A Codon-Optimized Bacterial Antibiotic Gene Used as Selection Marker for Stable Nuclear Transformation in the Marine Red Alga Pyropia yezoensis

Marine macroalgae play an important role in marine coastal ecosystems and are widely used as sea vegetation foodstuffs and for industrial purposes. Therefore, there have been increased demands for useful species and varieties of these macroalgae. However, genetic transformation in macroalgae has not yet been established. We have developed a dominant selection marker for stable nuclear transformation in the red macroalga Pyropia yezoensis. We engineered the coding region of the aminoglycoside phosphotransferase gene aph7″ from Streptomyces hygroscopicus to adapt codon usage of the nuclear genes of P. yezoensis. We designated this codon-optimized aph7″ gene as PyAph7. After bombarding P. yezoensis cells with plasmids containing PyAph7 under the control of their endogenous promoter, 1.9 thalli (or individuals) of hygromycin-resistant strains were isolated from a 10-mm square piece of the bombarded thallus. These transformants were stably maintained throughout the asexual life cycle. Stable expression of PyAph7was verified using Southern blot analysis and genomic PCR and RT-PCR analyses. PyAph7 proved to be a new versatile tool for stable nuclear transformation in P. yezoensis.     

The First Symbiont-Free Genome Sequence of Marine Red Alga,Susabi-nori (Pyropia yezoensis)

Nori, a marine red alga, is one of the most profitable mariculture crops in the world. However, the biological properties of this macroalga are poorly understood at the molecular level. In this study, we determined the draft genome sequence of susabi-nori (Pyropia yezoensis) using next-generation sequencing platforms. For sequencing, thalli of P. yezoensis were washed to remove bacteria attached on the cell surface and enzymatically prepared as purified protoplasts. The assembled contig size of the P. yezoensis nuclear genome was approximately 43 megabases (Mb), which is an order of magnitude smaller than the previously estimated genome size. A total of 10,327 gene models were predicted and about 60% of the genes validated lack introns and the other genes have shorter introns compared to large-genome algae, which is consistent with the compact size of the P. yezoensis genome. A sequence homology search showed that 3,611 genes (35%) are functionally unknown and only 2,069 gene groups are in common with those of the unicellular red alga, Cyanidioschyzon merolae. As color trait determinants of red algae, light-harvesting genes involved in the phycobilisome were predicted from the P. yezoensis nuclear genome. In particular, we found a second homolog of phycobilisome-degradation gene, which is usually chloroplast-encoded, possibly providing a novel target for color fading of susabi-nori in aquaculture. These findings shed light on unexplained features of macroalgal genes and genomes, and suggest that the genome of P. yezoensis is a promising model genome of marine red algae.     

A CELLULOSE SYNTHASE (CESA) GENE FROM THE RED ALGA PORPHYRA YEZOENSIS (RHODOPHYTA)1

The cell walls of Porphyra species, like those of land plants, contain cellulose microfibrils that are synthesized by clusters of cellulose synthase enzymes (“terminal complexes”), which move in the plasma membrane. However, the morphologies of the Porphyra terminal complexes and the cellulose microfibrils they produce differ from those of land plants. To characterize the genetic basis for these differences, we have identified, cloned, and sequenced a cellulose synthase (CESA) gene from Porphyra yezoensis Ueda strain TU‐1. A partial cDNA sequence was identified in the P. yezoensis expressed sequence tag (EST) index using a land plant CESA sequence as a query. High‐efficiency thermal asymmetric interlaced PCR was used to amplify sequences upstream of the cDNA sequence from P. yezoensis genomic DNA. Using the resulting genomic sequences as queries, we identified additional EST sequences and a full‐length cDNA clone, which we named PyCESA1. The conceptual translation of PyCESA1 includes the four catalytic domains and the N‐ and C‐terminal transmembrane domains that characterize CESA proteins. Genomic PCR demonstrated that PyCESA1 contains no introns. Southern blot analysis indicated that P. yezoensis has at least three genomic sequences with high similarity to the cloned gene; two of these are pseudogenes based on analysis of amplified genomic sequences. The P. yezoensis CESA peptide sequence is most similar to cellulose synthase sequences from the oomycete Phytophthora infestans and from cyanobacteria. Comparing the CESA genes of P. yezoensis and land plants may facilitate identification of sequences that control terminal complex and cellulose microfibril morphology.     

Isolation and evaluation of a strain of Pyropia yezoensis (Bangiales,Rhodophyta) resistant to red rot disease

Red rot disease (Pythium porphyrae) resistant strains of Pyropia yezoensis, AP1 and AP2, were isolated from live cells taken from lesions on infected P. yezoensis blades. The degree of resistance of these strains to red rot disease was evaluated over a range of environmental conditions including temperature (10, 15, and 20 °C), salinity (20, 26, and 32 ppt), and pH (7.5, 8.0, and 8.5). These conditions are within the range that red rot disease naturally occurs on Pyropia blades. P. yezoensis and Pyropia suborbiculata with low and high partial resistance to red rot disease, respectively, were used as controls. Infection with red rot disease occurred under all environmental conditions. The incidence, severity, and expansion of the disease increased with increasing temperature and decreasing salinity and pH. The resistance of the strains P. yezoensis-AP1 and P. yezoensis-AP2 was higher than that of P. yezoensis, but lower than that of P. suborbiculata. The degree of resistance was not significantly different between the AP1 and AP2 strains. These strains can therefore be considered to exhibit stable partial resistance to P. porphyrae, and as a good starting point for the development of more resistant strains that will prevent or reduce the incidence of red rot disease on Pyropia farms.     

Complete mitochondrial genome sequence of Pyropia yezoensis (Bangiales,Rhodophyta) from Korea

Species of the genera Porphyra and Pyropia are the major seaweed cultivars in Korea, Japan, and China. Genomic data from Porphyra/Pyropia species is now being used for molecular breeding and bioengineering. We determined the complete mitochondrial genome (mtDNA) sequence of Pyropia yezoensis (Bangiales, Rhodophyta) from Korea. Pyropia yezoensis mtDNA was 35,596 bp in size and exhibited high sequence similarity to Py. yezoensis mtDNA reported from China (NC_017837; 41,688 bp). However, the Korean Py. yezoensis differed from the Chinese strain in the presence/absence of introns and intronic open reading frames of the ribosomal RNA large subunit gene (rnl) and cytochrome c oxidase subunit 1 gene (cox1). These differences result in large variations between the mtDNA of the two strains of Py. yezoensis. Moreover, the Korean strain had a single copy of the trnfM–trnQ region, as compared to a duplicate copy of Py. yezoensis (NC_017837). Pyropia mtDNA exhibited unique genetic features at the intra- and interspecies levels. Therefore, mtDNA genomics can provide novel knowledge for red algal molecular systematics and help to differentiate between cultivars of Pyropia species with new molecular markers.     

Study of photosynthetic characteristics of the Pyropia yezoensis thallus during the cultivation process

The photosynthetic characteristics of thalli of cultured Pyropia yezoensis strains collected in January, February, and March in seaweed cultivation area of South China Yellow Sea were studied. Results showed that the maximum quantum efficiency (F _v/F _m) of all P. yezoensis thallus collected at different times was 0.65. The actual quantum efficiency (ΔF/F _m′) of samples in January was the lowest of all samples, while the ΔF/F _m′ of samples in March was significantly higher than those in January and February. The increase of temperature and photosynthetic pigments ratios of phycoerythrin and chlorophyll a (PE/Chla) and phycocyanin and chlorophyll a (PC/Chla) from January to March may be the important reasons for the increase in light use efficiency of thallus; although the thallus in March was significantly thicker than in January which may have reduced the light energy absorbed by photosynthetic pigments, the increase of relative high energy use efficiency also helped to maintain the photosynthetic oxygen evolution rate in March. The thicker thallus also reduced photodamage, and the thallus area was increased obviously in March, so the growth rate of thallus in March was over 35 % higher than that in February. Our research indicates that the photosynthetic characteristics of P. yezoensis strains thalli have a close relationship with their growth stage and environmental factors especially temperature, and those photosynthetic characteristics are also reflected in the growth rate of the thalli.     

Construction and characterization of a bacterial artificial chromosome library of marine macroalga<Emphasis Type="Italic">Porphyra yezoensis</Emphasis> (Rhodophyta)

A large-DNA-fragment library is necessary for research into thePorphyra genome. In this study, a bacterial artificial chromosome (BAC) library ofPorphyra yezoensis was constructed and characterized. The library contains 54,144 BAC clones with an average insert size of about 65 kb and fewer than 0.7% of clones without large inserts. Therefore, its capacity is more than 6.6P. yezoensis genome equivalents, and the probability of recovering any nuclear DNA sequence from the library is higher than 99%. The library shows good fidelity and stability. A putative trehalose-6-phosphate synthase (TPS) gene was successfully screened out from the library. The above results show that the library is useful for gene cloning and genomic research inP. yezoensis. These authors contributed equally to this work.     

Variation of Expression Levels of Seven Housekeeping Genes at Different Life-History Stages in Porphyra yezoensis

In order to identify the optimal internal control for relative real-time PCR when studying target gene expression in the red alga Porphyra yezoensis, we quantified the expression of seven housekeeping genes (18S ribosomal RNA, 30S ribosomal protein S8, Polyubiquitin-2, Glyceraldehyde-3-phosphate dehydrogenase, Elongation factor 1-alpha, Beta-tubulin and Actin 3) at different life-history stages. Absolute quantification was done by normalization to total RNA quantity and by normalization to genomic DNA quantity. We used these two normalization approaches, comparing the differences of expression levels of all candidate housekeeping genes between any two generations and across three life-history stages (filamentous sporophytes, leafy gametophytes and conchospores). We found GAPDH had the best stability in all cases and we recommend that GAPDH be considered as a potential internal control for gene expression studies at different life-history stages in P. yezoensis.     

Overexpression of a peroxiredoxin Q gene,SsPrxQ, in Eustoma grandiflorum Shinn enhances its tolerance to salt and high light intensity

Abiotic stress, such as salt, high light intensity and extreme temperature, can result in enhanced production of reactive oxygen species (ROS). One of the major ROS-scavenging enzymes of plants is peroxiredoxin (Prx). Peroxiredoxin Q (PrxQ), a member of the Prx gene family, was recently cloned from plants. To investigate the protective role of PrxQ during abiotic stress, we increased the capacity for its biosynthesis in Eustoma grandiflorum Shinn by overexpression of the PrxQ gene (SsPrxQ) from Suaeda salsa. The SsPrxQ gene driven by CaMV 35S promoter was expressed via E. grandiflorum. The rPrxQ protein shows antioxidant activity and thioredoxin-dependent peroxidase activity in vitro. Additionally, overexpression of SsPrxQ in E. grandiflorum leads to an increase in salt and high light intensity tolerance. These results indicate that SsPrxQ might act as an oxidative stress defensive gene in plants and could be useful for engineering stress-resistant plants.     

Development of a nuclear transformation system with a codon-optimized selection marker and reporter genes in Pyropia yezoensis (Rhodophyta)

Genetic transformation systems using reporter genes in whole plants have a wide variety of applications for molecular biological study including the visualization of expression patterns of particular genes and intracellular biological phenomena as well as the identification of novel genes. In this study, we assessed co-expression of each three codon-optimized reporter genes and a selectable marker in the nuclear transformation system of whole Pyropia yezoensis, a red marine alga. With the use of an endogenous promoter, both the codon-optimized hygromycin resistance gene and ß-glucuronidase gene (PyGUS) were co-expressed in P. yezoensis cells. A high level of GUS activity was observed in 60 % of the individuals in hygromycin-resistant lines. A histochemical GUS assay revealed that the PyGUS reporter gene was stably introduced and expressed throughout the algae's life cycle. In addition, two live cell reporters, humanized cyan fluorescent protein from Anemonia majano and luciferase from Gaussia princeps, were successfully expressed in whole P. yezoensis. The development of this transformation system involving three types of reporter genes provides opportunities for monitoring temporal changes in gene expression and for genetic screening in red marine algae.     

Development of cyan fluorescent protein (CFP) reporter system in green alga Chlamydomonas reinhardtii and macroalgae Pyropia sp.

Various fluorescent proteins have been developed for in vivo reporter systems in diverse prokaryotes and eukaryotes. However, few in vivo imaging systems have been reported for the model algae Chlamydomonas reinhardtii or Pyropia sp. In this study, an effective imaging system using cyan fluorescent protein (CFP) was developed for the green alga C. reinhardtii, and its application was also successful in the red macroalgae Pyropia tenera and P. yezoensis. For optimization of CFP expression in C. reinhardtii and Pyropia sp., we modified codon usage in the CFP gene (CFP), generating PtCrCFP (Pyropia tenera/Chlamydomonas reinhardtii CFP). PtCrCFP was successfully expressed in PtCrCFP-expressing UVM11 transgenic lines, and high accumulation levels of PtCrCFP were found by western blotting. Consistent with these results, PtCrCFP fluorescence was clearly detected with a low level of chlorophyll background fluorescence in PtCrCFP-expressing UVM11 transgenic lines. In Pyropia sp. gametophytic cells, transient expression of PtCrCFP fluorescence was distinctly visualized. PtCrCFP fluorescence was also observed during the regeneration of monospores and young gametophytes from PtCrCFP-expressing P. yezoensis gametophytic cells. These results suggest that PtCrCFP may be useful as an in vivo reporter in green algae due to the short emission wavelength of CFP, which provides a low level of chlorophyll background fluorescence. This study also presents the possibility of PtCrCFP’s use as a visible selection marker for the generation of transgenic lines in the red algae Pyropia sp. Thus, PtCrCFP as an in vivo visualization tool may offer new opportunities for the functional analysis of genetic studies in both green and red algae.     

Characterizing the microbial culprit of white spot disease of the conchocelis stage of Porphyra yezoensis (Bangiales, Rhodophyta)

White spot disease is the most frequent and harmful disease affecting the shell-boring conchocelis stage of the economically important red alga Porphyra yezoensis (= Pyropia yezoensis (Ueda) Hwang & Choi) (Bangiales, Rhodophyta). To date, its potential pathogens and pathogenesis remain poorly understood. In this study, we isolated 7 bacteria, 26 fungi, and 10 actinomycetes from sick conchocelis. Re-infection experiments revealed that only one fungus, GF014, caused disease. Morphological characteristics of the colony, mycelium, sporangium, and spore of GF014 suggested that the strain belongs to the order Pleosporales and the genus Phoma. Classification based on internal transcribed spacer (ITS) sequences supported the results of morphological identification. These results indicate that GF014 is a species of Phoma and a specific pathogen for Porphyra. GF014 preferred organic nitrogen sources, and 20–28 °C and low salinity water were its optimal living conditions. Our findings will play an active role in preventing and treating white spot disease in P. yezoensis.