Biology subjects

Trick, H. N.

Publications and source records attributed to Trick, H. N..

6 recordsLinked to original sources

bioRxiv2024

Non-additive dosage-dependent effects of TaGS3 gene editing on grain size and weight in wheat

The grain size in cereals is one of the main component traits contributing to yield. Previous studies showed that loss-of-function (LOF) mutations in GS3, encoding G{gamma} subunit of the multimeric G protein complex, increase grain size and weight in rice. While association between allelic variation in GS3 homologs of wheat and grain weight/size was detected previusly, the effects of LOF alleles on these traits remain unknown. We used genome editing to create the TaGS3 mutant lines with the LOF homeo-allele dosage variation. Contrary to results obtained for rice, editing of all three TaGS3 copies result in significant decrease in grain length, width, grain area and weight, without affecting number of grains per spike. Compared to wild type, the highest increase in grain weight and area was observed in mutants with the intermediate dosage of the LOF alleles, indicating that suppressive effects of TaGS3 on grain size and weight in wheat are dosage-dependent and non-additive. Our results suggest that TaGS3 likely represents a functionally diverged homolog of GS3 evolved in the wheat lineage. The newly developed LOF alleles of TaGS3 expand the set of CRISPR-Cas9-induced variants of yield component genes that could be used for increasing grain weight in wheat.

genetics↗

bioRxiv2022

Wild-type allele of TaHRC suppresses calcium-mediated plant immune response by hijacking TaCAXIP4 to trigger FHB susceptibility in wheat

Fusarium head blight (FHB) is one of the most destructive diseases of wheat worldwide. Epidemics of FHB cause a serious reduction in grain yield and quality of wheat and result in significant economic losses to wheat producers. Recently, we have cloned a histidine-rich calcium-binding protein gene (TaHRC) as the causal gene for Fhb1 and demonstrated that the wild-type allele of TaHRC conditions FHB susceptibility and a large deletion including the start codon resulted in FHB resistance. However, the molecular mechanisms on how TaHRC regulating FHB susceptibility remains unknown. In this study, we conducted yeast two-hybrid screening (Y2H) against the wheat cDNA expression libraries using TaHRC as bait and identified a cation exchanger (CAX)-interacting protein 4 (TaCAXIP4) as the candidate protein that interacts with TaHRC to affect calcium transport activity. The strong interaction was further confirmed by Bimolecular fluorescence complementation (BiFC) assays. Using gene editing, we edited three different sites (one before and one within and one after the NLS domain) of TaHRC in a susceptible wheat cultivar Bobwhite using the CRISPR/Cas9 gene editing technology and demonstrated the N-terminus carrying NLS domain of TaHRC plays a critical role for the interaction and conditions TaHRC function on FHB susceptibility. We determined that the interaction between TaCAXIP4 and TaHRC occurs in the nuclei of cells by subcellular colocalization assay. Intriguingly, we found TaHRC can sequester TaCAXIP4 to suppress the Ca2+ transporting activity of TaCAX1 (a H+/Ca2+ antiporter) through yeast calcium suppression assay and suggested wild-type TaHRC may hijack TaCAXIP4 to suppresses calcium-mediated plant immune response during Fusarium infection in wheat. Furthermore, we performed the reactive oxygen species (ROS) assays and further showed that TaHRC might suppress the chitin-triggered plant immune responses during Fusarium infection by sequestering TaCAXIP4 to trigger FHB susceptibility, which facilitates the pathogen spread within a wheat spike. This work provides first line of evidence to support wild type Fhb1 is a susceptible gene and how Fhb1 wild type allele regulate FHB susceptibility.

plant biology↗

bioRxiv2022

Multiplexed promoter and gene editing in wheat using the virus-based guide RNA delivery system

The low efficiency of genetic transformation and gene editing across diverse cultivars hinders the broad application of CRISPR technology for crop improvement. The development of virus-based methods of CRISPR-Cas system delivery into the plant cells holds great promise to overcome these limitations. Here, we applied the barley stripe mosaic virus (BSMV) for delivering guide RNAs (sgRNA) into the Cas9-expressing wheat lines to create targeted deletions in the promoter of a transcription factor and to perform multiplexed editing of agronomic genes. We demonstrate that pooled BSMV-sgRNAs could be used to generate heritable targeted deletions and multiple mutations in the genome. We transferred the high-expressing allele of Cas9 into adapted spring and winter cultivars and successfully performed the BSMV-sgRNA-based editing of two agronomic genes. The strategies presented in our study could be applied to any cultivar for creating new cis-regulatory diversity or targeting multiple genes in biological pathways or QTL regions, opening possibilities for the effective engineering of crop genomes and accelerating gene discovery and trait improvement efforts.

genetics↗

bioRxiv2022

Development and optimization of a Barley stripe mosaic virus (BSMV)-mediated gene editing system to improve Fusarium head blight (FHB) resistance in wheat

Fusarium head blight (FHB) is a devastating disease in wheat that causes million dollars of wheat yield losses annually in the U.S.A. Recently we demonstrated that wheat carry FHB susceptible genes and knocking out the susceptible gene increased FHB resistance, which suggests that manipulating susceptible genes using gene editing may open a new avenue to create new sources of FHB resistance. However, wheat genome editing uses gene transformation to deliver CRISPR/Cas9 and gRNA into plants, and most wheat genotypes are not suitable for transformation due to low rates of callus induction and regeneration, therefore it cannot be used in practical wheat breeding. In this study, we developed a new Barley stripe mosaic virus (BSMV)-mediated gRNA delivery system that does not need the gene transformation and tissue culture and it can be used in any genotypes for gene function validation and editing. We used this system edited the susceptible allele of Fhb1, a major FHB resistance gene. We demonstrated that the edited trait is heritable in different genetic backgrounds and knocking out Fhb1 susceptible allele improved FHB resistance in wheat. We also modified system to improve editing efficiency by using floral dip agroinfiltration and adding RNA mobility sequences to the gRNA in the viral vectors. We believe this work will facilitate wheat FHB resistance research and gene editing in cereal crops and will benefit cereal crop researchers and breeders worldwide.

plant biology↗

bioRxiv2021

Bacterium-Enabled Transient Gene Activation by Artificial Transcription Factor for Resolving Gene Regulation in Maize

Cellular functions are diversified through intricate transcription regulations, and an understanding gene regulation networks is essential to elucidating many developmental processes and environmental responses. Here, we employed the Transcriptional-Activator Like effectors (TALes), which represent a family of transcription factors that are synthesized by members of the {gamma}-proteobacterium genus Xanthomonas and secreted to host cells for activation of targeted host genes. Through delivery by the maize pathogen, Xanthomonas vasicola pv. vasculorum, designer TALes (dTALes), which are synthetic TALes, were used to induce the expression of the maize gene glossy3 (gl3), a MYB transcription factor gene involved in the cuticular wax biosynthesis. RNA-Seq analysis of leaf samples identified 146 gl3 downstream genes. Eight of the nine known genes known to be involved in the cuticular wax biosynthesis were up-regulated by at least one dTALe. A top-down Gaussian graphical model predicted that 68 gl3 downstream genes were directly regulated by GL3. A chemically induced mutant of the gene Zm00001d017418 from the gl3 downstream gene, encoding aldehyde dehydrogenase, exhibited a typical glossy leaf phenotype and reduced epicuticular waxes. The bacterial protein delivery of artificial transcription factors, dTALes, proved to be a straightforward and powerful approach for the revelation of gene regulation in plants.

plant biology↗

bioRxiv2018

Gene editing and mutagenesis reveal inter-cultivar differences and additivity in the contribution of TaGW2 homoeologues to grain size and weight in wheat

The TaGW2 gene homoeologues have been reported to be negative regulators of grain size (GS) and thousand grain weight (TGW) in wheat. However, the contribution of each homoeologue to trait variation among different wheat cultivars is not well documented. We used the CRISPR-Cas9 system and TILLING to mutagenize each homoeologous gene copy in cultivars Bobwhite and Paragon, respectively. Plants carrying single-copy nonsense mutations in different genomes showed different levels of GS/TGW increase, with TGW increasing by an average of 5.5% (edited lines) and 5.3% (TILLING mutants). In any combination, the double homoeologue mutants showed higher phenotypic effects than the respective single-genome mutants. The double mutants had on average 12.1% (edited) and 10.5% (TILLING) higher TGW with respect to wild-type lines. The highest increase in GS and TGW was shown for triple mutants of both cultivars, with increases of 16.3% (edited) and 20.7% (TILLING) in TGW. The additive effects of the TaGW2 homoeologues were also demonstrated by the negative correlation between the functional gene copy number and GS/TGW in Bobwhite mutants and an F2 population. The highest single-genome increases in GS and TGW in Paragon and Bobwhite were obtained by mutations in the B and D genomes, respectively. These inter-cultivar differences in the phenotypic effects between the TaGW2 gene homoeologues coincide with inter-cultivar differences in the homoeologue expression levels. These results indicate that GS/TGW variation in wheat can be modulated by the dosage of homoeologous genes with inter-cultivar differences in the magnitude of the individual homoeologue effects.

genetics↗