Deep sequencing-based comparative transcriptional profiles of Cymbidium hybridum roots in response to mycorrhizal and non-mycorrhizal beneficial fungi

Abstract

Background: The Orchidaceae is one of the largest families in the plant kingdom and orchid mycorrhizae (OM) are indispensable in the life cycle of all orchids under natural conditions. In spite of this, little is known concerning the mechanisms underlying orchid- mycorrhizal fungi interactions. Our previous work demonstrated that the non-mycorrhizal fungus Umbelopsis nana ZH3A-3 could improve the symbiotic effects of orchid mycorrhizal fungus Epulorhiza repens ML01 by co-cultivation with Cymbidium hybridum plantlets. Thus, we investigated the C. hybridum transcript profile associated with different beneficial fungi.

Results: More than 54,993,972 clean reads were obtained from un-normalized cDNA library prepared from fungal- and mock- treated Cymbidium roots at four time points using RNA-seq technology. These reads were assembled into 16,798 unique transcripts, with a mean length of 1127 bp. A total of 10,971 (65.31%) sequences were annotated based on BLASTX results and over ninety percent of which were assigned to plant origin. The digital gene expression profiles in Cymbidium root at 15 days post inoculation revealed that 1674, 845 and 1743 genes were sigificantly regulated in response to ML01, ZH3A-3 and ML01+ ZH3A-3 treatments, respectively. Twenty-six genes in different regulation patterns were validated using quantitative RT-PCR. Our analysis showed that general defense responses were co- induced by three treatments, including cell wall modification, reactive oxygen species detoxification, secondary biosynthesis and hormone balance. Genes involved in phosphate transport and root morphogenesis were also detected to be up-regulated collectively. Among the OM specifically induced transcripts, genes related to signaling, protein metabolism and processing, defense, transport and auxin response were identifed. Aside from these orchid transcripts, some putative fungal genes were also identified in symbiotic roots related to plant cell wall degradation, remodeling the fungal cell wall and nutrient transport.

Conclusion: The orchid root transcriptome will facilitate our understanding of orchid-associated biological mechanism. The comparative expression profiling revealed that the transcriptional reprogramming by OM symbiosis generally overlapped that of arbuscular mycorrhizas and ectomycorrhizas. The molecular basis of OM formation and function will improve our knowledge of plant-mycorrhzial fungi interactions, and their effects on plant and fungal growth, development and differentiation.

Figure 1

Microscopic structure of the symbionts formed by different C. hybridum - beneficial fungi interaction at 15 dpi. Mock-inoculated control (A) and inoculated with isolate ZH3A-3 (B), ML01 + ZH3A-3 (C) and ML01 (D). AH, aggregated hyphae of isolate ZH3A-3; Co, cortex cells; dH, degenerated hyphae; Ex, exodermis; Mh, fungal hyphae of isolate ML01; Pe, pelotons; Rh, root hair; Va, vascular cylinder; Ve, velamen cells. Bar, 50 μm

Figure 2

GO classification of C. hybridum unigenes.

Figure 3

COG function classification of C. hybridum unigenes.

Figure 4

Venn diagram of differentially expressed genes in the C. hybridum - beneficial fungi interaction. Mock-inoculated (CyEX20), ML01-treated (CyEX21), ZH3A-3-treated (CyEX22) or ML01 + ZH3A-3 -treated (CyEX23).

Figure 5

Twenty regulatory patterns of all the DEGs expressed in different C. hybridum - beneficial fungi interactions based on K-means method.

Figure 6

Phylogenetic tree for the amino acid sequences of plant phosphate transporters (PiTs) described as mycorrhiza-specific or -induced transporters, and two putative PiTs of C. hybridum (Unigene6086_All, Unigene6124_All) in this study were highlighted in red color. The dendrogram was generated by Mega 4.0 software using ClustlW for the alignment and the neighbor-joining method for the construction of phylogeny. The plant Pi-transporters accession numbers could be obtained in [49].