al 2002; http://rerst.gene-quantification.info/).…”

“…Recently, Gallou et al (2010) investigated the defence gene expression in potato plant during the pre-, early and late stages of root colonization. They used the mycelium donor plant (MDP) in vitro culture system (Voets et al 2009) which allowed to synchronize the development of the AM fungus in the roots and demonstrated the efficiency of this system to study the changes in defence gene expression during the different stages of AM fungal root colonization in potato plantlets.…”

“…Many studies have described the colonization process, from the early signalling in noncontacting organisms to root surface contact and development of a pre-penetration apparatus (Genre et al 2005), followed by fungal penetration and proliferation within and between cells. From a simplified perspective, the AM fungal root colonization may be divided in three main stages, all of which being finely modulated by a signalling dialogue between the two partners of the symbiosis: the pre-stage referring to the stage before contact of the AM fungus with the root, (2) the early stage referring to the stage of hyphopodium formation at the root surface and root colonization before arbuscules/vesicles development and (3) the late stage referring to the stage of intense root colonization with arbuscules and vesicles formed (Gallou et al 2010).…”