Transcriptome Analysis of Arbuscular Mycorrhizal Roots during Development of the Prepenetration Apparatus

Abstract

Information on changes in the plant transcriptome during early interaction with arbuscular mycorrhizal (AM) fungi is still limited since infections are usually not synchronized and plant markers for early stages of colonization are not yet available. A prepenetration apparatus (PPA), organized in epidermal cells during appressorium development, has been reported to be responsible for assembling a trans-cellular tunnel to accommodate the invading fungus. Here, we used PPAs as markers for cell responsiveness to fungal contact to investigate gene expression at this early stage of infection with minimal transcript dilution. PPAs were identified by confocal microscopy in transformed roots of Medicago truncatula expressing green fluorescent protein-HDEL, colonized by the AM fungus Gigaspora margarita. A PPA-targeted suppressive-subtractive cDNA library was built, the cDNAs were cloned and sequenced, and, consequently, 107 putative interaction-specific genes were identified. The expression of a subset of 15 genes, selected by reverse northern dot blot screening, and five additional genes, potentially involved in PPA formation, was analyzed by real-time reverse transcription-polymerase chain reaction and compared with an infection stage, 48 h after the onset of the PPA. Comparison of the expression profile of G. margarita-inoculated wild type and the mycorrhiza-defective dmi3-1 mutant of M. truncatula revealed that an expansin-like gene, expressed in wild-type epidermis during PPA development, can be regarded as an early host marker for successful mycorrhization. A putative Avr9/Cf-9 rapidly elicited gene, found to be up-regulated in the mutant, suggests novel regulatory roles for the DMI3 protein in the early mycorrhization process.Arbuscular mycorrhizal (AM) symbiosis represents a unique interaction between a symbiotic fungus and its host plant. The fungus, an obligate biotroph belonging to Glomeromycota (Schü ßler et al., 2001), is provided by the host plant with carbon sources required to complete its life cycle, whereas it facilitates the plant with the uptake of nutrients, such as phosphate (Smith and Read, 1997). Cellular, physiological, biochemical, and molecular research on mycorrhization is currently performed in many laboratories because positive effects of symbiotic fungi on the metabolism of their host plants are recognized as being beneficial to many ecosystems.The AM-plant associations are a result of a long coevolutionary process (Karandashov and Bucher, 2005) that required profound morphological readjustments in the plant to accommodate the fungus and to facilitate the exchange of nutrients between the two partners. In both epidermal and cortical cells, the fungus is surrounded by a membrane of host origin. The formation of this apoplastic interface causes a dramatic structural re-organization of the plant cells with all their organelles (Bonfante, 1984;Bonfante and Perotto, 1995).Development of new technological (in vivo confocal microscopy) and molecular (transformed plants expressing GUS or GFP marke...

Immunolocalization of α-Expansin Protein (NtEXPA5) in Tobacco Roots in the Presence of the Arbuscular Mycorrhizal Fungus Glomus Mosseae Nicol. & Gerd

“…It is known that there are a large number of structural and functional similarities between AM and N-fixing nodule formation. The assumption that AM-specific expansins exist was recently supported by studies showing that expansin and expansin-like genes are up-regulated during the early events of AMF infection (Weidmann et al, 2004, Siciliano et al, 2007, Genre et al, 2009, Dermatsev et al, 2010. These findings suggest that fungal contact and/or fungal enzymatic activity may stimulate plant cell wall plasticity.…”

“…Our results confirm that various cell wall protein-encoding genes appear to be differentially regulated upon mycorrhization. Among genes predicted to be up-regulated in mycorrhized roots are those coding for extensins, endo-beta-1,4-glucanases (Journet et al, 2002), cellulases (Liu et al, 2003), xyloglucan endo-transglycosylates (MaldondoMandoza and Harrison, 1998) and expansins (Weidmann et al, 2004;Siciliano et al, 2007). Endoglucanases and xyloglucan transglycosylases modify cell wall components enzymatically, whereas expansins have been identified as proteins modulating cell wall extension due to their ability to weaken noncovalent bonding between cell wall polysaccharides (Cosgrove, 1999).…”

“…These findings suggest that fungal contact and/or fungal enzymatic activity may stimulate plant cell wall plasticity. Comparison of expression profiles revealed that an expansin-like gene expressed in Gigaspora margarita-inoculated wildtype epidermis during PPA development can be regarded as an early host marker for successful mycorrhization (Siciliano et al, 2007). Expansinlike transcript (EXLB1) localization suggests local induction of EXLB1 transcription in AMF-tomato interaction (Dermatsev et al, 2010).…”

The exudate from an arbuscular mycorrhizal fungus induces nitric oxide accumulation in Medicago truncatula roots

“…This intriguing possibility, which requires further investigation, is also supported by the well-known presence of NO in root meristems and at sites of lateral root emergence (Correa-Aragunde et al 2004. Another clue to the possible role of NO in AM comes from the recent observation that variations in NO levels influence cellulose synthesis in roots (Correa-Aragunde et al 2008); upregulation of a putative cellulose synthase has been observed at very early stages of the AM interaction (Siciliano et al 2007) and related to cell wall remodeling during the process of fungal accommodation within the epidermal cell. A second open question concerns the routes of NO production.…”

Application of Laser Microdissection to plant pathogenic and symbiotic interactions

“…Recent observations have shown that the hypha enters through a single root epidermal cell, leading to the creation of a novel tunnel-like structure called the pre-penetration apparatus, PPA (Genre et al 2005). This process is accompanied by important changes in gene expression (Siciliano et al 2007). Subsequently, the cortical tissue is colonized with coils and intercellular hyphae which spread the infection.…”