Advance Publication J. Gen. Appl. Microbiol. doi 10.2323/jgam.2020.02.003 ©2020 Applied Microbiology, Molecular and Cellular Biosciences Research Foundation 1 Short Communication 2 Establishment of a firefly luciferase reporter assay system in the unicellular red alga 3 Cyanidioschyzon merolae 4 (Received January 25, 2020; Accepted February 11, 2020; J-STAGE Advance publication date: September 16, 2020) 5 Running Head: Luciferase reporter system in C. merolae 6 Baifeng Zhou1,2, Sota Takahashi1,3, Tokiaki Takemura1,2, Kan Tanaka1,*, Sousuke Imamura1,* 7 1Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute 8 of Technology, Nagatsuta, Midori-ku, Yokohama, Japan 9 2School of Life Science and Technology, Tokyo Institute of Technology, Nagatsuta, Midori- 10 ku, Yokohama, Japan 11 3Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of 12 Technology, Nagatsuta, Midori-ku, Yokohama, Japan 13 14 Key Words: Cyanidioschyzon merolae; luciferase; nitrogen; reporter assay; transcription 15 factor 16 *To whom correspondence should be addressed. E-mail: [email protected] (K. 17 Tanaka); [email protected] (S. Imamura) 18 The firefly luciferase (Luc) reporter assay is a powerful tool used to analyze promoter 19 activities in living cells. In this report, we established a firefly Luc reporter assay system in 20 the unicellular model red alga Cyanidioschyzon merolae. A nitrite reductase (NIR) promoter- 21 Luc fusion gene was integrated into the URA5.3 genomic region to construct the C. merolae 22 NIR-Luc strain. Luc activities in the NIR-Luc strain were increased, correlating with the 23 accumulation of endogenous NIR transcripts in response to nitrogen depletion. Luc activity 24 was also significantly increased by the overexpression of the MYB1 gene, which encodes a 1 25 transcription factor responsible for NIR promoter activation. Thus, our results demonstrate 26 the utility of the Luc reporter system in C. merolae. 27 Abbreviations: GFP, green fluorescent protein; GUS, β-glucuronidase; kbp, kilobase pair; 28 kDa, kilodalton; Luc, firefly luciferase; ORF, open reading frame; PCR, polymerase chain 29 reaction; PEG, polyethylene glycol; SD, standard deviation; TFs, transcription factors; −/+ N, 30 nitrogen depleted/replete condition 31 Reporter genes are marker genes used to study the regulatory elements of the genes of 32 interest, where the expression of reporter genes can be easily detected and measured. Several 33 reporter genes, such as firefly luciferase (Luc), β-glucuronidase (GUS), and green fluorescent 34 protein (GFP), are widely used in plants (Jefferson, 1987; Koncz et al., 1990; Millar et al., 35 1992; Naylor, 1999). The GUS system has the advantage of no background noise in most 36 plant species and is easily quantifiable using a substrate. Therefore, the GUS reporter system 37 has been used for the quantitative analysis of promoter activity (Quaedvlieg et al., 1998). 38 GUS activity can also be used for histochemical localization of GUS-tagged target proteins 39 (Guivarc’H et al., 1996). The GFP system is used for analyzing the subcellular localization of 40 target proteins since fluorescence from GFP can be directly detected in living cells (Haseloff 41 and Amos, 1995; Moriguchi et al., 2005). On the other hand, the Luc system is used to study 42 gene transcription regulation because of its high sensitivity, time resolution, and accurate 43 quantitative characteristics (Velten et al., 2008). 44 Cyanidioschyzon merolae is a unicellular red alga with a completely sequenced and 45 annotated genome (Matsuzaki et al., 2004; Nozaki et al., 2007). Because of the small number 46 of transcription factors (TFs; less than 100 TFs are estimated for the 16.5 Mbp of the nuclear 47 genome) as well as several genetic and molecular biology related tools, C. merolae has been 48 considered as an ideal photosynthetic model eukaryote for studying fundamental 49 transcriptional networks (Imamura et al., 2009, 2010; Matsuzaki et al., 2004). However, to 50 date, only the GFP reporter system has been established in C. merolae for monitoring the 51 expression of nitrogen (N) assimilation genes (Fujiwara et al., 2015). In the case of the 52 unicellular model green alga, Chlamydomonas reinhardtii, the luciferase system has been 53 established and used for monitoring expression of nuclear genes (Fuhrmann et al., 2004; 54 Ruecker et al., 2008; Shao and Bock, 2008). In this study, we constructed the Luc reporter 55 system to analyze transcriptional regulation in C. merolae. 2 56 To examine the utility of the Luc reporter system in C. merolae, we used MYB1, an 57 R2R3-type MYB TF, and the nitrite reductase (NIR) promoter region, since MYB1 58 positively regulates the expression of N assimilation genes, including NIR, under the N 59 depleted (−N) condition (Imamura et al., 2009). To examine MYB1-dependent NIR 60 transcription through Luc activity, we first constructed the NIR-Luc strain; in this strain, the 61 NIR promoter-driven Luc gene was used to replace the UMP synthase gene URA5.3 in the 62 genome of C. merolae (Fig. 1A). The NIR-Luc strain exhibits uracil auxotrophic phenotype 63 and, therefore, can be used as a host strain for expressing exogenous TFs, including MYB1. 64 The NIR promoter fragment (−1,200 to +61; +1 represents the translation start site) was 65 generated by a polymerase chain reaction (PCR) using the primers NIR_pKTL_F1 (5′- 66 CGCGAAGATCTCATATGGATTTACCGTCGTTCAACTCAAA-3′) and NIR_pKTL1_R1 67 (5′-ACCGGAATGCCAAGCTAGATTGGTGGGTGCCAAACCTCTGC-3′), C. merolae 68 genomic DNA (template), and KOD-Plus Neo DNA polymerase (Toyobo, Tokyo, Japan). 69 The PCR product was then cloned into EcoRV-digested pKTL1 vector (Imamura et al., 2017), 70 which contains the open reading frame (ORF) of Luc, URA5.3 upstream region (URA5.3 up), 71 and URA5.3 downstream region (URA5.3 down), using Gibson assembly (Gibson et al., 72 2009) to create the pKTL1-NIR construct. Finally, a fragment containing URA5.3 up, NIR 73 promoter, Luc, URA5.3 down, in this order from 5′ to 3′ (hereafter this fragment is referred to 74 as NIR-Luc), was amplified by PCR with the primers URA_F1 and URA_R1 (Imamura et al., 75 2017) using pKTL1-NIR DNA as the template. The purified 7.1 kbp fragment was used for 76 polyethylene glycol (PEG)-mediated transformation of C. merolae wild-type (WT) (Taki et 77 al., 2015). To confirm the replacement of the URA5.3 gene by the NIR-Luc fragment, 78 transformants were further screened by PCR using two primer sets. First, primers F1 and R1 79 (Imamura et al., 2017), which anneal outside of the integration region, were used to obtain a 80 7.2 kbp fragment from the positive stain and a 6.1 kbp fragment from the WT strain, as 81 predicted (Fig. 1B, left). Then, primers F2 (5′-ACGGAAAAAGAGATCGTGGATTAC-3′) 82 and R1 were used to amplify a 2.3 kbp fragment from the positive stain, where no 83 amplification was obtained from the WT strain, as predicted (Fig. 1B, right). Taken together, 84 these data indicated that the NIR-Luc fragment was integrated into the URA5.3 locus in the 85 positive strain; this strain is hereafter referred to as the NIR-Luc strain. 86 To examine NIR promoter activity, we performed a Luc reporter assay using proteins 87 extracted from the NIR-Luc stain and T1 strain (lacking the URA5.3 gene) before and after 88 exposure to -N conditions. The T1 strain, which carries a complete deletion of the URA5.3 89 gene was used as a control (Taki et al., 2015). Protein samples were prepared as previously 3 90 described (Imamura et al., 2008). The total protein (120 µg) in 100 µl of lysis buffer 91 (Imamura et al., 2008) was incubated with 100 µl of luciferin-containing ONE-Glo™ 92 Reagent (Promega, Tokyo, Japan) for 5 min in the dark. Luc activity was then measured by 93 Lumat LB 9507 (EG & G Berthold) and estimated as relative fluorescence units (RFU) 94 divided by the amount of total input protein (RFU/Protein). The results showed that Luc 95 activity was significantly increased at 2 h after exposure to -N and peaked at 4 h in the NIR- 96 Luc strain; however, the T1 strain showed no increase of the luminescence (Fig. 2A). To 97 compare the Luc activity with mRNA levels of Luc and endogenous NIR in the NIR-Luc 98 strain, we performed quantitative reverse transcription PCR (qRT-PCR) using mRNA 99 extracted from cells under the same conditions as those described in Fig. 2A with sequence- 100 specific primers (for NIR, NIR_F1: 5′-ATCCGTTGACCGAGGTACTG-3′ and NIR_R1: 5′- 101 TGCAGTCATCGGAGATGAAG-3′; for Luc, Luc_F1: 5′- 102 GGTTTTGGAATGTTTACTACACTCG-3′ and Luc_R1: 5′- 103 CTCAGAAACAGCTCTTCTTCAAATC-3′). The transcript levels of both genes were 104 increased at 2 h after exposure to -N, and their patterns corresponded well with the Luc 105 activity (Fig. 2A and 2B). These results indicate that the NIR-Luc strain can be used to 106 monitor NIR promoter activity. Furthermore, these results also demonstrate that the NIR 107 promoter region used in this study contains a cis-acting element for the -N responsive 108 transcription. 109 Next, to evaluate whether the Luc reporter assay system can be used to identify trans- 110 acting factor(s), we constructed a FLAG-tagged MYB1 overexpression (MYB1-OE) strain 111 using NIR-Luc as the host strain. Previously, we showed that MYB1 specifically binds to the 112 promoter of N assimilation genes, including NIR, under the -N condition as a positive 113 transcriptional regulator (Imamura et al., 2009). Therefore, we hypothesized that 114 overexpression of MYB1 would increase Luc activity in the MYB1-OE strain compared with 115 that in the control strain (MYB1-C, see below) even under the +N condition.