# New Insights into the Symbiotic Relationship between Orchids and Fungi
## 4 Molecular Studies on Orchid Mycorrhizal Symbiosis by Next-Generation Sequencing and Proteomics
(2017) demonstrated that the albino _Epipactis heleborine_ is fully mycoheterotrophic by stable isotope analysis and the green and albino individuals of _E. heleborine_ can form mycorrhizas with same fungi which are mainly belong to the _Wilcoxina_ genus. Gene expression analyses according to RNA-seq and cDNA _de novo_ assembly revealed that the albino variants may suffer greater oxidative stress than the green ones due to the higher expression of the genes involved in antioxidant metabolism, such as glutathione peroxidase and catalase-peroxidase thioredoxin genes, in the albino individuals (Table 1). In addition, the fungal DNA level was found to be lower in the albino plants. [PERSON] et al. (1976) reported that activation of antioxidant enzymes is coincident with digestion of pelotons in the mycorrhizal protocorms of orchids [68]. Therefore, the authors speculated that the upregulation of antioxidant enzymes in the albino _E. heleborine_ may cause more frequent peloton digestion, leading to the promotion of the C flux from the fungus to the orchid [62]. As shown in other orchid mycorrhizalas, the gene ontology (GO) analysis represents an enrichment in transport genes in the albino variants and the fungal partner. In the albino _E. heleborine_, similar to the finding in the mycorrhizal protocorms of _S. vomeracea_, an upregulation of a putative _SWEET_ gene was found [58,62]. As for the fungal genes, the major facilitator superfamily (MFS) genes with transporter activities for sugars, nucleosides, N and P were significantly upregulated in the albino variants [62,69], suggesting that the fully mycheterotrophic _E. heleborine_ may also transport some nutrients to its fungal partner. The results are somewhat consistent with the finding for _T. calospora-S. vomeracea_ symbiosis [29,37]. It is worth noting that a glycerol-3-phosphate acyl transferase (GPAT) gene, _RAM2_, and a subtilisin-like protease gene required for arbuscular mycorrhizal symbiosis are up-regulated in the albino orchids. In contrast, two genes encoding gibberell 20 oxidase and cytokinin dehydrogenase precursor which can reduce arbuscular mycorrhizal colonization are down-regulated. Furthermore, up-regulation of auxin efflux carrier component genes important for arbuscular mycorrhizal colonization is also detected in the albino variants (Table 1) [62]. This indicates that a common mechanism may be involved in both orchid mycorrhizal and arbuscular mycorrhizal symbioses. In addition to isotope tracking analysis, the transcriptome-wide identification of transporter genes in orchid mycorrhizalas has now been widely applied to investigate nutrient exchange between orchids and orchid mycorrhizal fungi. As we have summarized from the studies done in _C. hybridum_ and _E. helebore_, various transporter genes involved in the uptake of N, P, sugars and nucleosides have been identified [57,62]. In the _T. calospora-S. vomeracea_ symbiosis, several genes putatively encoding transporters for ammonium and amino acids were isolated by RNA-seq using the Illumina HiSeq2000 system including _TcAMTT_1 and _TcAMT_2 in the fungus _T. calospora_ and _SvAMTT_, _SvAMTT_2, _SvAAP1_, _SvAAP2_ and _SvLHT_ in the orchid _S. vomeracea_[37]. The expression levels of these transporter genes were further confirmed by reverse transcription-quantitative PCR analysis and the ammonium transporter activities of TcAMT1 and TcAMT2 were verified in a yeast _mep_ mutant with defects in all the three endogenous Mep ammonium transporters [37; 70]. Not only transcriptome analysis but also proteomic tools have been applied to investigate orchid mycorrhizas. The studies on the orchid-fungus symbiosis mainly focused on terrestrial species. The epiphytic orchid _Oncidium sphauclatum_ was firstly investigated by the 2D-LC-MS/MS and 2D-MALDI-TOF MS analysis for the symbiotic relationships with the mycorrhizal fungi _Certobasidium_ sp. isolate and _Thanatephorus_ sp. strain RG26, respectively [65; 66].