OsGEX3 affects anther development and improves osmotic stress tolerance in rice.

MAIN CONCLUSION: Overexpression and loss of function of OsGEX3 reduce seed setting rates and affect pollen fertility in rice. OsGEX3 positively regulates osmotic stress response by regulating ROS scavenging. GEX3 proteins are conserved in plants. AtGEX3 encodes a plasma membrane protein that plays a crucial role in pollen tube guidance. However, the function of its homolog in rice, OsGEX3, has not been determined. Our results demonstrate that OsGEX3 is localized in the plasma membrane and the nucleus as shown by a transiently transformed assay using Nicotiana benthamiana leaves. The up-regulation of OsGEX3 was detected in response to treatments with polyethylene glycol (PEG) 4000, hydrogen peroxide, and abscisic acid (ABA) via RT-qPCR analysis. Interestingly, we observed a significant decline in the seed setting rates of OsGEX3-OE lines and mutants, compared to the wild type. Further investigations reveal that overexpression and loss of function of OsGEX3 affect pollen maturation. TEM observation revealed a significant decrease in the fertile pollen rates of OsGEX3-OE transgenic lines and Osgex3 mutants due to a delay in pollen development at the late vacuolated stage. Overexpression of OsGEX3 improved osmotic stress and oxidative stress tolerance by enhancing reactive oxygen species (ROS) scavenging in rice seedlings, whereas Osgex3 mutants exhibited an opposite phenotype in osmotic stress. These findings highlight the multifunctional roles of OsGEX3 in pollen development and the response to abiotic stress. The functional characterization of OsGEX3 provides a fundamental basis for rice molecular breeding and can facilitate efforts to cultivate drought resistance and yield-related varieties.

OsNAC103, an NAC transcription factor negatively regulates plant height in rice.

MAIN CONCLUSION: OsNAC103 negatively regulates rice plant height by influencing the cell cycle and crosstalk of phytohormones. Plant height is an important characteristic of rice farming and is directly related to agricultural yield. Although there has been great progress in research on plant growth regulation, numerous genes remain to be elucidated. NAC transcription factors are widespread in plants and have a vital function in plant growth. Here, we observed that the overexpression of OsNAC103 resulted in a dwarf phenotype, whereas RNA interference (RNAi) plants and osnac103 mutants showed no significant difference. Further investigation revealed that the cell length did not change, indicating that the dwarfing of plants was caused by a decrease in cell number due to cell cycle arrest. The content of the bioactive cytokinin N6-Δ2-isopentenyladenine (iP) decreased as a result of the cytokinin synthesis gene being downregulated and the enhanced degradation of cytokinin oxidase. OsNAC103 overexpression also inhibited cell cycle progression and regulated the activity of the cell cyclin OsCYCP2;1 to arrest the cell cycle. We propose that OsNAC103 may further influence rice development and gibberellin-cytokinin crosstalk by regulating the Oryza sativa homeobox 71 (OSH71). Collectively, these results offer novel perspectives on the role of OsNAC103 in controlling plant architecture.

Cytosolic isocitrate dehydrogenase regulates plant stem cell maintenance in response to nutrient deficiency.

WUSCHEL (WUS) and WUSCHEL-RELATED HOMEOBOX (WOX) proteins determine stem cell maintenance for continual plant growth and development under changing environmental conditions. Nutrient availability is an environmental factor that substantially controls plant growth and development. However, how plant stem cell homeostasis is regulated under nutrient deficiency remains to be elucidated. Here, we showed that cytosolic isocitrate dehydrogenase (ICDH) plays an important role in nutrient sensing of stem cells in Arabidopsis (Arabidopsis thaliana). Nutrient deficiency induced the cytoplasmic-to-nuclear translocation of cytosolic ICDH protein. ICDH can interact with WUS/WOX protein as a complex that further promotes WUS/WOX expression by binding to its promoter. WUS/WOX expression in the icdh-2 mutant was lower than that of wild-type plants under nutrient deficiency. Consistently, loss of ICDH function caused a more serious growth repression under nutrient deficiency that was independent of ICDH's catalytic activity. Therefore, cytosolic ICDH regulates stem cell homeostasis of plants in response to nutrient deficiency.

The RNA binding protein OsLa influences grain and anther development in rice.

La proteins are found widely in eukaryotes and play a variety of vital roles. AtLa1 has been identified as an La protein that is necessary for embryogenesis in Arabidopsis; however, the existence and biological functions of La proteins in rice (Oryza sativa L.) remain unclear. In this study, we identified and characterized two La proteins in rice that are homologous to AtLa1 and named them OsLa1 and OsLa2. Both the OsLa1 and OsLa2 genes encode RNA-binding proteins with an La domain and two RNA-binding domains. Mutant OsLa1 reduced grain length and pollen fertility, whereas OsLa1 overexpression caused the opposite phenotypes. Further experiments indicated that OsLa1 modulates grain size by influencing cell expansion. Interestingly, mutant OsLa2 resulted in thin grains with decreased weight and a low seed-setting rate. We also found that OsLa1 interacted with OsLa2 and that both OsLa1 and OsLa2 interacted with OseIF6.1, a eukaryotic translation initiation factor involved in ribosome biogenesis. In addition, OsLa1 was able to bind to OseIF6.1 mRNA to modulate its expression. Complete OseIF6.1 knockout caused lethality and OseIF6.1/oseif6.1 heterozygous plants displayed low fertility and low seed setting. Together, our results enrich our knowledge of the role of La proteins in rice growth and development, as well as the relationship between La and eIF6 in rice.

Identification of natural allelic variation in TTL1 controlling thermotolerance and grain size by a rice super pan-genome.

Continuously increasing global temperatures present great challenges to food security. Grain size, one of the critical components determining grain yield in rice (Oryza sativa L.), is a prime target for genetic breeding. Thus, there is an immediate need for genetic improvement in rice to maintain grain yield under heat stress. However, quantitative trait loci (QTLs) endowing heat stress tolerance and grain size in rice are extremely rare. Here, we identified a novel negative regulator with pleiotropic effects, Thermo-Tolerance and grain Length 1 (TTL1), from the super pan-genomic and transcriptomic data. Loss-of-function mutations in TTL1 enhanced heat tolerance, and caused an increase in grain size by coordinating cell expansion and proliferation. TTL1 was shown to function as a transcriptional regulator and localized to the nucleus and cell membrane. Furthermore, haplotype analysis showed that hapL and hapS of TTL1 were obviously correlated with variations of thermotolerance and grain size in a core collection of cultivars. Genome evolution analysis of available rice germplasms suggested that TTL1 was selected during domestication of the indica and japonica rice subspecies, but still had much breeding potential for increasing grain length and thermotolerance. These findings provide insights into TTL1 as a novel potential target for the development of high-yield and thermotolerant rice varieties.

NbJAZ3 is required for jasmonate-meditated glandular trichome development in Nicotiana benthamiana.

Exogenous methyl jasmonate (MeJA) treatment induces glandular trichome development in Nicotiana benthamiana, but the function of JAZ proteins, acting as core repressors, and their downstream genes have not been clearly shown in plants. Here, a bioinformatics analysis of 71 JAZ genes from tobacco, Arabidopsis thaliana, and tomato was carried out and shown to share highly conserved domains. Then, the expression profile of 17 NbJAZs in different tissues was analyzed, and NbJAZ3 was highly expressed in trichome. Through transgenic technology, we demonstrated that the glandular trichome density of NbJAZ3-overexpression lines significantly decreased with lower expression levels of NbWo, NbCycB2, and NbMIXTA. In contrast, the trichome density of NbJAZ3 RNAi lines slightly increased with higher expression level of NbWo. Given the negative protein feedback regulation relationship between NbCycB2 and NbWo, we verified that MeJA induced NbWo expression. NbWo was a direct target gene of NbJAZ3 and further demonstrated that NbJAZ3 inhibited the transcriptional activation of NbCycB2 by NbWo. Together, our findings outline a novel JA-meditated glandular trichome development model consisting of the NbJAZ3-NbWo-NbCycB2 axis.

Derepression of specific miRNA-target genes in rice using CRISPR/Cas9.

MicroRNAs (miRNAs) target specific mRNA molecules based on sequence complementarity for their degradation or repression of translation, thereby regulating various developmental and physiological processes in eukaryotic organisms. Expressing the target mimicry (MIM) and short tandem target mimicry (STTM) can block endogenous activity of mature miRNAs and eliminate the inhibition of their target genes, resulting in phenotypic changes due to higher expression of the target genes. Here, we report a strategy to achieve derepression of interested miRNA-target genes through CRISPR/Cas9-based generation of in-frame mutants within the miRNA-complementary sequence of the target gene. We show that two rice genes, OsGRF4 (GROWTH REGULATING FACTOR 4) and OsGRF8 carrying in-frame mutants with disruption of the miR396 recognition sites, escape from miR396-mediated post-transcriptional silencing, resulting in enlarged grain size and increase in brown planthopper (BPH) resistance, in their respective transgenic rice lines. These results demonstrate that CRISPR/Cas9-mediated disruption of miRNA target sites can be effectively employed to precisely derepress particular target genes of functional importance for trait improvement in plants.

NbCycB2 represses Nbwo activity via a negative feedback loop in tobacco trichome development.

The transcription factor Woolly (Wo) and its downstream gene CycB2 have been shown to regulate trichome development in tomato (Solanum lycopersicum). It has been demonstrated that only the gain-of-function allele of Slwo (SlWoV, the Slwo woolly motif mutant allele) can increase the trichome density; however, it remains unclear why the two alleles function differently in trichome development. In this study, we used Nicotiana benthamiana as a model and cloned the homologues of Slwo and SlCycB2 (named Nbwo and NbCycB2). We also constructed a Nbwo gain-of-function allele with the same mutation site as SlWoV (named NbWoV). We found that both Nbwo and NbWoV directly regulate NbCycB2 and their own expression by binding to the promoter of NbCycB2 and their own genomic sequences. As form of a feedback regulation, NbCycB2 negatively regulates trichome formation by repressing Nbwo activity at the protein level. We also found that mutations in the Nbwo woolly motif can prevent repression of NbWoV by NbCycB2, which results in a significant increase in the amount of active Nbwo proteins and in increases in trichome density and the number of branches. Our results reveal a novel reciprocal regulation mechanism between NbCycB2 and Nbwo during trichome formation in N. benthamiana.

Comparison of immune responses induced by porcine parvovirus virus-like particles and inactivated vaccine.

Laboratory-prepared inactivated porcine parvovirus (PPV) vaccines and VP2 virus-like particles (VLPs) were utilized to immunize gilts. PPV BQ strain and SP2/0 cells were used. Hemagglutination-inhibiting (HI) antibody titers were measured in the immunized gilts and the differences in cytokine production of interferon gamma (IFN-γ, IL-2 and IL-4) were compared. CD4+ and CD8+ T cells proliferation were compared by flow cytometry. The variation between the immune response level induced by inactivated PPV vaccine and VP2 VLPs were determined. The results showed that all vaccinated gilts had HI antibody titers reaching 1:256 for at least one month post immunization and the peak level of antibody could be sustained for one month; further, PPV antibodies could be detected in the second week post immunization with VP2 VLPs. We also found that the level of cytokines (IFN-γ, IL-2 and IL-4) were all increased post immunization and continued to rise after the booster immunization; the level of increase in IFN-γ and IL-2 were significantly higher than IL-4. The flow cytometry results showed that the numbers of the CD4+ and CD8+ T cells subsets were significantly higher in the groups immunized with inactivated PPV vaccine or VP2 VLPs than those of negative control group (p<0.01); additionally, the number of CD4+ cells in the gilts that received VP2 VLP immunization was significantly higher than the inactivated vaccine group (p<0.01). In summary, the inactivated PPV vaccine and PPV VP2 VLPs were both able to induce humoral and cellular immunity, but the VP2 VLPs lead to better cellular immune responses in gilts compared to those of the inactivated vaccine..

AtLa1 protein initiates IRES-dependent translation of WUSCHEL mRNA and regulates the stem cell homeostasis of Arabidopsis in response to environmental hazards.

Plant stem cells are hypersensitive to environmental hazards throughout their life cycle, but the mechanism by which plants safeguard stem cell homeostasis in response to environmental hazards is largely unknown. The homeodomain transcription factor WUSCHEL (WUS) protein maintains the stem cell pool in the shoot apical meristem of Arabidopsis. Here, we demonstrate that the translation of WUS mRNA is directed by an internal ribosomal entry site (IRES) located in the 5'-untranslated region. The AtLa1 protein, an RNA-binding factor, binds to the 5'-untranslated region and initiates the IRES-dependent translation of WUS mRNA. Knockdown of AtLa1 expression represses the WUS IRES-dependent translation and leads to the arrest of growth and development. The AtLa1 protein is mainly located in the nucleoplasm. However, environmental hazards promote the nuclear-to-cytoplasmic translocation of the AtLa1 protein, which further enhances the IRES-dependent translation of WUS mRNA. Genetic evidence indicates that the WUS protein increases the tolerance of the shoot apical meristem to environmental hazards. Based on these results, we conclude that the stem cell niche in Arabidopsis copes with environmental hazards by enhancing the IRES-dependent translation of WUS mRNA under the control of the AtLa1 protein.

OsSIDP301, a Member of the DUF1644 Family, Negatively Regulates Salt Stress and Grain Size in Rice.

As a major environmental factor, salt stress substantially retards growth and reduces the productivity of rice (Oryza sativa). Members of the DUF1644 family, "the domains of unknown function 1644 motif" are predicted to play an essential regulatory role in response to abiotic stress. However, the specific molecular mechanisms of most members of this family remain elusive. Here, we report that the OsSIDP301 (stress-induced DUF1644 protein) was induced by salt stress and abscisic acid (ABA). We found that overexpression of OsSIDP301 (OE) in plants conferred salt hypersensitivity and reduced grain size, whereas plants with OsSIDP301 RNA interference (RNAi) exhibited salt tolerance and increased grain size in rice. OsSIDP301 determines salt stress tolerance by modulating genes involved in the salt-response and ABA signaling pathways. Further studies suggest that OsSIDP301 regulates grain size by influencing cell expansion in spikelet hulls. Moreover, OsSIDP301 interacts with OsBUL1 COMPLEX1 (OsBC1), which positively regulates grain size in rice. Our findings reveal that OsSIDP301 functions as a negative regulator of salt stress and grain size, and repressing its expression represents a promising strategy for improving salt stress tolerance and yield in rice.

RNA Binding Protein OsTZF7 Traffics Between the Nucleus and Processing Bodies/Stress Granules and Positively Regulates Drought Stress in Rice.

Tandem CCCH zinc finger (TZF) proteins are the essential components of processing bodies (PBs) and stress granules (SGs), which play critical roles in growth development and stress response in both animals and plants through posttranscriptional regulation of target mRNA. In this study, we characterized the biological and molecular functions of a novel tandem zinc finger protein, OsTZF7. The expression of OsTZF7 was upregulated by abiotic stresses, including polyethylene glycol (PEG) 4000, NaCl, and abscisic acid (ABA) in rice. Accordingly, the overexpression of OsTZF7 increased drought tolerance and enhanced sensitivity to exogenous ABA in rice, whereas the knockdown of OsTZF7 resulted in the opposite phenotype. RNA-seq analysis revealed that genes related to "response to stress," "abscisic acid signaling," "methylated histone binding," and "cytoplasmic mRNA processing body" are regulated by OsTZF7. We demonstrated that OsTZF7 can traffic between the nucleus and PBs/SGs, and the leucine-rich nuclear export signal (NES) mediates the nuclear export of OsTZF7. Additionally, we revealed that OsTZF7 can bind adenine- and uridine-rich (AU-rich) element (ARE) or ARE-like motifs within the 3' untranslated region of downregulated mRNAs, and interact with PWWP family proteins in vitro. Together, these results indicate that OsTZF7 positively regulates drought response in rice via ABA signaling and may be involved in mRNA turnover.

APICAL SPIKELET ABORTION (ASA) Controls Apical Panicle Development in Rice by Regulating Salicylic Acid Biosynthesis.

Panicle degradation causes severe yield reduction in rice. There are two main types of panicle degradation: apical spikelet abortion and basal degeneration. In this study, we isolated and characterized the apical panicle abortion mutant apical spikelet abortion (asa), which exhibits degeneration and defects in the apical spikelets. This mutant had a pleiotropic phenotype, characterized by reduced plant height, increased tiller number, and decreased pollen fertility. Map-based cloning revealed that OsASA encodes a boric acid channel protein that showed the highest expression in the inflorescence, peduncle, and anther. RNA-seq analysis of the asa mutant vs wild-type (WT) plants revealed that biological processes related to reactive oxygen species (ROS) homeostasis and salicylic acid (SA) metabolism were significantly affected. Furthermore, the asa mutants had an increased SA level and H2O2 accumulation in the young panicles compared to the WT plants. Moreover, the SA level and the expression of OsPAL3, OsPAL4, and OsPAL6 genes (related to SA biosynthesis) were significantly increased under boron-deficient conditions in the asa mutant and in OsASA-knockout plants. Collectively, these results suggest that the boron distribution maintained by OsASA is required for normal panicle development in a process that involves modulating ROS homeostasis and SA biosynthesis.

The AtMC4 regulates the stem cell homeostasis in Arabidopsis by catalyzing the cleavage of AtLa1 protein in response to environmental hazards.

The AtLa1 protein is an RNA binding factor that initiates the translation of WUSCHEL (WUS) mRNA in Arabidopsis. The AtLa1 protein can regulate the stem cell homeostasis via the nuclear-to-cytoplasmic translocation in response to environmental hazards. However, the translocation mechanism of AtLa1 protein remains to be elucidated. In the present study, we have explored the role of AtMC4 protein in the subcellular translocation of AtLa1 protein. Our results showed that the AtLa1 protein is a substrate of AtMC4 protein. The AtMC4 protein can interact with AtLa1 protein and catalyze the cleavage of the C-terminal nuclear localization signal peptide of AtLa1 protein. The AtMC4 protein is mainly distributed in the cytoplasm. In the presence of environmental stresses, the cytoplasmic-to-nuclear translocation of AtMC4 protein is enhanced with the result that more AtLa1 protein can be cleaved and transported from nucleus to cytoplasm, where AtLa1 protein further initiates the translation of WUS mRNA. By contrast, knockdown of AtMC4 expression inhibits the nuclear-to-cytoplasmic translocation of AtLa1 protein and the WUS protein translation. Based on these results, we conclude that the AtMC4 protein regulates stem cell homeostasis by catalyzing the cleavage of AtLa1 protein in response to environmental hazards.

The complete mitochondrial genome sequence of Drepanidotaenia lanceolata (Cyclophyllidea: Hymenolepididae).

The complete mitochondrial genome of Drepanidotaenia lanceolata was sequenced. The complete mtDNA sequence is 13 573 bp long and contains 12 protein-coding genes (cox1-3, nad1-6, nad4L, atp6, and cytb), two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and 2 non-coding regions (NCR). Phylogenetic analysis using three methods (Bayesian inference, maximum parsimony, and maximum likelihood) demonstrates with high statistical support that D. lanceolata is closely related to the genus Hymenolepis. This study reports the first complete mt genome sequence of a representative species of the genus Drepanidotaenia and should provide novel genetic data for further studies of the taxonomy, systematics, and population genetics of these and other related cestodes of socio-economic significance.

A new nanoPCR molecular assay for detection of porcine bocavirus.

Nanoparticle-assisted polymerase chain reaction (nanoPCR) is a novel method for the rapid amplification of DNA and has been used for the detection of virus. For detection of porcine bocavirus (PBoV), a sensitive and specific nanoPCR assay was developed with a pair of primers that were designed based on NS1 gene sequences available in GenBank. Under the optimized conditions of the PBoV nanoPCR assay, the nanoPCR assay was 100-fold more sensitive than a conventional PCR assay. The lower detection limit of the nanoPCR assay was about 6.70×10(1) copies. The nanoPCR assay amplified the specific 482-bp fragment of the PBoV NS1 recombinant plasmid but did not produce any product with genomic DNA or cDNA of porcine parvovirus, porcine circovirus type II, porcine reproductive and respiratory syndrome virus, pseudorabies virus, classic swine fever virus, Encephalomyocarditis virus, Porcine Teschovirus or African swine fever virus plasmid. Of 65 clinical samples collected from diseased pigs, 73.8% and 86.2% were determined to be PBoV positive by PBoV conventional PCR and PBoV nanoPCR assay, respectively. Of 36 clinical samples from healthy pigs, 27.8% and 44.4% were PBoV positive by PBoV conventional PCR and PBoV nanoPCR assay, respectively. The nanoPCR assay will be useful for diagnosing PBoV and for studying its epidemiology and pathology.

Development and evaluation of the rVP-ELISA for detection of antibodies against porcine parvovirus.

The gene encoding the VP2 protein of porcine parvovirus (PPV) was expressed in an insect-baculovirus system. The recombinant (r) VP2 was similar antigenically/functionally to the native capsid protein as demonstrated by hemagglutination (HA), Western blotting using PPV positive sera. The purified rVP2 proteins were used as coating antigen to establish a rVP-ELISA method for detection of PPV positive and negative sera from pigs. The optimal operating conditions of the rVP-ELISA were: the concentration of rVP2 proteins coated on the wells was 2 µg/mL; the diluted concentration of serum was 1: 150 and that of the enzyme-labeled antibody was 1: 6000. A total of 596 sera were detected by this assay, and the average positive rate was 87%. Compared with France LSI kit, the result showed that the coincidence rate was 96.7%. In conclusion, the rVP2-ELISA is a sensitive and specific method for detecting antibodies against PPV.

A nanoparticle-assisted PCR assay to improve the sensitivity for rapid detection and differentiation of wild-type pseudorabies virus and gene-deleted vaccine strains.

Nanoparticle-assisted polymerase chain reaction (nanoPCR) is a novel method for the rapid amplification of DNA and has been adopted for the detection of virus because of its simplicity, rapidity, and specificity. A nanoPCR assay was developed to detect and differentiate wild-type and gene-deleted pseudorabies virus (PRV). Three pairs of primers for nanoPCR developed in this study were selected from conserved regions of PRV, producing specific amplicons of 431 bp (gB), 316 bp (gE), and 202 bp (gG). The sensitivity of this assay using purified plasmid constructs containing the specific gene fragments was 100-1000 fold higher than conventional PCR. The PRV nanoPCR assay did not amplify porcine parvovirus, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, porcine teschovirus, or African swine fever virus but produced three bands of expected size with PRV and two bands of expected size with the gene-deleted PRV-Bartha-K61. Of 110 clinical samples collected from seven provinces in China, 53% and 48% were positive for wild-type PRV according to the nanoPCR assay and virus isolation, respectively.