Paramutation involves homologous sequence communication that leads to meiotically heritable transcriptional silencing. We demonstrate that mop2 (mediator of paramutation2), which alters paramutation ...at multiple loci, encodes a gene similar to Arabidopsis NRPD2/E2, the second-largest subunit of plant-specific RNA polymerases IV and V. In Arabidopsis, Pol-IV and Pol-V play major roles in RNA-mediated silencing and a single second-largest subunit is shared between Pol-IV and Pol-V. Maize encodes three second-largest subunit genes: all three genes potentially encode full length proteins with highly conserved polymerase domains, and each are expressed in multiple overlapping tissues. The isolation of a recessive paramutation mutation in mop2 from a forward genetic screen suggests limited or no functional redundancy of these three genes. Potential alternative Pol-IV/Pol-V-like complexes could provide maize with a greater diversification of RNA-mediated transcriptional silencing machinery relative to Arabidopsis. Mop2-1 disrupts paramutation at multiple loci when heterozygous, whereas previously silenced alleles are only up-regulated when Mop2-1 is homozygous. The dramatic reduction in b1 tandem repeat siRNAs, but no disruption of silencing in Mop2-1 heterozygotes, suggests the major role for tandem repeat siRNAs is not to maintain silencing. Instead, we hypothesize the tandem repeat siRNAs mediate the establishment of the heritable silent state-a process fully disrupted in Mop2-1 heterozygotes. The dominant Mop2-1 mutation, which has a single nucleotide change in a domain highly conserved among all polymerases (E. coli to eukaryotes), disrupts both siRNA biogenesis (Pol-IV-like) and potentially processes downstream (Pol-V-like). These results suggest either the wild-type protein is a subunit in both complexes or the dominant mutant protein disrupts both complexes. Dominant mutations in the same domain in E. coli RNA polymerase suggest a model for Mop2-1 dominance: complexes containing Mop2-1 subunits are non-functional and compete with wild-type complexes.
Abstract
The selection and introduction of disease resistance genes in livestock not only provide health benefits to animals but opportunities for breeders and farmers to meet the growing demand for ...high-quality meat and milk while reducing agriculture’s footprint on the environment. As traditional methods of classical breeding and selection for trait improvement are slow, recent progress in several areas of biology including (a) understanding host–pathogen interactions, (b) inexpensive and rapid DNA sequencing, and (c) robust gene editing like CRISPR-Cas provide geneticists tools to accelerate discovery and deployment of disease resistance alleles in livestock. Using these advances, the introduction of resistance genes into commercially relevant germplasm requires access to genetically superior livestock, an infrastructure for scalable allele deployment, freedom to operate, global regulatory approvals, and acceptance of gene edited livestock by producers and consumers. Importantly, academic researchers have recently discovered that modification of the CD163 gene in pigs can confer resistance to the virus that causes porcine reproductive and respiratory syndrome (PRRS). While this achievement represents a major step towards solving an important disease in livestock, to realize the positive impact on animal health while benefiting the pork industry and consumers, it is necessary to introduce this recessive disease resistance allele into commercial breeding populations. Rather than backcrossing the resistance gene from a few non-commercial founders, as a global supplier of high genetic merit livestock genetics, Genus plc and its pig division PIC (Pig Improvement Company) with Genus R&D have mobilized advances in reproductive biology, gene editing, DNA sequencing, and bioinformatics to simultaneously generate and introduce a single modified CD163 allele across four genetically diverse porcine lines of commercial importance that prevents PRRS virus (PRRSV) infection. This report focuses on technical aspects for a scaled gene editing program to consider for rapid and efficient generation and advancement of a small population of non-transgenic founder pigs for commercial breeding. This high genetic merit herd containing a PRRS disease resistance allele will provide important benefits to animal health and food chain value once approved for commercial sale and export.
Transcriptional gene silencing has broad applications for studying gene function in planta. In maize, a large number of genes have been identified as tassel-preferred in their expression pattern, ...both by traditional genetic methods and by recent high-throughput expression profiling platforms. Approaches using RNA suppression may provide a rapid alternative means to identify genes directly related to pollen development in maize. The male fertility gene Ms45 and several anther-expressed genes of unknown function were used to evaluate the efficacy of generating male-sterile plants by transcriptional gene silencing. A high frequency of male-sterile plants was obtained by constitutively expressing inverted repeats (IR) of the Ms45 promoter. These sterile plants lacked MS45 mRNA due to transcriptional inactivity of the target promoter. Moreover, fertility was restored to these promoter IR-containing plants by expressing the Ms45 coding region using heterologous promoters. Transcriptional silencing of other anther-expressed genes also significantly affected male fertility phenotypes and led to increased methylation of the target promoter DNA sequences. These studies provide evidence of disruption of gene activity in monocots by RNA interference constructs directed against either native or transformed promoter regions. This approach not only enables the correlation of monocot anther-expressed genes with functions that are important for reproduction in maize, but may also provide a tool for studying gene function and identifying regulatory components unique to transcriptional gene control.
Modern maize hybrids often contain biotech and native traits. To-date all biotech traits have been randomly inserted in the genome. Consequently, developing hybrids with multiple traits is expensive, ...time-consuming, and complex. Here we report using CRISPR-Cas9 to generate a complex trait locus (CTL) to facilitate trait stacking. A CTL consists of multiple preselected sites positioned within a small well-characterized chromosomal region where trait genes are inserted. We generated individual lines, each carrying a site-specific insertion landing pad (SSILP) that was targeted to a preselected site and capable of efficiently receiving a transgene via recombinase-mediated cassette exchange. The selected sites supported consistent transgene expression and the SSILP insertion had no effect on grain yield. We demonstrated that two traits residing at different sites within a CTL can be combined via genetic recombination. CTL technology is a major step forward in the development of multi-trait maize hybrids.
Targeted mutagenesis, editing of endogenous maize (Zea mays) genes, and site-specific insertion of a trait gene using clustered regularly interspaced short palindromic repeats (CRISPR)-associated ...(Cas)-guide RNA technology are reported in maize. DNA vectors expressing maize codon-optimizedStreptococcus pyogenesCas9 endonuclease and single guide RNAs were cointroduced with or without DNA repair templates into maize immature embryos by biolistic transformation targeting five different genomic regions: upstream of the liguleless1 (LIG1) gene, male fertility genes (Ms26andMs45), and acetolactate synthase (ALS) genes (ALS1andALS2). Mutations were subsequently identified at all sites targeted, and plants containing biallelic multiplex mutations atLIG1,Ms26, andMs45were recovered. Biolistic delivery of guide RNAs (as RNA molecules) directly into immature embryo cells containing preintegrated Cas9 also resulted in targeted mutations. Editing theALS2gene using either single-stranded oligonucleotides or double-stranded DNA vectors as repair templates yielded chlorsulfuron-resistant plants. Double-strand breaks generated by RNA-guided Cas9 endonuclease also stimulated insertion of a trait gene at a site nearLIG1by homology-directed repair. Progeny showed expected Mendelian segregation of mutations, edits, and targeted gene insertions. The examples reported in this study demonstrate the utility of Cas9-guide RNA technology as a plant genome editing tool to enhance plant breeding and crop research needed to meet growing agriculture demands of the future.
Development of transgenic cell lines or organisms for industrial, agricultural, or medicinal applications involves inserting DNA into the target genome in a way that achieves efficacious transgene ...expression without a deleterious impact on fitness. The genomic insertion site is widely recognized as an important determinant of success. However, the effect of chromosomal location on transgene expression and fitness has not been systematically investigated in plants. Here we evaluate the importance of transgene insertion site in maize and soybean using both random and site-specific transgene integration. We have compared the relative contribution of genomic location on transgene expression levels with other factors, including cis-regulatory elements, neighboring transgenes, genetic background, and zygosity. As expected, cis-regulatory elements and the presence/absence of nearby transgene neighbors can impact transgene expression. Surprisingly, we determined not only that genomic location had the least impact on transgene expression compared to the other factors that were investigated but that the majority of insertion sites recovered supported transgene expression levels that were statistically not distinguishable. All 68 genomic sites evaluated were capable of supporting high-level transgene expression, which was also consistent across generations. Furthermore, multilocation field evaluation detected no to little decrease in agronomic performance as a result of transgene insertion at the vast majority of sites we evaluated with a single construct in five maize hybrid backgrounds.
Key message
Hexaploid bread wheat is not readily amenable to traditional mutagenesis approaches. In this study, we show efficient utilization of CRISPR-Cas system and Next Generation Sequencing for ...mutant analysis in wheat.
Identification and manipulation of male fertility genes in hexaploid bread wheat is important for understanding the molecular basis of pollen development and to obtain novel sources of nuclear genetic male sterility (NGMS). The maize
Male sterile 45
(
Ms45
) gene encodes a strictosidine synthase-like enzyme and has been shown to be required for male fertility. To investigate the role of
Ms45
gene in wheat, mutations in the A, B and D homeologs were produced using CRISPR-Cas9. A variety of mutations in the three homeologs were recovered, including a plant from two different genotypes each with mutations in all three homeologs. Genetic analysis of the mutations demonstrated that all three wheat
Ms45
homeologs contribute to male fertility and that triple homozygous mutants are required to abort pollen development and achieve male sterility. Further, it was demonstrated that a wild-type copy of
Ms45
gene from rice was able to restore fertility to these wheat mutant plants. Taken together, these observations provide insights into the conservation of MS45 function in a polyploid species.
Ms45
based NGMS can be potentially utilized for a Seed Production Technology (SPT)-like hybrid seed production system in wheat.
Recently discovered bacteria and archaea adaptive immune system consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) endonuclease has been ...explored in targeted genome editing in different species.Streptococcus pyogenesCas9-guide RNA (gRNA) was successfully applied to generate targeted mutagenesis, gene integration, and gene editing in soybean (Glycine max). Two genomic sites,DD20andDD43on chromosome 4, were mutagenized with frequencies of 59% and 76%, respectively. Sequencing randomly selected transgenic events confirmed that the genome modifications were specific to the Cas9-gRNA cleavage sites and consisted of small deletions or insertions. Targeted gene integrations through homology-directed recombination were detected by border-specific polymerase chain reaction analysis for both sites at callus stage, and oneDD43homology-directed recombination event was transmitted to T1 generation. T1 progenies of the integration event segregated according to Mendelian laws and clean homozygous T1 plants with the donor gene precisely inserted at theDD43target site were obtained. The Cas9-gRNA system was also successfully applied to make a directed P178S mutation of acetolactate synthase1 gene through in planta gene editing.
Summary
Transcriptional gene silencing (TGS) can offer a straightforward tool for functional analysis of plant genes, particularly in polyploid species such as wheat, where genetic redundancy poses a ...challenge in applying mutagenesis approaches, including CRISPR gene editing. In this study, we demonstrate efficient TGS in wheat, mediated by constitutive RNA expression of a promoter inverted repeat (pIR). pIR‐mediated TGS of two anther‐specific genes, TaMs45 and TaMs1, abolished their function resulting in male sterility. Whilst TGS of TaMs45 required transcriptional silencing of all three homoeologs, a B‐genome‐specific pIR for TaMs1 was sufficient to confer male sterility. We further show that the pIRs effect TGS of TaMs45 gene through DNA methylation of homologous promoter sequence, successfully suppressing transcription of all three homoeologs. Applying pIR‐mediated TGS in wheat, we have generated a dominant male fertility system for production of hybrid seed and demonstrated the efficacy of this system under greenhouse and field conditions. This report describes the first successful TGS in wheat, whilst providing a dominant negative approach as alternative to gene knockout strategies for hybrid wheat breeding and seed production.
We have developed a novel hybridization platform that utilizes nuclear male sterility to produce hybrids in maize and other cross‐pollinating crops. A key component of this platform is a process ...termed Seed Production Technology (SPT). This process incorporates a transgenic SPT maintainer line capable of propagating nontransgenic nuclear male‐sterile lines for use as female parents in hybrid production. The maize SPT maintainer line is a homozygous recessive male sterile transformed with a SPT construct containing (i) a complementary wild‐type male fertility gene to restore fertility, (ii) an α‐amylase gene to disrupt pollination and (iii) a seed colour marker gene. The sporophytic wild‐type allele complements the recessive mutation, enabling the development of pollen grains, all of which carry the recessive allele but with only half carrying the SPT transgenes. Pollen grains with the SPT transgenes exhibit starch depletion resulting from expression of α‐amylase and are unable to germinate. Pollen grains that do not carry the SPT transgenes are nontransgenic and are able to fertilize homozygous mutant plants, resulting in nontransgenic male‐sterile progeny for use as female parents. Because transgenic SPT maintainer seeds express a red fluorescent protein, they can be detected and efficiently separated from seeds that do not contain the SPT transgenes by mechanical colour sorting. The SPT process has the potential to replace current approaches to pollen control in commercial maize hybrid seed production. It also has important applications for other cross‐pollinating crops where it can unlock the potential for greater hybrid productivity through expanding the parental germplasm pool.
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