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RNA-seq Transcriptional Profiling of an Arbuscular Mycorrhiza Provides Insights into
Regulated and Coordinated Gene Expression in Lotus japonicus and Rhizophagus irregularis
Yoshihiro Handa
1
, Hiroyo Nishide
2
, Naoya Takeda
1,3
, Yutaka Suzuki
4
, Masayoshi Kawaguchi
1,3
and
Katsuharu Saito
5,
*
1
Division of Symbiotic Systems, National Institute for Basic Biology, Okazaki, Aichi 444-8585, Japan
2
Data Integration and Analysis Facility, National Institute for Basic Biology, Okazaki, Aichi 444-8585,
Japan
3
School of Life Science, SOKENDAI (Graduate University for Advanced Studies), Okazaki, Aichi
444-8585, Japan
4
Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of
Tokyo, Kashiwa, Chiba 277-8561, Japan
5
Faculty of Agriculture, Shinshu University, Minamiminowa, Nagano 399-4598, Japan
*Corresponding author: E-mail, saitok@shinshu-u.ac.jp; Tel, +81-265-77-1407.
Abbreviations: AM, arbuscular mycorrhiza, AM; B&D, Broughton and Dilworth; CRP, cysteine-rich
peptide; CYP, cytochrome P450; dat, days after transplanting; DEFL, defensin-like peptide; DEG,
differentially expressed gene; dpi, days post-inoculation; FDR, false discovery rate; Gloin1,
Rhizophagus irregularis genome assembly; GLP, germin-like protein; GO, gene ontology; GPAT,
glycerol-3-phosphate acyltransferase; Lj2.5, Lotus japonicus genome assembly build 2.5; LTP, lipid
transfer protein; NCR, nodule-specific cysteine-rich; Pi, orthophosphate; RN, root nodule; RPKM,
reads per kilobase per million reads; VAMP, vesicle-associated membrane protein.
2
Footnotes: Data sets of short reads have been deposited in the DNA Data Bank of Japan Sequence
Read Archive under the accession number DRA000535, DRA001845 and DRA002581.
3
Abstract
Gene expression during arbuscular mycorrhizal development is highly orchestrated in both plants and
arbuscular mycorrhizal fungi. To elucidate the gene expression profiles of the symbiotic association, we
performed a digital gene expression analysis of Lotus japonicus and Rhizophagus irregularis using a
HiSeq 2000 next-generation sequencer with a Cufflinks assembly and de novo transcriptome assembly.
There were 3,641 genes differentially expressed during arbuscular mycorrhizal development,
approximately 80% of which were upregulated. The upregulated genes included secreted proteins,
transporters, proteins involved in lipid and amino acid metabolism, ribosomes, and histones. We also
detected many genes that were differentially expressed in small-secreted peptides and transcription
factors, which may be involved in signal transduction or transcription regulation during symbiosis.
Co-regulated genes between arbuscular mycorrhizal and root nodule symbiosis were not particularly
abundant, but transcripts encoding for membrane traffic-related proteins, transporters and iron
transport-related proteins were found to be highly co-upregulated. In transcripts of arbuscular
mycorrhizal fungi, expansion of cytochrome P450 was observed, which may contribute to various
metabolic pathways required to accommodate roots and soil. The comprehensive gene expression
data of both plants and arbuscular mycorrhizal fungi provide a powerful platform for investigating the
functional and molecular mechanisms underlying arbuscular mycorrhizal symbiosis.
Key words: Arbuscular mycorrhiza · Lotus japonicus · Rhizophagus irregularis · Root nodule ·
Symbiosis · Transcriptome
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Introduction
Plant–microbe symbioses are established through a complex and highly coordinated gene network of
both the plant and the microbe. Among the plant–microbe interactions, arbuscular mycorrhizas (AM)
are symbiotic associations between plants and symbionts of AM fungi that belong to the phylum
Glomeromycota (Schüßler et al. 2001). Their system of symbiosis is considered to be ancient on the
basis of fossil records of the early land plants and analyses of the molecular clock of AM fungi (Simon
et al. 1993; Remy et al. 1994; Taylor et al. 1995; Redecker et al. 2000). Currently AM symbiosis is
found in the majority of land plants (Brundrett 2009). AM fungal hyphae attach to the plant root surface
and traverse the epidermis through the pre-penetration apparatus, a host-derived, tunnel-like structure
formed in epidermal cells (Genre et al. 2005; Genre et al. 2008). The hyphae spread into the
intercellular space in the root cortex and further penetrate into inner cortical cells to develop a highly
branched structure termed an arbuscule, where nutrient exchange occurs between the plant and the
fungus (Bonfante and Perotto 1995; Harrison 1999).
A key step in AM symbiosis is the recognition of signal molecules between the host plant and the AM
fungus. The plant roots release strigolactones into the soil, and the AM fungus recognizes the
molecules that promote germination of spores, branching of hyphae, and activation of the metabolism
(Akiyama et al. 2005; Besserer et al. 2006; Besserer et al. 2008). On the other hand, the AM fungus
releases soluble molecules such as chitooligosaccharides (CO) (Genre et al. 2013) and
lipochitooligosaccharides (LCO), which have structural characteristics similar to those of Nod factors
produced by rhizobia (Maillet et al. 2011). The CO and LCO signaling molecules are likely to be
recognized by the plant LysM receptor-like kinases NFR1/LYK3/CERK1 and NFR5/NFP like proteins,
which trigger the signaling pathways in plant cells that are required for AM symbiosis (Op den Camp et
al. 2011; Miyata et al. 2014; Zhang et al. 2015). The signal transduction mechanism in the early phase
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of symbiosis has been analyzed extensively in model legume plants. Several genes are found to be
necessary for the formation of both AM and root nodules (RN). The signaling pathway involving these
genes was recently designated the common symbiosis signaling pathway (Kistner and Parniske 2002;
Parniske 2008) and was shown to comprise leucine-rich repeat receptor kinases (Endre et al. 2002;
Stracke et al. 2002), cation channels (Ané et al. 2004; Imaizumi-Anraku et al. 2005), nucleoporins
(Kanamori et al. 2006; Saito et al. 2007; Groth et al. 2010), calcium and calmodulin-dependent kinases
(Lévy et al. 2004; Mitra et al. 2004; Tirichine et al. 2006), and CYCLOPS/IPD3 (Messinese et al. 2007;
Yano et al. 2008). These findings provide strong support for the hypothesis that a part of the early
signaling pathway in RN symbiosis is recruited from the more ancient genetic system of AM symbiosis
(Parniske 2008). Recently, the GRAS transcription factors NSP1 and NSP2, which were initially
identified as being required for RN symbiosis, have been shown to play roles in the common symbiosis
pathway (Liu et al. 2011; Maillet et al. 2011; Lauressergues et al. 2012; Delaux et al. 2013; Takeda et al.
2013). In Medicago truncatula, the novel GRAS transcription factor RAM1 has been shown to be
indispensable for Myc factor signaling but not for Nod factor signaling (Gobbato et al. 2012). The RAM1
gene regulates the transcript level of RAM2, which codes for a glycerol-3-phosphate acyltransferase
that enhances cutin production to promote fungal hyphopodia formation on the root surface (Wang et al.
2012).
AM are not accompanied by clear de novo organ formation, unlike RN, which exhibit nodule
organogenesis during symbiosis. However, specialized hyphal structures known as arbuscules are
formed within the cortical cells of roots. Arbuscule development proceeds with at least five distinct
stages: formation of a pre-penetration apparatus, fungal cell entry, formation of a birdsfoot-like structure
(trunk hyphae), maturation of arbuscules, and arbuscule collapse (Gutjahr and Parniske 2013). The
processes preceding the formation of a birdsfoot-like structure are known to require the common
