Rice Protoplasts Research Articles

High-Efficient Transient Gene Transfection for Transcriptional Activity Assay Using Rice Protoplasts on Large-Scale Platform.

Transient gene expression in protoplasts offers a straightforward and efficient approach for investigating gene function. Here, I introduce a method that utilizes a 96-well plate format to assess transcriptional activation capacity of transcription factors through transient expression system in rice protoplasts. This method includes plant cultivation, protoplast preparation, transfection, and transactivation assay and allows for the simultaneous measurement and validation of 23 samples with 4 replicates per sample at a time.

Heading Date 3a Stimulates Tiller Bud Outgrowth in Oryza sativa L. through Strigolactone Signaling Pathway.

Heading date 3a (Hd3a, a FLOWERING LOCUS T (FT) ortholog from rice) is well known for its important role in rice (Oryza sativa L.), controlling floral transition under short-day (SD) conditions. Although the effect of Hd3a on promoting branching has been found, the underlying mechanism remains largely unknown. In this report, we overexpressed an Hd3a and BirAG (encoding a biotin ligase) fusion gene in rice, and found that early flowering and tiller bud outgrowth was promoted in BHd3aOE transgenic plants. On the contrary, knockout of Hd3a delayed flowering and tiller bud outgrowth. By using the BioID method, we identified multiple Hd3a proximal proteins. Among them, D14, D53, TPR1, TPR2, and TPRs are central components of the strigolactone signaling pathway, which has an inhibitory effect on rice tillering. The interaction between Hd3a, on the one hand, and D14 and D53 was further confirmed by the bimolecular fluorescence complementation (BiFC), yeast two-hybrid (Y2H), and co-immunoprecipitation (Co-IP) methods. We also found that Hd3a prevented the degradation of D53 induced by rac-GR24 (a strigolactone analog) in rice protoplasts. RT-qPCR assay showed that the expression levels of genes involved in strigolactone biosynthesis and signal transduction were altered significantly between WT and Hd3a overexpression (Hd3aOE) or mutant (hd3a) plants. OsFC1, a downstream target of the strigolactone signaling transduction pathway in controlling rice tillering, was downregulated significantly in Hd3aOE plants, whereas it was upregulated in hd3a lines. Collectively, these results indicate that Hd3a promotes tiller bud outgrowth in rice by attenuating the negative effect of strigolactone signaling on tillering and highlight a novel molecular network regulating rice tiller outgrowth by Hd3a.

Enhancing Plant Stress Tolerance: The Role of LcWRKY40 Gene in Drought and Alkaline Salt Resistance in Tobacco and Yeast.

Leymus chinensis, a halophytic perennial grass belonging to the Poaceae family, thrives in saline-alkali grasslands and harbors a rich repository of resistance-related genetic resources. This study focused on deciphering the stress-responsive mechanisms of L. chinensis by conducting transcriptomic sequencing under NaHCO3 stress, which resulted in the annotation of a segment corresponding to the 51WRKY gene. The alkali-induced gene LcWRKY40 (QIG37591) was identified by phylogenetic analysis. Real-time quantitative PCR analysis was performed on L. chinensis plants subjected to PEG6000 and alkaline salt (NaHCO3) stress, and the results indicated that the LcWRKY40 gene was upregulated in both the leaves and roots. The localization of the LcWRKY40 protein was confirmed by the use of green fluorescent protein (GFP) fusion technology in transformed rice protoplast cells. The GAL4-driven transformation of the LcWRKY40 gene in INVScI yeast cells, which exhibited enhanced tolerance upon overexpression of the LcWRKY40 gene under mannitol and alkaline salt (NaHCO3) stress conditions. Under drought stress using mannitol, the fresh weight of Nicotiana tabacum overexpressing the LcWRKY40 gene was significantly higher than that of wild-type(WT) tobacco. Through drought and salt alkali stress, we found that overexpressed tobacco at different stages always outperformed the wild type in terms of fresh weight, SOD, MDA, and Fv/Fm. This study provides preliminary insights into the involvement of the LcWRKY40 gene in responding to drought and alkaline salt stresses, highlighting its role in enhancing plant resistance to drought and saline-alkaline conditions. These findings lay the foundation for future molecular breeding strategies aimed at improving grass resistance from different aspects.

Improving photosynthetic efficiency of rice via over-expressing a ferredoxin-like protein gene from Methanothermobacter thermautotrophicus.

Ferredoxins (Fds) are crucial in various essential plant metabolic processes, including photosynthesis, fermentation and aerobic nitrogen fixation, due to their role in electron transport rate (ETR). However, the full scope of ferredoxin's function across prokaryotes and eukaryotic plants remains less understood. This study investigated the effect of MtFd from Methanothermobacter thermoautotrophicus on rice photosynthetic efficiency. We found that MtFd was localized in the chloroplasts of rice protoplasts. Transgenic analysis showed that MtFd significantly enhanced the photosynthetic capacity compared to the wild-type plants. This enhancement was evident through increased ETR, NADPH content and net photosynthetic rates, as well as decreased non-photochemical quenching (NPQ). Despite similar biomass to wild type plants, MtFd transgenic plants exhibited a marked increase in grain size and the 1000-grian weight. The elevated ETR and surplus free electrons in transgenic plants result in a considerable rise in cellular ROS content, which in turn enhances the enzymatic activity of the antioxidant system. In summary, our findings suggest that introducing the Fd protein from M. thermoautotrophicus into transgenic rice improves photosynthetic efficiency by accelerating ETR, which triggers the cellular oxidative stress response.

CRISPR/Cas9 effectively generate chromosome structural variations in rice protoplasts

AbstractChromosome structural variations (SVs), such as deletion, duplication, inversion, and translocation, are important contributors to genetic diversification and crop improvement. Using genome editing tools such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated nuclease (Cas9), desired SVs involving large DNA fragments have been created in rice (Oryza sativa L.), maize (Zea mays L.), and Arabidopsis (Arabidopsis thaliana L.). However, it is still uncertain whether the size of DNA fragment involved could be a prohibiting factor to generate Cas9‐mediated SVs. In this study, we constructed five CRISPR/Cas9 vectors, each expressing two single‐guide RNAs (sgRNAs), to cut two sites spacing at 0.5, 5, 10, 20, and 30 Mb on rice chromosome 4 (Chr4), respectively. Meanwhile, another CRISPR/Cas9 vector cutting two sites, one on Chr4 and the other on Chr1, was also constructed for creation of chromosomal translocation between Chr1 and Chr4. These vectors were transfected into rice protoplasts by polyethylene glycol–mediated transformation. Specific primers were designed to detect desired SV events. The results showed that all designed SVs could be effectively generated by CRISPR/Cas9 in rice protoplasts. This study suggested that the size of DNA fragment involved is unlikely a prohibiting factor for creation of desired SV events.

Efficient and multiplex gene upregulation in plants through CRISPR-Cas-mediated knockin of enhancers

Gene upregulation through genome editing is important for plant research and breeding. Targeted insertion of short transcriptional enhancers (STEs) into gene promoters may offer a universal solution akin to transgene-mediated overexpression while avoiding the drawbacks associated with transgenesis. Here, we introduce an “in locus activation” technique in rice that leverages well-characterized STEs for refined, heritable, and multiplexed gene upregulation. To address the scarcity of potent enhancers, we developed a large-scale mining approach and discovered a suite of STEs that are capable of enhancing gene expression in rice protoplasts. The in locus integration of these STEs into eight rice genes resulted in substantial transcriptional upregulation in the edited plants, with up to 869.1-fold increases in their transcript levels. Employing a variety of STEs, we achieved delicate control of gene expression, enabling the fine-tuning of key phenotypic traits such as plant height. Our approach also enabled efficient multiplexed gene upregulation, with up to four genes activated simultaneously, significantly enhancing the nicotinamide mononucleotide metabolic pathway. Importantly, heritability studies from the T0 to T3 generations confirmed the stable and heritable nature of STE-driven gene activation. Collectively, our work demonstrates that coupled with STE mining, leveraging genome editing for in locus activation and gene upregulation holds great promise to be widely adopted in fundamental plant research and crop breeding.

Comparing the Mechanical Properties of Rice Cells and Protoplasts under PEG6000 Drought Stress Using Double Resonator Piezoelectric Cytometry.

Plant cells' ability to withstand abiotic stress is strongly linked to modifications in their mechanical characteristics. Nevertheless, the lack of a workable method for consistently tracking plant cells' mechanical properties severely restricts our comprehension of the mechanical alterations in plant cells under stress. In this study, we used the Double Resonator Piezoelectric Cytometry (DRPC) method to dynamically and non-invasively track changes in the surface stress (ΔS) generated and viscoelasticity (storage modulus G' and loss modulus G″) of protoplasts and suspension cells of rice under a drought stress of 5-25% PEG6000. The findings demonstrate that rice suspension cells and protoplasts react mechanically differently to 5-15% PEG6000 stress, implying distinct resistance mechanisms. However, neither of them can withstand 25% PEG6000 stress; they respond mechanically similarly to 25% PEG6000 stress. The results of DRPC are further corroborated by the morphological alterations of rice cells and protoplasts observed under an optical microscope. To sum up, the DRPC technique functions as a precise cellular mechanical sensor and offers novel research tools for the evaluation of plant cell adversity and differentiating between the mechanical reactions of cells and protoplasts under abiotic stress.

Genome editing in rice and tomato with a small Un1Cas12f1 nuclease.

The clustered regularly interspaced short palindromic repeats (CRISPR) systems have been demonstrated to be the foremost compelling genetic tools for manipulating prokaryotic and eukaryotic genomes. Despite the robustness and versatility of Cas9 and Cas12a/b nucleases in mammalian cells and plants, their large protein sizes may hinder downstream applications. Therefore, investigating compact CRISPR nucleases will unlock numerous genome editing and delivery challenges that constrain genetic engineering and crop development. In this study, we assessed the archaeal miniature Un1Cas12f1 type-V CRISPR nuclease for genome editing in rice and tomato protoplasts. By adopting the reengineered guide RNA modifications ge4.1 and comparing polymerase II (Pol II) and polymerase III (Pol III) promoters, we demonstrated uncultured archaeon Cas12f1 (Un1Cas12f1) genome editing efficacy in rice and tomato protoplasts. We characterized the protospacer adjacent motif (PAM) requirements and mutation profiles of Un1Cas12f1 in both plant species. Interestingly, we found that Pol III promoters, not Pol II promoters, led to higher genome editing efficiency when they were used to drive guide RNA expression. Unlike in mammalian cells, the engineered Un1Cas12f1-RRA variant did not perform better than the wild-type Un1Cas12f1 nuclease, suggesting continued protein engineering and other innovative approaches are needed to further improve Un1Cas12f1 genome editing in plants.

Rice SRO1a Contributes to Xanthomonas TAL Effector-mediated Expression of Host Susceptible Genes.

Xanthomonas species infect many important crops and cause huge yield loss. These pathogens deliver transcription activator-like (TAL) effectors into the cytoplasm of plant cells. TAL effectors move to host nuclei, directly bind to the promoters of host susceptible genes, and activate their transcription. However, the molecular mechanisms by which TAL effectors induce host transcription remain unclear. We herein demonstrated that TAL effectors interacted with the SIMILAR TO RCD ONE (SRO) family proteins OsSRO1a and OsSRO1b in nuclei. A transactivation assay using rice protoplasts indicated that OsSRO1a and OsSRO1b enhanced the activation of the OsSWEET14 promoter by the TAL effector AvrXa7. The AvrXa7-mediated expression of OsSWEET14 was significantly reduced in ossro1a mutants. However, the overexpression of OsSRO1a increased disease resistance by up-regulating the expression of defense-related genes, such as WRKY62 and PBZ1. This was attributed to OsSRO1a and OsSRO1b also enhancing the transcriptional activity of WRKY45, a direct regulator of WRKY62 expression. Therefore, OsSRO1a and OsSRO1b appear to positively contribute to transcription mediated by bacterial TAL effectors and rice transcription factors.

Real-time monitoring abscisic acid release from single rice protoplast by amperometry at microelectrodes modified with abscisic acid receptor PYL2

It was previously reported that stress induces a cellular production of abscisic acid in plants, but no direct method shows the evidence. Here, an electrochemical microsensor involving an abscisic acid receptor PYL2 modified carbon fiber microelectrode was fabricated by self-assembly method, where the Cu2+ combined with the histidine tag of PYL2 on the surface of microelectrode was used as the detection probe, the mediated reaction between Cu+ and ferricyanide realized the amplification responses and provided the microsensor with a high sensitivity for detection of abscisic acid with a detection limit of 0.8 nM. With use of this microsensor, an increase of extracellular abscisic acid from single rice protoplast induced by sulfate, osmotic and salinity stress was real-time monitored. Direct measurement of free extracellular abscisic acid in single plant cells might offer important new insights into its role in plants challenged by abiotic stresses.

OsBBP1, a newly identified protein containing DUF630 and DUF632 domains confers drought tolerance in rice

Domain of unknown function (DUF) protein families, which are uncharacterized and numerous within the Pfam database. Recently, studies have demonstrated that DUFs played crucial roles in plant development, but whether, or how, they function in drought resistance remain unclear. In this study, we identified the Os03g0321500 gene, encoding OsbZIP72 binding protein 1 (OsBBP1), as a target of OsbZIP72 using chromatin immunoprecipitation sequencing in rice. OsBBP1 is a novel member of DUFs, which localize both in the nuclei and cytoplasm of rice protoplasts. Furthermore, yeast one-hybrid and electrophoretic mobility shift assays confirmed the specific binding between OsbZIP72 and OsBBP1. Additionally, a luciferase reporter analysis illustrated that OsbZIP72 activated the expression of OsBBP1. Drought tolerance experiments demonstrate that the OsBBP1 CRISPER-CAS9 transgenic mutants were sensitive to drought stress, but the transgenic OsBBP1 over-expressing rice plants showed enhanced drought resistance. Moreover, drought tolerance experiments in a paddy field suggested that OsBBP1 contributed to less yield or yield-related losses under drought conditions. Mechanistically, OsBBP1 might confer drought resistance by inducing more efficient reactive oxygen species (ROS) scavenging. Several ROS scavenging-related genes showed increased expression levels in OsBBP1 overexpression lines and decreased expression levels in OsBBP1 CRISPER-CAS9 mutants under drought conditions. Thus, OsBBP1, acting downstream of OsbZIP72, contributes to drought resistance and causes less yield or yield-related losses under drought conditions.

Rice Serine Hydroxymethyltransferases: Evolution, Subcellular Localization, Function and Perspectives.

In rice, there is a lack of comprehensive research on the functional aspects of the members of the serine hydroxymethyltransferase (SHMT) gene family. This study provides a comprehensive investigation of the SHMT gene family, covering phylogeny, gene structure, promoter analysis, expression analysis, subcellular localization, and protein interaction. Remarkably, we discovered a specific gene loss event occurred in the chloroplast-localized group IIa SHMTs in monocotyledons. However, OsSHMT3, which originally classified within cytoplasmic-localized group Ib, was found to be situated within chloroplasts in rice protoplasts. All five OsSHMTs are capable of forming homodimers, with OsSHMT3 being the only one able to form dimers with other OsSHMTs, except for OsSHMT1. It is proposed that OsSHMT3 functions as a mobile protein, collaborating with other OsSHMT proteins. Furthermore, the results of cis-acting element prediction and expression analysis suggested that members of the OsSHMT family could be involved in diverse stress responses and hormone regulation. Our study aims to provide novel insights for the future exploration of SHMTs.

Optimized protoplast isolation and transfection with a breakpoint: accelerating Cas9/sgRNA cleavage efficiency validation in monocot and dicot

The CRISPR-Cas genome editing tools are revolutionizing agriculture and basic biology with their simplicity and precision ability to modify target genomic loci. Software-predicted guide RNAs (gRNAs) often fail to induce efficient cleavage at target loci. Many target loci are inaccessible due to complex chromatin structure. Currently, there is no suitable tool available to predict the architecture of genomic target sites and their accessibility. Hence, significant time and resources are spent on performing editing experiments with inefficient guides. Although in vitro-cleavage assay could provide a rough assessment of gRNA efficiency, it largely excludes the interference of native genomic context. Transient in-vivo testing gives a proper assessment of the cleavage ability of editing reagents in a native genomic context. Here, we developed a modified protocol that offers highly efficient protoplast isolation from rice, Arabidopsis, and chickpea, using a sucrose gradient, transfection using PEG (polyethylene glycol), and validation of single guide RNAs (sgRNAs) cleavage efficiency of CRISPR-Cas9. We have optimized various parameters for PEG-mediated protoplast transfection and achieved high transfection efficiency using our protocol in both monocots and dicots. We introduced plasmid vectors containing Cas9 and sgRNAs targeting genes in rice, Arabidopsis, and chickpea protoplasts. Using dual sgRNAs, our CRISPR-deletion strategy offers straightforward detection of genome editing success by simple agarose gel electrophoresis. Sanger sequencing of PCR products confirmed the editing efficiency of specific sgRNAs. Notably, we demonstrated that isolated protoplasts can be stored for up to 24/48 h with little loss of viability, allowing a pause between isolation and transfection. This high-efficiency protocol for protoplast isolation and transfection enables rapid (less than 7 days) validation of sgRNA cleavage efficiency before proceeding with stable transformation. The isolation and transfection method can also be utilized for rapid validation of editing strategies, evaluating diverse editing reagents, regenerating plants from transfected protoplasts, gene expression studies, protein localization and functional analysis, and other applications.

Fusion of a rice endogenous N-methylpurine DNA glycosylase to a plant adenine base transition editor ABE8e enables A-to-K base editing in rice plants

Engineering of a new type of plant base editor for simultaneous adenine transition and transversion within the editing window will greatly expand the scope and potential of base editing in directed evolution and crop improvement. Here, we isolated a rice endogenous hypoxanthine excision protein, N-methylpurine DNA glycosylase (OsMPG), and engineered two plant A-to-K (K = G or T) base editors, rAKBE01 and rAKBE02, for simultaneous adenine transition and transversion base editing in rice by fusing OsMPG or its mutant mOsMPG to a plant adenine transition base editor, ABE8e. We further coupled either OsMPG or mOsMPG with a transactivation factor VP64 to generate rAKBE03 and rAKBE04, respectively. Testing these four rAKBEs, at five endogenous loci in rice protoplasts, indicated that rAKBE03 and rAKBE04 enabled higher levels of A-to-G base transitions when compared to ABE8e and ABE8e-VP64. Furthermore, whereas rAKBE01 only enabled A-to-C/T editing at one endogenous locus, in comparison with rAKBE02 and rAKBE03, rAKBE04 could significantly improve the A-to-C/T base transversion efficiencies by up to 6.57- and 1.75-fold in the rice protoplasts, respectively. Moreover, although no stable lines with A-to-C transversion were induced by rAKBE01 and rAKBE04, rAKBE04 could enable simultaneous A-to-G and A-to-T transition and transversion base editing, at all the five target loci, with the efficiencies of A-to-G transition and A-to-T transversion editing ranging from 70.97 to 92.31% and 1.67 to 4.84% in rice stable lines, respectively. Together, these rAKBEs enable different portfolios of editing products and, thus, now expands the potential of base editing in diverse application scenario for crop improvement.

Rice NLR protein XinN1, induced by a pattern recognition receptor XA21, confers enhanced resistance to bacterial blight.

Rice CC-type NLR XinN1, specifically induced by a PRR XA21, activates defense pathways against Xoo. Plants have evolved two layers of immune systems regulated by two different types of immune receptors, cell surface located pattern recognition receptors (PRRs) and intracellular nucleotide-binding domain leucine-rich repeat-containing receptors (NLRs). Plant PRRs recognize conserved molecular patterns from diverse pathogens, resulting in pattern-triggered immunity (PTI), whereas NLRs are activated by effectors secreted by pathogens into plant cells, inducing effector-triggered immunity (ETI). Rice PRR, XA21, recognizes a tyrosine-sulfated RaxX peptide (required for activation of XA21-mediated immunity X) as a molecular pattern secreted by Xanthomonas oryzae pv. oryzae (Xoo). Here, we identified a rice NLR gene, XinN1, that is specifically induced during the XA21-mediated immune response against Xoo. Transgenic rice plants overexpressing XinN1 displayed increased resistance to infection by Xoo with reduced lesion length and bacterial growth. Overexpression of autoactive mutant of XinN1 (XinN1D543V) also displayed increased resistance to Xoo, accompanied with severe growth retardation and cell death. In rice protoplast system, overexpression of XinN1 or XinN1D543V significantly elevated reactive oxygen species (ROS) production and cytosolic-free calcium (Ca2+) accumulations. In addition, XinN1 overexpression additionally elevated the ROS burst caused by the interaction between XA21 and RaxX-sY and induced the transcription of PTI signaling components, including somatic embryogenesis receptor kinases (OsSERKs) and receptor-like cytoplasmic kinases (OsRLCKs). Our results suggest that XinN1 induced by the PRR XA21 activates defense pathways and provides enhanced resistance to Xoo in rice.

The synthetic NLR RGA5HMA5 requires multiple interfaces within and outside the integrated domain for effector recognition

Some plant sensor nucleotide-binding leucine-rich repeat (NLR) receptors detect pathogen effectors through their integrated domains (IDs). Rice RGA5 sensor NLR recognizes its corresponding effectors AVR-Pia and AVR1-CO39 from the blast fungus Magnaporthe oryzae through direct binding to its heavy metal-associated (HMA) ID to trigger the RGA4 helper NLR-dependent resistance in rice. Here, we report a mutant of RGA5 named RGA5HMA5 that confers complete resistance in transgenic rice plants to the M. oryzae strains expressing the noncorresponding effector AVR-PikD. RGA5HMA5 carries three engineered interfaces, two of which lie in the HMA ID and the other in the C-terminal Lys-rich stretch tailing the ID. However, RGA5 variants having one or two of the three interfaces, including replacing all the Lys residues with Glu residues in the Lys-rich stretch, failed to activate RGA4-dependent cell death of rice protoplasts. Altogether, this work demonstrates that sensor NLRs require a concerted action of multiple surfaces within and outside the IDs to both recognize effectors and activate helper NLR-mediated resistance, and has implications in structure-guided designing of sensor NLRs.

Co-Immunoprecipitation (Co-IP) using pAN580 in Nicotiana benthamiana or Rice Protoplasts

Co-Immunoprecipitation (Co-IP) using pAN580 in Nicotiana benthamiana or Rice Protoplasts

Antiviral Activity of Ailanthone from Ailanthus altissima on the Rice Stripe Virus.

Rice stripe disease caused by the rice stripe virus (RSV), which infects many Poaceae species in nature, is one of the most devastating plant viruses in rice that causes enormous losses in production. Ailanthone is one of the typical C20 quassinoids synthesized by the secondary metabolism of Ailanthus altissima, which has been proven to be a biologically active natural product with promising prospects and great potential for use as a lead structure for pesticide development. Based on the achievement of the systemic infection and replication of RSV in Nicotiana benthamiana plants and rice protoplasts, the antiviral properties of Ailanthone were investigated by determining its effects on viral-coding RNA gene expression using reverse transcription polymerase chain reaction, and Western blot analysis. Ailanthone exhibited a dose-dependent inhibitory effect on RSV NSvc3 expression in the assay in both virus-infected tobacco plants and rice protoplasts. Further efforts revealed a potent inhibitory effect of Ailanthone on the expression of seven RSV protein-encoding genes, among which NS3, NSvc3, NS4, and NSvc4 are the most affected genes. These facts promoted an extended and greater depth of understanding of the antiviral nature of Ailanthone against plant viruses, in addition to the limited knowledge of its anti-tobacco mosaic virus properties. Moreover, the leaf disc method introduced and developed in the study for the detection of the antiviral activity of Ailanthone facilitates an available and convenient screening method for anti-RSV natural products or synthetic chemicals.

The seven rice vacuolar sorting receptors localize to prevacuolar compartments

Vacuolar sorting is critically important in plants as it regulates the mobilization of proteins and plays a major role in important agricultural traits like yield and seed protein content. Vacuolar sorting receptors (VSRs) are integral membrane proteins that mediate protein trafficking from the Golgi apparatus to the vacuole via the intermediate membrane-bound prevacuolar compartment (PVC)/multivesicular body (MVB). VSR proteins, such as an 80 kD (BP-80) from pea, also serve as markers for PVC/MVB. Dissecting VSR-mediated protein trafficking pathways may provide ways to enhance agronomic traits and crop yield. Green fluorescence protein (GFP) fusions with the seven Arabidopsis (Arabidopsis thaliana) VSRs were previously shown to localize to PVCs in transgenic tobacco BY-2 cells. The Rice (Oryza sativa) genome contains seven VSRs (OsVSR1-7), but little is known about their subcellular localizations. Here we studied the subcellular localization of OsVSR1-7 b y using a reporter approach, in which GFP-OsVSR1-7 fusions containing the transmembrane domain (TMD) and cytoplasmic tail (CT) of individual OsVSR were expressed in the protoplasts of rice, transgenic tobacco BY-2 cells and transgenic rice plants. Immunofluorescent labelling studies and confocal laser scanning microscope observation demonstrated that the seven OsVSRs are localized to PVCs and form ring-like structures upon wortmannin treatment. Therefore, we have verified the subcellular localization of OsVSR1-7 in this study. The OsVSRs tagged with GFP can serve as PVCs/MVBs markers in rice for the future studies.

MiR2118 Negatively Regulates Bacterial Blight Resistance through Targeting Several Disease Resistance Genes in Rice.

Plant miRNAs are a class of noncoding RNA with a length of 21-24 nt that play an important role in plant responses to biotic and abiotic stresses. Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most serious bacterial diseases in rice. Our previous work showed that osa-miR2118b/n was induced by Xoo infection. However, the biological function of miR2118 has not yet been characterized in experiments. Herein, we constructed MIR2118b OE, as well as single and double mutants of MIR2118b/n using CRISPR/Cas9. Further results showed that osa-MIR2118b OE plants exhibited longer lesion lengths than the wild type after Xoo inoculation, while MIR2118 CRISPR plants exhibited shorter lesion lengths than the wild type after Xoo inoculation. Co-transformation experiments in rice protoplasts indicated that osa-miR2118 negatively regulated the transcripts of three nucleotide-binding sites and leucine-rich repeat (NLR) genes (LOC_Os08g42700.1, LOC_Os01g05600.1, and LOC_Os12g37290.1) which are predicted target genes of miR2118, but not the mutated NLR genes with a 3 bp insertion at the center of the binding sites. The transcriptional level of the three NLR genes was reversed relative to osa-miR2118 in the MIR2118b OE and MIR2118b CRISPR plants. The above results demonstrate that osa-miR2118b/n negatively regulates the resistance to bacterial blight through negatively regulating several NLR genes.