Establishment of an efficient Agrobacterium ‐mediated transformation system for the economically important macroalga Pyropia yezoensis (Rhodophyta)

Macroalgae possess significant ecological benefits and economic value (Ortega et al. , 2019). Pyropia yezoensis is one of the most economically valuable red macroalgae and is extensively cultivated in China, Korea, and Japan as the primary source of the nutrient-rich and widely consumed seafood product ‘nori’ (Uji et al. , 2014). As an intertidal seaweed species, P. yezoensis plays a fundamental role in coastal ecosystems. Besides its economic value, P. yezoensis has attracted considerable scientific interest as a model organism for studying marine red algae physiology and genetics, owing to its distinct evolutionary lineage, complete life cycle under laboratory conditions, diverse reproductive systems, and high-quality genomic resources (Wang et al. , 2020; Zheng et al. , 2021). Recent progress in genetic engineering has further enhanced its utility (Izumi et al. , 2025; Wang et al. , 2025), yet establishing an efficient and stable nuclear transformation system in P. yezoensis remains a major technical challenge. Previous studies have employed molecular strategies to improve transformation efficiency, such as reporter gene screening, promoter optimization, and codon optimization, along with various physical delivery methods, including particle bombardment and electroporation (Mizukami et al. , 2004; Wang et al. , 2010; Hirata et al. , 2014; Uji et al. , 2014; Kong et al. , 2017; Cao et al. , 2022; Zheng et al. , 2022; Wang et al. , 2024; Izumi et al. , 2025). However, these approaches have generally yielded low transformation efficiencies, impeding stable transgenesis and functional genomic analysis. For P. yezoensis, a mariculture seaweed, functional analysis and genetic modification of economically important genes are vital for targeted breeding. Agrobacterium tumefaciens-mediated transformation is widely used in higher plants due to its efficiency and simplicity (Gutierrez-Valdes et al. , 2020; Zhang et al. , 2021). Previous studies have explored its potential in red seaweeds, including P. yezoensis, but these investigations were largely preliminary (Cheney et al. , 2001; Wang et al. , 2010), with outcomes limited to transient expression or lacking sufficient evidence of genetic stability (Alimuddin et al. , 2014; Triana et al. , 2016; Ramessur et al. , 2018). Major obstacles include the absence of phenolic inducers, incompatibility with saline conditions, and, crucially for P. yezoensis, a dense surface polysaccharide layer that severely impedes bacterial attachment and T-DNA delivery (Stachel et al. , 1985). Nonetheless, biological features of P. yezoensis, such as its haploid thallus and wound-induced archeospore release, enable rapid generation of genetically uniform transgenic materials (Chen et al. , 2020). In this study, we used vacuum infiltration to overcome P. yezoensis physical barriers, systematically optimized key transformation parameters, and established a high-throughput screening strategy. Following hygromycin selection, the final positive rate of stable transformation reached nearly 100%. This represents the first complete, efficient, stable, low-cost, and high-throughput Agrobacterium-mediated transformation system for P. yezoensis. We successfully applied this system to functionally characterize the nitrate reductase (NR) gene PyNR, and we generated transgenic lines exhibiting enhanced growth under nitrogen (N) -limited conditions. Additionally, an effective RNAi system was established, enabling targeted silencing of endogenous genes. This highly efficient and cost-effective transformation system provides a powerful toolbox for investigating fundamental biological processes in P. yezoensis and offers a valuable methodological framework for developing similar transformation systems in other red algae. Previous studies identified codon-optimized PyGUS and the hygromycin resistance gene (Hyg) as reliable reporter and selection markers in P. yezoensis (Fukuda et al. , 2008; Uji et al. , 2014). Building on this foundation, we constructed an expression cassette for Agrobacterium-mediated infiltration, in which both PyGUS (reporter) and Hyg (selection marker) were driven by the endogenous PyACTIN4 (ACT4) promoter, previously shown to effectively initiate gene expression in this system (Cao et al. , 2022). Initial Agro-infiltration attempts using intact or wounded thalli yielded no detectable transformants. Consequently, we employed thalli chopped into small fragments and precultured for 0–15 d as the starting material. Among the five Agrobacteria strains tested, four achieved successful transformation and PyGUS expression in thallus fragments cultured for 10–13 d post chopping, as confirmed by a β-glucuronidase (GUS) activity assay. Strain LBA4404 exhibited the highest transformation efficiency. Subsequently, several key parameters were optimized, including Agrobacteria concentration, vacuum infiltration durations, and coculture periods. The optimal infection conditions were determined as follows: chopped thallus fragments cultured for 10–13 d were used as the starting material, infiltrated with the LBA4404 strain, resuspended to OD600 = 0. 5 under vacuum for 30 min, and followed by a 2-d coculture period (Fig. 1a, b). Under these optimized conditions, strong GUS activity was detected in multiple transformed materials, including thallus fragments, released archeospores, and developing thallus seedlings. Archeospores exhibited the highest proportion of positive transformants (88%), followed by seedlings (58%) and fragments (29%). Furthermore, when these optimized conditions were applied to evaluate the exogenous 2 × CaMV35S promoter, no GUS expression was detected (Supporting Information Fig. S1). Although hygromycin selection has been applied previously in particle bombardment transformation in P. yezoensis, the resulting positive rates obtained after antibiotic screening were low (Cao et al. , 2022; Wang et al. , 2024). To improve the efficiency of post-transformation selection, transformed materials were initially treated with 2 mg ml−1 hygromycin. Under these conditions, wild-type (WT) thallus fragments died within 8 d (Fig. S2a), whereas some Agro-infiltrated fragments retained viable cells, while a previously generated Hyg-expressing transgenic line (Cao et al. , 2022), used as a positive control, exhibited nearly complete cell survival (Figs 1c, S2c). After a one-wk recovery, the surviving fragments were subjected to secondary screening with 4 mg ml−1 hygromycin for 12 d. During the subsequent four-wk recovery, viable cells released archeospores that subsequently developed into thallus seedlings. Nearly 100% of these archeospore-derived seedlings exhibited GUS activity (Figs 1c, S2b, c). The two-stage screening strategy thus ensured that only stably transformed cells could survive, achieving a near 100% positive rate among the surviving cells and significantly improving screening efficiency. PCR amplification of the GUS sequence in the asexually derived offspring confirmed the stable maintenance of the GUS-expressing cassette in thallus cells (Fig. S3a). Western blot analysis using an anti-GUS antibody further confirmed its active expression (Fig. S3b). PyGUS transcription and robust GUS activity were observed throughout the life cycle, including both sexual and asexual reproduction (Figs 1d, S4), confirming the successful generation of stable GUS transgenic lines in P. yezoensis. Furthermore, genome resequencing revealed multicopy T-DNA integration in the nuclear genome of these transgenic lines. Three randomly selected lines, Py-#1, Py-#2, and Py-#3, contained three, two, and two integration sites, respectively (Fig. 1e) ; each integration site was validated by site-specific PCR (Tables S1, S2; Fig. S5). Notably, genetic mosaicism is inherently avoided in P. yezoensis due to its haploid thallus and asexual reproductive mode. Following antibiotic selection, each surviving positive cell, originating from an independent transformation event, developed into an asexual archeospore and subsequently into a genetically uniform thallus. In summary, we have established an efficient Agrobacterium-mediated genetic transformation method for P. yezoensis, achieving stable expression of exogenous genes. Compared with particle bombardment, Agro-infiltration offers distinct advantages, including reduced cost, simpler operation, shorter culture cycles, a significantly higher initial transformation efficiency, postselection positive rate, and stable transformation efficiency. In this study, c. 9 × 105 cells, isolated from a single thallus measuring 4 cm by 0. 5 cm, were used with the Agro-infiltration method, achieving an initial transformation efficiency between 30% and 40%. Following a stringent two-stage hygromycin selection (2 mg ml−1 for 8 d; 4 mg ml−1 for 12 d), we recovered c. 200 stable transformants, representing a stable transformation frequency of 0. 02%, and a postselection positivity rate of 100% (Table S3; Fig. 1c). Conversely, the biolistic transformation method previously reported by Cao et al. (2022) in our laboratory employed 4 × 106 cells (derived from one 3 cm × 3 cm thallus) and achieved an initial transformation efficiency of only 0. 8%, yielding c. 32 000 positive cells. After one round of hygromycin selection, only two stable transformants were recovered, resulting in a stable transformation efficiency of 5 × 10−5% and a postselection positive rate of 1. 3%. When normalized to an equivalent initial biomass of 4 × 106 cells, the Agrobacterium-mediated infiltration method used in this study would yield c. 900 stable transformants, representing a c. 450-fold increase compared with the two transformants obtained via the biolistic method (Table S3). Along with greater efficiency, the Agro-infiltration method removes the need for expensive, specialized equipment. The operational cost per transformation is approximately one-fifth that of particle bombardment, which itself requires an initial capital investment of c. 50 000 to 70 000 USD. Consequently, within an equivalent budget and timeframe, the Agro-infiltration method enables the generation of a substantially larger number of transformants, enabling high-throughput applications such as insertional mutant library construction. To broaden the genetic toolkit for P. yezoensis, we constructed an RNAi vector to stably silence the endogenous transcriptional adapter gene ADA2b in the WT strain. The RNAi-PyADA2b construct contained a sense fragment of the PyADA2b target sequence (1689 bp) and a corresponding antisense fragment linked by a spacer sequence, both driven by the ACT4 promoter (Fig. 1f). We introduced the RNAi-PyADA2b vector into WT thallus fragments via Agro-infiltration and obtained three RNAi-PyADA2b lines after antibiotic screening. Transformation with an empty vector was used as a control. Transcriptional expression of PyADA2b in these RNAi-PyADA2b lines was significantly lower than that in the EV line (Fig. 1g), demonstrating that the RNAi-PyADA2b vector effectively suppressed PyADA2b gene expression at the transcriptional level. To further verify the functional impact of RNAi-mediated gene silencing at the protein level, we transformed an RNAi-PyGUS vector into the stable OE-PyGUS line to silence PyGUS. The transformed materials were subjected directly to GUS staining at 2 d post cocultivation, without hygromycin selection, as the recipient OE-PyGUS line already carries the HygR marker. All of the three transformed replicates showed lighter blue staining than the OE-PyGUS controls, indicating reduced PyGUS activity resulting from transient siRNA expression (Fig. 1h). Transcriptional expression of PyGUS was also significantly reduced (Fig. S6). These results confirm the effectiveness of the RNAi-based gene silencing system and establish a valuable tool for functional analysis of endogenous genes in P. yezoensis. Nitrate represents the primary nitrogen source for most photosynthetic plants and algae, and enhancing nitrate uptake can promote growth and improve productivity and yields in both agriculture and aquaculture (Zayed et al. , 2023). In Pyropia, thallus discoloration (Fig. S7), often triggered by nitrogen deficiency due to reduced riverine input, phytoplankton blooms, and low temperatures, can result in substantial economic losses in aquaculture. NR, which catalyzes nitrate reduction into nitrite, plays a central role in nitrogen assimilation and is typically induced under N-limited conditions (Paine et al. , 2021). We identified the NR gene PyNR (py08787) in the P. yezoensis genome and generated homozygous OE-PyNR lines expressing a PyNR-GUS fusion via Agrobacterium-mediated transformation. Stable integration of the OE-PyNR: GUS cassette was confirmed by genomic PCR (Fig. 2a), and successful PyNR overexpression was verified by transcriptional quantification and GUS activity assays (Fig. 2b, c). Furthermore, PyNR expression remained stable throughout both the sexual and asexual stages of the P. yezoensis life cycle (Fig. S8). To investigate the impact of PyNR overexpression on nitrogen metabolism in P. yezoensis thalli, nitrate and nitrite levels were measured in both WT and OE-PyNR thalli, as well as in medium nitrate-limited conditions. The nitrate and nitrite contents in OE-PyNR thalli were significantly higher than WT thalli, and the magnitude of this difference increased over the course of the treatment period (Fig. 2d, e). Correspondingly, the nitrate concentration in the culture medium of OE-PyNR was consistently and significantly lower than that in the WT medium throughout the experiment (Fig. 2f). Moreover, OE-PyNR thalli exhibited improved growth under low nitrate (L-N) conditions, as evidenced by a larger and more rapidly expanding thallus surface area compared with WT thalli of the same developmental stage (Fig. 2g). Under N-replete (+N) conditions, the maximum quantum yield of PSII (FV/FM) and the maximum quantum yield (QYₘax) showed no significant difference between OE-PyNR and WT. However, under Nn limitation, both parameters decreased in WT but remained stable in OE-PyNR (Fig. 2h, i). Additionally, OE-PyNR thalli exhibited lower nonphotochemical quenching values than WT thalli, especially under N-limited conditions (Fig. 2j), indicating that OE-PyNR exhibits stronger photosynthetic adaptability and photoprotective capacity compared with the WT under N-limited conditions. Collectively, these results demonstrate that PyNR overexpression enhances nitrate uptake and assimilation in P. yezoensis, thereby facilitating the maintenance of nitrogen metabolism and overall growth under nitrogen-deficient conditions. Agro-infiltration-based transformation effectively enabled functional analysis of the PyNR gene, highlighting its value for genomics research and molecular breeding in P. yezoensis and other red algae. We greatly appreciate Dr. Zhenghong Sui, Dr. Guoying Du, Dr. Fanna Kong, Dr. Xianghai Tang, and Dr. Fuli Liu from Ocean University of China for discussion about this study. This work was supported by the National Key Research and Development Program of China (2023YFD2400101), the National Natural Science Foundation of China (32503184), the Key Technology Research and Development Program of Shandong Province (2025CXPT148), the China Postdoctoral Science Foundation (2024M763093), the Shandong Postdoctoral Science Foundation (SDCX-ZG-202400097), and the Hainan Special PhD Scientifc Research Foundation of Sanya Yazhou Bay Science and Technology City (HSPHDSRF-2024-02-011). None declared. DW and YM designed this research. QS and QW performed the experiments and data analysis. JD and KZ collected the materials. QX and JW analyzed the resequencing data. ZZ screened the relative genes. XG provided suggestions on experiments. DW and QS write the manuscript. All the authors read and approved this manuscript submission. QS and QW contributed equally to this work. Resequencing data have been deposited with the National Center for Genome Resources (NCBI, Sequence Read Archive, BioProject ID: PRJNA1441821), and the vector sequences are provided in the Supporting Information (Datasets S1–S3). Dataset S1 Sequence and annotation of the T-DNA region of the OE-PyGUS vector. Dataset S2 Sequence and annotation of T-DNA region of the RNAi- PyADA2b and RNAi- PyGUS vector. Dataset S3 Sequence and annotation of the T-DNA region of the OE-PyNR vector. Fig. S1 Transformation efficiency of proPyACT4 and 2 × CaMV35S promoters at different developmental stages of P. yezoensis. Fig. S2 Hygromycin sensitivity testing of P. yezoensis fragments and thallus. Fig. S3 DNA level (a) and protein level (b) identification of OE-PyGUS transgenic lines. Fig. S4 Identification of transgenic P. yezoensis across different generations. Fig. S5 Validation of 6 T-DNA integration sites (TISs) by PCR and Sanger sequencing analysis. Fig. S6 The transcriptional expression of PyGUS in three RNAi-PyGUS lines. Fig. S7 Effects of different nitrogen source deficiencies on P. yezoensis cells. Fig. S8 Transcriptional expression levels of PyNR in different generations of OE-PyNR transgenic P. yezoensis. Fig. S9 Schematic representation of the vectors used in this study. Table S1 T-LOC outputs the detailed sequences of the left and right split reads that define TIS. Table S2 Primers used in this study. Table S3 Comparison of Agro-Infiltration and traditional biolistic bombardment methods in P. yezoensis. Please note: Wiley is not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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