# New Insights into the Symbiotic Relationship between Orchids and Fungi
## 4 Molecular Studies on Orchid Mycorrhizal Symbiosis by Next-Generation Sequencing and Proteomics  
In the past decade, the next-generation sequencing (NGS) tools were widely applied in whole genome sequencing and RNA sequencing (RNA-seq). The first whole genome sequence of the orchid _Phalaenopsis equestris_ was published in 2015 [51] and the number of orchid genomes has continuously increased [52; 53; 54; 55; 56]. The whole genome sequencing of Orchidaceae provides a gateway into orchid research, including flower morphology, sexual deception, environmental stress responses and symbiosis. The platforms of 454-pyrosequencing and Illumina were adopted to generate the transcriptomes from _Cumbidium hybridum_[57], _Serapias vomeraceae_[37; 58], _Anoectochilus roxburghii_[59], _Gastrodia elata_[60], _Dendrobium officinale_[61], _Epipactis heleborine_[62], and _Bletilla striata_[63] to identify the molecular processes involved in the symbiosis between orchids and orchid mycorrhizal fungi. Owing to the NGS technologies, the orchid-fungus symbiosis for facilitating seed germination and protocorm development is now better and comprehensively understood. Recent studies have focused on comparative transcriptomes, nutrient transfer, and regulatory module between the symbiotic and asymbiotic orchids during seed germination and protocorm development stages. Transcriptome analysis of _S. vomeracea_ by 454-pyrosequencing indicated that no significant induction of defense genes, such as pathogenesis-related genes, was found in the mycorrhizal protocorms (Table 1) [58]. Similarly, two wound response marker genes, _SvWound2_ and _SvWound3_, which code enzymes involved in the biosynthesis of many signaling compounds and the cellular processes against pathogen infection and oxidative stress, were not up-regulated in the mycorrhizal tissues. In contrast, several nodulin-like genes, such as _SvNod1_ and _SvNod9_, were apparently up-regulated. _SvNod9_ encodes a putative bidirectional sugar transporter belonging to the SWEET family which possesses activity for sugar export from cells and can be controlled by pathogens to acquire carbohydrates for C and energy [58; 64]. Their preliminary data using laser microdissection analysis indicates that _SvNod1_ could be used as a marker gene of orchid symbiosis because it only accumulates in the fungi-colonized cells. The up-regulation of nodulin genes and the lack of induction of defense genes in mycorrhizal protocorms of _S. vomeracea_ suggest a high level of orchid-fungus compatibility [58]. \begin{table}
\begin{tabular}{c c c c c} \hline
**Orchid Species/Lifestyle** & **Fungi Strain** & **Platform** & **Differentially Expressed Genes/proteins** & **Ref. ** \\ \hline  & & & **Up-regulation under all fungal** \\  & & & **treatments (ML01, ZH3A-3, ML01+ZH3A-3): phosphate transport, root morphogenesis, cell wall modification, ROS detoxification, secondary metabolism and hormone biosynthesis and signaling. ** \\ _hybridium_ (terrestrial) & **(mycorrhizal);** & Illumina HiSeq 2000 (transcriptomics) & **Up-regulation under ML01 treatment only: signaling (LysM domain receptor-like kinase 3-like), cell wall degradation and reinforcement, protein metabolism and processing, defense (chitinase and mannose-specific lectin), N and Fe transport and auxin response. \\ \hline  & & & **Up-regulation of marker genes of** \\ _Senapias nomenclature_ (terrestrial) & _Tulasnella calospora_ & 454-pyrosequencing (transcriptomics) & _SuNad1, SuNad9, SuNact3, SuNact5. No significant induction of pathogenesis and wound/stress-related genes._ \\ \hline  & & & **Up-regulation of the GA-GD1-DELLA module: 2 GA biosynthesis genes (GA20-oxidase: c99861\_g1, \_g0/765\_g1), 2** \\ _Anoectochilus_ & **Isolate Ar-34 (unpublished) & **Illumina HiSeq 4000 (transcriptomics) & GA catabolism genes (GA2-oxidase: c99070\_g1, c106997\_g1), 2 GA signaling genes (DELLA family protein SLR1: c85242\_g1, c93049\_g1)**.