Barley Stripe Mosaic Virus Research Articles

Puccinia triticina avirulence protein AvrLr21 directly interacts with wheat resistance protein Lr21 to activate wheat immune response

Wheat leaf rust, caused by Puccinia triticina (Pt), remains a constant threat to wheat production worldwide. Deployment of race-specific leaf rust (Lr) resistance genes in wheat provides effective protection against leaf rust, but often leads to selective pressures that drive the rapid emergence of new virulent Pt isolates in nature. However, the molecular mechanisms underlying the evasion of Lr-delivered resistance by leaf rust remain largely unknown. Here, we identify an avirulence gene AvrLr21 in Pt that triggers Lr21-dependent immune responses. BSMV (Barley stripe mosaic virus)-mediated host-induced gene silencing assay shows that silencing AvrLr21 compromises Lr21-mediated immunity. AvrLr21 interacts directly with Lr21 protein to induce a hypersensitive response in tobacco leaves. The evolved Lr21-breaking Pt isolates can suppress Lr21-mediated immunity. Our data provide a basis for studying the molecular determinants in Pt-wheat incompatible interaction and monitoring natural Pt populations to prioritize the deployment of Lr resistance genes in the field.

DEAD-box RNA helicase RH20 positively regulates RNAi-based antiviral immunity in plants by associating with SGS3/RDR6 bodies.

RNA silencing plays a crucial role in defending against viral infections in diverse eukaryotic hosts. Despite extensive studies on core components of the antiviral RNAi pathway such as DCLs, AGOs and RDRs proteins, host factors involved in antiviral RNAi remain incompletely understood. In this study, we employed the proximity labelling approach to identify the host factors required for antiviral RNAi in Nicotiana benthamiana. Using the barley stripe mosaic virus (BSMV)-encoded γb, a viral suppressor of RNA silencing (VSR), as the bait protein, we identified the DEAD-box RNA helicase RH20, a broadly conserved protein in plants and animals with a homologous human protein known as DDX5. We demonstrated the interaction between RH20 and BSMV γb. Knockdown or knockout of RH20 attenuates the accumulation of viral small interfering RNAs, leading to increased susceptibility to BSMV, while overexpression of RH20 enhances resistance to BSMV, a process requiring the cytoplasmic localization and RNA-binding activity of RH20. In addition to BSMV, RH20 also negatively regulates the infection of several other positive-sense RNA viruses, suggesting the broad-spectrum antiviral activity of RH20. Mechanistic analysis revealed the colocalization and interaction of RH20 with SGS3/RDR6, and disruption of either SGS3 or RDR6 undermines the antiviral function of RH20, suggesting RH20 as a new component of the SGS3/RDR6 bodies. As a counter-defence, BSMV γb VSR subverts the RH20-mediated antiviral defence by interfering with the RH20-SGS3 interaction. Our results uncover RH20 as a new positive regulator of antiviral RNAi and provide new potential targets for controlling plant viral diseases.

Electroless Deposition of Noble Metals on Rod-Shape Plant Viruses in Various Aqueous Metal Precursor Solutions.

The challenge of synthesizing noble metal nanostructures sustainably has encouraged researchers to explore biological routes for nanostructure production, such as biotemplating. Plant viruses with rod-shape morphology, such as tobacco mosaic virus (TMV) and barley stripe mosaic virus (BSMV), offer promising biotemplates to produce metal nanorods. TMV and BSMV can be incubated in aqueous metal precursor solutions to mineralize metals on the coat proteins (CPs) of the viruses. Previous studies have primarily examined palladium (Pd) mineralization on TMV and BSMV using Na2PdCl4 as the Pd precursor. There is limited scientific literature on the effect of using alternative Pd precursor solutions besides Na2PdCl4 such as K2PdCl4 and PdCl2 to mineralize Pd on TMV and BSMV. Past attempts at mineralizing other noble metals such as platinum (Pt) and gold (Au) required an initial layer of Pd to be deposited on the TMV and BSMV biotemplates. In this study, we aimed to expand the understanding of using alternative Pd precursor solutions to mineralize Pd on TMV and BSMV. Additionally, the deposition of Pt and Au onto TMV and BSMV without the need for an initial Pd mineralization layer was achieved using alternative Pt and Au precursors, including K2PtCl4 and AuCl3, respectively. Pd, Pt, and Au were successfully deposited on TMV and BSMV by incubation in aqueous solutions of Na2PdCl4, K2PdCl4, PdCl2, K2PtCl4, and AuCl3. Kinetic studies were also conducted using ultraviolet-visible (UV-vis) spectroscopy to examine the rates at which Pd, Pt, and Au precursor ions were reduced during the mineralization process, mimicking their adsorption onto TMV and BSMV CPs. BSMV adsorbed noble metal precursor ions faster than TMV as determined by UV-vis spectroscopy. While palladium nanorods (PdNRs) offer high electrical conductivity desirable for electronic applications, Pd-coated TMV and BSMV may face limitations due to their organic cores, potentially compromising conductivity. To address this, one approach is to convert the organic core into conductive amorphous carbon through thermal annealing. In this study, in situ transmission electron microscopy was utilized to thermally anneal Pd-TMV2Cys, thereby transforming them into PdNRs with amorphous carbon cores.

Zn2+-dependent association of cysteine-rich protein with virion orchestrates morphogenesis of rod-shaped viruses.

The majority of rod-shaped and some filamentous plant viruses encode a cysteine-rich protein (CRP) that functions in viral virulence; however, the roles of these CRPs in viral infection remain largely unknown. Here, we used barley stripe mosaic virus (BSMV) as a model to investigate the essential role of its CRP in virus morphogenesis. The CRP protein γb directly interacts with BSMV coat protein (CP), the mutations either on the His-85 site in γb predicted to generate a potential CCCH motif or on the His-13 site in CP exposed to the surface of the virions abolish the zinc-binding activity and their interaction. Immunogold-labeling assays show that γb binds to the surface of rod-shaped BSMV virions in a Zn2+-dependent manner, which enhances the RNA binding activity of CP and facilitates virion assembly and stability, suggesting that the Zn2+-dependent physical association of γb with the virion is crucial for BSMV morphogenesis. Intriguingly, the tightly binding of diverse CRPs to their rod-shaped virions is a general feature employed by the members in the families Virgaviridae (excluding the genus Tobamovirus) and Benyviridae. Together, these results reveal a hitherto unknown role of CRPs in the assembly and stability of virus particles, and expand our understanding of the molecular mechanism underlying virus morphogenesis.

Development of a Csy4-Processing TRV-Based CRISPR/Cas9 Genome Editing System in Nicotiana benthamiana

AbstractThe CRISPR/Cas9 system is a site-specific genome editing tool that has been widely used in various plant species. The plant virus-based gRNA (guide RNA) delivery system, which differs from the typical Agrobacterium-mediated transformation, is an attractive method to facilitate the application of CRISPR/Cas9. The virally delivered gRNA is usually driven by heterologous plant U6 or viral promoters (e.g., pea early-browning virus, PEBV; barley stripe mosaic virus, BSMV). However, heterologous promoters may have poor performance in some cases. In this paper, a feasible option to detach gRNA(s) from the virus genome is employed. Specifically, the Csy4-RNA processing system is used to release gRNA(s) from the tobacco rattle virus (TRV). The coding sequences of Cas9 and Csy4 nucleases are cloned into a single polycistronic expression cassette under an estrogen-inducible promoter in a binary vector, and the gRNA is cloned into the TRV genome flanked by two 20 bp Csy4 recognition sites. The results show that the Csy4-processing TRV-based delivery system works effectively in targeting single and multiple sites, nucleotide replacement, and large fragment deletion in Cas9-mediated genome editing via transient expression in Nicotiana benthamiana. The Csy4-TRV is a promising gRNA(s) processing and delivery system for CRISPR/Cas9 genome editing. This method can be easily adapted to other plant RNA viruses, facilitating the application of the CRISPR/Cas9 system in plants.

Development of an NLR-ID Toolkit and Identification of Novel Disease-Resistance Genes in Soybean.

The recognition of pathogen effectors through the nucleotide-binding leucine-rich repeat receptor (NLR) family is an important component of plant immunity. In addition to typical domains such as TIR, CC, NBS, and LRR, NLR proteins also contain some atypical integrated domains (IDs), the roles of which are rarely investigated. Here, we carefully screened the soybean (Glycine max) genome and identified the IDs that appeared in the soybean TNL-like proteins. Our results show that multiple IDs (36) are widely present in soybean TNL-like proteins. A total of 27 Gm-TNL-ID genes (soybean TNL-like gene encoding ID) were cloned and their antiviral activity towards the soybean mosaic virus (SMV)/tobacco mosaic virus (TMV) was verified. Two resistance (R) genes, SRA2 (SMV resistance gene contains AAA_22 domain) and SRZ4 (SMV resistance gene contains zf-RVT domain), were identified to possess broad-spectrum resistance characteristics towards six viruses including SMV, TMV, plum pox virus (PPV), cabbage leaf curl virus (CaLCuV), barley stripe mosaic virus (BSMV), and tobacco rattle virus (TRV). The effects of Gm-TNL-IDX (the domain of the Gm-TNL-ID gene after the TN domain) on the antiviral activity of a R protein SRC7TN (we previously reported the TN domain of the soybean broad-spectrum resistance gene SRC7) were validated, and most of Gm-TNL-IDX inhibits antiviral activity mediated by SRC7TN, possibly through intramolecular interactions. Yeast-two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that seven Gm-TNL-IDX interacted with SMV-component proteins. Truncation analysis on a broad-spectrum antiviral protein SRZ4 indicated that SRZ4TIR is sufficient to mediate antiviral activity against SMV. Soybean cDNA library screening on SRZ4 identified 48 interacting proteins. In summary, our results indicate that the integration of IDs in soybean is widespread and frequent. The NLR-ID toolkit we provide is expected to be valuable for elucidating the functions of atypical NLR proteins in the plant immune system and lay the foundation for the development of engineering NLR for plant-disease control in the future.

Analysis of the Time Course of the Establishment of Systemic Gene Silencing by Barley Stripe Mosaic Virus Virus-Induced Gene Silencing in Wheat

Wheat is one of the major sources of protein worldwide. Its hexaploidy significantly complicates the identification of genes that may be crucial for improving wheat production to meet the challenges of an increased world population and climate change. Virus-induced gene silencing (VIGS) using Barley stripe mosaic virus (BSMV)-based constructs has proven to be a very useful tool in the analysis of gene function in the hexaploid plant, wheat. However, most published applications of this technique focus on phenotypes that can be observed in the leaves of wheat. A few studies have reported successful VIGS in the spikes of wheat, but this has proven to be more difficult than the seedling leaf assays. This study reports a time course analysis of the movement of BSMV from the site of inoculation into the meristematic region of wheat. It also describes how the photobleaching phenotype resulting from silencing phytoene desaturase (PDS), which is often used as a reporter for VIGS, does not indicate the full extent of where VIGS occurs, and this can mislead scientists as they design silencing studies. These findings provide guidance for more effective VIGS studies to determine the function of genes expressed in the spikes of wheat and may be important for wheat improvement.

Minimizing the Formation of Polynuclear Pd(II) Hydroxo Complex Clusters in Biomineralization of Barley Stripe Mosaic Virus

Minimizing the Formation of Polynuclear Pd(II) Hydroxo Complex Clusters in Biomineralization of Barley Stripe Mosaic Virus

Distribution of Wheat-Infecting Viruses and Genetic Variability of Wheat Streak Mosaic Virus and Barley Stripe Mosaic Virus in Kazakhstan.

Wheat is an essential cereal crop for the economy and food safety of Kazakhstan. In the present work, a screening of wheat and barley from different regions of Kazakhstan was conducted using newly developed specific primers for reverse transcription PCR and loop-mediated isothermal amplification (LAMP) assays. In total, 82 and 19 of 256 samples of wheat and barley tested positive for wheat streak mosaic virus (WSMV) and barley stripe mosaic virus (BSMV), respectively. A phylogenetic analysis using two independent methods revealed that most of the analyzed isolates had a European origin. Molecular data on the distribution and diversity of cereal viruses in Kazakhstan were obtained for the first time and will help lay a foundation for the implementation of genetics and genomics in wheat phyto-epidemiology in the country.

Occurrence and diversity of barley yellow dwarf virus in Algeria

AbstractCereals are prone to viral infections and the economic impact of these has increased in recent years. Among these diseases barley yellow dwarf (BYD) is one of the most destructive diseases of cereals today. For three consecutive years (2014–2015–2016) surveys were carried out in order to search for BYDV species (BYDV-PAV and -MAV) as well as other cereal viruses, wheat spindle streak mosaic virus (WSSMV), southern bean mosaic virus (SBMV) and barley stripe mosaic virus (BSMV) in seven regions of Algeria (Algiers, Boumerdes, Tipaza Médéa, Adrar, Khenchla and Batna).Targeted samples were taken randomly from plants of different cereal species (wheat, barley, oats). The sample were analyzed by DAS-ELISA and RT-PCR.The results of ELISA and PCR tests showed the presence of BYDV-PAV in barley, durum wheat, soft wheat and oats. Thus, this viral species were found in all the cereal regions surveyed (North, South, East and West).Seven samples (durum wheat, barley and oats) were sequenced and phylogenetic analyses performed revealed that the Algerian sequences clustered in group I and group II.

Loss of Foreign DNA Inserts from Barley Stripe Mosaic Virus Vectors—Potential Consequences for Use in Functional Genomics Studies

Barley stripe mosaic virus (BSMV)-based vectors are frequently used in virus-induced gene silencing (VIGS) and, more recently, viral overexpression (VOX) studies in wheat. Two general strategies are employed to initiate infection in wheat plants with BSMV in VIGS and VOX studies. One method involves the direct infection of wheat using viral RNA produced via the in vitro transcription of BSMV constructs. The second class utilizes viral replication in an intermediate host plant to produce large amounts of BSMV viral particles that are then used to inoculate wheat plants. This study was designed to examine the potential for BSMV-VIGS constructs to rearrange during replication in the intermediate host and result in initiating the VIGS studies with a virus that is significantly different from the original experimental construct. It is shown that in the case of BSMV-VIGS constructs harboring a PDS-silencing fragment, significant rearrangement can occur during replication in the intermediate host that has the potential to introduce artifactual experimental outcomes.

Identifying Critical Regulators in the Viral Stress Response of Wheat (Triticum aestivum L.) Using Large-Scale Transcriptomics Data

Wheat (Triticum aestivum L.) cultivation has been globally restricted by many plant viruses such as the Wheat streak mosaic virus (WSMV), Barley stripe mosaic virus (BSMV), and Brome mosaic virus (BMV). Herein, the transcriptome of wheat was in silico analyzed under mono- (WSMV, BSMV, or BMV), bi- (BMV&BSMV, BMV&WSMV, and BSMV&WSMV), and tripartite (WSMV, BSMV, and BMV) infections using the RNA-seq technique. Total numbers of 1616/270, 5243/690 and 5589/2183 differentially expressed genes (DEGs) were up/down-regulated during the bipartite infection of BMV&BSMV, BMV&WSMV and BSMV&WSMV, respectively, while the tripartite infection resulted in the up/down-regulation of 6110/2424 DEGs. The NAC and bHLH were the most commonly presented transcription factor (TF) families in WSMV, BMV, and BSMV infection, while C2H2, bHLH, and NAC were the TF families involved in BMV&WSMV, BMV&BSMV, and BSMV&WSMV infections, respectively. The RLK-Pelle_DLSV was the most commonly expressed protein kinase (PK) family in all infection patterns. Promoter analysis showed that the motifs involved in gene expression, CUL4 RING ubiquitin ligase complex, stress response, brassinosteroid response, and energy-related pathways were significantly induced in wheat plants under bipartite infections. The gene expression network analysis showed that a defense-related gene, i.e., allene oxide synthase (AOS) gene, serves as a crucial hub in tripartite infections.

Barley stripe mosaic virus-mediated somatic and heritable gene editing in barley (Hordeum vulgare L.).

Genome editing strategies in barley (Hordeum vulgare L.) typically rely on Agrobacterium-mediated genetic transformation for the delivery of required genetic reagents involving tissue culture techniques. These approaches are genotype-dependent, time-consuming, and labor-intensive, which hampers rapid genome editing in barley. More recently, plant RNA viruses have been engineered to transiently express short guide RNAs facilitating CRISPR/Cas9-based targeted genome editing in plants that constitutively express Cas9. Here, we explored virus-induced genome editing (VIGE) based on barley stripe mosaic virus (BSMV) in Cas9-transgenic barley. Somatic and heritable editing in the ALBOSTRIANS gene (CMF7) resulting in albino/variegated chloroplast-defective barley mutants is shown. In addition, somatic editing in meiosis-related candidate genes in barley encoding ASY1 (an axis-localized HORMA domain protein), MUS81 (a DNA structure-selective endonuclease), and ZYP1 (a transverse filament protein of the synaptonemal complex) was achieved. Hence, the presented VIGE approach using BSMV enables rapid somatic and also heritable targeted gene editing in barley.

First Report of Barley Stripe Mosaic Virus Occurrence in Wheat in Tibet, China

First Report of Barley Stripe Mosaic Virus Occurrence in Wheat in Tibet, China

A Novel Mitovirus PsMV2 Facilitates the Virulence of Wheat Stripe Rust Fungus.

Wheat stripe rust, caused by the obligate biotrophic fungus Puccinia striiformis f. sp. tritici (Pst), seriously affects wheat production. Here, we report the complete genome sequence and biological characterization of a new mitovirus from P. striiformis strain GS-1, which was designated as "Puccinia striiformis mitovirus 2" (PsMV2). Genome sequence analysis showed that PsMV2 is 2658 nt in length with an AU-rich of 52.3% and comprises a single ORF of 2348 nt encoding an RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis indicated that PsMV2 is a new member of the genus Unuamitovirus within the family Mitoviridae. In addition, PsMV2 multiplied highly during Pst infection and it suppresses programmed cell death (PCD) triggered by Bax. Silencing of PsMV2 in Pst by barley stripe mosaic virus (BSMV)-mediated Host Induced Gene Silencing (HIGS) reduced fungal growth and decreased pathogenicity of Pst. These results indicate PsMV2 promotes host pathogenicity in Pst. Interestingly, PsMV2 was detected among a wide range of field isolates of Pst and may have coevolved with Pst in earlier times. Taken together, our results characterized a novel mitovirus PsMV2 in wheat stripe rust fungus, which promotes the virulence of its fungal host and wide distribution in Pst which may offer new strategies for disease control.

Rapid Screening of Barley Stripe Mosaic Virus Via Microfluidic Chip Method

<i>Barley stripe mosaic virus</i>, the type member of <i>Hordeivirus</i>, constitutes an important seed-transmitted pathogen distributed in almost every barley growing area worldwide. Researches on efficient detection of BSMV suitable in wild application are in urgent need. New platforms like microfluidic technology keep progressing rapidly in past decades and gains increasing influence on life sciences. Thus in this study, one rapid screening method for BSMV via microfluidic chip was established. Through method validity, we succeeded in demonstrating the amplification of BSMV within 30 min at one constant temperature using a microfluidic chip assay. Sensitivity analysis conformed that this method obtained a detection limit at 1.00×102 copies/μL, which was even more sensitive than real-time RT-PCR. <i>Maize chlorotic mottle virus</i>, <i>Maize dwarf mosaic virus</i>, <i>Oat mosaic virus</i>, and <i>Wheat streak mosaic virus</i> were chosen in specificity test along with BSMV, but only BSMV expressed typical amplification curves and Ct. Microfluidic analytic systems realize miniaturization by reducing the reaction volume to just 5 μL, which has several superiorities such as saving expensive reagents and reducing inspection costs. Moreover this microfluidic chip assay can simultaneously detect a variety of viruses, and the whole process takes just 1 hour, which greatly speeds up the detection and improves efficiency. All in all, this microfluidic chip method has the potential to be further implemented by phytosanitary services for routine diagnose as well as rapid screening in places like ports.

A Chloroplast-Localized Glucose-6-Phosphate Dehydrogenase Positively Regulates Stripe Rust Resistance in Wheat.

Glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme of the pentose phosphate pathway (PPP), plays a pivotal role in plant stress responses. However, the function and mechanism of G6PDHs in crop plants challenged by fungal pathogens remain poorly understood. In this study, a wheat G6DPH gene responding to infection by Puccinia striiformis f. sp. tritici (Pst), designated TaG6PDH2, was cloned and functionally identified. TaG6PDH2 expression was significantly upregulated in wheat leaves inoculated with Pst or treated with abiotic stress factors. Heterologous mutant complementation and enzymatic properties indicate that TaG6PDH2 encodes a G6PDH protein. The transient expression of TaG6PDH2 in Nicotiana benthamiana leaves and wheat protoplasts revealed that TaG6PDH2 is a chloroplast-targeting protein. Silencing TaG6PDH2 via the barley stripe mosaic virus (BSMV)-induced gene silencing (VIGS) system led to compromised wheat resistance to the Pst avirulent pathotype CYR23, which is implicated in weakened H2O2 accumulation and cell death. In addition, TaG6PDH2 was confirmed to interact with the wheat glutaredoxin TaGrxS4. These results demonstrate that TaG6PDH2 endows wheat with increased resistance to stripe rust by regulating reactive oxygen species (ROS) production.

The C2H2 zinc finger protein TaZFP13D increases drought stress tolerance in wheat

Wheat is one of the most cultivated crops, and its production is greatly affected by abiotic stresses such as drought. Transcription factors that regulate multiple downstream genes have a good potential to improve stress tolerance due to their ability to modulate various response pathways. Many members of the C1-2i C2H2 zinc finger protein (ZFP) sub-family are up-regulated in response to abiotic stress and specific members were shown to improve oxidative and abiotic stress tolerance in different plant species. Our previous work demonstrated that the most up-regulated TaZFP, TaZFP1B, improves drought tolerance and oxidative stress protection. Based on their strong response to different abiotic stresses, five additional candidate TaZFPs were selected for functional characterization by reverse genetics in three wheat lines, using the four-component Barley Stripe Mosaic Virus system. Of the five candidates studied, TaZFP13D was the only gene that increased drought stress tolerance (resistance to wilting and improved relative water content) in three different wheat lines and significantly improved antioxidant enzymes activities resulting in lower accumulation of superoxide and hydrogen peroxide. Moreover, TaZFP13D down-regulation decreased drought stress tolerance suggesting that this gene does not have a redundant function within the TaZFPs family.

The CC-NB-LRR protein BSR1 from Brachypodium confers resistance to Barley stripe mosaic virus in gramineous plants by recognising TGB1 movement protein.

Although some nucleotide binding, leucine-rich repeat immune receptor (NLR) proteins conferring resistance to specific viruses have been identified in dicot plants, NLR proteins involved in viral resistance have not been described in monocots. We have used map-based cloning to isolate the CC-NB-LRR (CNL) Barley stripe mosaic virus (BSMV) resistance gene barley stripe resistance 1 (BSR1) from Brachypodium distachyon Bd3-1 inbred line. Stable BSR1 transgenic Brachypodium line Bd21-3, barley (Golden Promise) and wheat (Kenong 199) plants developed resistance against BSMV ND18 strain. Allelic variation analyses indicated that BSR1 is present in several Brachypodium accessions collected from countries in the Middle East. Protein domain swaps revealed that the intact LRR domain and the C-terminus of BSR1 are required for resistance. BSR1 interacts with the BSMV ND18 TGB1 protein in planta and shows temperature-sensitive antiviral resistance. The R390 and T392 residues of TGB1ND (ND18 strain) and the G196 and K197 residues within the BSR1 P-loop motif are key amino acids required for immune activation. BSR1 is the first cloned virus resistance gene encoding a typical CNL protein in monocots, highlighting the utility of the Brachypodium model for isolation and analysis of agronomically important genes for crop improvement.

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.