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The spatiotemporal control of meristem identity is critical for determining inflorescence architecture, and thus yield, of cereal plants. However, the precise mechanisms underlying inflorescence and spikelet meristem determinacy in cereals are still largely unclear. We have generated loss-of-function and overexpression mutants of the paralogous OsMADS5 and OsMADS34 genes in rice (Oryza sativa), and analysed their panicle phenotypes. Using chromatin immunoprecipitation, electrophoretic mobility-shift and dual-luciferase assays, we have also identified RICE CENTRORADIALIS 4 (RCN4), a TFL1-like gene, as a direct downstream target of both OsMADS proteins, and have analysed RCN4 mutants. The osmads5 osmads34 mutant lines had significantly enhanced panicle branching with increased secondary, and even tertiary and quaternary, branches, compared to wild type and osmads34 plants. The osmads34 mutant phenotype could largely be rescued by also knocking out RCN4. Moreover, transgenic panicles overexpressing RCN4 had significantly increased branching, and initiated development of ~7× more spikelets than wild type. Our results reveal a role for OsMADS5 in panicle development, and show that OsMADS5 and OsMADS34 play similar functions in limiting branching and promoting the transition to spikelet meristem identity, in part by repressing RCN4 expression. These findings provide new insights to better understand the molecular regulation of rice inflorescence architecture.

Panicle architecture is one of the major factors influencing productivity of rice crops. The regulatory mechanisms underlying this complex trait are still unclear and genetic resources for rice breeders to improve panicle architecture are limited. Here, we have performed a genome-wide association study (GWAS) to analyze and identify genetic determinants underlying three panicle architecture traits. A population of 340 rice accessions from the 3000 Rice Genomes Project was phenotyped for panicle length, primary panicle number and secondary branch number over two years; GWAS was performed across the whole panel, and also across the japonica and indica sub-panels. A total of 153 quantitative trait loci (QTLs) were detected, of which 5 were associated with multiple traits, 8 were unique to either indica or japonica sub-panels, while 37 QTLs were stable across both years. Using haplotype and expression analysis, we reveal that genetic variations in the OsSPL18 promoter significantly affect gene expression and correlate with panicle length phenotypes. Three new candidate genes with putative roles in determining panicle length were also identified. Haplotype analysis of OsGRRP and LOC_Os03g03480 revealed high association with panicle length variation. Gene expression of DSM2, involved in abscisic acid biosynthesis, was up-regulated in long panicle accessions. Our results provide valuable information and resources for further unravelling the genetic basis determining rice panicle architecture. Identified candidate genes and molecular markers can be used in marker-assisted selection to improve rice panicle architecture through molecular breeding.

Seed germination is critical for plant survival and agricultural production, which is affected by both internal seed factors and external environmental conditions. However, the genetic basis and underlying molecular mechanisms of early seed germination in crops remain largely unclear. Here, we report that R2R3 MYB transcription factor Carbon Starved Anther (CSA) is expressed specifically in Oryza sativa embryo and aleurone in response to seed imbibition, peaking at 3–6 h and undetectable by 24-h post-imbibition. CSA seeds germinated more quickly than wild-type rice seeds and had higher levels of amylase activity, glucose, and inactive abscisic acid-glucose ester (ABA-GE), but lower levels of ABA. Through analyzing the CSA-associated transcriptome and CSA binding to downstream target genes, we identified two glycolytic genes as direct CSA targets. CSA inhibits Amylase 3A expression to limit glucose production from starch and activates Os3BGlu6 expression to promote de-conjugation of ABA-GE to ABA; these functions serve to slow germination and improve seedling resilience to abiotic stress in the first 3 weeks of growth. Therefore, this study unveils a protection mechanism conferred by CSA during early seed germination by balancing glucose and ABA metabolism to optimize seed germination and stress response fitness.

Several QTLs and genes responsible for seed dormancy were detected and SNP candidates were shown to cause changes in seed germination. Seed dormancy is a key agricultural trait to prevent pre-harvest sprouting in crop plants such as rice (Oryza sativa L.), wheat (Triticum aestivum), and barley (Hordeum vulgare L.). However, our knowledge of seed dormancy is hampered by the complexities of studying a trait that changes over time after seed harvest, and is complicated by interactions between phytohormones, seed coat components and the environment. Here, we have conducted a genome-wide association study using a panel of 311 natural accessions of cultivated rice, examining a total of 519,158 single nucleotide polymorphisms (SNPs). Eight quantitative trait loci (QTLs) were found to associate with seed dormancy in the whole panel and five in the Japonica and Indica subpanel; expression of candidate genes within 100 kb of each QTL was examined in two published, germination-specific transcriptomic datasets. Ten candidate genes, differentially expressed within the first four days post-imbibition, were identified. Five of these genes had previously been associated with awn length, heading date, yield, and spikelet length phenotypes. Two candidates were validated using Quantitative Reverse Transcription (qRT)-PCR. In addition, previously identified genes involved in hormone signaling during germination were found to be differentially expressed between a japonica and an indica line; SNPs in the promoter of Os9BGlu33 were associated with germination index, with qRT-PCR validation. Collectively, our results are useful for future characterization of seed dormancy mechanism and crop improvement, and suggest haplotypes for further analysis that may be of use to boost PHS resistance in rice.

Temperature stresses affect plant phenotypic diversity. The developmental stability of the inflorescence, required for reproductive success, is tightly regulated by the interplay of genetic and environmental factors. However, the mechanisms underpinning how plant inflorescence architecture responds to temperature are largely unknown. We demonstrate that the barley SEPALLATA MADS-box protein HvMADS1 is responsible for maintaining an unbranched spike architecture at high temperatures, while the loss-of-function mutant forms a branched inflorescence-like structure. HvMADS1 exhibits increased binding to target promoters via A-tract CArG-box motifs, which change conformation with temperature. Target genes for high-temperature-dependent HvMADS1 activation are predominantly associated with inflorescence differentiation and phytohormone signalling. HvMADS1 directly regulates the cytokinin-degrading enzyme HvCKX3 to integrate temperature response and cytokinin homeostasis, which is required to repress meristem cell cycle/division. Our findings reveal a mechanism by which genetic factors direct plant thermomorphogenesis, extending the recognized role of plant MADS-box proteins in floral development.

Photoperiod-dependent male fertility is a critical enabler of modern hybrid breeding. A MYB transcription factor, CSA, is a key regulator of sugar partitioning in rice anthers, disruption of which causes photoperiod-sensitive male sterility. However, little is known about the molecular mechanisms governing plant fertility in response to photoperiod. Here, we have obtained another rice photoperiod-sensitive male sterile mutant, csa2, which exhibits semi-sterility under long-day (LD) conditions, with normal fertility under short-day (SD) conditions. CSA2 specifically expressed in anthers, and here is shown to be indispensable for sugar partitioning to anthers under LD conditions. The CSA2 protein can restore the fertility of csa mutants under SD conditions when expressed in a CSA-specific pattern, indicating that the two proteins share common downstream regulatory targets. Transcriptomic analyses also reveal discrete regulatory targets in anthers. Furthermore, the regulatory role of CSA2 in sugar transport was influenced by the photoperiod conditions during floral initiation, not simply during anther development. Collectively, we propose that rice evolved at least two MYB proteins, CSA2 and CSA, that regulate sugar transport in anthers under LD and SD conditions, respectively. This finding provides insight into the molecular mechanisms that regulate male fertility in response to photoperiod.

Increasing crop production to meet the demands of a growing population depends largely on crop improvement through new plant-breeding techniques (NPBT) such as genome editing. CRISPR/Cas systems are NPBTs that enable efficient target-specific gene editing in crops, which is supposed to accelerate crop breeding in a way that is different from genetically modified (GM) technology. Herein, we review the applications of CRISPR/Cas systems in crop breeding focusing on crop domestication, heterosis, haploid induction, and synthetic biology, and summarize the screening methods of CRISPR/Cas-induced mutations in crops. We highlight the importance of molecular characterization of CRISPR/Cas-edited crops, and pay special attentions to emerging highly specific genome-editing tools such as base editors and prime editors. We also discuss future improvements of CRISPR/Cas systems for crop improvement.

The spikelet is a unique structure of inflorescence in grasses that generates one to many flowers depending on its determinate or indeterminate meristem activity. The growth patterns and number of spikelets, furthermore, define inflorescence architecture and yield. Therefore, understanding the molecular mechanisms underlying spikelet development and evolution are attractive to both biologists and breeders. Based on the progress in rice and maize, along with increasing numbers of genetic mutants and genome sequences from other grass families, the regulatory networks underpinning spikelet development are becoming clearer. This is particularly evident for domesticated traits in agriculture. This review focuses on recent progress on spikelet initiation, and spikelet and floret fertility, by comparing results from Arabidopsis with that of rice, sorghum, maize, barley, wheat, Brachypodium distachyon, and Setaria viridis. This progress may benefit genetic engineering and molecular breeding to enhance grain yield.

Cytochrome P450 monooxygenase 704B (CYP704B), a member of the CYP86 clan, was found to be needed in Arabidopsis and rice to biosynthesize precursors of sporopollenin through oxidizing fatty acids. In the present study, we cloned and characterized a CYP704B gene in Panax ginseng, named PgCYP704B1. It shared high sequence identity (98-99%) with CYP704 of Arabidopsis, Theobroma cacao, and Morus notabilis. The phylogenetic comparison of ginseng and higher plants between the members of CYP86 clan revealed that ginseng CYP704 was categorized as a group of CYP704B with dicot plants. The expression of PgCYP704B1 is low in the stem, leaf, and fruit, and high in flower buds, particularly detected in the young gametic cell and tapetum layer of the developing anther. Arabidopsis plants overexpressing PgCYP704B1 improved plant biomass such as plant height, siliques and seed number and size. A cytological observation by transverse and longitudinal semi-thin sections of the siliques cuticles revealed that the cell length increased. Furthermore a chemical analysis showed that PgCYP704B1ox lines increased their cutin monomers contents in the siliques. Our results suggest that PgCYP704B1 has a conserved role during male reproduction for fatty acid biosynthesis and its overexpression increases cutin monomers in siliques that eventually could be used for seed production.

The aperture on the pollen surface provides an exit for the emerging pollen tube. Apertures exhibit huge morphological variation across plant species-grasses, including rice, possess a complex aperture consisting of an annulus and an operculum-but little is known about how this species-specific cell-surface pattern forms. Here, we report a lectin receptor-like kinase in Oryza sativa, OsDAF1, which is essential for annulus formation and thus for fertility. OsDAF1 is evenly distributed in early microsporocytes but localizes to the distal pre-aperture site at the tetrad stage. We further reveal that the rice orthologue of a key aperture factor in Arabidopsis, OsINP1, has conserved and diversified roles in rice aperture formation. Disruption of OsINP1 prevents formation of the aperture, precluding pollen-tube germination. Furthermore, our results demonstrate that OsINP1 is required for polarization of OsDAF1 via direct protein interaction, suggesting that OsINP1 has an additional role in the formation of annulus that is absent in Arabidopsis. Our study reveals the importance of the aperture for rice grain yield and reveals mechanisms controlling pollen aperture development in cereal species.

The shape of an inflorescence varies among cereals, ranging from a highly branched panicle in rice to a much more compact spike in barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.). However, little is known about the molecular basis of cereal inflorescence architecture. We profiled transcriptomes at three developmental stages of the barley main shoot apex — spikelet initiation, floral organ differentiation, and floral organ growth — and compared them with those from vegetative seedling tissue. Transcript analyses identified 3688 genes differentially transcribed between the three meristem stages, with a further 1394 genes preferentially expressed in reproductive compared with vegetative tissue. Co-expression assembly and Gene Ontology analysis classified these 4888 genes into 28 clusters, revealing distinct patterns for genes such as transcription factors, histone modification, and cell-cycle progression specific for each stage of inflorescence development. We also compared expression patterns of VRS (SIX-ROWED SPIKE) genes and auxin-, gibberellic acid- and cytokinin-associated genes between two-rowed and six-rowed barley to describe regulators of lateral spikelet fertility. Our findings reveal barley inflorescence phase-specific gene expression, identify new candidate genes that regulate barley meristem activities and flower development, and provide a new genetic resource for further dissection of the molecular mechanisms of spike development. Keywords: Inflorescence meristem, Transcriptome, Gene expression, Hormones, Barley

Understanding the molecular basis of pollen germination in cereals holds great potential to improve yield. Pollen, a highly specialized haploid male gametophyte, transports sperm cells through a pollen tube to the female ovule for fertilization, directly determining grain yield in cereal crops. Although insights into the regulation of pollen germination and gamete interaction have advanced rapidly in the model Arabidopsis thaliana (arabidopsis), the molecular mechanisms in monocot cereals remain largely unknown. Recently, pollen-specific genome-wide and mutant analyses in rice and maize have extended our understanding of monocot regulatory components. We highlight conserved and diverse mechanisms underlying pollen hydration, germination, and tube growth in cereals that provide ideas for translating this research from arabidopsis. Recent developments in gene-editing systems may facilitate further functional genetic research.

Duck viral hepatitis (DVH) is an acute, highly lethal infectious disease of ducklings that causes huge losses in the duck industry. Duck hepatitis A virus genotype 3 (DHAV-3) has been one of the most prevalent DVH pathogen in the Asian duck industry in recent years. Here, we investigated the genetic basis of the resistance and susceptibility of ducks to DVH by comparing the genomes and transcriptomes of a resistant Pekin duck flock (Z8) and a susceptible Pekin duck flock (SZ7). Our comparative genomic and transcriptomic analyses suggested that NOD1 showed a strong signal of association with DVH susceptibility in ducks. Then, we found that NOD1 showed a significant expression difference between the livers of susceptible and resistant individuals after infection with DHAV-3, with higher expression in the SZ7 flock. Furthermore, suppression and overexpression experiments showed that the number of DHAV-3 genomic copies in primary duck hepatocytes was influenced by the expression level of NOD1. In addition, in situ RNAscope analysis showed that the localization of NOD1 and DHAV-3 in liver cells was consistent. Altogether, our data suggested that NOD1 was likely associated with DHAV-3 susceptibility in ducks, which provides a target for future investigations of the pathogenesis of DVH.

Organ size is mainly determined by cell division and cell expansion. Several genetic factors regulating development of plant lateral organs have been characterised, but those involved in determining reproductive organ size and separation in rice (Oryza sativa) remain unknown. We have isolated the rice gene SMALL REPRODUCTIVE ORGANS (SRO) encoding a nucleus-localised C2 H2 zinc finger protein orthologous to Arabidopsis transcription factor SUPERMAN (SUP). Combined developmental, genetic, histological and transcriptomic analyses were used to determine the function of SRO in regulating reproductive organ size. SRO affects genes involved in cell division, cell expansion and phytohormone signalling in the rice flower. SRO is specifically expressed in the first stages of stamen filament development to regulate their correct formation and separation. In addition, SRO non-cell-autonomously regulates the size and functionality of male and female reproductive organs. The B-class MADS-box gene OsMADS16/SPW1 is epistatic to SRO, while SRO regulates reproductive organ specification and floral meristem determinacy synergistically with C-class genes OsMADS3 and OsMADS58. These findings provide insights into how an evolutionarily conserved transcription factor has a pivotal role in reproductive organ development in core eudicots and monocots, through partially conserved expression, function, and regulatory network.

Molecular characterisation of genetically modified organisms (GMOs) yields basic information on exogenous DNA integration, including integration sites, entire inserted sequences and structures, flanking sequences and copy number, providing key data for biosafety assessment. However, there are few effective methods for deciphering transgene integration, especially for large DNA fragment integration with complex rearrangement, inversion, and tandem repeats. Herein, we developed a universal Large Integrated DNA Fragments Enrichment strategy combined with PacBio Sequencing (LIFE-Seq) for deciphering transgene integration in GMOs. Universal tilling DNA probes targeting transgenic elements and exogenous genes facilitate specific enrichment of large inserted DNA fragments associated with transgenes from plant genomes, followed by PacBio sequencing. LIFE-Seq were evaluated using six GM events and four crop species. Target DNA fragments averaging ∼6275 bp were enriched and sequenced, generating ∼26,352 high fidelity reads for each sample. Transgene integration structures were determined with high repeatability and sensitivity. Compared with whole-genome sequencing, LIFE-Seq achieved better data integrity and accuracy, greater universality, and lower cost, especially for transgenic crops with complex inserted DNA structures. LIFE-Seq could be applied in molecular characterisation of transgenic crops and animals, and complex DNA structure analysis in genetics research.

Jasmonate (JA) critically regulates plant development and stress response, but its spatio-temporal distribution at the cellular level remains unclear. A JA biosensor consisting of a JA degron motif Jas9 fused with the fluorescent protein VENUS was developed in Arabidopsis (Larrieu et al., 2015), but its 35S promoter has low activity in reproductive tissues and does not express well in monocotyledons, thus limiting its application in crops and reproductive development.

Previous studies resulted in the isolation of a low-virulence plaque-purified variant from the third passage (P3) in BHK-21 cells of a Tembusu virus (TMUV) isolate, suggesting the presence of viral quasispecies in the P3 culture. To confirm this notion, the fourth passage virus (P4) was prepared by infecting BHK-21 cells with P3 for isolation of more variants. We isolated 10 plaque-purified viruses. Comparative genome sequence analysis identified six of the 10 viruses as genetically different variants, which harbored a total of eight amino acid differences in the envelope, NS1, NS3, and NS5 proteins. When tested in a 2-day-old Pekin duck model, P4 caused 80 % mortality, belonging to a high-virulence TMUV strain. Out of the six genetically different variants, two presented high-virulence, one exhibited moderate-virulence, and three displayed low-virulence, causing 60 %–70 %, 40 %, and 10 % mortalities, respectively. These results demonstrate that P4 contains at least three groups of variants with distinct virulence phenotypes. Analysis of links between the eight residues and virulence of the six variants identified NS1 protein residue 183 and NS5 protein residues 275 and/or 287 as novel determinants of TMUV virulence. The analysis also provided a new clue for future studies on the molecular basis of TMUV virulence in terms of genetic interaction of different proteins. Overall, our study provides direct evidence to suggest that TMUV exists in in vitro culture of a virulent isolate as a quasispecies, which may enhance our understanding of molecular mechanism of TMUV virulence.

Glycoside hydrolase family1 β-glucosidases play a variety of roles in plants, but their in planta functions are largely unknown in rice (Oryza sativa). In this study, the biological function of Os12BGlu38, a rice β-glucosidase, expressed in bicellular to mature pollen, was examined. Genotype analysis of progeny of the self-fertilized heterozygous Os12BGlu38 T-DNA mutant, os12bglu38-1, found no homozygotes and a 1:1 ratio of wild type to heterozygotes. Reciprocal cross analysis demonstrated that Os12BGlu38 deficiency cannot be inherited through the male gamete. In cytological analysis, the mature mutant pollen appeared shrunken and empty. Histochemical staining and TEM showed that mutant pollen lacked intine cell wall, which was rescued by introduction of wild-type Os12BGlu38 genomic DNA. Metabolite profiling analysis revealed that cutin monomers and waxes, the components of the pollen exine layer, were increased in anthers carrying pollen of os12bglu38-1 compared with wild type and complemented lines. Os12BGlu38 fused with green fluorescent protein was localized to the plasma membrane in rice and tobacco. Recombinant Os12BGlu38 exhibited β-glucosidase activity on the universal substrate p-nitrophenyl β-d-glucoside and some oligosaccharides and glycosides. These findings provide evidence that function of a plasma membrane-associated β-glucosidase is necessary for proper intine development.

We recently developed a Tembusu virus (TMUV)-specific monoclonal antibody (MAb) 12F11, which was found to recognize a long amino acid sequence between residues 8 and 77 of domain III of the envelope protein (EDIII). Here, the epitope recognized by MAb 12F11 was mapped using alanine substitutions combined with dissociation constant analysis. The findings, and prediction of tertiary structure of TMUV EDIII, showed that the MAb 12F11 epitope contained one critical residue and 13 peripheral residues. Moreover, the antigenic site was shown to span four loops (N-terminal region, AB, BC, and CD) and three β-strands (A, B, and D). The present work contributes to the understanding of antigenic structure of TMUV envelope protein.

The timely programmed cell death (PCD) of the tapetum, the innermost somatic anther cell layer in flowering plants, is critical for pollen development, including the deposition and patterning of the pollen wall. Although several genes involved in tapetal PCD and pollen wall development have been characterized, the underlying regulatory mechanism remains elusive. Here we report that PERSISTENT TAPETAL CELL2 (PTC2), which encodes an AT-hook nuclear localized protein in rice (Oryza sativa), is required for normal tapetal PCD and pollen wall development. The mutant ptc2 showed persistent tapetal cells and abnormal pollen wall patterning including absent nexine, collapsed bacula, and disordered tectum. The defective tapetal PCD phenotype of ptc2 was similar to that of a PCD delayed mutant, ptc1, in rice, while the abnormal pollen wall patterning resembled that of a pollen wall defective mutant, Transposable Element Silencing Via AT-Hook, in Arabidopsis (Arabidopsis thaliana). Levels of anther cutin monomers in ptc2 anthers were significantly reduced, as was expression of a series of lipid biosynthetic genes. PTC2 transcript and protein were shown to be present in the anther after meiosis, consistent with the observed phenotype. Based on these data, we propose a model explaining how PTC2 affects anther and pollen development. The characterization of PTC2 in tapetal PCD and pollen wall patterning expands our understanding of the regulatory network of male reproductive development in rice and will aid future breeding approaches.

In flowering plants, pollen wall is a specialized extracellular cell-wall matrix surrounding male gametophytes and acts as a natural protector of pollen grains against various environmental and biological stresses. The formation of pollen wall is a complex but well-regulated process, which involves the action of many different genes. However, the genetic and molecular mechanisms underlying this process remain largely unknown. In this study, we isolated and characterized a novel rice male sterile mutant, defective pollen wall3 (dpw3), which displays smaller and paler anthers with aborted pollen grains. DPW3 encodes a novel membrane-associated alpha integrin-like protein conserved in land plants. DPW3 is ubiquitously expressed in anther developmental stages and its protein is localized to the plasma membrane, endoplasmic reticulum (ER) and Golgi. Anthers of dpw3 plants exhibited unbalanced anther cuticular profile, abnormal Ubisch bodies, disrupted callose deposition, defective pollen wall formation such as abnormal microspore plasma membrane undulation and defective primexine formation, resulting in pollen abortion and complete male sterility. Our findings revealed a novel and vital role of alpha integrin-like proteins in plant male reproduction.

Since January 2019, abnormal molting has been observed frequently in approximately 40-day-old Pekin ducks in China. To investigate the possible involvement of a virus, we tested the prevalence of duck circovirus (DuCV), goose hemorrhagic polyomavirus (GHPyV), and goose parvovirus (GPV) in 11 molt cases in two provinces. GPV was detected in all cases, particularly in all samples collected from the feather area. The complete genome sequences of three GPV strains were determined and found to have 52 nucleotide changes relative to GPVs associated with short beak and dwarfism syndrome of Pekin ducks. These data will enhance our understanding of GPV diversity and outcomes of GPV infection in Pekin ducks.

In flowering plants, male reproduction occurs within the male organ anther with a series of complex biological events including de novo specification of germinal cells and somatic cells, male meiosis, and pollen development and maturation. Particularly, unlike other tissue, anther lacks a meristem, therefore, both germinal and somatic cell types are derived from floral stem cells within anther lobes. Here, we review the molecular mechanism specifying the identity of somatic cells and reproductive microsporocytes by redox homoeostasis during rice anther development. Factors such as glutaredoxins (GRXs), TGA transcription factors, receptor-like protein kinase signaling pathway, and glutamyl-tRNA synthetase maintaining the redox status are discussed. We also conceive the conserved and divergent aspect of cell identity specification of anther cells in plants via changing redox status.

Two methods, PCR and amplicon labeling based, were developed and successfully applied to reliably detect CRISPR/Cas9 induced indels in rice. The use of CRISPR/Cas9 has emerged as a powerful nuclease-based genome editing tool in several model organisms including plants for mutagenesis by inducing precise gene editing through efficient double strand DNA breaks (DSBs) at the target site and subsequent error-prone non-homologous end joining (NHEJ) repair, leading to indel mutations. Different molecular methods including enzymatic mismatch cleavage (EMC), high-resolution melting curve analysis (HRMA) and conventional polymerase chain reaction (PCR) combined with ligation detection reaction (LDR) have been developed to quick identify CRISPR/Cas9 induced mutations. However, their intrinsic drawbacks limit their application in the identification of indel mutants in plants. Here we present two methods (one simple PCR based and the other amplicon labeling based) for effective and sensitive detection of CRISPR/Cas9 induced indels in rice. In PCR-based method, targets were amplified using two pairs of primers for each target locus and visualized on gel electrophoresis, while in amplicon labeling-based method, targets were amplified using tri-primers (with one a universal 6-FAM 5'-labelled) and detected by DNA capillary electrophoresis. Both methods can accurately define indel sizes down to ± 1 bp, and are amenable for high throughput analysis, therefore, will significantly facilitate the identification of indel mutants generated by CRISPR/Cas9 for further functional analysis and breeding in rice and other plants.

Jasmonates (JAs) are key phytohormones that regulate plant responses and development. JASMONATE-ZIM DOMAIN (JAZ) proteins safeguard JA signaling by repressing JA-responsive gene expression in the absence of JA. However, the interaction and cooperative roles of JAZ repressors remain unclear during plant development. Here, we found that OsJAZ6 interacts with OsJAZ1 depending on a single amino acid in the so-called ZIM domain of OsJAZ6 in rice JA signaling transduction and JA-regulated rice spikelet development. In vivo protein distribution analysis revealed that the OsJAZ6 content is efficiently regulated during spikelet development, and biochemical and genetic evidence showed that OsJAZ6 is more sensitive to JA-mediated degradation than OsJAZ1. Through over- and mis-expression experiments, we further showed that the protein stability and levels of OsJAZ6 orchestrate the output of JA signaling during rice spikelet development. A possible mechanism, which outlines how OsJAZ repressors interact and function synergistically in specifying JA signaling output through degradation titration, is also discussed.

MADS-box genes have a wide range of functions in plant reproductive development and grain production. The ABCDE model of floral organ development shows that MADS-box genes are central players in these events in dicotyledonous plants but the applicability of this model remains largely unknown in many grass crops. Here, we show that transcript analysis of all MIKCc MADS-box genes through barley (Hordeum vulgare L.) inflorescence development reveals co-expression groups that can be linked to developmental events. Thirty-four MIKCc MADS-box genes were identified in the barley genome and single-nucleotide polymorphism (SNP) scanning of 22,626 barley varieties revealed that the natural variation in the coding regions of these genes is low and the sequences have been extremely conserved during barley domestication. More detailed transcript analysis showed that MADS-box genes are generally expressed at key inflorescence developmental phases and across various floral organs in barley, as predicted by the ABCDE model. However, expression patterns of some MADS genes, for example HvMADS58 (AGAMOUS subfamily) and HvMADS34 (SEPALLATA subfamily), clearly deviate from predicted patterns. This places them outside the scope of the classical ABCDE model of floral development and demonstrates that the central tenet of antagonism between A- and C-class gene expression in the ABC model of other plants does not occur in barley. Co-expression across three correlation sets showed that specifically grouped members of the barley MIKCc MADS-box genes are likely to be involved in developmental events driving inflorescence meristem initiation, floral meristem identity and floral organ determination. Based on these observations, we propose a potential floral ABCDE working model in barley, where the classic model is generally upheld, but that also provides new insights into the role of MIKCc MADS-box genes in the developing barley inflorescence.

Flag leaf angle impacts the photosynthetic capacity of densely grown plants and is thus an important agronomic breeding trait for crop architecture and yield. The hormone auxin plays a key role in regulating this trait, yet the underlying molecular and cellular mechanisms remain unclear. Here, we report that two rice (Oryza sativa) auxin response factors (ARFs), OsARF6 and OsARF17, which are highly expressed in lamina joint tissues, control flag leaf angle in response to auxin. Loss-of-function double osarf6 osarf17 mutants displayed reduced secondary cell wall levels of lamina joint sclerenchymatous cells (Scs), resulting in an exaggerated flag leaf angle and decreased grain yield under dense planting conditions. Mechanical measurements indicated that the mutant lamina joint tissues were too weak to support the weight of the flag leaf blade, resembling the phenotype of the rice increased leaf angle1 (ila1) mutant. We demonstrate that OsARF6 and OsARF17 directly bind to the ILA1 promoter independently and synergistically to activate its expression. In addition, auxin-induced ILA1 expression was dependent on OsARF6 and OsARF17. Collectively, our study reveals a mechanism that integrates auxin signaling with the secondary cell wall composition to determine flag leaf angle, providing breeding targets in rice, and potentially other cereals, for this key trait., Two auxin response genes influence the secondary cell wall biosynthesis and the strength of lamina joints, thereby contributing to the adjustment of flag leaf angles.

Carpel is the ovule-bearing female reproductive organ of flowering plants and is required to ensure its protection, an efficient fertilization, and the development of diversified types of fruits, thereby it is a vital element of most food crops. The origin and morphological changes of the carpel are key to the evolution and adaption of angiosperms. Progresses have been made in elucidating the developmental mechanisms of carpel establishment in the model eudicot plantArabidopsis thaliana, while little and fragmentary information is known in grasses, a family that includes many important crops such as rice (Oryza sativa), maize (Zea mays), barley (Hordeum vulgare), and wheat (Triticum aestivum). Here, we highlight recent advances in understanding the mechanisms underlying potential pathways of carpel development in grasses, including carpel identity determination, morphogenesis, and floral meristem determinacy. The known role of transcription factors, hormones, and miRNAs during grass carpel formation is summarized and compared with the extensively studied eudicot model plantArabidopsis. The genetic and molecular aspects of carpel development that are conserved or diverged between grasses and eudicots are therefore discussed.

Environmental conditions, such as photoperiod and temperature, can affect male fertility in plants. While this feature is heavily exploited in rice to generate male-sterile lines for hybrid breeding, the underlying molecular mechanisms remain largely unknown. In this study, we use a transcriptomics approach to identify key genes and regulatory networks affecting pollen maturation in rice anthers in response to different day lengths. A total of 11,726 differentially expressed genes (DEGs) were revealed, of which 177 were differentially expressed at six time points over a 24-h period. GO enrichment analysis revealed that genes at all time points were enriched in transport, carbohydrate, and lipid metabolic processes, and signaling pathways, particularly phytohormone signaling. In addition, co-expression network analysis revealed four modules strongly correlated with photoperiod. Within these four modules, 496 hub genes were identified with a high degree of connectivity to other photoperiod-sensitive DEGs, including two previously reported photoperiod- and temperature-sensitive genes affecting male fertility, Carbon Starved Anther and UDP-glucose pyrophosphorylase, respectively. This work provides a new understanding on photoperiod-sensitive pollen development in rice, and our gene expression data will provide a new, comprehensive resource to identify new environmentally sensitive genes regulating male fertility for use in crop improvement.

Panax ginseng, a medicinally important perennial herb, has been widely used as the medicinal plant in South East Asian countries such as Korea, Japan, and China. Various pharmacological efficacies of ginseng are mainly correlated to its unique triterpenoid saponins ginsenosides. The backbone of ginsenosides are synthesized as triterpene saponins via the isoprenoid pathway, and the various ginsenosides are formed by glycosyl transferases. Ginseng plant has a long life cycle over 4–6 years, and it is frequently exposed to environmental stresses during this long-term cultivation. Therefore, ginseng needs to activate an array of defense mechanisms controlled by defense-related genes to confer the enhanced resistance with minimal fitness cost. Infection, wound, irradiation, and other abiotic stresses induce several defense-related genes. Such responses are caused by salicylates and jasmonates, although their roles and interactions in development and signaling are not yet fully studied in ginseng. This chapter summarizes the genes involved in the synthesis of ginseng saponins and defense-related genes in ginseng in response to biotic and abiotic stresses. Moreover, this chapter discusses the kinetics of metabolic pathways throughout the ginseng development supporting the therapeutic significance and bioactive ingredients of ginseng during plant growth and development.

The Tembusu virus (TMUV) PS strain, derived by several passages and plaque purifications in BHK-21 cells, displays markedly lower virulence in Pekin ducklings relative to a natural isolate of TMUV, but the potential virulence determinants and the in vivo mechanisms for substantial virulence attenuation of the passage variant remain unknown. Here, we constructed a series of chimeric and mutant viruses and assessed their virulence using a 2-day-old Pekin duckling model. We showed that residue 304 in the envelope (E) protein is the molecular determinant of TMUV virulence. Further investigations with mutant and parental viruses demonstrated that acquisition of positive charges at E protein residue 304 plays a critical role in substantial attenuation of neurovirulence and neuroinvasiveness, which is linked to enhanced binding affinity for glycosaminoglycans (GAGs). In Pekin ducklings infected by subcutaneous inoculation, an Arg at residue 304 in the E protein was shown to contribute to more rapid virus clearance from the circulation, markedly reduced viremia, and significantly decreased viral growth in the extraneural tissues and the central nervous system, relative to a Met at the corresponding residue. These findings suggest that the in vivo mechanism of virulence attenuation of the TMUV passage variant closely resembles that proposed previously for GAG-binding variants of other flaviviruses. Overall, our study provides insight into the molecular basis of TMUV virulence and the in vivo consequences of acquisition of a GAG-binding determinant at residue 304 in the E protein of TMUV. IMPORTANCE TMUV-related disease emerged in 2010 and has a significant economic impact on the duck industry. Although the disease was originally recognized to affect adult ducks, increasing evidence has shown that TMUV also causes severe disease of young ducklings. It is, therefore, essential to investigate the pathogenesis of TMUV infection in a young duckling model. The significance of our studies is in identifying E protein residue Arg304 as the molecular determinant for TMUV virulence and in clarifying the crucial role of positive charges at E protein residue 304 in virulence attenuation of a TMUV passage variant. These data will greatly enhance our understanding of the pathogenesis of TMUV infection in ducklings and have implications for development of a safe and efficient vaccine.

Ability to tolerate low salinity is a key factor affecting the distribution of the Chinese shrimp (Fenneropenaeus chinensis). Although previous studies have investigated the mechanisms underlying adaptations to low salinity in some crustaceans, little is known about low-salinity adaptations inF. chinensis, particularly at the molecular level. Here, to identify genes potentially associated with the molecular response ofF. chinensisto low-salinity exposure, we compared the transcriptomes ofF. chinensisin low-salinity (5 ppt) and normal-salinity (20 ppt) environments. In total, 45,297,936 and 44,685,728 clean reads were acquired from the low-salinity and control groups, respectively. De novo assembly of the clean reads yielded 159,130 unigenes, with an average length of 662.82 bp. Of these unigenes, only a small fraction (10.5% on average) were successfully annotated against six databases. We identified 3,658 differentially expressed genes (DEGs) between the low-salinity group and the control group: 1,755 DEGs were downregulated in the low-salinity group as compared to the control, and 1,903 were upregulated. Of these DEGs, 282 were significantly overrepresented in 38 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways. Notably, several DEGs were associated with pathways important for osmoregulation, including the mineral absorption pathway (ATP1A, Sodium/potassium-transporting ATPase subunit alpha;CLCN2, Chloride channel 2;HMOX2, Heme oxygenase 2;SLC40A1/FPN1, Solute carrier family 40 iron-regulated transporter, member 1), the vasopressin-regulated water reabsorption pathway (AQP4, Aquaporin-4;VAMP2, Vesicle-associated membrane protein 2;RAB5, Ras-related protein Rab-5) and the ribosome pathway. Our results help to clarify the molecular basis of low-salinity adaptations inF. chinensis.

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive organs/gametes. Recent advances in CRISPR-Cas9 genome editing technology allow for high editing efficacy and timesaving knockout mutations of endogenous candidate genes at specific sites. Additionally, Agrobacterium-mediated genetic transformation of rice is also a key method for gene modification, which has been widely adopted by many public and private laboratories. In this study, we applied CRISPR-Cas9 genome editing tools and successfully generated three male sterile mutant lines by targeted genome editing of OsABCG15 in a japonica cultivar. We used a modified Agrobacterium-mediated rice transformation method that could provide excellent means of genetic emasculation for hybrid seed production in rice. Transgenic plants can be obtained within 2-3 months and homozygous transformants were screened by genotyping using PCR amplification and Sanger sequencing. Basic phenotypic characterization of the male sterile homozygous line was performed by microscopic observation of the rice male reproductive organs, pollen viability analysis by iodine potassium iodide (I2-KI) staining semi-thin cross-sectioning of developing anthers.

Tembusu virus (TMUV) is a mosquito-borne flavivirus that most commonly affects adult breeder and layer ducks. However, a TMUV-caused neurological disease has also been found in ducklings below 7 weeks of age, highlighting the need to develop a safe vaccine for young ducklings. In this study, a plaque-purified PS TMUV strain was attenuated by serial passage in BHK-21 cells. Using 1-day-old Pekin ducklings as a model, the virus was confirmed to be attenuated sufficiently after 180 passages, whereas the neutralizing antibody response elicited by the 180th passage virus (PS180) was substantially impaired compared with PS. The findings suggest that sufficient attenuation results in loss of immunogenicity in the development of the live-attenuated TMUV vaccine. Comparative sequence analysis revealed that PS180 acquired one mutation (V41M) in prM and four mutations (T70A, Y176H, K313R, and F408L) in the envelope (E) protein. To identify the amino acid substitution(s) associated with loss of immunogenicity of PS180, we rescued parental viruses, rPS and rPS180, and produced mutant viruses, rPS180-M41V, rPS180-A70T, rPS180-H176Y, rPS180-R313K, rPS180-L408F, and rPS180-M5, which contained residue 41V in prM, residues 70T, 176Y, 313K, and 408F in E, and combination of the five residues, respectively, of PS in the backbone of the rPS180 genome. The neutralizing antibody response elicited by rPS180-L408F and rPS180-M5 was significantly higher than those by other mutant viruses and comparable to that by rPS. Furthermore, we produced mutant virus rPS-F408L, which contained residue 408L of PS180 in the backbone of the rPS genome. The F408L mutation conferred significantly decreased neutralizing antibody response to rPS-F408L, which was comparable to that elicited by rPS180. Based on homologous modeling, residue 408 was predicted to be located within the first helical domain of the stem region of the E protein (EH1). Together, these data demonstrate that a single mutation within the EH1 domain exerts a dramatical impact on the TMUV neutralizing antibody response. The present work may enhance our understanding of molecular basis of the TMUV neutralizing antibody response, and provides an important step for the development of a safe and efficient live-attenuated TMUV vaccine.

Floral patterning is regulated by intricate networks of floral identity genes. The peculiar MADS32 subfamily genes, absent in eudicots but prevalent in monocots, control floral organ identity. However, how the MADS32 family genes interact with other floral homeotic genes during flower development is mostly unknown. We show here that the rice homeotic transcription factor OsMADS32 regulates floral patterning by interacting synergistically with E class protein OsMADS6 in a dosage-dependent manner. Furthermore, our results indicate important roles for OsMADS32 in defining stamen, pistil, and ovule development through physical and genetic interactions with OsMADS1, OsMADS58, and OsMADS13, and in specifying floral meristem identity with OsMADS6, OsMADS3, and OsMADS58, respectively. Our findings suggest that OsMADS32 is an important factor for floral meristem identity maintenance and that it integrates the action of other MADS-box homeotic proteins to sustain floral organ specification and development in rice. Given that OsMADS32 is an orphan gene and absent in eudicots, our data substantially expand our understanding of flower development in plants.

Phosphate is an essential macro-element for plant growth accumulated in the topsoil. The improvement of phosphate uptake efficiency via manually manipulating root system architecture is of vital agronomic importance. This review discusses the molecular mechanisms of root patterning in response to external phosphate availability, which could be applied on the alleviation of phosphate-starvation stress. During the long time evolution, plants have formed sophisticated mechanisms to adapt to environmental phosphate conditions. In terms of root systems, plants would adjust their root system architecture via the regulation of the length of primary root, the length/density of lateral root and root hair and crown root growth angle to cope with different phosphate conditions. Finally, plants develop shallow or deep root system in low or high phosphate conditions, respectively. The plasticity of root system architecture responds to the local phosphate concentrations and this response was regulated by actin filaments, post-translational modification and phytohormones such as auxin, ethylene and cytokinin. This review summarizes the recent progress of adaptive response to external phosphate with focus on integrated physiological, cellular and molecular signaling transduction in rice and Arabidopsis.

Tembusu virus (TMUV) infection most commonly affects breeder and layer ducks during laying period, and can also affect young ducks below 7 weeks of age. Here, we report our investigation of a TMUV-caused fatal disease of Jingding ducklings (Anas platyrhynchos domesticus) in Northeast China. The disease resulted in mortalities of up to 40 % in 2 to 4-week-old ducks, up to 25 % in 5 to 6-week-old ducks, and less than 10 % in 7 to 8-week-old ducks. Using a TMUV-specific reverse transcription-PCR assay, all 44 ducks collected from 10 different farms were found positive for TMUV. Phylogenetic analysis of the E nucleotide sequence revealed that five of the six TMUV strains detected from three young ducks and three laying ducks were grouped within cluster 2.1. Inoculation of the liver sample of a 40-day-old sick duck in BHK-21 cells resulted in isolation of cluster 2.1 TMUV strain H. In experimental infections performed using 3-week-old Pekin ducklings (Anas platyrhynchos domesticus) (n = 30; 10 birds/group), high mortality (60 %) was caused by strain H, in sharp contrast with a very low mortality (10 %) caused by strain Y which was isolated during outbreaks of the TMUV-related disease of young Jinding ducks in 2014 in the same region. These findings clearly demonstrated that the cluster 2.1 TMUV strain H is more pathogenic for 3-week-old ducklings as compared to the cluster 2.2 TMUV strain Y. The present study may enhance our understanding of pathogenicity of TMUV in young ducks, and will stimulate further studies on the pathogenesis of TMUV infection.

Domain III of the envelope protein (EDIII) is the major target of flavivirus neutralizing antibody. To date, little is known regarding antibody-mediated neutralization of Tembusu virus (TMUV), a novel flavivirus emerging in duck in 2010. Here, a novel monoclonal antibody (MAb), designated 12F11, was prepared by immunization of mice with recombinant EDIII (rEDIII) protein. Using virus neutralization test, 12F11 in undiluted ascites neutralized the TMUV infectivity to induce the development of cytopathic effects in BHK-21 cells, indicating that 12F11 exhibits a neutralizing activity. The neutralizing activity of 12F11 was confirmed by plaque reduction neutralization test, in which 12F11 reduced significantly the number of plaques produced by TMUV in BHK-21 cells. Western blot analyses of a series of truncated rEDIII proteins showed that the epitope recognized by 12F11 includes amino acids between residues 8 and 77 of EDIII protein. Function analysis demonstrated that 12F11 neutralizes TMUV infection at virus adsorption and at a step after adsorption to a certain extent. The present study provides an important step towards elucidating antibody-mediated neutralization of TMUV.

The GRAS (named after first three identified proteins within this family, GAI, RGA, and SCR) family contains plant-specific genes encoding transcriptional regulators that play a key role in gibberellin (GA) signaling, which regulates plant growth and development. Even though GRAS genes have been characterized in some plant species, little research is known about the GRAS genes in barley (Hordeum vulgare L.). In this study, we observed 62 GRAS members from barley genome, which were grouped into 12 subgroups by using phylogenomic analysis together with the GRAS genes from Arabidopsis (Arabidopsis thaliana), maize (Zea mays), and rice (Oryza sativa). Chromosome localization and gene structure analysis suggested that duplication events and abundant presence of intronless genes might account for the massive expansion of GRAS gene family in barley. The analysis of RNA-seq data indicates the expression pattern of GRAS genes in various tissues at different stages in barley. Noteworthy, our qRT-PCR analysis showed the expression of 18 candidate GRAS genes abundantly in the developing inflorescence, indicating their potential roles in the barley inflorescence development and reproduction. Collectively, our evolutionary and expression analysis of GRAS family are useful for future functional characterization of GA signaling in barley and agricultural improvement.

Background In plants, the key roles played by RopGEF-mediated ROP signaling in diverse processes, including polar tip growth, have been identified. Despite their important roles in reproduction, a comprehensive analysis of RopGEF members has not yet been performed in rice (Oryza sativa). To determine whether RopGEF regulators are involved in rice pollen tube growth, we performed genome-wide analysis of this family in rice. Results Phylogenomic and meta-expression analysis of eleven RopGEFs in rice showed that four genes were preferentially expressed in mature pollen. These four genes contain the plant-specific Rop nucleotide exchanger (PRONE) domain and possible phosphorylated residues, suggesting a conserved role in polar tip growth with Arabidopsis thaliana. In subcellular localization analysis of the four RopGEFs through tobacco (Nicotiana benthamiana) infiltration, four proteins were predominantly identified in plasma membrane. Moreover, double mutants of RopGEF2/8 exhibited reduced pollen germination, causing partial male sterility. These genes possess unique cis-acting elements in their promoters compared with the other RopGEF genes. Conclusions In this study, four RopGEF genes were identified as pollen-specific gene in eleven members of rice, and the expression pattern, promoter analysis, and evolutionary relationship of the RopGEF family were studied compared with Arabidopsis. Our study indicated that four RopGEF genes might function during pollen germination in distinct subcellular localization. Our study could provide valuable information on the functional study of RopGEF in rice.

Although Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has been widely used for basic research in model plants, its application for applied breeding in crops has faced strong regulatory obstacles, due mainly to a poor understanding of the authentic output of this system, particularly in higher generations. In this study, different from any previous studies, we investigated in detail the molecular characteristics and production performance of CRISPR/Cas9-generated SD1 (semi-dwarf 1) mutants from T2 to T4 generations, of which the selection of T1 and T2 was done only by visual phenotyping for semidwarf plants. Our data revealed not only on- and off-target mutations with small or lager indels but also exogenous elements in T2 plants. All indel mutants passed stably to T3 or T4 without additional modifications independent on the presence of Cas9, while some lines displayed unexpected hereditary patterns of Cas9 or some exogenous elements. In addition, effects of various SD1 alleles on rice height and yield differed depending on genetic backgrounds. Taken together, our data showed that the CRISPR/Cas9 system is effective in producing homozygous mutants for functional analysis, but it may be not as precise as expected in rice, and that early and accurate molecular characterization and screening must be carried out for generations before transitioning of the CRISPR/Cas9 system from laboratory to field.

A multiplex ligation-dependent probe amplification (MLPA)-based method was developed and successfully utilized to efficiently detect both CRISPR/Cas9-induced and naturally occurred mutations in rice. The site-specific nuclease-based CRISPR/Cas9 system has emerged as one of the most efficient genome editing tools to modify multiple genomic targets simultaneously in various organisms, including plants for both fundamental and applied researches. Screening for both on-target and off-target mutations in CRISPR/Cas9-generated mutants at the early stages is an indispensable step for functional analysis and subsequent application. Various methods have been developed to detect CRISPR/Cas9-induced mutations in plants. Still, very few have focused on the detection of both on- and off-targets simultaneously, let alone the detection of natural mutations. Here, we report a multiplex capable method that allows to detect CRISPR/Cas9 induced on- and off-target mutations as well as naturally occurred mutation based on a multiplex ligation-dependent probe amplification (MLPA) method. We demonstrated that unlike other methods, the modified target-specific MLPA method can accurately identify any INDELs generated naturally or by the CRISPR/Cas9 system and that it can detect natural variation and zygosity of the CRISPR/Cas9-generated mutants in rice as well. Furthermore, its high sensitivity allowed to define INDELs down to 1 bp and substitutions to a single nucleotide. Therefore, this sensitive, reliable, and cheap method would further accelerate functional analysis and marker-assisted breeding in plants, including rice.

Rice flag leaves are major source organs providing more than half of the nutrition needed for rice seed development. The dynamic metabolic changes in rice flag leaves and the detailed metabolic relationship between source and sink organs in rice, however, remain largely unknown. In this study, the metabolic changes of flag leaves in two japonica and two indica rice cultivars were investigated using non-targeted metabolomics approach. Principal component analysis (PCA) revealed that flag leaf metabolomes varied significantly depending on both species and developmental stage. Only a few of the metabolites in flag leaves displayed the same change pattern across the four tested cultivars along the process of seed development. Further association analysis found that levels of 45 metabolites in seeds that are associated with human nutrition and health correlated significantly with their levels in flag leaves. Comparison of metabolomics of flag leaves and seeds revealed that some flavonoids were specific or much higher in flag leaves while some lipid metabolites such as phospholipids were much higher in seeds. This reflected not only the function of the tissue specific metabolism but also the different physiological properties and metabolic adaptive features of these two tissues.

Among the causative agents of duck viral hepatitis, duck hepatitis A virus genotype 1 (DHAV-1) is the most common virus reported in most outbreaks worldwide. How to propagate DHAV-1 in cell cultures efficiently remains a problem to be explored. Here, we aimed to test the effect of serum type on DHAV-1 replication in duck embryo fibroblast (DEF) cells. Comparative studies involved virus culture and passage, observation of cytopathic effect (CPE), virus quantification, and plaque formation assay. From the results of these investigations, we conclude that use of chicken serum (CS) in maintenance medium allows DHAV-1 to establish productive, cytocidal infection in DEF cells, whereas FCS exerts inhibitory effects on DHAV-1 replication, CPE development, and plaque formation. By using a neutralization test, we found that the direct action of FCS on virions is likely to play a key role in inhibiting DHAV-1 replication in DEF cells. Mechanism analyses revealed that FCS inhibits DHAV-1 replication at virus adsorption and reduces extracellular virus yields. The present work may shed light on a new perspective for antiviral agent development, and have provided a virus&ndash, host cell system for further studies on molecular mechanism involved DHAV-1 replication and pathogenesis.

Aux/IAA genes are early auxin-responsive genes and essential for auxin signaling transduction. There is little information about Aux/IAAs in the agriculturally important cereal, barley. Using in silico method, we identified and subsequently characterized 36 Aux/IAAs from the barley genome. Based on their genomic sequences and the phylogenic relationship with Arabidopsis and rice Aux/IAA, the 36 HvIAAs were categorized into two major groups and 14 subgroups. The indication of the presence or absence of these domains for the biological functions and acting mechanisms was discussed. The cis-element distributions in HvIAA promoters suggests that the HvIAAs expressions may not only regulated by auxin (the presence of AuxREs and TGA-element) but also by other hormones and developmental and environmental cues. We then studied the HvIAAs expression in response to NAA (1-Naphthaleneacetic acid) using quantitative real-time PCR (qRT-PCR). Like the promoter analysis, only 14 HvIAAs were upregulated by NAA over two-fold at 4 h. HvIAAs were clustered into three groups based on the spatiotemporal expression data. We confirmed by qRT-PCR that most HvIAAs, especially HvIAA3, HvIAA7, HvIAA8, HvIAA18, HvIAA24 and HvIAA34, are expressed in the developing barley spike compared within seedling, suggesting their roles in regulating spike development. Taken together, our data provide a foundation for further revealing the biological function of these HvIAAs.

OsMADS6, an ancient AGAMOUS-LIKE6 (AGL6)-like gene, has essential functions in specifying floral organ and meristem identity in rice (Oryza sativa). However, how AGL6 genes control flower development remains largely unknown. In this study, we report that OsMADS6 directly targets FACTOR OF DNA METHYLATION LIKE 1 (OsFDML1), a rice homolog of the SUPPRESSOR OF GENE SILENCING3-like gene FACTOR OF DNA METHYLATION 1 (FDM1) from Arabidopsis (Arabidopsis thaliana). Arabidopsis FDM1 is involved in RNA-directed DNA methylation and OsFDML1 regulates flower development. The expression of OsFDML1 overlaps with that of OsMADS6 in the palea primordia and the ovule, and OsMADS6 directly promotes OsFDML1 expression through binding to regions containing putative CArG motifs within the OsFDML1 promoter during rice spikelet development. Consistent with the phenotypes of osmads6 mutants, the osfdml1 mutants showed floral defects, including altered palea identity with lemma-like shape containing no marginal region of palea, increased numbers of stigmas and fused carpels, and meristem indeterminacy. Moreover, transgenic plants overexpressing OsFDML1 displayed floral defects, such as abnormal paleae. Phylogenetic analysis showed that OsFDML1 homologs exist only in terrestrial plants. In addition, protein-protein interaction assays showed that OsFDML1 interacts with its close paralog OsFDML2, similar to the activity of OsFDML1 homologs in Arabidopsis. These results provide insight into how the ancient AGL6 gene regulates floral development.

This study made the first attempt to relate the production of deoxynivalenol (DON) to the expression of TRI5 gene in Fusarium graminearum as a function of interacting environmental factors (water activity (aw) (0.95-0.98), temperature (20-30 °C) and incubation time (7 day-28 day)), so as to investigate its production mechanisms in Lentinula edodes. Changes in temperature, water activity and incubation time could significantly (P−ΔΔCt=38.8±4.8) were observed when the cultures were incubated at 20 °C and 0.98 aw for 21 days. Multi-regression analysis was performed and nonlinear models based on polynomial equations were established to uncover the individual effects of temperature, water activity and incubation time as well as their interactions on DON production and TRI5 gene expression. The established model was further used to develop contour maps to predict the DON production and TRI5 gene expression in relation to storage conditions in L. edodes. Highly significant positive correlation between DON production and fold TRI5 gene expression (R=0.5534, P

The pathogenicity of a variant goose parvovirus (GPV), isolated from short beak and dwarfism syndrome of Pekin ducks (strain Cherry Valley), was investigated in embryonating goose eggs and goslings. The virus was easily grown in GPV antibody-free goose embryos and caused high mortality and severe lesions of goose embryos, indicating that the variant GPV has good adaptation and high pathogenicity to embryonated goose eggs similar to the classical GPV. Like the third egg-passage virus (strain H) of a classical GPV, the third egg-passage virus (strain JS1) of the variant GPV caused Derzsy's disease in 2-day-old goslings with high mortality. The findings suggest that the variant GPV strain, which had specifically adapted to Pekin ducks, still retained high pathogenicity for its original host. The mortality (73.3-80%) caused by the first and third egg-passages of the variant GPV was somewhat lower than that (93.3%) caused by the third passage virus of the classical GPV, reflecting the higher pathogenicity of the classical GPV for its original host. These findings are likely to reinforce the importance of surveillance for parvoviruses in different waterfowl species and stimulate further study to elucidate the impact of mutations in the GPV genome on its pathogenicity to goslings and ducks.

We performed an international collaborative ring trial to validate gene-specific loop-mediated isothermal amplification (LAMP) assays targeting four exogenous transgenes commonly utilized in the development of genetically modified (GM) crops: bar, pat, cp4-epsps, and Cry1Ac, respectively. A total of 12 laboratories participated in the ring trial and returned their test results. Statistical analysis of the returned results showed high specificity and low limit of detection (LOD) of the four LAMP assays. The LOD for cp4-epsps, Cry1Ac, bar and pat genes assay was 5, 10, 20 and 20 haploid genome equivalents (HGE), respectively. Furthermore, good performance of the four assays was further demonstrated in the analysis of practical samples in which all participating laboratories returned expected and accurate test results. Altogether, the ring trial results confirmed that the four gene-specific LAMP assays can be utilized for quick screening of transgenic traits in routine laboratory analysis.

SEPALLATA (SEP)-like genes, which encode a subfamily of MADS-box transcription factors, are essential for specifying floral organ and meristem identity in angiosperms. Rice (Oryza sativa) has five SEP-like genes with partial redundancy and overlapping expression domains, yet their functions and evolutionary conservation are only partially known. Here, we describe the biological role of one of the SEP genes of rice, OsMADS5, in redundantly controlling spikelet morphogenesis. OsMADS5 belongs to the conserved LOFSEP subgroup along with OsMADS1 and OsMADS34OsMADS5 was expressed strongly across a broad range of reproductive stages and tissues. No obvious phenotype was observed in the osmads5 single mutants when compared with the wild type, which was largely due to the functional redundancy among the three LOFSEP genes. Genetic and molecular analyses demonstrated that OsMADS1, OsMADS5, and OsMADS34 together regulate floral meristem determinacy and specify the identities of spikelet organs by positively regulating the other MADS-box floral homeotic genes. Experiments conducted in yeast also suggested that OsMADS1, OsMADS5, and OsMADS34 form protein-protein interactions with other MADS-box floral homeotic members, which seems to be a typical, conserved feature of plant SEP proteins.