Rice Protoplasts Research Articles

Hsp70, sHsps and ubiquitin proteins modulate HsfA6a-mediated Hsp101 transcript expression in rice (Oryza sativa L.).

Hsp100 chaperones disaggregate the aggregated proteins and are vital for maintenance of protein homeostasis. The level of Hsp100 synthesised in the cells has a bearing on the survival of plants under heat stress. The Hsp100 transcription machinery is activated within minutes of the onset of heat stress. The heat shock factor HsfA6a plays a major role in the transcriptional regulation of the Hsp101 gene in rice plants. Through yeast-2-hybrid library screening, we identified small heat shock proteins (sHSPs), Hsp70 and ubiquitin as HsfA6a interacting proteins (HIPs). The bimolecular fluorescence complementation assays showed the physical interaction of HsfA6a with Hsp16.9A-CI and Hsp18.0-CII in the cytosolic region and with cHsp70-1 in the nucleus. With the Hsp101 promoter: reporter gene assays, using yeast cells and rice protoplasts, we show that CI-sHsps and CII-sHsps are negative regulators and Hsp70 positive regulator of the HsfA6a activity in modulation of Hsp101 transcription. We also noted that the HsfA6a interactors, Hsp70 and CI-sHsps and CII-sHsps, physically interact with each other. We noted that HsfA6a binds with the CI-sHsp and Hsp70 promoters, implying that HsfA6a has a role in transcriptional regulation of its interacting proteins. Furthermore, we noted that the mutation of the ubiquitin/sumoylation acceptor site lysine 10 to alanine (K10A) of HsfA6a enhanced its DNA binding potential on the Hsp101 promoter, implying that these modifiers are possibly involved in modulation of HsfA6a activity. Our work shows that Hsp70, CI-sHsps and CII-sHsp, and ubiquitin proteins coordinate with HsfA6a in mediating the Hsp101 transcription process in rice.

Exploring C-To-G Base Editing in Rice, Tomato, and Poplar.

As a precise genome editing technology, base editing is broadly used in both basic and applied plant research. Cytosine base editors (CBEs) and adenine base editors (ABEs) represent the two commonly used base editor types that mediate C-to-T and A-to-G base transition changes at the target sites, respectively. To date, no transversion base editors have been described in plants. Here, we assessed three C-to-G base editors (CGBEs) for targeting sequences with SpCas9’s canonical NGG protospacer adjacent motifs (PAMs) as well as three PAM-less SpRY-based CGBEs for targeting sequences with relaxed PAM requirements. The analyses in rice and tomato protoplasts showed that these CGBEs could make C-to-G conversions at the target sites, and they preferentially edited the C6 position in the 20-nucleotide target sequence. C-to-T edits, insertions and deletions (indels) were major byproducts induced by these CGBEs in the protoplast systems. Further assessment of these CGBEs in stably transformed rice and poplar plants revealed the preference for editing of non-GC sites, and C-to-T edits are major byproducts. Successful C-to-G editing in stably transgenic rice plants was achieved by rXRCC1-based CGBEs with monoallelic editing efficiencies up to 38% in T0 lines. The UNG-rAPOBEC1 (R33A)-based CGBE resulted in successful C-to-G editing in polar, with monoallelic editing efficiencies up to 6.25% in T0 lines. Overall, this study revealed that different CGBEs have different preference on preferred editing sequence context, which could be influenced by cell cycles, DNA repair pathways, and plant species.

Genome-Wide Identification, Expansion Mechanism and Expression Profiling Analysis of GLABROUS1 Enhancer-Binding Protein(GeBP) Gene Family in Gramineae Crops.

The GLABROUS1 enhancer-binding protein (GeBP) gene family encodes a typical transcription factor containing a noncanonical Leucine (Leu-)-zipper motif that plays an essential role in regulating plant growth and development, as well as responding to various stresses. However, limited information on the GeBP gene family is available in the case of the Gramineae crops. Here, 125 GeBP genes from nine Gramineae crops species were phylogenetically classified into four clades using bioinformatics analysis. Evolutionary analyses showed that whole genome duplication (WGD) and segmental duplication play important roles in the expansion of the GeBP gene family. The various gene structures and protein motifs revealed that the GeBP genes play diverse functions in plants. In addition, the expression profile analysis of the GeBP genes showed that 13 genes expressed in all tested organs and stages of development in rice, with especially high levels of expression in the leaf, palea, and lemma. Furthermore, the hormone- and metal-induced expression patterns showed that the expression levels of most genes were affected by various biotic stresses, implying that the GeBP genes had an important function in response to various biotic stresses. Furthermore, we confirmed that OsGeBP11 and OsGeBP12 were localized to the nucleus through transient expression in the rice protoplast, indicating that GeBPs function as transcription factors to regulate the expression of downstream genes. This study provides a comprehensive understanding of the origin and evolutionary history of the GeBP genes family in Gramineae, and will be helpful in a further functional characterization of the GeBP genes.

Rice aquaporin OsPIP2;2 is a water-transporting facilitator in relevance to drought-tolerant responses.

In rice (Oryza sativa), the PLASMA MEMBRANE INTRINSIC PROTEIN (PIP) family of aquaporin has 11 members, OsPIP1;1 to OsPIP1;3, and OsPIP2;1 to OsPIP2;8, which are hypothesized to facilitate the transport of H2O and other small compounds across cell membranes. To date, however, only OsPIP1;2, OsPIP2;1, and OsPIP2;4 have been demonstrated for substrate selectivity in their source plant (rice). In this study, OsPIP2;2 was characterized as the most efficient facilitator of H2O transport across cell membranes in comparison with the other 10 OsPIPs. In concomitant tests of all OsPIPs, four genes (OsPIP1;3, OsPIP2;1, OsPIP2;2, and OsPIP2;4) were induced to express in leaves of rice plants following a physiological drought stress, while OsPIP2;2 was expressed to the highest level. After de novo expression in frog oocytes and yeast cells, the four OsPIP proteins were localized to the plasma membranes in trimer and tetramer and displayed the activity to increase the membrane permeability to H2O. In comparison, OsPIP2;2 was most supportive to H2O import to oocytes and yeast cells. After de novo expression in tobacco protoplasts, OsPIP2;2 exceeded OsPIP1;3, OsPIP2;1, and OsPIP2;4 to support H2O transport across the plasma membranes. OsPIP2;2‐mediated H2O transport was accompanied by drought‐tolerant responses, including increases in concentrations of proline and polyamines, both of which are physiological markers of drought tolerance. In rice protoplasts, H2O transport and drought‐tolerant responses, which included expression of marker genes of drought tolerance pathway, were considerably enhanced by OsPIP2;2 overexpression but strongly inhibited by the gene silencing. Furthermore, OsPIP2;2 played a role in maintenance of the cell membrane integrity and effectively protected rice cells from electrolyte leakage caused by the physiological drought stress. These results suggest that OsPIP2;2 is a predominant facilitator of H2O transport in relevance to drought tolerance in the plant.

Regulation of Oryza sativa molybdate transporter1;3 degradation via RING finger E3 ligase OsAIR3

High concentrations of As in contaminated environments pose a serious threat to plant, human, and animal health. In this study, we characterized an As-responsive Really Interesting New Gene (RING) E3 ubiquitin ligase gene under arsenate (AsV) stress, named as Oryza sativa As-Induced RING E3 ligase 3 (OsAIR3). AsV treatment highly induced the expression of OsAIR3. OsAIR3-EYFP was localized to the nucleus in rice protoplasts and exhibited E3 ligase activity. Yeast two-hybrid screening and bimolecular fluorescence complementation and pull-down assays revealed the interaction of OsAIR3 with an O. sativa molybdate transporter (OsMOT1;3) in the plasma membrane and cytoplasm. In addition, an in vitro cell-free degradation assay was performed to demonstrate the degradation of OsMOT1;3 by OsAIR3 via the 26S proteasome system. Heterogeneous overexpression of OsAIR3 in Arabidopsis yielded AsV-tolerant phenotypes, as indicated by the comparison of cotyledon expansion, root elongation, shoot fresh weight, and As accumulation between the OsAIR3-overexpressing and control plants. Collectively, these findings suggest that OsAIR3 positively regulates plant response to AsV stress.

The OsERF115/AP2EREBP110 Transcription Factor Is Involved in the Multiple Stress Tolerance to Heat and Drought in Rice Plants.

The AP2/EREBP family transcription factors play important roles in a wide range of stress tolerance and hormone signaling. In this study, a heat-inducible rice ERF gene was isolated and functionally characterized. The OsERF115/AP2EREBP110 was categorized to Group-IIIc of the rice AP2/EREBP family and strongly induced by heat and drought treatment. The OsERF115/AP2EREBP110 protein targeted to nuclei and suppressed the ABA-induced transcriptional activation of Rab16A promoter in rice protoplasts. Overexpression of OsERF115/AP2EREBP110 enhanced thermotolerance of seeds and vegetative growth stage plants. The OsERF115/AP2EREBP110 overexpressing (OE) plants exhibited higher proline level and increased expression of a proline biosynthesis P5CS1 gene. Phenotyping of water use dynamics of the individual plant indicates that the OsERF115/AP2EREBP110-OE plant exhibited better water saving traits under heat and drought combined stress. Our combined results suggest the potential use of OsERF115/AP2EREBP110 as a candidate gene for genetic engineering approaches to develop heat and drought stress-tolerant crops.

Oryza sativa, C4HC3-type really interesting new gene (RING), OsRFPv6, is a positive regulator in response to salt stress by regulating Na+ absorption.

Salinity negatively affects plant growth, productivity, and metabolism. Therefore, plants have evolved diverse strategies to survive in saline environments. To identify such strategies involving the ubiquitin/26S proteasome system, we characterized molecular functions of a rice C4HC3 really interesting new gene (RING)-type E3-ubiquitin ligase gene. Oryza sativa RING finger protein v6 (OsRFPv6) was highly expressed under conditions of abiotic stress, induced by 100 mM NaCl and 20% PEG. The GFP-OsRFPv6 protein was localized in the plasma membrane and cytosol in rice protoplasts. In vitro ubiquitin assay revealed that OsRFPv6 possessed E3-ubiquitin ligase activity, but its variant OsRFPv6C100A did not. OsRFPv6-overexpressing plants were insensitive to salinity, but their growth was delayed under normal conditions. Under saline conditions, transgenic plants exhibited higher proline, soluble sugar, and chlorophyll content and lower H2 O2 accumulation than wild-type plants. Moreover, transgenic plants exhibited lower Na+ uptake, lower Na+ content, and higher K+ content in the xylem sap assay. Under saline conditions, the expression levels of nine Na+ /K+ transporter genes in roots and leaves were significantly different between transgenic and wild-type plants. Specifically, under both normal and saline conditions, the expression of OsHKT2;1, a Na+ transporter, in the roots of transgenic plants was lower than that in the roots of wild-type plants. These results suggest that OsRFPv6 E3-ubiquitin ligase serves as a positive regulator of salinity response via Na+ uptake.

Improved plant cytosine base editors with high editing activity, purity, and specificity.

SummaryCytosine base editors (CBEs) are great additions to the expanding genome editing toolbox. To improve C‐to‐T base editing in plants, we first compared seven cytidine deaminases in the BE3‐like configuration in rice. We found A3A/Y130F‐CBE_V01 resulted in the highest C‐to‐T base editing efficiency in both rice and Arabidopsis. Furthermore, we demonstrated this A3A/Y130F cytidine deaminase could be used to improve iSpyMacCas9‐mediated C‐to‐T base editing at A‐rich PAMs. To showcase its applications, we first applied A3A/Y130F‐CBE_V01 for multiplexed editing to generate microRNA‐resistant mRNA transcripts as well as pre‐mature stop codons in multiple seed trait genes. In addition, we harnessed A3A/Y130F‐CBE_V01 for efficient artificial evolution of novel ALS and EPSPS alleles which conferred herbicide resistance in rice. To further improve C‐to‐T base editing, multiple CBE_V02, CBE_V03 and CBE_V04 systems were developed and tested in rice protoplasts. The CBE_V04 systems were found to have improved editing activity and purity with focal recruitment of more uracil DNA glycosylase inhibitors (UGIs) by the engineered single guide RNA 2.0 scaffold. Finally, we used whole‐genome sequencing (WGS) to compare six CBE_V01 systems and four CBE_V04 systems for genome‐wide off‐target effects in rice. Different levels of cytidine deaminase‐dependent and sgRNA‐independent off‐target effects were indeed revealed by WGS among edited lines by these CBE systems. We also investigated genome‐wide sgRNA‐dependent off‐target effects by different CBEs in rice. This comprehensive study compared 21 different CBE systems, and benchmarked PmCDA1‐CBE_V04 and A3A/Y130F‐CBE_V04 as next‐generation plant CBEs with high editing efficiency, purity, and specificity.

A Salivary Odorant-Binding Protein Mediates Nilaparvata lugens Feeding and Host Plant Phytohormone Suppression.

Odorant-binding proteins (OBPs) typically act as transporters of odor molecules and play an important role in insect host location. Here, we identified an OBP in brown planthopper (BPH) Nilaparvata lugens salivary glands via transcriptome sequencing. Real-time quantitative PCR and Western blotting analysis results showed that NlugOBP11 was highly expressed in salivary glands and secreted into rice plant during feeding, suggesting that it assists in BPH feeding on rice. Functional analysis in N. lugens saliva revealed that silencing this gene by RNA interference decreased the BPH stylet performance in the phloem of rice plants, reduced sap sucking, and ultimately led to insect death. Moreover, overexpression of NlugOBP11 in rice protoplasts or Nicotiana benthamiana leaves inhibited the production of defense-related signaling molecule salicylic acid in rice plant. The results demonstrate that NlugOBP11 is not only essential for BPH feeding, but also acts as an effector that inhibits plant defense.

Rice Transcription Factor OsWRKY55 Is Involved in the Drought Response and Regulation of Plant Growth.

WRKY transcription factors (TFs) have been reported to respond to biotic and abiotic stresses and regulate plant growth and development. However, the molecular mechanisms of WRKY TFs involved in drought stress and regulating plant height in rice remain largely unknown. In this study, we found that transgenic rice lines overexpressing OsWRKY55 (OsWRKY55-OE) exhibited reduced drought resistance. The OsWRKY55-OE lines showed faster water loss and greater accumulation of hydrogen peroxide (H2O2) and superoxide radical (O2−·) compared to wild-type (WT) plants under drought conditions. OsWRKY55 was expressed in various tissues and was induced by drought and abscisic acid (ABA) treatments. Through yeast two-hybrid assays, we found that OsWRKY55 interacted with four mitogen-activated protein kinases (MAPKs) that could be induced by drought, including OsMPK7, OsMPK9, OsMPK20-1, and OsMPK20-4. The activation effects of the four OsMPKs on OsWRKY55 transcriptional activity were demonstrated by a GAL4-dependent chimeric transactivation assay in rice protoplasts. Furthermore, OsWRKY55 was able to reduce plant height under normal conditions by decreasing the cell size. In addition, based on a dual luciferase reporter assay, OsWRKY55 was shown to bind to the promoter of OsAP2-39 through a yeast one-hybrid assay and positively regulate OsAP2-39 expression. These results suggest that OsWRKY55 plays a critical role in responses to drought stress and the regulation of plant height in rice, further providing valuable information for crop improvement.

The tonoplast-localized transporter OsNRAMP2 is involved in iron homeostasis and affects seed germination in rice.

Vacuolar storage of iron (Fe) is important for Fe homeostasis in plants. When sufficient, excess Fe could be stored in vacuoles for remobilization in the case of Fe deficiency. Although the mechanism of Fe remobilization from vacuoles is critical for crop development under low Fe stress, the transporters that mediate vacuolar Fe translocation into the cytosol in rice remains unknown. Here, we showed that under high Fe2+ concentrations, the Δccc1 yeast mutant transformed with the rice natural resistance-associated macrophage protein 2 gene (OsNRAMP2) became more sensitive to Fe toxicity. In rice protoplasts and transgenic plants expressing Pro35S:OsNRAMP2-GFP, OsNRAMP2 was localized to the tonoplast. Vacuolar Fe content in osnramp2 knockdown lines was higher than in the wild type, while the growth of osnramp2 knockdown plants was significantly influenced by Fe deficiency. Furthermore, the germination of osnramp2 knockdown plants was arrested. Conversely, the vacuolar Fe content of Pro35S:OsNRAMP2-GFP lines was significantly lower than in the wild type, and overexpression of OsNRAMP2 increased shoot biomass under Fe deficiency. Taken together, we propose that OsNRAMP2 transports Fe from the vacuole to the cytosol and plays a pivotal role in seed germination.

Tonoplast-Localized OsMOT1;2 Participates in Interorgan Molybdate Distribution in Rice

Molybdenum (Mo) is an essential element for plant growth and is utilized by several key enzymes in biological redox processes. Rice assimilates molybdate ions via OsMOT1;1, a transporter with a high affinity for molybdate. However, other systems involved in the molecular transport of molybdate in rice remain unclear. Here, we characterized OsMOT1;2, which shares amino acid sequence similarity with AtMOT1;2 and functions in vacuolar molybdate export. We isolated a rice mutant harboring a complete deletion of OsMOT1;2. This mutant exhibited a significantly lower grain Mo concentration than the wild type (WT), but its growth was not inhibited. The Mo concentration in grains was restored by the introduction of WT OsMOT1;2. The OsMOT1;2-GFP protein was localized to the vacuolar membrane when transiently expressed in rice protoplasts. At the reproductive growth stage of the WT plant, OsMOT1;2 was highly expressed in the 2nd and lower leaf blades and nodes. The deletion of OsMOT1;2 impaired interorgan Mo allocation in aerial parts: relative to the WT, the mutant exhibited decreased Mo levels in the 1st and 2nd leaf blades and grains but increased Mo levels in the 2nd and lower leaf sheaths, nodes and internodes. When the seedlings were exposed to a solution with a high KNO3 concentration in the absence of Mo, the mutant exhibited significantly lower nitrate reductase activity in the shoots than the WT. Our results suggest that OsMOT1;2 plays an essential role in interorgan Mo distribution and molybdoenzyme activity in rice.

Reconstitution of Cytokinin Signaling in Rice Protoplasts.

The major components of the cytokinin (CK) signaling pathway have been identified from the receptors to their downstream transcription factors. However, since signaling proteins are encoded by multigene families, characterizing and quantifying the contribution of each component or their combinations to the signaling cascade have been challenging. Here, we describe a transient gene expression system in rice (Oryza sativa) protoplasts suitable to reconstitute CK signaling branches using the CK reporter construct TCSn:fLUC, consisting of a synthetic CK-responsive promoter and the firefly luciferase gene, as a sensitive readout of signaling output. We used this system to systematically test the contributions of CK signaling components, either alone or in various combinations, with or without CK treatment. The type-B response regulators (RRs) OsRR16, OsRR17, OsRR18, and OsRR19 all activated TCSn:fLUC strongly, with OsRR18 and OsRR19 showing the strongest induction by CK. Cotransfecting the reporter with OsHP01, OsHP02, OsHP05, or OsHK03 alone resulted in much weaker effects relative to those of the type-B OsRRs. When we tested combinations of OsHK03, OsHPs, and OsRRs, each combination exhibited distinct CK signaling activities. This system thus allows the rapid and high-throughput exploration of CK signaling in rice.

Novel assays to monitor gene expression and protein-protein interactions in rice using the bioluminescent protein, NanoLuc.

Luciferases have been widely utilized as sensitive reporters to monitor gene expression and protein-protein interactions. Compared to firefly luciferase (Fluc), a recently developed luciferase, Nanoluciferase (NanoLuc or Nluc), has several superior properties such as a smaller size and stronger luminescence activity. We compared the reporter properties of Nluc and Fluc in rice (Oryza sativa). In both plant-based two-hybrid and split luc complementation (SLC) assays, Nluc activity was detected with higher sensitivity and specificity than that with Fluc. To apply Nluc to research involving the photoperiodic regulation of flowering, we made a knock-in rice plant in which the Nluc coding region was inserted in-frame with the OsMADS15 gene, a target of the rice florigen Hd3a. Strong Nluc activity in response to Hd3a, and in response to change in day length, was detected in rice protoplasts and in a single shoot apical meristem, respectively. Our results indicate that Nluc assay systems will be powerful tools to monitor gene expression and protein-protein interaction in plant research.

OrMKK3 Influences Morphology and Grain Size in Rice.

Although morphology and grain size are important to rice growth and yield, the identity of abundant natural allelic variations that determine agronomically important differences in crops is unknown. Here, we characterized the function of mitogen-activated protein kinase 3 from Oryza officinalis Wall. ex Watt encoded by OrMKK3. Different alternative splicing variants occurred in OrMKK3. Green fluorescent protein (GFP)–OrMKK3 fusion proteins localized to the cell membrane and nuclei of rice protoplasts. Overexpression of OrMKK3 influenced the expression levels of the grain size-related genes SMG1, GW8, GL3, GW2, and DEP3. Phylogenetic analysis showed that OrMKK3 is well conserved in plants while showing large amounts of variation between indica, japonica, and wild rice. In addition, OrMKK3 slightly influenced brassinosteroid (BR) responses and the expression levels of BR-related genes. Our findings thus identify a new gene, OrMKK3, influencing morphology and grain size and that represents a possible link between mitogen-activated protein kinase and BR response pathways in grain growth.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12374-020-09290-2.

Rapid Vector Construction and Assessment of BE3 and Target-AID C to T Base Editing Systems in Rice Protoplasts.

CRISPR-Cas9 has revolutionized the field of genome engineering. Base editing, a new genome editing strategy, was recently developed to engineer nucleotide substitutions. DNA base editing systems use a catalytically impared Cas nuclease together with a nucleobase deaminase enzyme to specifically introduce point mutations without generating double-stranded breaks, which provide huge potential in crop improvement. Here, we describe fast and efficient preparation of user-friendly C to T base editors, BE3, and Target-AID. Presented are detailed protocols for T-DNA vector preparation with BE3 or modified Target-AID base editor based on Gateway assembly and efficiency assessment of base editing through a rice protoplast transient expression system.

OsPP2C09 Is a Bifunctional Regulator in Both ABA-Dependent and Independent Abiotic Stress Signaling Pathways.

Clade A Type 2C protein phosphatases (PP2CAs) negatively regulate abscisic acid (ABA) signaling and have diverse functions in plant development and in response to various stresses. In this study, we showed that overexpression of the rice ABA receptor OsPYL/RCAR3 reduces the growth retardation observed in plants exposed to osmotic stress. By contrast, overexpression of the OsPYL/RCAR3-interacting protein OsPP2C09 rendered plant growth more sensitive to osmotic stress. We tested whether OsPP2CAs activate an ABA-independent signaling cascade by transfecting rice protoplasts with luciferase reporters containing the drought-responsive element (DRE) or ABA-responsive element (ABRE). We observed that OsPP2CAs activated gene expression via the cis-acting drought-responsive element. In agreement with this observation, transcriptome analysis of plants overexpressing OsPP2C09 indicated that OsPP2C09 induces the expression of genes whose promoters contain DREs. Further analysis showed that OsPP2C09 interacts with DRE-binding (DREB) transcription factors and activates reporters containing DRE. We conclude that, through activating DRE-containing promoters, OsPP2C09 positively regulates the drought response regulon and activates an ABA-independent signaling pathway.

Rice Protoplast Isolation and Transfection for Transient Gene Expression Analysis.

Protoplasts are a versatile and powerful cell-based system to study different plant processes in vivo, due to their ability to maintain cell identity and carry out reactions and metabolic processes similar to intact plants. In rice, despite numerous reports, difficulties are encountered in protoplast isolation and transfection. These include insufficient numbers of protoplasts isolated and inefficient transfection. Such difficulties limit the use of this simple yet useful technology. The need to use protoplasts is particularly important when similar experiments may not work in yeast or Pichia, due to differences in functionally essential protein post-translation modifications. In this chapter, we describe a rice protoplast isolation and transfection method.

OsClo5 functions as a transcriptional co-repressor by interacting with OsDi19-5 to negatively affect salt stress tolerance in rice seedlings.

Caleosins constitute a small protein family with one calcium-binding EF-hand motif. They are involved in the regulation of development and response to abiotic stress in plants. Nevertheless, how they impact salt stress tolerance in rice is largely unknown. Thereby, biochemical and molecular genetic experiments were carried out, and the results revealed that OsClo5 was able to bind calcium and phospholipids in vitro and localized in the nucleus and endoplasmic reticulum in rice protoplasts. At the germination and early seedlings stages, overexpression transgenic lines and T-DNA mutant lines exhibited reduced and increased tolerance to salt stress, respectively, compared with the wild-type. Yeast two-hybrid, bimolecular fluorescence complementation and in vitro pull-down assays demonstrated that the EF-hand motif of OsClo5 was essential for the interactions with itself and OsDi19-5. Yeast one-hybrid, electrophoretic migration shift and dual-luciferase reporter assays identified OsDi19-5 as a transcriptional repressor via the TACART cis-element in the promoters of two salt stress-related target genes, OsUSP and OsMST. In addition, OsClo5 enhanced the inhibitory effect of OsDi19-5 in the tobacco transient system, which was confirmed by qRT-PCR analysis in rice seedlings under salt stress. The collective results deepen the understanding of the molecular mechanism underlying the roles of caleosin in the salt stress response. These findings will also inform efforts to improve salt tolerance of rice.

Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assays

BackgroundAn efficient in vivo transient transfection system using protoplasts is an important tool to study gene expression, metabolic pathways, and multiple mutagenesis parameters in plants. Although rice protoplasts can be isolated from germinated seedlings or cell suspension culture, preparation of those donor tissues can be inefficient, time-consuming, and laborious. Additionally, the lengthy process of protoplast isolation and transfection needs to be completed in a single day.ResultsHere we report a protocol for the isolation of protoplasts directly from rice calli, without using seedlings or suspension culture. The method is developed to employ discretionary pause points during protoplast isolation and before transfection. Protoplasts maintained within a sucrose cushion partway through isolation, for completion on a subsequent day, per the first pause point, are referred to as S protoplasts. Fully isolated protoplasts maintained in MMG solution for transfection on a subsequent day, per the second pause point, are referred to as M protoplasts. Both S and M protoplasts, 1 day after initiation of protoplast isolation, had minimal loss of viability and transfection efficiency compared to protoplasts 0 days after isolation. S protoplast viability decreases at a lower rate over time than that of M protoplasts and can be used with added flexibility for transient transfection assays and time-course experiments. The protoplasts produced by this method are competent for transfection of both plasmids and ribonucleoproteins (RNPs). Cas9 RNPs were used to demonstrate the utility of these protoplasts to assay genome editing in vivo.ConclusionThe current study describes a highly effective and accessible method to isolate protoplasts from callus tissue induced from rice seeds. This method utilizes donor materials that are resource-efficient and easy to propagate, permits convenience via pause points, and allows for flexible transfection days after protoplast isolation. It provides an advantageous and useful platform for a variety of in vivo transient transfection studies in rice.