Immunolocalization and Changes of Hydroxyproline-Rich Glycoproteins During Symbiotic Germination of Dendrobium officinale

Abstract

Hydroxyproline-rich glycoproteins (HRGPs) are abundant cell wall components involved in mycorrhizal symbiosis, but little is known about their function in orchid mycorrhizal association. To gain further insight into the role of HRGPs in orchid symbiosis, the location and function of HRGPs were investigated during symbiotic germination of Dendrobium officinale. The presence of JIM11 epitope in developing protocorms was determined using immunodot blots and immunohistochemical staining procedures. Real-time PCR was also employed to verify the expression patterns of genes coding for extensin-like genes selected from the transcriptomic database. The importance of HRGPs in symbiotic germination was further investigated using 3,4-dehydro-L-proline (3,4-DHP), an inhibitor of HRGP biosynthesis. In symbiotic cultures, immunodot blots of JIM11 signals were moderate in mature seeds, and the signals became stronger in swollen embryos. After germination, signal intensities decreased in developing protocorms. In contrast, in asymbiotic cultures, JIM11 signals were much lower as compared with those stages in symbiotic cultures. Immunofluorescence staining enabled the visualization of JIM11 epitope in mature embryo and protocorm cells. Positive signals were initially localized in the larger cells near the basal (suspensor) end of uninfected embryos, marking the future colonization site of fungal hyphae. After 1 week of inoculation, the basal end of embryos had been colonized, and a strong signal was detected mostly at the mid- and basal regions of the enlarging protocorm. As protocorm development progressed, the signal was concentrated in the colonized cells at the basal end. In colonized cells, signals were present in the walls and intracellularly associated with hyphae and the pelotons. The precise localization of JIM11 epitope is further examined by immunogold labeling. In the colonized cells, gold particles were found mainly in the cell wall and the interfacial matrix near the fungal cell wall. Four extensin-like genes were verified to be highly up-regulated in symbiotically germinated protocorms as compared to asymbiotically germinated ones. The 3,4-DHP treatment inhibited the accumulation of HRGPs and symbiotic seed germination. In these protocorms, fungal hyphae could be found throughout the protocorms. Our results indicate that HRGPs play an important role in symbiotic germination. They can serve as markers for fungal colonization, establishing a symbiotic compartment and constraining fungal colonization inside the basal cells of protocorms.

Role of Protein Glycosylation in Host-Pathogen Interaction

“…In plants, hydroxyproline-rich glycoproteins (HRGPs) which are characterized by a rigid polyproline type II (PPII) conformation and extensive O-glycosylation of 4 (R)-hydroxy-l-proline residues [87], can physically inhibit pathogen penetration [88]. HRGPs play a leading role in plant defense, e.g., HRGPs were induced in incompatible plant-pathogen interactions [18,89] and rapidly deposited in the cell wall after pathogen infection [90,91]. Such a defense ability is a result of the intra-and inter-molecular cross-linking in plant cell walls after HRGPs become insolubilized [88,92].…”

Effect of different mycobionts on symbiotic germination and seedling growth of Dendrobium officinale, an important medicinal orchid

“…In our laboratory, we have developed efficient methods for fungal isolation from D. officinale protocorms and roots (Li et al 2018a). In this study, Sebacina strains S2 and S3 were isolated from mycorrhizal protocorms by the technique of in situ seed baiting in the natural habitat in Yunnan Province of Southwestern China, and Tulasnella strains S4, S5, S6 and S7 were isolated from roots of mature plants in the same area.…”

An Overview of Orchid Protocorm-Like Bodies: Mass Propagation, Biotechnology, Molecular Aspects, and Breeding

“…Asymbiotic germination involves the in vitro inoculation and germination of orchid seeds with the aid of a sucrose-containing culture medium [18,19], under conditions free of microorganisms; including those symbionts that assist in germination, especially under natural conditions, a technique known as symbiotic germination, which can be done in vitro [19,20], ex vitro, or in situ and which, unlike asymbiotic, considers the use of symbiotic microorganisms to assist in the germination and early development of newly germinated seedlings, and lacking nutritional reserves to support early seedling development [20,21].…”