A systematic analysis of the subtilase gene family and expression and subcellular localization investigation of anther-specific members in maize.

Subtilases (SBTs), also known as Subtilisin-like serine proteases, are extracellular alkaline protease proteins. SBTs function in all stages of plant growth, development and stress responses. Maize (Zea mays L.) is a crop widely used worldwide as food, feed, and industrial materials. However, information about the members and their functions of the SBT proteins in maize is lacking. In this study, we identified 58 ZmSBT genes from the maize genome and conducted a comprehensive investigation of ZmSBTs by phylogenetic, gene duplication event, gene structure, and protein conserved motif analyses. The ZmSBT proteins were phylogenetically classified into seven groups, and collinearity analysis indicated that many ZmSBTs originate from tandem or segmental duplications. Structural and homolog protein comparison revealed ZmSBTs have conserved protein structures with reported subtilase proteins, suggesting the conserved functions. Further analysis showed that ZmSBTs are expressed in different tissues, and many are responses to specific abiotic stress. Analysis of the anther-specific ZmSBT genes showed their expression peaked at different developmental stages of maize anthers. Subcellular localization analysis of selected maize ZmSBTs showed they are located in different cellular compartments. The information provided in this study is valuable for further functional study of ZmSBTs.

ZmMS1/ZmLBD30-orchestrated transcriptional regulatory networks precisely control pollen exine development.

Because of its significance for plant male fertility and, hence, direct impact on crop yield, pollen exine development has inspired decades of scientific inquiry. However, the molecular mechanism underlying exine formation and thickness remains elusive. In this study, we identified that a previously unrecognized repressor, ZmMS1/ZmLBD30, controls proper pollen exine development in maize. Using an ms1 mutant with aberrantly thickened exine, we cloned a male-sterility gene, ZmMs1, which encodes a tapetum-specific lateral organ boundary domain transcription factor, ZmLBD30. We showed that ZmMs1/ZmLBD30 is initially turned on by a transcriptional activation cascade of ZmbHLH51-ZmMYB84-ZmMS7, and then it serves as a repressor to shut down this cascade via feedback repression to ensure timely tapetal degeneration and proper level of exine. This activation-feedback repression loop regulating male fertility is conserved in maize and sorghum, and similar regulatory mechanism may also exist in other flowering plants such as rice and Arabidopsis. Collectively, these findings reveal a novel regulatory mechanism of pollen exine development by which a long-sought master repressor of upstream activators prevents excessive exine formation.

Promoting genotype-independent plant transformation by manipulating developmental regulatory genes and/or using nanoparticles.

KEY MESSAGE: This review summarizes the molecular basis and emerging applications of developmental regulatory genes and nanoparticles in plant transformation and discusses strategies to overcome the obstacles of genotype dependency in plant transformation. Plant transformation is an important tool for plant research and biotechnology-based crop breeding. However, Plant transformation and regeneration are highly dependent on species and genotype. Plant regeneration is a process of generating a complete individual plant from a single somatic cell, which involves somatic embryogenesis, root and shoot organogeneses. Over the past 40 years, significant advances have been made in understanding molecular mechanisms of embryogenesis and organogenesis, revealing many developmental regulatory genes critical for plant regeneration. Recent studies showed that manipulating some developmental regulatory genes promotes the genotype-independent transformation of several plant species. Besides, nanoparticles penetrate plant cell wall without external forces and protect cargoes from degradation, making them promising materials for exogenous biomolecule delivery. In addition, manipulation of developmental regulatory genes or application of nanoparticles could also bypass the tissue culture process, paving the way for efficient plant transformation. Applications of developmental regulatory genes and nanoparticles are emerging in the genetic transformation of different plant species. In this article, we review the molecular basis and applications of developmental regulatory genes and nanoparticles in plant transformation and discuss how to further promote genotype-independent plant transformation.

Overexpression of HLH4 Inhibits Cell Elongation and Anthocyanin Biosynthesis in Arabidopsis thaliana.

In plants, many basic helix-loop-helix (bHLH) transcription factors are involved in controlling cell elongation. Three bHLH proteins, PACLOBTRAZOL RESISTANCE1 (PRE1), Cryptochrome Interacting Basic Helix-loop-helix 5 (CIB5), and Arabidopsis ILI1 binding bHLH1 (IBH1) form a triantagonistic system that antagonistically regulates cell elongation in a competitive manner. In this study, we identified a new player, HLH4, related to IBH1, that negatively regulates cell elongation in Arabidopsis thaliana. Overexpression of HLH4 causes dwarf and dark green phenotypes and results in the downregulation of many key regulatory and enzymatic genes that participate in the anthocyanin biosynthetic pathway. HLH4 interacts with CIB5 and PRE1. By interacting with CIB5, HLH4 interferes with the activity of CIB5, and thus inhibiting the transcription of cell elongation-related genes regulated by CIB5, including EXPANSINS8 and 11 (EXP8 and EXP11) and indole-3-acetic acid 7 and 17 (IAA7 and IAA17). The interference of HLH4 on CIB5 is counteracted by PRE1, in which these bHLH proteins form a new tri-antagonistic system.

Mining of Potential Gene Resources for Breeding Nutritionally Improved Maize.

Maize is one of the leading food crops and its kernel is rich in starch, lipids, protein and other energy substances. In addition, maize kernels also contain many trace elements that are potentially beneficial to human health, such as vitamins, minerals and other secondary metabolites. However, gene resources that could be applied for nutrient improvement are limited in maize. In this review, we summarized 107 genes that are associated with nutrient content from different plant species and identified 246 orthologs from the maize genome. In addition, we constructed physical maps and performed a detailed expression pattern analysis for the 246 maize potential gene resources. Combining expression profiles and their potential roles in maize nutrient improvement, genetic engineering by editing or ectopic expression of these genes in maize are expected to improve resistant starch, oil, essential amino acids, vitamins, iron, zinc and anthocyanin levels of maize grains. Thus, this review provides valuable gene resources for maize nutrient improvement.

Use of CRISPR/Cas9-Based Gene Editing to Simultaneously Mutate Multiple Homologous Genes Required for Pollen Development and Male Fertility in Maize.

Male sterility represents an important trait for hybrid breeding and seed production in crops. Although the genes required for male fertility have been widely studied and characterized in many plant species, most of them are single genic male-sterility (GMS) genes. To investigate the role of multiple homologous genes in anther and pollen developments of maize, we established the CRISPR/Cas9-based gene editing method to simultaneously mutate the homologs in several putative GMS gene families. By using the integrated strategies of multi-gene editing vectors, maize genetic transformation, mutation-site analysis of T0 and F1 plants, and genotyping and phenotyping of F2 progenies, we further confirmed gene functions of every member in ZmTGA9-1/-2/-3 family, and identified the functions of ZmDFR1, ZmDFR2, ZmACOS5-1, and ZmACOS5-2 in controlling maize male fertility. Single and double homozygous gene mutants of ZmTGA9-1/-2/-3 did not affect anther and pollen development, while triple homozygous gene mutant resulted in complete male sterility. Two single-gene mutants of ZmDFR1/2 displayed partial male sterility, but the double-gene mutant showed complete male sterility. Additionally, only the ZmACOS5-2 single gene was required for anther and pollen development, while ZmACOS5-1 had no effect on male fertility. Our results show that the CRISPR/Cas9 gene editing system is a highly efficient and convenient tool for identifying multiple homologous GMS genes. These findings enrich GMS genes and mutant resources for breeding of maize GMS lines and promote deep understanding of the gene family underlying pollen development and male fertility in maize.

Genome-Wide Identification and Characterization of Polygalacturonase Gene Family in Maize (Zea mays L.).

Polygalacturonase (PG, EC 3.2.1.15) is a crucial enzyme for pectin degradation and is involved in various developmental processes such as fruit ripening, pollen development, cell expansion, and organ abscission. However, information on the PG gene family in the maize (Zea mays L.) genome and the specific members involved in maize anther development are still lacking. In this study, we identified 55 PG family genes from the maize genome and further characterized their evolutionary relationship and expression patterns. Phylogenetic analysis revealed that ZmPGs are grouped into six Clades, and gene structures of the same Clade are highly conserved, suggesting their functional conservation. The ZmPGs are randomly distributed across maize chromosomes, and collinearity analysis showed that many ZmPGs might be derived from tandem duplications and segmental duplications, and these genes are under purifying selection. Furthermore, gene expression analysis provided insights into possible functional divergence among ZmPGs. Based on the RNA-seq data analysis, we found that many ZmPGs are expressed in various tissues while 18 ZmPGs are highly expressed in maize anther, and their detailed expression profiles in different anther developmental stages were further investigated by using RT-qPCR analysis. These results provide valuable information for further functional characterization and application of the ZmPGs in maize.

ZmFAR1 and ZmABCG26 Regulated by microRNA Are Essential for Lipid Metabolism in Maize Anther.

The function and regulation of lipid metabolic genes are essential for plant male reproduction. However, expression regulation of lipid metabolic genic male sterility (GMS) genes by noncoding RNAs is largely unclear. Here, we systematically predicted the microRNA regulators of 34 maize white brown complex members in ATP-binding cassette transporter G subfamily (WBC/ABCG) genes using transcriptome analysis. Results indicate that the ZmABCG26 transcript was predicted to be targeted by zma-miR164h-5p, and their expression levels were negatively correlated in maize B73 and Oh43 genetic backgrounds based on both transcriptome data and qRT-PCR experiments. CRISPR/Cas9-induced gene mutagenesis was performed on ZmABCG26 and another lipid metabolic gene, ZmFAR1. DNA sequencing, phenotypic, and cytological observations demonstrated that both ZmABCG26 and ZmFAR1 are GMS genes in maize. Notably, ZmABCG26 proteins are localized in the endoplasmic reticulum (ER), chloroplast/plastid, and plasma membrane. Furthermore, ZmFAR1 shows catalytic activities to three CoA substrates in vitro with the activity order of C12:0-CoA > C16:0-CoA > C18:0-CoA, and its four key amino acid sites were critical to its catalytic activities. Lipidomics analysis revealed decreased cutin amounts and increased wax contents in anthers of both zmabcg26 and zmfar1 GMS mutants. A more detailed analysis exhibited differential changes in 54 monomer contents between wild type and mutants, as well as between zmabcg26 and zmfar1. These findings will promote a deeper understanding of miRNA-regulated lipid metabolic genes and the functional diversity of lipid metabolic genes, contributing to lipid biosynthesis in maize anthers. Additionally, cosegregating molecular markers for ZmABCG26 and ZmFAR1 were developed to facilitate the breeding of male sterile lines.

ZmMs25 encoding a plastid-localized fatty acyl reductase is critical for anther and pollen development in maize.

Fatty acyl reductases (FARs) catalyse the reduction of fatty acyl-coenzyme A (CoA) or -acyl carrier protein (ACP) substrates to primary fatty alcohols, which play essential roles in lipid metabolism in plants. However, the mechanism by which FARs are involved in male reproduction is poorly defined. Here, we found that two maize allelic mutants, ms25-6065 and ms25-6057, displayed defective anther cuticles, abnormal Ubisch body formation, impaired pollen exine formation and complete male sterility. Based on map-based cloning and CRISPR/Cas9 mutagenesis, Zm00001d048337 was identified as ZmMs25, encoding a plastid-localized FAR with catalytic activities to multiple acyl-CoA substrates in vitro. Four conserved residues (G101, G104, Y327 and K331) of ZmMs25 were critical for its activity. ZmMs25 was predominantly expressed in anther, and was directly regulated by transcription factor ZmMYB84. Lipidomics analysis revealed that ms25 mutation had significant effects on reducing cutin monomers and internal lipids, and altering the composition of cuticular wax in anthers. Moreover, loss of function of ZmMs25 significantly affected the expression of its four paralogous genes and five cloned lipid metabolic male-sterility genes in maize. These data suggest that ZmMs25 is required for anther development and male fertility, indicating its application potential in maize and other crops.

CRISPR/Cas9-based discovery of maize transcription factors regulating male sterility and their functional conservation in plants.

Identifying genic male-sterility (GMS) genes and elucidating their roles are important to unveil plant male reproduction and promote their application in crop breeding. However, compared with Arabidopsis and rice, relatively fewer maize GMS genes have been discovered and little is known about their regulatory pathways underlying anther and pollen development. Here, by sequencing and analysing anther transcriptomes at 11 developmental stages in maize B73, Zheng58 and M6007 inbred lines, 1100 transcription factor (TF) genes were identified to be stably differentially expressed among different developmental stages. Among them, 14 maize TF genes (9 types belonging to five TF families) were selected and performed CRISPR/Cas9-mediated gene mutagenesis, and then, 12 genes in eight types, including ZmbHLH51, ZmbHLH122, ZmTGA9-1/-2/-3, ZmTGA10, ZmMYB84, ZmMYB33-1/-2, ZmPHD11 and ZmLBD10/27, were identified as maize new GMS genes by using DNA sequencing, phenotypic and cytological analyses. Notably, ZmTGA9-1/-2/-3 triple-gene mutants and ZmMYB33-1/-2 double-gene mutants displayed complete male sterility, but their double- or single-gene mutants showed male fertility. Similarly, ZmLBD10/27 double-gene mutant displayed partial male sterility with 32.18% of aborted pollen grains. In addition, ZmbHLH51 was transcriptionally activated by ZmbHLH122 and their proteins were physically interacted. Molecular markers co-segregating with these GMS mutations were developed to facilitate their application in maize breeding. Finally, all 14-type maize GMS TF genes identified here and reported previously were compared on functional conservation and diversification among maize, rice and Arabidopsis. These findings enrich GMS gene and mutant resources for deeply understanding the regulatory network underlying male fertility and for creating male-sterility lines in maize.

Complete genome analysis of PaGz-1 and PaZq-1, two novel phages belonging to the genus Pakpunavirus.

Pseudomonas phages PaGz-1 and PaZq-1, two new phages infecting Pseudomonas aeruginosa, were isolated from fresh water in Guangdong province, China. The genomes of these two phages consist of 93,975 bp and 94,315 bp and contain 175 and 172 open reading frames (ORFs), respectively. The genome sequences of PaGz-1 and PaZq-1 share 95.8% identity with a query coverage of 94%, suggesting that these two phages belong to two different species. Based on results of nucleotide sequence alignment, gene annotation, and phylogenetic analysis, we propose PaGz-1 and PaZq-1 as representative isolates of two species in the genus Pakpunavirus within the family Myoviridae.

Characterization and Genomic Analysis of ValSw3-3, a New Siphoviridae Bacteriophage Infecting Vibrio alginolyticus.

A novel lytic bacteriophage, ValSw3-3, which efficiently infects pathogenic strains of Vibrio alginolyticus, was isolated from sewage water and characterized by microbiological and in silico genomic analyses. Transmission electron microscopy indicated that ValSw3-3 has the morphology of siphoviruses. This phage can infect four species in the Vibrio genus and has a latent period of 15 min and a burst size of 95 ± 2 PFU/infected bacterium. Genome sequencing results show that ValSw3-3 has a 39,846-bp double-stranded DNA genome with a GC content of 43.1%. The similarity between the genome sequences of ValSw3-3 and those of other phages recorded in the GenBank database was below 50% (42%), suggesting that ValSw3-3 significantly differs from previously reported phages at the DNA level. Multiple genome comparisons and phylogenetic analysis based on the major capsid protein revealed that phage ValSw3-3 is grouped in a clade with five other phages, including Listonella phage phiHSIC (GenBank accession no. NC_006953.1), Vibrio phage P23 (MK097141.1), Vibrio phage pYD8-B (NC_021561.1), Vibrio phage 2E1 (KX507045.1), and Vibrio phage 12G5 (HQ632860.1), and is distinct from all known genera within the Siphoviridae family that have been ratified by the International Committee on Taxonomy of Viruses (ICTV). An in silico proteomic comparison of diverse phages from the Siphoviridae family supported this clustering result and suggested that ValSw3-3, phiHSIC, P23, pYD8-B, 2E1, and 12G5 should be classified as a novel genus cluster of Siphoviridae A subsequent analysis of core genes also revealed the common genes shared within this new cluster. Overall, these results provide a characterization of Vibrio phage ValSw3-3 and support our proposal of a new viral genus within the family SiphoviridaeIMPORTANCE Phage therapy has been considered a potential alternative to antibiotic therapy in treating bacterial infections. For controlling the vibriosis-causing pathogen Vibrio alginolyticus, well-documented phage candidates are still lacking. Here, we characterize a novel lytic Vibrio phage, ValSw3-3, based on its morphology, host range and infectivity, growth characteristics, stability under various conditions, and genomic features. Our results show that ValSw3-3 could be a potent candidate for phage therapy to treat V. alginolyticus infections due to its stronger infectivity and better pH and thermal stability than those of previously reported Vibrio phages. Moreover, genome sequence alignments, phylogenetic analysis, in silico proteomic comparison, and core gene analysis all support that this novel phage, ValSw3-3, and five unclassified phages form a clade distant from those of other known genera ratified by the ICTV. Thus, we propose a new viral genus within the Siphoviridae family to accommodate this clade, with ValSw3-3 as a representative member.

Isolation and Characterization of Specific Phages to Prepare a Cocktail Preventing Vibrio sp. Va-F3 Infections in Shrimp (Litopenaeus vannamei).

Vibrio is one of the most detrimental agents of shrimp premature death syndrome. Phage therapy for prevention and treatment of Vibrio infections has attracted increasing attentions due to the emergence of antibiotic-resistant bacterial variants. Here, we describe a workflow of preparing a phage cocktail against Vibrio infections for practical applications. Twenty Vibrio strains were isolated from the gut of diseased shrimp and aquaculture wastewater, and five of them were identified as pathogens causing shrimp vibriosis. Twenty-two lytic phages were then isolated using the above five pathogens as hosts, and five of them showed broad host ranges and high lytic capability against the Vibrio strains. Whole genomic sequencing and phylogenetic analysis of the five phages indicated that they are novel and belong to the Siphoviridae family. The phage cocktail consisting of these five phages showed higher efficiency in inhibiting the growth of pathogenic Vibrio sp. Va-F3 than any single phage in vitro. We then evaluated the performance of the phage cocktail in protecting shrimp against Vibrio sp. Va-F3 infections in situ. The results showed that shrimp survival rates could reach 91.4 and 91.6% in 7 days, for the cocktail-treated and the antibiotic-treated groups, respectively. By contrast, the shrimp survival rate of the group without any treatment was only 20.0%. Overall, this study describes a general workflow of how to prepare a phage cocktail and apply it in controlling bacterial infections in the shrimp aquaculture. Knowledge gained from this study will not only help fight against the shrimp vibriosis in practical but also facilitate the design of phage cocktails with a satisfying performance in controlling other animal diseases in aquaculture.

Discovering and Constructing ceRNA-miRNA-Target Gene Regulatory Networks during Anther Development in Maize.

The "competing endogenous RNA (ceRNA) hypothesis" has recently been proposed for a new type of gene regulatory model in many organisms. Anther development is a crucial biological process in plant reproduction, and its gene regulatory network (GRN) has been gradually revealed during the past two decades. However, it is still unknown whether ceRNAs contribute to anther development and sexual reproduction in plants. We performed RNA and small RNA sequencing of anther tissues sampled at three developmental stages in two maize lines. A total of 28,233 stably transcribed loci, 61 known and 51 potentially novel microRNAs (miRNAs) were identified from the transcriptomes. Predicted ceRNAs and target genes were found to conserve in sequences of recognition sites where their corresponding miRNAs bound. We then reconstructed 79 ceRNA-miRNA-target gene regulatory networks consisting of 51 known miRNAs, 28 potentially novel miRNAs, 619 ceRNA-miRNA pairs, and 869 miRNA-target gene pairs. More than half of the regulation pairs showed significant negative correlations at transcriptional levels. Several well-studied miRNA-target gene pairs associated with plant flower development were located in some networks, including miR156-SPL, miR159-MYB, miR160-ARF, miR164-NAC, miR172-AP2, and miR319-TCP pairs. Six target genes in the networks were found to be orthologs of functionally confirmed genes participating in anther development in plants. Our results provide an insight that the ceRNA-miRNA-target gene regulatory networks likely contribute to anther development in maize. Further functional studies on a number of ceRNAs, miRNAs, and target genes will facilitate our deep understanding on mechanisms of anther development and sexual plants reproduction.

Characterization and complete genomic analysis of two Salmonella phages, SenALZ1 and SenASZ3, new members of the genus Cba120virus.

Salmonella phages SenALZ1 and SenASZ3, two novel phages infecting Salmonella enterica, were isolated and analyzed. The genomes of these two phages consist of 154,811 and 157,630 base pairs (bp), with G+C contents of 44.56% and 44.74%, respectively. Fifty-nine of 199 open reading frames (ORFs) in the SenALZ1 genome, and 60 of the 204 in the SenASZ3 genome show similarity to reference sequences in the NCBI nr database that encode putative phage proteins with predicted functions. Based on the results of transmission electron microscopy (TEM) examination, complete genome sequence alignment, phylogenetic analysis, and gene annotation, we propose that these two phages are representative isolates of two new species of the genus Cba120virus, subfamily Cvivirinae, family Ackermannviridae.

ZmMs30 Encoding a Novel GDSL Lipase Is Essential for Male Fertility and Valuable for Hybrid Breeding in Maize.

Genic male sterility (GMS) is very useful for hybrid vigor utilization and hybrid seed production. Although a large number of GMS genes have been identified in plants, little is known about the roles of GDSL lipase members in anther and pollen development. Here, we report a maize GMS gene, ZmMs30, which encodes a novel type of GDSL lipase with diverged catalytic residues. Enzyme kinetics and activity assays show that ZmMs30 has lipase activity and prefers to substrates with a short carbon chain. ZmMs30 is specifically expressed in maize anthers during stages 7-9. Loss of ZmMs30 function resulted in defective anther cuticle, irregular foot layer of pollen exine, and complete male sterility. Cytological and lipidomics analyses demonstrate that ZmMs30 is crucial for the aliphatic metabolic pathway required for pollen exine formation and anther cuticle development. Furthermore, we found that male sterility caused by loss of ZmMs30 function was stable in various inbred lines with different genetic background, and that it didn't show any negative effect on maize heterosis and production, suggesting that ZmMs30 is valuable for cross-breeding and hybrid seed production. We then developed a new multi-control sterility system using ZmMs30 and its mutant line, and demonstrated it is feasible for generating desirable GMS lines and valuable for hybrid maize seed production. Taken together, our study sheds new light on the mechanisms of anther and pollen development, and provides a valuable male-sterility system for hybrid breeding maize.