Reduction of Fusarium head blight and trichothecene contamination in transgenic wheat expressing Fusarium graminearum trichothecene 3-O-acetyltransferase.

Fusarium graminearum, the causal agent of Fusarium head blight (FHB), produces various mycotoxins that contaminate wheat grains and cause profound health problems in humans and animals. Deoxynivalenol (DON) is the most common trichothecene found in contaminated grains. Our previous study showed that Arabidopsis-expressing F. graminearum trichothecene 3-O-acetyltransferase (FgTRI101) converted DON to 3-acetyldeoxynivalenol (3-ADON) and excreted it outside of Arabidopsis cells. To determine if wheat can convert and excrete 3-ADON and reduce FHB and DON contamination, FgTRI101 was cloned and introduced into wheat cv Bobwhite. Four independent transgenic lines containing FgTRI101 were identified. Gene expression studies showed that FgTRI101 was highly expressed in wheat leaf and spike tissues in the transgenic line FgTri101-1606. The seedlings of two FgTri101 transgenic wheat lines (FgTri101-1606 and 1651) grew significantly longer roots than the controls on media containing 5 µg/mL DON; however, the 3-ADON conversion and excretion was detected inconsistently in the seedlings of FgTri101-1606. Further analyses did not detect 3-ADON or other possible DON-related products in FgTri101-1606 seedlings after adding deuterium-labeled DON into the growth media. FgTri101-transgenic wheat plants showed significantly enhanced FHB resistance and lower DON content after they were infected with F. graminearum, but 3-ADON was not detected. Our study suggests that it is promising to utilize FgTRI101, a gene that the fungus uses for self-protection, for managing FHB and mycotoxin in wheat production.

Metabolism of Tembotrione, a Triketone Herbicide, confers Differential Sensitivity in Winter Wheat (Triticum aestivum).

Tembotrione is a triketone herbicide widely used for broad-spectrum weed control in corn but not registered for use in wheat. A wide collection of spring, winter, and EMS-derived mutant lines of wheat was evaluated for their response to tembotrione treatment. Two winter wheat (WW) genotypes (WW-1 and WW-2) were found to be least sensitive to this herbicide, surviving >6 times the field recommended dose (92 g ai ha-1) compared to the most sensitive genotype (WW-24). Further, HPLC analysis using [14C] tembotrione suggested that both WW-1 and WW-2 metabolized tembotrione rapidly to nontoxic metabolites. Pretreatment with a P450 inhibitor (malathion) followed by tembotrione application increased the sensitivity of WW-1 and WW-2 genotypes to this herbicide, suggesting likely involvement of P450 enzymes in metabolizing tembotrione similar to corn. Overall, our results suggest that the genotypes WW-1 and WW-2 can potentially be used to develop tembotrione-resistant wheat varieties.

Transformative Approaches for Sustainable Weed Management: The Power of Gene Drive and CRISPR-Cas9.

Weeds can negatively impact crop yields and the ecosystem's health. While many weed management strategies have been developed and deployed, there is a greater need for the development of sustainable methods for employing integrated weed management. Gene drive systems can be used as one of the approaches to suppress the aggressive growth and reproductive behavior of weeds, although their efficacy is yet to be tested. Their popularity in insect pest management has increased, however, with the advent of CRISPR-Cas9 technology, which provides specificity and precision in editing the target gene. This review focuses on the different types of gene drive systems, including the use of CRISPR-Cas9-based systems and their success stories in pest management, while also exploring their possible applications in weed species. Factors that govern the success of a gene drive system in weeds, including the mode of reproduction, the availability of weed genome databases, and well-established transformation protocols are also discussed. Importantly, the risks associated with the release of weed populations with gene drive-bearing alleles into wild populations are also examined, along with the importance of addressing ecological consequences and ethical concerns.

Bacterium-enabled transient gene activation by artificial transcription factors for resolving gene regulation in maize.

Understanding gene regulatory networks is essential to elucidate developmental processes and environmental responses. Here, we studied regulation of a maize (Zea mays) transcription factor gene using designer transcription activator-like effectors (dTALes), which are synthetic Type III TALes of the bacterial genus Xanthomonas and serve as inducers of disease susceptibility gene transcription in host cells. The maize pathogen Xanthomonas vasicola pv. vasculorum was used to introduce 2 independent dTALes into maize cells to induced expression of the gene glossy3 (gl3), which encodes a MYB transcription factor involved in biosynthesis of cuticular wax. RNA-seq analysis of leaf samples identified, in addition to gl3, 146 genes altered in expression by the 2 dTALes. Nine of the 10 genes known to be involved in cuticular wax biosynthesis were upregulated by at least 1 of the 2 dTALes. A gene previously unknown to be associated with gl3, Zm00001d017418, which encodes aldehyde dehydrogenase, was also expressed in a dTALe-dependent manner. A chemically induced mutant and a CRISPR-Cas9 mutant of Zm00001d017418 both exhibited glossy leaf phenotypes, indicating that Zm00001d017418 is involved in biosynthesis of cuticular waxes. Bacterial protein delivery of dTALes proved to be a straightforward and practical approach for the analysis and discovery of pathway-specific genes in maize.

Fusarium graminearum Effector FgNls1 Targets Plant Nuclei to Induce Wheat Head Blight.

Fusarium head blight (FHB) caused by Fusarium graminearum is one of the most devastating diseases of wheat and barley worldwide. Effectors suppress host immunity and promote disease development. The genome of F. graminearum contains hundreds of effectors with unknown function. Therefore, investigations of the functions of these effectors will facilitate developing novel strategies to enhance wheat resistance to FHB. We characterized a F. graminearum effector, FgNls1, containing a signal peptide and multiple eukaryotic nuclear localization signals. A fusion protein of green fluorescent protein and FgNls1 accumulated in plant cell nuclei when transiently expressed in Nicotiana benthamiana. FgNls1 suppressed Bax-induced cell death when co-expressed in N. benthamiana. We revealed that the expression of FgNLS1 was induced in wheat spikes infected with F. graminearum. The Fgnls1 mutants significantly reduced initial infection and FHB spread within a spike. The function of FgNLS1 was restored in the Fgnls1-complemented strains. Wheat histone 2B was identified as an interacting protein by FgNls1-affinity chromatography. Furthermore, transgenic wheat plants that silence FgNLS1 expression had significantly lower FHB severity than control plants. This study demonstrates a critical role of FgNls1 in F. graminearum pathogenesis and indicates that host-induced gene silencing targeting F. graminearum effectors is a promising approach to enhance FHB resistance. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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

The low efficiency of genetic transformation and gene editing across diverse cultivars hinder 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 perform direct inoculation of wheat leaves with the barley stripe mosaic virus (BSMV) transcripts to deliver guide RNAs (sgRNA) into the Cas9-expressing wheat. We demonstrate that wheat inoculation with the pool of BSMV-sgRNAs could be used to generate heritable precise deletions in the promoter region of a transcription factor and to perform multiplexed editing of agronomic genes. We transfer the high-expressing locus of Cas9 into adapted spring and winter cultivars by marker-assisted introgression and use of the BSMV-sgRNAs to edit two agronomic genes. A strategy presented in our study could be applied to any adapted cultivar for creating new cis-regulatory diversity or large-scale editing of 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.

Cloning of the broadly effective wheat leaf rust resistance gene Lr42 transferred from Aegilops tauschii.

The wheat wild relative Aegilops tauschii was previously used to transfer the Lr42 leaf rust resistance gene into bread wheat. Lr42 confers resistance at both seedling and adult stages, and it is broadly effective against all leaf rust races tested to date. Lr42 has been used extensively in the CIMMYT international wheat breeding program with resulting cultivars deployed in several countries. Here, using a bulked segregant RNA-Seq (BSR-Seq) mapping strategy, we identify three candidate genes for Lr42. Overexpression of a nucleotide-binding site leucine-rich repeat (NLR) gene AET1Gv20040300 induces strong resistance to leaf rust in wheat and a mutation of the gene disrupted the resistance. The Lr42 resistance allele is rare in Ae. tauschii and likely arose from ectopic recombination. Cloning of Lr42 provides diagnostic markers and over 1000 CIMMYT wheat lines carrying Lr42 have been developed documenting its widespread use and impact in crop improvement.

Effector Genes in Magnaporthe oryzae Triticum as Potential Targets for Incorporating Blast Resistance in Wheat.

Wheat blast (WB), caused by Magnaporthe oryzae Triticum pathotype, recently emerged as a destructive disease that threatens global wheat production. Because few sources of genetic resistance have been identified in wheat, genetic transformation of wheat with rice blast resistance genes could expand resistance to WB. We evaluated the presence/absence of homologs of rice blast effector genes in Triticum isolates with the aim of identifying avirulence genes in field populations whose cognate rice resistance genes could potentially confer resistance to WB. We also assessed presence of the wheat pathogen AVR-Rmg8 gene and identified new alleles. A total of 102 isolates collected in Brazil, Bolivia, and Paraguay from 1986 to 2018 were evaluated by PCR using 21 pairs of gene-specific primers. Effector gene composition was highly variable, with homologs to AvrPiz-t, AVR-Pi9, AVR-Pi54, and ACE1 showing the highest amplification frequencies (>94%). We identified Triticum isolates with a functional AvrPiz-t homolog that triggers Piz-t-mediated resistance in the rice pathosystem and produced transgenic wheat plants expressing the rice Piz-t gene. Seedlings and heads of the transgenic lines were challenged with isolate T25 carrying functional AvrPiz-t. Although slight decreases in the percentage of diseased spikelets and leaf area infected were observed in two transgenic lines, our results indicated that Piz-t did not confer useful WB resistance. Monitoring of avirulence genes in populations is fundamental to identifying effective resistance genes for incorporation into wheat by conventional breeding or transgenesis. Based on avirulence gene distributions, rice resistance genes Pi9 and Pi54 might be candidates for future studies.

Expanding the range of editable targets in the wheat genome using the variants of the Cas12a and Cas9 nucleases.

The development of CRISPR-based editors recognizing distinct protospacer-adjacent motifs (PAMs), or having different spacer length/structure requirements broadens the range of possible genomic applications. We evaluated the natural and engineered variants of Cas12a (FnCas12a and LbCas12a) and Cas9 for their ability to induce mutations in endogenous genes controlling important agronomic traits in wheat. Unlike FnCas12a, LbCas12a-induced mutations in the wheat genome, even though with a lower rate than that reported for SpCas9. The eight-fold improvement in the gene editing efficiency was achieved for LbCas12a by using the guides flanked by ribozymes and driven by the RNA polymerase II promoter from switchgrass. The efficiency of multiplexed genome editing (MGE) using LbCas12a was mostly similar to that obtained using the simplex RNA guides and showed substantial increase after subjecting transgenic plants to high-temperature treatment. We successfully applied LbCas12a-MGE for generating heritable mutations in a gene controlling grain size and weight in wheat. We showed that the range of editable loci in the wheat genome could be further expanded by using the engineered variants of Cas12a (LbCas12a-RVR) and Cas9 (Cas9-NG and xCas9) that recognize the TATV and NG PAMs, respectively, with the Cas9-NG showing higher editing efficiency on the targets with atypical PAMs compared to xCas9. In conclusion, our study reports a set of validated natural and engineered variants of Cas12a and Cas9 editors for targeting loci in the wheat genome not amenable to modification using the original SpCas9 nuclease.

Comparative analyses of transcriptional responses of Dectes texanus LeConte (Coleoptera: Cerambycidae) larvae fed on three different host plants and artificial diet.

Dectes texanus is an important coleopteran pest of soybeans and cultivated sunflowers in the Midwestern United States that causes yield losses by girdling stems of their host plants. Although sunflower and giant ragweed are primary hosts of D. texanus, they began colonizing soybeans approximately 50 years ago and no reliable management method has been established to prevent or reduce losses by this pest. To identify genes putatively involved when feeding soybean, we compared gene expression of D. texanus third-instar larvae fed soybean to those fed sunflower, giant ragweed, or artificial diet. Dectes texanus larvae differentially expressed 514 unigenes when fed on soybean compared to those fed the other diet treatments. Enrichment analyses of gene ontology terms from up-regulated unigenes in soybean-fed larvae compared to those fed both primary hosts highlighted unigenes involved in oxidoreductase and polygalacturonase activities. Cytochrome P450s, carboxylesterases, major facilitator superfamily transporters, lipocalins, apolipoproteins, glycoside hydrolases 1 and 28, and lytic monooxygenases were among the most commonly up-regulated unigenes in soybean-fed larvae compared to those fed their primary hosts. These results suggest that D. texanus larvae differentially expressed unigenes involved in biotransformation of allelochemicals, digestion of plant cell walls and transport of small solutes and lipids when feeding in soybean.

A Lipid Transfer Protein has Antifungal and Antioxidant Activity and Suppresses Fusarium Head Blight Disease and DON Accumulation in Transgenic Wheat.

Trichothecene mycotoxins such as deoxynivalenol (DON) are virulence factors of Fusarium graminearum, which causes Fusarium head blight, one of the most important diseases of small grain cereals. We previously identified a nonspecific lipid transfer protein (nsLTP) gene, AtLTP4.4, which was overexpressed in an activation-tagged Arabidopsis line resistant to trichothecin, a type B trichothecene in the same class as DON. Here we show that overexpression of AtLTP4.4 in transgenic wheat significantly reduced F. graminearum growth in 'Bobwhite' and 'RB07' lines in the greenhouse and reduced fungal lesion size in detached leaf assays. Hydrogen peroxide accumulation was attenuated on exposure of transgenic wheat plants to DON, indicating that AtLTP4.4 may confer resistance by inhibiting oxidative stress. Field testing indicated that disease severity was significantly reduced in two transgenic 'Bobwhite' lines expressing AtLTP4.4. DON accumulation was significantly reduced in four different transgenic 'Bobwhite' lines expressing AtLTP4.4 or a wheat nsLTP, TaLTP3, which was previously shown to have antioxidant activity. Recombinant AtLTP4.4 purified from Pichia pastoris exhibited potent antifungal activity against F. graminearum. These results demonstrate that overexpression of AtLTP4.4 in transgenic wheat suppresses DON accumulation in the field. Suppression of DON-induced reactive oxygen species by AtLTP4.4 might be the mechanism by which fungal spread and mycotoxin accumulation are inhibited in transgenic wheat plants.

Identification of candidate chromosome region of Sbwm1 for Soil-borne wheat mosaic virus resistance in wheat.

Soil-borne wheat mosaic virus (SBWMV) causes a serious viral disease that can significantly reduce grain yield in winter wheat worldwide. Using resistant cultivars is the only feasible strategy to reduce the losses caused by SBWMV. To fine map the resistance gene Sbwm1, 205 wheat accessions was genotyped using wheat Infinium iSelect Beadchips with 90 K SNPs. Association analysis identified 35 SNPs in 12 wheat genes and one intergenic SNP in the Sbwm1 region that showed a significant association with SBWMV resistance. Those SNPs were converted into Kompetitive Allele-Specific Polymerase assays (KASP) and analyzed in two F6-derived recombinant inbred line (RIL) populations derived from the crosses between two resistant cultivars 'Wesley' and 'Deliver' and a susceptible line 'OK03825-5403-6'. Linkage analysis mapped this gene on chromosome 5D at intervals of 5.1 cM and 3.4 cM in the two populations, respectively. The two flanking markers in both populations delimited the gene to a 620 kb region where 19 genes were annotated. Comparative analysis identified a syntenic region of 660 kb in Ae. tauschii with 18 annotated genes and a syntenic region in chromosome 1 of B. distachyon. The candidate region includes several disease resistance related genes and we identified a PTI1-like tyrosine-protein kinase 1 gene as a putative candidate gene for Sbwm1. The two flanking SNPs for Sbwm1 can effectively separate the resistant and susceptible lines in a new diversity panel of 159 wheat germplasm. The results from this study lay a solid foundation for the cloning, functional characterization and marker-assisted selection of Sbwm1.

A simplified method for producing laboratory grade recombinant TEV protease from E. coli.

The tobacco etch virus (TEV) protease has become a popular choice for cleaving fusion proteins because of its high stringency in sequence recognition. Procedures for isolating recombinant protein from the cytoplasm of E. coli require rupturing of the cell wall via enzymatic treatment combined with sonication or French press. Here we present an expedited method for producing laboratory-grade TEV protease in E. coli using a freeze-thaw method, followed by purification with immobilized metal affinity chromatography. Protease is obtained by expression from the pDZ2087 plasmid in BL21 (DE3) cells. Proteolysis resulting from this product, cleaves a maltose-binding protein fusion to completion at a fusion-to-protease molar ratio of 50:1.

Allelochemicals targeted to balance competing selections in African agroecosystems.

Among major cereals domesticated as staple food, only sorghum has a high proportion of cultivars with condensed tannins in grain, which can trigger bitter taste perception in animals by binding to type 2 taste receptors (TAS2Rs). Here, we report the completion of uncovering of a pair of duplicate recessive genes (Tannin1 and Tannin2) underlying tannin presence. Three loss-of-function alleles from each gene were identified in non-tannin sorghum desired as palatable food. Condensed tannins effectively prevented sparrows from consuming sorghum grain. Parallel geographic distributions between tannin sorghum and Quelea quelea supported the role of tannins in fighting against this major herbivore threat. Association between geographic distributions of human TAS2R variants and tannin sorghum across Africa suggested that different causes had probably driven this bidirectional selection according to varied local herbivore threats and human taste sensitivity. Our investigation uncovered coevolution among humans, plants and environments linked by allelochemicals.

Host-derived gene silencing of parasite fitness genes improves resistance to soybean cyst nematodes in stable transgenic soybean.

KEY MESSAGE: Soybean expressing small interfering RNA of SCN improved plant resistance to SCN consistently, and small RNA-seq analysis revealed a threshold of siRNA expression required for resistance ability. Soybean cyst nematode (SCN), Heterodera glycines, is one of the most destructive pests limiting soybean production worldwide, with estimated losses of $1 billion dollars annually in the USA alone. RNA interference (RNAi) has become a powerful tool for silencing gene expression. We report here that the expression of hairpin RNAi constructs, derived from two SCN genes related to reproduction and fitness, HgY25 and HgPrp17, enhances resistance to SCN in stably transformed soybean plants. The analyses of T3 to T5 generations of stable transgenic soybeans by molecular strategies and next-generation sequencing confirmed the presence of specific short interfering RNAs complementary to the target SCN genes. Bioassays performed on transgenic soybean lines targeting SCN HgY25 and HgPrp17 fitness genes showed significant reductions (up to 73%) for eggs/g root in the T3 and T4 homozygous transgenic lines. Targeted mRNAs of SCN eggs collected from the transgenic soybean lines were efficiently down-regulated, as confirmed by quantitative RT-PCR. Based on the small RNA-seq data and bioassays, it is our hypothesis that a threshold of small interfering RNA molecules is required to significantly reduce SCN populations feeding on the host plants. Our results demonstrated that host-derived gene silencing of essential SCN fitness genes could be an effective strategy for enhancing resistance in crop plants.

Gene editing of the wheat homologs of TONNEAU1-recruiting motif encoding gene affects grain shape and weight in wheat.

Grain size and weight are important components of a suite of yield-related traits in crops. Here, we showed that the CRISPR-Cas9 gene editing of TaGW7, a homolog of rice OsGW7 encoding a TONNEAU1-recruiting motif (TRM) protein, affects grain shape and weight in allohexaploid wheat. By editing the TaGW7 homoeologs in the B and D genomes, we showed that mutations in either of the two or both genomes increased the grain width and weight but reduced the grain length. The effect sizes of mutations in the TaGW7 gene homoeologs coincided with the relative levels of their expression in the B and D genomes. The effects of gene editing on grain morphology and weight traits were dosage dependent with the double-copy mutant showing larger effect than the respective single copy mutants. The TaGW7-centered gene co-expression network indicated that this gene is involved in the pathways regulating cell division and organ growth, also confirmed by the cellular co-localization of TaGW7 with α- and ß-tubulin proteins, the building blocks of microtubule arrays. The analyses of exome capture data in tetraploid domesticated and wild emmer, and hexaploid wheat revealed the loss of diversity around TaGW7-associated with domestication selection, suggesting that TaGW7 is likely to play an important role in the evolution of yield component traits in wheat. Our study showed how integrating CRISPR-Cas9 system with cross-species comparison can help to uncover the function of a gene fixed in wheat for allelic variants targeted by domestication selection and select targets for engineering new gene variants for crop improvement.

Targeting the pattern-triggered immunity pathway to enhance resistance to Fusarium graminearum.

Fusarium head blight (FHB) is a disease of the floral tissues of wheat and barley for which highly resistant varieties are not available. Thus, there is a need to identify genes/mechanisms that can be targeted for the control of this devastating disease. Fusarium graminearum is the primary causal agent of FHB in North America. In addition, it also causes Fusarium seedling blight. Fusarium graminearum can also cause disease in the model plant Arabidopsis thaliana. The Arabidopsis-F. graminearum pathosystem has facilitated the identification of targets for the control of disease caused by this fungus. Here, we show that resistance against F. graminearum can be enhanced by flg22, a bacterial microbe-associated molecular pattern (MAMP). flg22-induced resistance in Arabidopsis requires its cognate pattern recognition receptor (PRR) FLS2, and is accompanied by the up-regulation of WRKY29. The expression of WRKY29, which is associated with pattern-triggered immunity (PTI), is also induced in response to F. graminearum infection. Furthermore, WRKY29 is required for basal resistance as well as flg22-induced resistance to F. graminearum. Moreover, constitutive expression of WRKY29 in Arabidopsis enhances disease resistance. The PTI pathway is also activated in response to F. graminearum infection of wheat. Furthermore, flg22 application and ectopic expression of WRKY29 enhance FHB resistance in wheat. Thus, we conclude that the PTI pathway provides a target for the control of FHB in wheat. We further show that the ectopic expression of WRKY29 in wheat results in shorter stature and early heading time, traits that are important to wheat breeding.

Biolistic Transformation of Wheat.

Biolistic transformation of wheat is one of the most commonly used methods for gene function study and trait discovery. It has been widely adapted as a fundamental platform to generate wheat plants with new traits and has become a powerful tool for facilitating the crop improvement. In this chapter, we present a complete and straightforward protocol for wheat transformation via biolistic bombardment system. Although wheat is still one of the hardest plant species to transform, this protocol offers an optimized and efficient system to produce transgenic plants. To demonstrate the application of this protocol, in this chapter we describe an example of obtaining transgenic wheat by the co-bombardment of two plasmids, containing a green fluorescent protein gene and a glufosinate herbicide selection gene, respectively. In addition, procedures for the screening and testing of putative transgenic plants are described. This protocol has been successfully applied to generate stable transgenic bread wheat (Triticum aestivum) in both spring and winter varieties.