Application of RNA Interference Technology to Acroporid Juvenile Corals

Abstract

Numerous genes involved in calcification, algal endosymbiosis, and the stress response have been identified in corals by large-scale gene expression analysis, but functional analysis of those genes is lacking. There are few experimental examples of gene expression manipulation in corals, such as gene knockdown by RNA interference (RNAi). The purpose of this study is to establish an RNAi method for coral juveniles. As a first trial, the genes encoding green fluorescent protein (GFP, an endogenous fluorophore expressed by corals) and thioredoxin (TRX, a stress response gene) were selected for knockdown. Synthesized double-stranded RNAs (dsRNAs) corresponding to GFP and TRX were transformed into planula larvae by lipofection method to attempt RNAi. Real-time PCR analysis to verify knockdown showed that GFP and TRX expression levels tended to decrease with each dsRNA treatment (not significant). In addition, stress exposure experiments following RNAi treatment revealed that planulae with TRX knockdown exhibited increased mortality at elevated temperatures. In GFP-knockdown corals, decreased GFP fluorescence was observed. However, the effect of GFP-knockdown was confirmed only in the coral at the initial stages of larval metamorphosis into polyps, but not in planulae and 1 month-old budding polyps. This study showed that lipofection RNAi can be applied to coral planulae and polyps after settlement, and that this method provides a useful tool to modify expression of genes involved in stress tolerance and fluorescence emission of the corals.

Thermal preconditioning in a reef-building coral alleviates oxidative damage through a BI-1 mediated antioxidant response

“…Existing protocols in cnidarians were not technically feasible to adult reef-building corals, relying on microinjection of juveniles or electroporation of anemones as the delivery technique [47][48][49]78 . Other studies using liposomal compounds transferred long dsRNA into anemones or coral larva, where the animal's RNAi machinery was allowed to process it into siRNA 48,79,80 . The skeletal structure of adult reef-building corals prevents these approaches from being effective.…”

“…Gene knockdown assays have shown limited efficiency in Cnidarians and have only been used in a few studies, representing a major obstacle for functional studies of these non-model organisms. Existing protocols in cnidarians were not technically feasible to adult reef-building corals, relying on microinjection of juveniles or electroporation of anemones as the delivery technique [47][48][49]78 . Other studies using liposomal compounds transferred long dsRNA into anemones or coral larva, where the animal's RNAi machinery was allowed to process it into siRNA 48,79,80 .…”

“…In this experiment, we used processed siRNA designed to specifically target the gene of interest and delivered it to the coral tissue using a lipidic vesicle INTERFERin (Polyplus) which proved to be the most efficient and the only repeatable method of those tested. We first targeted the family of green fluorescent proteins, a standard strategy to validate gene-knockdown in nonmodel organisms owing to its ease of monitoring success in real time [47][48][49] . We were able to achieve a significant downregulation of GFP proteins in the majority of test corals and we hypothesize that coral mucus likely interferes with the siRNA transfection procedure in some individuals, meaning that individuals undergoing siRNA-involved experiments must be first tested for the successful knockdown before further functional analyses take place.…”

“…We developed and validated a siRNA-mediated gene knockdown method in living adult corals by targeting proteins of the GFP family, using a common approach for developing similar technologies in non-model organisms [47][48][49] .…”

Thermal preconditioning in a reef-building coral alleviates oxidative damage through a BI-1-mediated antioxidant response

“…We developed and validated a siRNA-mediated gene knockdown method in living adult corals by targeting proteins of the GFP family, using a common approach for developing similar technologies in non-model organisms (Cleves et al, 2018;Karabulut et al, 2019;Yuyama et al, 2021). We identified 12 predicted Pocillopora GFP-like fluorescent chromoproteins in the NCBI mRNA database and used BLOCK-iT ™ RNAi Designer (Thermo Fisher Scientific) to design siRNA molecules with the best predicted success rate to inhibit expression of individual predicted GFP-like genes.…”