Premeiotic 24-nt phasiRNAs are present in the Zea genus and unique in biogenesis mechanism and molecular function.

Reproductive phasiRNAs (phased, small interfering RNAs) are broadly present in angiosperms and play crucial roles in sustaining male fertility. While the premeiotic 21-nt (nucleotides) phasiRNAs and meiotic 24-nt phasiRNA pathways have been extensively studied in maize (Zea mays) and rice (Oryza sativa), a third putative category of reproductive phasiRNAs-named premeiotic 24-nt phasiRNAs-have recently been reported in barley (Hordeum vulgare) and wheat (Triticum aestivum). To determine whether premeiotic 24-nt phasiRNAs are also present in maize and related species and begin to characterize their biogenesis and function, we performed a comparative transcriptome and degradome analysis of premeiotic and meiotic anthers from five maize inbred lines and three teosinte species/subspecies. Our data indicate that a substantial subset of the 24-nt phasiRNA loci in maize and teosinte are already highly expressed at the premeiotic phase. The premeiotic 24-nt phasiRNAs are similar to meiotic 24-nt phasiRNAs in genomic origin and dependence on DCL5 (Dicer-like 5) for biogenesis, however, premeiotic 24-nt phasiRNAs are unique in that they are likely i) not triggered by microRNAs, ii) not loaded by AGO18 proteins, and iii) not capable of mediating PHAS precursor cleavage. In addition, we also observed a group of premeiotic 24-nt phasiRNAs in rice using previously published data. Together, our results indicate that the premeiotic 24-nt phasiRNAs constitute a unique class of reproductive phasiRNAs and are present more broadly in the grass family (Poaceae) than previously known.

NAKED ENDOSPERM1, NAKED ENDOSPERM2, and OPAQUE2 interact to regulate gene networks in maize endosperm development.

NAKED ENDOSPERM1 (NKD1), NKD2, and OPAQUE2 (O2) are transcription factors important for cell patterning and nutrient storage in maize (Zea mays) endosperm. To study the complex regulatory interrelationships among these 3 factors in coregulating gene networks, we developed a set of nkd1, nkd2, and o2 homozygous lines, including all combinations of mutant and wild-type genes. Among the 8 genotypes tested, we observed diverse phenotypes and gene interactions affecting cell patterning, starch content, and storage proteins. From ∼8 to ∼16 d after pollination, maize endosperm undergoes a transition from cellular development to nutrient accumulation for grain filling. Gene network analysis showed that NKD1, NKD2, and O2 dynamically regulate a hierarchical gene network during this period, directing cellular development early and then transitioning to constrain cellular development while promoting the biosynthesis and storage of starch, proteins, and lipids. Genetic interactions regulating this network are also dynamic. The assay for transposase-accessible chromatin using sequencing (ATAC-seq) showed that O2 influences the global regulatory landscape, decreasing NKD1 and NKD2 target site accessibility, while NKD1 and NKD2 increase O2 target site accessibility. In summary, interactions of NKD1, NKD2, and O2 dynamically affect the hierarchical gene network and regulatory landscape during the transition from cellular development to grain filling in maize endosperm.

Coexpression network and trans-activation analyses of maize reproductive phasiRNA loci.

The anther-enriched phased, small interfering RNAs (phasiRNAs) play vital roles in sustaining male fertility in grass species. Their long non-coding precursors are synthesized by RNA polymerase II and are likely regulated by transcription factors (TFs). A few putative transcriptional regulators of the 21- or 24-nucleotide phasiRNA loci (referred to as 21- or 24-PHAS loci) have been identified in maize (Zea mays), but whether any of the individual TFs or TF combinations suffice to activate any PHAS locus is unclear. Here, we identified the temporal gene coexpression networks (modules) associated with maize anther development, including two modules highly enriched for the 21- or 24-PHAS loci. Comparisons of these coexpression modules and gene sets dysregulated in several reported male sterile TF mutants provided insights into TF timing with regard to phasiRNA biogenesis, including antagonistic roles for OUTER CELL LAYER4 and MALE STERILE23. Trans-activation assays in maize protoplasts of individual TFs using bulk-protoplast RNA-sequencing showed that two of the TFs coexpressed with 21-PHAS loci could activate several 21-nucleotide phasiRNA pathway genes but not transcription of 21-PHAS loci. Screens for combinatorial activities of these TFs and, separately, the recently reported putative transcriptional regulators of 24-PHAS loci using single-cell (protoplast) RNA-sequencing, did not detect reproducible activation of either 21-PHAS or 24-PHAS loci. Collectively, our results suggest that the endogenous transcriptional machineries and/or chromatin states in the anthers are necessary to activate reproductive PHAS loci.

Phylogenetic analyses of seven protein families refine the evolution of small RNA pathways in green plants.

Several protein families participate in the biogenesis and function of small RNAs (sRNAs) in plants. Those with primary roles include Dicer-like (DCL), RNA-dependent RNA polymerase (RDR), and Argonaute (AGO) proteins. Protein families such as double-stranded RNA-binding (DRB), SERRATE (SE), and SUPPRESSION OF SILENCING 3 (SGS3) act as partners of DCL or RDR proteins. Here, we present curated annotations and phylogenetic analyses of seven sRNA pathway protein families performed on 196 species in the Viridiplantae (aka green plants) lineage. Our results suggest that the RDR3 proteins emerged earlier than RDR1/2/6. RDR6 is found in filamentous green algae and all land plants, suggesting that the evolution of RDR6 proteins coincides with the evolution of phased small interfering RNAs (siRNAs). We traced the origin of the 24-nt reproductive phased siRNA-associated DCL5 protein back to the American sweet flag (Acorus americanus), the earliest diverged, extant monocot species. Our analyses of AGOs identified multiple duplication events of AGO genes that were lost, retained, or further duplicated in subgroups, indicating that the evolution of AGOs is complex in monocots. The results also refine the evolution of several clades of AGO proteins, such as AGO4, AGO6, AGO17, and AGO18. Analyses of nuclear localization signal sequences and catalytic triads of AGO proteins shed light on the regulatory roles of diverse AGOs. Collectively, this work generates a curated and evolutionarily coherent annotation for gene families involved in plant sRNA biogenesis/function and provides insights into the evolution of major sRNA pathways.

Rearrangement with the nkd2 promoter contributed to allelic diversity of the r1 gene in maize (Zea mays).

The maize red1 (r1) locus regulates anthocyanin accumulation and is a classic model for allelic diversity; changes in regulatory regions are responsible for most of the variation in gene expression patterns. Here, an intrachromosomal rearrangement between the distal upstream region of r1 and the region of naked endosperm 2 (nkd2) upstream to the third exon generated a nkd2 null allele lacking the first three exons, and the R1-st (stippled) allele with a novel r1 5' promoter region homologous to 5' regions from nkd2-B73. R1-sc:124 (an R1-st derivative) shows increased and earlier expression than a standard R1-g allele, as well as ectopic expression in the starchy endosperm compartment. Laser capture microdissection and RNA sequencing indicated that ectopic R1-sc:124 expression impacted expression of genes associated with RNA modification. The expression of R1-sc:124 resembled nkd2-W22 expression, suggesting that nkd2 regulatory sequences may influence the expression of R1-sc:124. The r1-sc:m3 allele is derived from R1-sc:124 by an insertion of a Ds6 transposon in intron 4. This insertion blocks anthocyanin regulation by causing mis-splicing that eliminates exon 5 from the mRNA. This allele serves as an important launch site for Ac/Ds mutagenesis studies, and two Ds6 insertions believed to be associated with nkd2 mutant alleles were actually located in the r1 5' region. Among annotated genomes of teosinte and maize varieties, the nkd2 and r1 loci showed conserved overall gene structures, similar to the B73 reference genome, suggesting that the nkd2-r1 rearrangement may be a recent event.

Soybean DICER-LIKE2 Regulates Seed Coat Color via Production of Primary 22-Nucleotide Small Interfering RNAs from Long Inverted Repeats.

In plants, 22-nucleotide small RNAs trigger the production of secondary small interfering RNAs (siRNAs) and enhance silencing. DICER-LIKE2 (DCL2)-dependent 22-nucleotide siRNAs are rare in Arabidopsis (Arabidopsis thaliana) and are thought to function mainly during viral infection; by contrast, these siRNAs are abundant in many crops such as soybean (Glycine max) and maize (Zea mays). Here, we studied soybean 22-nucleotide siRNAs by applying CRISPR-Cas9 to simultaneously knock out the two copies of soybean DCL2, GmDCL2a and GmDCL2b, in the Tianlong1 cultivar. Small RNA sequencing revealed that most 22-nucleotide siRNAs are derived from long inverted repeats (LIRs) and disappeared in the Gmdcl2a/2b double mutant. De novo assembly of a Tianlong1 reference genome and transcriptome profiling identified an intronic LIR formed by the chalcone synthase (CHS) genes CHS1 and CHS3 This LIR is the source of primary 22-nucleotide siRNAs that target other CHS genes and trigger the production of secondary 21-nucleotide siRNAs. Disruption of this process in Gmdcl2a/2b mutants substantially increased CHS mRNA levels in the seed coat, thus changing the coat color from yellow to brown. Our results demonstrated that endogenous LIR-derived transcripts in soybean are predominantly processed by GmDCL2 into 22-nucleotide siRNAs and uncovered a role for DCL2 in regulating natural traits.

Opaque-2 Regulates a Complex Gene Network Associated with Cell Differentiation and Storage Functions of Maize Endosperm.

Development of the cereal endosperm involves cell differentiation processes that enable nutrient uptake from the maternal plant, accumulation of storage products, and their utilization during germination. However, little is known about the regulatory mechanisms that link cell differentiation processes with those controlling storage product synthesis and deposition, including the activation of zein genes by the maize (Zea mays) bZIP transcription factor Opaque-2 (O2). Here, we mapped in vivo binding sites of O2 in B73 endosperm and compared the results with genes differentially expressed in B73 and B73o2 We identified 186 putative direct O2 targets and 1677 indirect targets, encoding a broad set of gene functionalities. Examination of the temporal expression patterns of O2 targets revealed at least two distinct modes of O2-mediated gene activation. Two O2-activated genes, bZIP17 and NAKED ENDOSPERM2 (NKD2), encode transcription factors, which can in turn coactivate other O2 network genes with O2. NKD2 (with its paralog NKD1) was previously shown to be involved in regulation of aleurone development. Collectively, our results provide insights into the complexity of the O2-regulated network and its role in regulation of endosperm cell differentiation and function.

RNA sequencing of laser-capture microdissected compartments of the maize kernel identifies regulatory modules associated with endosperm cell differentiation.

Endosperm is an absorptive structure that supports embryo development or seedling germination in angiosperms. The endosperm of cereals is a main source of food, feed, and industrial raw materials worldwide. However, the genetic networks that regulate endosperm cell differentiation remain largely unclear. As a first step toward characterizing these networks, we profiled the mRNAs in five major cell types of the differentiating endosperm and in the embryo and four maternal compartments of the maize (Zea mays) kernel. Comparisons of these mRNA populations revealed the diverged gene expression programs between filial and maternal compartments and an unexpected close correlation between embryo and the aleurone layer of endosperm. Gene coexpression network analysis identified coexpression modules associated with single or multiple kernel compartments including modules for the endosperm cell types, some of which showed enrichment of previously identified temporally activated and/or imprinted genes. Detailed analyses of a coexpression module highly correlated with the basal endosperm transfer layer (BETL) identified a regulatory module activated by MRP-1, a regulator of BETL differentiation and function. These results provide a high-resolution atlas of gene activity in the compartments of the maize kernel and help to uncover the regulatory modules associated with the differentiation of the major endosperm cell types.

Premeiotic 24-nt phasiRNAs are present in the Zea genus and unique in biogenesis mechanism and molecular function.

Reproductive phasiRNAs are broadly present in angiosperms and play crucial roles in sustaining male fertility. While the premeiotic 21-nt phasiRNAs and meiotic 24-nt phasiRNA pathways have been extensively studied in maize (Zea mays) and rice (Oryza sativa), a third putative category of reproductive phasiRNAs-named premeiotic 24-nt phasiRNAs-have recently been reported in barley (Hordeum vulgare) and wheat (Triticum aestivum). To determine whether premeiotic 24-nt phasiRNAs are also present in maize and related species and begin to characterize their biogenesis and function, we performed a comparative transcriptome and degradome analysis of premeiotic and meiotic anthers from five maize inbred lines and three teosinte species/subspecies. Our data indicate that a substantial subset of the 24-nt phasiRNA loci in maize and teosinte are already highly expressed at premeiotic phase. The premeiotic 24-nt phasiRNAs are similar to meiotic 24-nt phasiRNAs in genomic origin and dependence on DCL5 for biogenesis, however, premeiotic 24-nt phasiRNAs are unique in that they are likely (i) not triggered by microRNAs, (ii) not loaded by AGO18 proteins, and (iii) not capable of mediating cis-cleavage. In addition, we also observed a group of premeiotic 24-nt phasiRNAs in rice using previously published data. Together, our results indicate that the premeiotic 24-nt phasiRNAs constitute a unique class of reproductive phasiRNAs and are present more broadly in the grass family (Poaceae) than previously known.

Plant Small RNAs: Their Biogenesis, Regulatory Roles, and Functions.

Plant cells accumulate small RNA molecules that regulate plant development, genome stability, and environmental responses. These small RNAs fall into three major classes based on their function and mechanisms of biogenesis-microRNAs, heterochromatic small interfering RNAs, and secondary small interfering RNAs-plus several other less well-characterized categories. Biogenesis of each small RNA class requires a pathway of factors, some specific to each pathway and others involved in multiple pathways. Diverse sequenced plant genomes, along with rapid developments in sequencing, imaging, and genetic transformation techniques, have enabled significant progress in understanding the biogenesis, functions, and evolution of plant small RNAs, including those that had been poorly characterized because they were absent or had low representation in Arabidopsis (Arabidopsis thaliana). Here, we review recent findings about plant small RNAs and discuss our current understanding of their biogenesis mechanisms, targets, modes of action, mobility, and functions in Arabidopsis and other plant species, including economically important crops.

Multiantigen epitope fusion recombinant proteins from capsids of serotype 4 fowl adenovirus induce chicken immunity against avian Angara disease.

Avian Angara disease caused by fowl adenovirus serotype 4 (FAdV-4) has spread widely and brought economic losses to the poultry industry in some countries. Effective vaccines for Angara disease control are currently lacking. In this study, four capsid proteins (hexon, penton, fiber1 and fiber2) from FAdV-4 were selected, and their optimal efficient antigenic epitopes predicted by bioinformatics software were tandemly linked with the flexible linker GGGGS. Based on their amino acid sequences, the DNA sequences for the genes encoding the multiantigen epitope tandem proteins (MAETPs) FAdV4:F1, FAdV4:P, FAdV4:F2 and FAdV4:H were chemosynthesized and then ligated by T4 ligases at the cleavage sites of restriction endonucleases to construct DNAs encoding the multilinked fusion recombinant proteins (MLFRPs) used as protective antigens from avian Angara disease. These genes ligated into the expression vector pET-28a were successfully expressed using the Escherichia coli prokaryotic expression system to prepare five kinds of MLFRPs (FAdV4:F1-P-F2-H, FAdV4:F1-F2-P-H, FAdV4:F1-F2-H-P, FAdV4:F1-P-H-F2 and FAdV4:F1-H-F2-P) for use to immunize chicks. FAdV-4 was injected into MLFRP-immunized chickens, and the challenge protection rate was evaluated. FAdV4:F1-P-F2-H produced the best protection against FAdV-4, with a single immunization resulting in a 100 % protection rate, followed by FAdV4:F1-F2-P-H (83.33 %) and FAdV4:F1-F2-H-P (66.67 %). FAdV4:F1-P-H-F2 and FAdV4:F1-H-F2-P were not able to induce a good immune protection effect after one immunization. However, all of the MLFRPs were capable of protecting the host from FAdV-4 infection after two immunizations. In conclusion, these MLFRPs generated based on capsid proteins of FAdV-4 are promising candidate subunit vaccines against Angara disease.

A recombinase polymerase amplification-SYBR Green I assay for the rapid and visual detection of Brucella.

Brucellosis is a zoonosis caused by Brucella, which poses a great threat to human health and animal husbandry. Pathogen surveillance is an important measure to prevent brucellosis, but the traditional method is time-consuming and not suitable for field applications. In this study, a recombinase polymerase amplification-SYBR Green I (RPAS) assay was developed for the rapid and visualized detection of Brucella in the field by targeting BCSP31 gene, a conserved marker. The method was highly specific without any cross-reactivity with other common bacteria and its detection limit was 2.14 × 104 CFU/mL or g of Brucella at 40 °C for 20 min. It obviates the need for costly instrumentation and exhibits robustness towards background interference in serum, meat, and milk samples. In summary, the RPAS assay is a rapid, visually intuitive, and user-friendly detection that is highly suitable for use in resource-limited settings. Its simplicity and ease of use enable swift on-site detection of Brucella, thereby facilitating timely implementation of preventive measures.