Agrobacterium- mediated transformation relies on binary vectors in which T-DNA and virulence genes are maintained on separate replicons. While Golden Gate cloning has become standard for T-DNA assembly, no modular framework exists for systematic construction of Agrobacterium vector backbones. Here, we present BackBone Builder (B3), a Golden Gate-based platform for combinatorial backbone assembly. B3 uses the Type IIS enzyme PaqCI to minimize domestication and enables one-pot assembly of nine backbone modules plus a selectable cloning cassette. The system is compatible with GreenGate and remains independent of downstream cloning strategies. We generated a library of 42 backbone components, supporting a theoretical design space exceeding 370,000 constructs. A 4 x 4 origin-of-replication (ORI) matrix combining four Escherichia coli and four Agrobacterium ORIs assembled with 100% efficiency and functioned robustly in bacterial and plant contexts. Reporter expression reflected expected ORI-dependent patterns in E. coli, Agrobacterium , and Nicotiana benthamiana . A B3-derived maize transformation backbone achieved stable transformation efficiencies comparable to established vectors. B3 establishes a standardized and extensible framework for rational engineering of Agrobacterium binary vector architecture.

The high efficiency of grass stomatal movement is believed to be due to their unique four-celled structure, with two dumbbell-shaped guard cells (GCs) flanked by two subsidiary cells (SCs). The molecular interaction between these cells requires further investigation. To investigate genes, including those encoding aquaporins (AQPs), expressed in maize GCs and SCs, RNA sequencing was performed on microdissected GCs and SCs from epidermis harvested during day and night. A CRISPR-based tissue-specific knockout (KO) system was then used to target ZmPIPs belonging to the plasma membrane intrinsic protein1 (PIP1) subfamily in maize GCs or SCs. A general transcriptomic analysis first revealed that genes preferentially expressed in SCs compared with GCs during the day were primarily involved in lipid transport, localization, and metabolism, while genes more highly expressed in GCs were associated with photosynthesis, chloroplasts, and plastids. Interestingly, the expression of several AQP genes varied both spatially and temporally. The KO of ZmPIP1s in GCs resulted in greater stomatal opening than wild-type plants, particularly under mild water-deficit conditions. Overall, these findings reveal transcriptomic specificities between maize GCs and SCs, providing valuable insights into the regulation of rapid stomatal movements in grasses and highlight the role of ZmPIP1s in these processes.

2. Abstract Hairy root disease (HRD), caused by rhizogenic Agrobacterium , is an economically important disease affecting hydroponic tomato ( Solanum lycopersicum L.) production worldwide. HRD-affected plants show extensive root proliferation, resulting in decreased energy expenditure towards fruit production. Host plant susceptibility to rhizogenic Agrobacterium is typically evaluated through artificial wounding-based infection bioassays. However, under natural infection settings, rhizogenic Agrobacterium can induce disease symptoms without deliberate, artificial wounding. We developed a soil-based, non-wounding bioassay that closely mimics natural rhizosphere interactions and permits quantitative and qualitative assessment of HRD symptoms. The assay measured root dry weight, documented agravitropic root development typical of HRD and confirmed in planta T-DNA gene expression using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). We used this bioassay to evaluate disease symptoms towards rhizogenic Agrobacterium in tomato cv. ‘Moneymaker’ and the rootstocks ‘Optifort’, ‘Maxifort’, and ‘Arnold’. ‘Optifort’ and ‘Maxifort’ exhibited significantly higher root biomass than ‘Arnold’ and ‘Moneymaker’, indicating more pronounced symptom development. The bioassay also differentiated virulence levels amongst various rhizogenic Agrobacterium strains isolated from HRD-affected plants. Together, these results show that our soil-based bioassay provides a robust and ecologically relevant platform for screening tomato genotypes and comparing virulence levels of rhizogenic Agrobacterium strains supporting resistance breeding and disease management efforts.

The endosperm is a transient nutritive tissue in plant seeds. During maize (Zea mays) grain development, 2 distinct endosperm cell death processes occur: in 1 process, the endosperm adjacent to the embryo scutellum (EAS) is completely dismantled; in the other, the starchy endosperm (SE) retains nutrient-packed cell corpses after grain filling. Here, we show that SE cell death degrades some organelles including the mitochondria and the endoplasmic reticulum, while preserving protein bodies, starch granules, and chromatin. In contrast, EAS cells undergo lytic cell death to remobilize stored nutrients through a complex corpse clearance process. Using single-cell transcriptome analysis, we identified 2 NAC transcription factors, KIRA-LIKE 1 (KIL1) and 2 (KIL2), as specifically upregulated in the EAS. Analyses using dominant and recessive loss-of-function kil mutants demonstrate that these genes redundantly promote cell death and corpse clearance in the EAS, but are not required for SE cell death. Reduced EAS cell death in kil loss-of-function mutants strongly impeded embryo growth, indicating that EAS elimination is crucial for optimal embryo development. Notably, kil1 and kil2 expression is regulated by DOSAGE-EFFECT DEFECTIVE1, an imprinted paternally expressed endosperm transcription factor. Our findings suggest paternal control over EAS cell death and the embryo-endosperm size ratio in maize, providing new leads to modulate this agronomically important trait.

Hairy roots induced by Agrobacterium spp. are widely used in plant biotechnology. Composite plants, consisting of a wild-type shoot and a mix of transformed hairy roots and non-transformed roots, offer a versatile platform for investigating gene function, plant-microbe interactions, and systemic signaling. However, existing protocols for composite plant generation in the model Arabidopsis thaliana remain underdeveloped and inefficient limiting their broader application in basic research. Here, we optimized an Agrobacterium strain K599-mediated hairy root transformation protocol for A. thaliana Columbia-0 using a fractional factorial design to systematically evaluate key transformation parameters. These included bacterial growth medium, inoculation strategy, acetosyringone supplementation, and photoperiod. The optimized protocol, combining ATGN medium and a cut-and-coat inoculation strategy, consistently achieved high average transformation efficiencies ( 80

Somatic embryogenesis allows a somatic plant cell to develop into an embryo, and potentially into a fertile plant. Transcription factors such as BABY BOOM can induce somatic embryogenesis when ectopically expressed and are widely used for aiding regeneration in tissue culture for transformation and gene editing of crops. Nevertheless, regeneration remains a bottleneck and alternative morphogenic genes are highly desired. Here, we co-expressed BABY BOOM and WUSCHEL2 in zygotic maize ( Zea may s L.) embryos and studied gene regulatory networks in induced somatic embryos at the single-cell level. By inferring cell-type-specific regulons, we prioritized candidate regulators and confirmed functionality of four transcription factors, bHLH48, EREB152, GRF4, and HB77, for enhanced maize transformation frequency, leading to fertile, transgenic plants. Interestingly, the basic helix-loop-helix and homeodomain-leucine zipper families had previously not been associated with induced somatic embryogenesis. Our work will contribute to more efficient transformation, much needed to deliver on the promise of gene editing for agriculture. ### Competing Interest Statement The authors (J.R., S.L., K.V., and L.P.) declare that a patent application related to the content of this manuscript has been filed. Industrieel Onderzoeksfonds, F2020/IOF-StarTT/151; IOF.PRO.2021.0017

Valorization of lignocellulosic biomass for sustainable production of high-value chemicals is challenged by the complexity of lignin, a phenolic biopolymer. Beyond the classical lignin monomers derived from p-coumaryl, coniferyl, and sinapyl alcohol, grass lignins incorporate substantial amounts of monolignol p-coumarates that are produced by p-COUMAROYL-CoA:MONOLIGNOL TRANSFERASE (PMT). Here, the CRISPR/Cas9-mediated mutation of ZmPMT1 in maize enabled the design of biomass depleted in p-coumaroylated lignin and enriched in guaiacyl lignin. Lignin-first biorefining of stem biomass from zmpmt1 mutants by reductive catalytic fractionation (RCF) generated a lignin oil depleted in carboxylates and enriched in guaiacyl-derived alcohols, which are desirable substrates for bio-based polyurethane synthesis. The reported lignin engineering in maize is a promising strategy for designing a dual-purpose crop, providing both food and feed, along with a renewable feedstock for the production of plant-based chemicals.

The plasma membrane aquaporin ZmPIP1;6 is expressed in maize stomatal complexes, with higher expression during the day than at night. To elucidate the role of ZmPIP1;6 in gas exchange and stomatal movement, it was expressed in maize (inbred line B104) under the control of the p35S promoter (overexpression) or its native promoter fused with mYFP (monomeric yellow fluorescent protein) cDNA (mYFP-ZmPIP1;6). In stomatal complexes of the mature zone of the leaf, mYFP-ZmPIP1;6 showed higher expression in subsidiary cells than in guard cells, and light and dark treatments influenced its subcellular localization. Notably, ZmPIP1;6 internalization increased in dark conditions compared with light. Stomatal opening was greater in ZmPIP1;6 overexpression plants than in the wild type, while closure exhibited greater sensitivity to elevated CO2 concentration or abscisic acid (ABA) treatment. Our findings revealed that reactive oxygen species (H2O2) were involved in ABA-induced stomatal closure, while ZmPIP1;6 was unable to facilitate H2O2 diffusion when expressed in yeast. Finally, ZmPIP1;6 overexpression and mYFP-ZmPIP1;6 transgenic plants exhibited higher abaxial stomatal density than the wild type. Overall, these results indicate that ZmPIP1;6 plays important roles in stomatal opening and CO2- and ABA-induced stomatal closure.

Maize (Zea mays L.) is an important crop that has been widely studied for its agronomic and industrial applications and is one of the main classical model organisms for genetic research. Agrobacterium-mediated transformation of immature maize embryos is a commonly used method to introduce transgenes, but a low transformation frequency remains a bottleneck for many gene-editing applications. Previous approaches to enhance transformation included the improvement of tissue culture media and the use of morphogenic regulators such as BABY BOOM and WUSCHEL2. Here, we show that the frequency can be increased using a pVS1-VIR2 virulence helper plasmid to improve T-DNA delivery, and/or expressing a fusion protein between a GROWTH-REGULATING FACTOR (GRF) and GRF-INTERACTING FACTOR (GIF) protein to improve regeneration. Using hygromycin as a selection agent to avoid escapes, the transformation frequency in the maize inbred line B104 significantly improved from 2.3 to 8.1% when using the pVS1-VIR2 helper vector with no effect on event quality regarding T-DNA copy number. Combined with a novel fusion protein between ZmGRF1 and ZmGIF1, transformation frequencies further improved another 3.5- to 6.5-fold with no obvious impact on plant growth, while simultaneously allowing efficient CRISPR-/Cas9-mediated gene editing. Our results demonstrate how a GRF-GIF chimera in conjunction with a ternary vector system has the potential to further improve the efficiency of gene-editing applications and molecular biology studies in maize.

Drought is one of the most devastating causes of yield losses in crops like maize, and the anticipated increases in severity and duration of drought spells due to climate change pose an imminent threat to agricultural productivity. To understand the drought response, phenotypic and molecular studies are typically performed at a given time point after drought onset, representing a steady-state adaptation response. Because growth is a dynamic process, we monitored the drought response with high temporal resolution and examined cellular and transcriptomic changes after rehydration at 4 and 6 days after leaf four appearance. These data showed that division zone activity is a determinant for full organ growth recovery upon rehydration. Moreover, a prolonged maintenance of cell division by the ectopic expression of PLASTOCHRON1 extends the ability to resume growth after rehydration. The transcriptome analysis indicated that GROWTH-REGULATING FACTORS (GRFs) affect leaf growth by impacting cell division duration, which was confirmed by a prolonged recovery potential of the GRF1-overexpression line after rehydration. Finally, we used a multiplex genome editing approach to evaluate the most promising differentially expressed genes from the transcriptome study and as such narrowed down the gene space from 40 to seven genes for future functional characterization.

Sorghum bicolor (sorghum) is a vital C4 monocotyledon crop cultivated in arid regions worldwide, valued for its significance in both human and animal nutrition. Despite its agricultural prominence, sorghum research has been hindered by low transformation frequency. In this study, we examined sorghum transformation using the pVS1-VIR2 ternary vector system for Agrobacterium, combined with the morphogenic genes BABY BOOM and WUSCHEL2 and selection using G418. We optimized Agrobacterium-mediated infection, targeting key parameters such as bacterial optical density, co-cultivation time, and temperature. Additionally, an excision-based transformation system enabled us to generate transgenic plants free of morphogenic regulators. The method yielded remarkable transformation frequencies, reaching up to 164.8% based on total isolated plantlets. The same combination of ternary vector, morphogenic genes and geneticin-based selection also resulted in a marked increase in transformation efficiency of the Zea mays (maize) inbred line B104. The potential for genomic editing using this approach positions it as a valuable tool for the development of sorghum and maize varieties that comply with evolving European regulations. Our work marks a significant stride in sorghum biotechnology and holds promise for addressing global food security challenges in a changing climate.

A major advantage of using CRISPR/Cas9 for gene editing is multiplexing, that is, the simultaneous targeting of many genes. However, primary transformants typically contain hetero-allelic mutations or are genetic mosaic, while genetically stable lines that are homozygous are desired for functional analysis. Currently, a dedicated and labor-intensive effort is required to obtain such higher-order mutants through several generations of genetic crosses and genotyping. We describe the design and validation of a rapid and efficient strategy to produce lines of genetically identical plants carrying various combinations of homozygous edits, suitable for replicated analysis of phenotypical differences. This approach was achieved by combining highly multiplex gene editing in Zea mays (maize) with in vivo haploid induction and efficient in vitro generation of doubled haploid plants using embryo rescue doubling. By combining three CRISPR/Cas9 constructs that target in total 36 genes potentially involved in leaf growth, we generated an array of homozygous lines with various combinations of edits within three generations. Several genotypes show a reproducible 10% increase in leaf size, including a septuple mutant combination. We anticipate that our strategy will facilitate the study of gene families via multiplex CRISPR mutagenesis and the identification of allele combinations to improve quantitative crop traits.

The HD-ZIP III transcription factor REVOLUTA (REV) is involved in early leaf development, as well as in leaf senescence. REV directly binds to the promoters of senescence-associated genes, including the central regulator WRKY53. As this direct regulation appears to be restricted to senescence, we aimed to characterize protein-interaction partners of REV which could mediate this senescence-specificity. The interaction between REV and the TIFY family member TIFY8 was confirmed by yeast two-hybrid assays, as well as by bimolecular fluorescence complementation in planta. This interaction inhibited REV's function as an activator of WRKY53 expression. Mutation or overexpression of TIFY8 accelerated or delayed senescence, respectively, but did not significantly alter early leaf development. Jasmonic acid (JA) had only a limited effect on TIFY8 expression or function; however, REV appears to be under the control of JA signaling. Accordingly, REV also interacted with many other members of the TIFY family, namely the PEAPODs and several JAZ proteins in the yeast system, which could potentially mediate the JA-response. Therefore, REV appears to be under the control of the TIFY family in two different ways: a JA-independent way through TIFY8, which controls REV function in senescence, and a JA-dependent way through PEAPODs and JAZ proteins.

To meet the increasing global demand for food, feed, fibre and other plant-derived products, a steep increase in crop productivity is a scientifically and technically challenging imperative. The CropBooster-P project, a response to the H2020 call 'Future proofing our plants', is developing a roadmap for plant research to improve crops critical for the future of European agriculture by increasing crop yield, nutritional quality, value for non-food applications and sustainability. However, if we want to efficiently improve crop production in Europe and prioritize methods for crop trait improvement in the coming years, we need to take into account future socio-economic, technological and global developments, including numerous policy and socio-economic challenges and constraints. Based on a wide range of possible global trends and key uncertainties, we developed four extreme future learning scenarios that depict complementary future developments. Here, we elaborate on how the scenarios could inform and direct future plant research, and we aim to highlight the crop improvement approaches that could be the most promising or appropriate within each of these four future world scenarios. Moreover, we discuss some key plant technology options that would need to be developed further to meet the needs of multiple future learning scenarios, such as improving methods for breeding and genetic engineering. In addition, other diverse platforms of food production may offer unrealized potential, such as underutilized terrestrial and aquatic species as alternative sources of nutrition and biomass production. We demonstrate that although several methods or traits could facilitate a more efficient crop production system in some of the scenarios, others may offer great potential in all four of the future learning scenarios. Altogether, this indicates that depending on which future we are heading toward, distinct plant research fields should be given priority if we are to meet our food, feed and non-food biomass production needs in the coming decades.

Ensuring food security for an ever-growing global population while adapting to climate change is the main challenge for agriculture in the 21st century. Although new technologies are being applied to tackle this problem, we are approaching a plateau in crop improvement using conventional breeding. Recent advances in CRISPR/Cas9-mediated gene engineering have paved the way to accelerate plant breeding to meet this increasing demand. However, many traits are governed by multiple small-effect genes operating in complex interactive networks. Here, we present the gene discovery pipeline BREEDIT, which combines multiplex genome editing of whole gene families with crossing schemes to improve complex traits such as yield and drought tolerance. We induced gene knockouts in 48 growth-related genes into maize (Zea mays) using CRISPR/Cas9 and generated a collection of over 1,000 gene-edited plants. The edited populations displayed (on average) 5%-10% increases in leaf length and up to 20% increases in leaf width compared with the controls. For each gene family, edits in subsets of genes could be associated with enhanced traits, allowing us to reduce the gene space to be considered for trait improvement. BREEDIT could be rapidly applied to generate a diverse collection of mutants to identify promising gene modifications for later use in breeding programs.

Recent advances in genome editing have enormously enhanced the effort to develop biotechnology crops for more sustainable food production. CRISPR/Cas, the most versatile genome-editing tool, has shown the potential to create genome modifications that range from gene knockout and gene expression pattern modulations to allele-specific changes in order to design superior genotypes harboring multiple improved agronomic traits. However, a frequent bottleneck is the delivery of CRISPR/Cas to crops that are less amenable to transformation and regeneration. Several technologies have recently been proposed to overcome transformation recalcitrance, including HI-Edit/IMGE and ectopic/transient expression of genes encoding morphogenic regulators. These technologies allow the eroding of the barriers that make crops inaccessible for genome editing. In this review, we discuss the advances in genome editing in crops with a particular focus on the use of technologies to improve complex traits such as water use efficiency, drought stress, and yield in maize.

Phytopathogenic bacteria play important roles in plant productivity, and developments in gene editing have potential for enhancing the genetic tools for the identification of critical genes in the pathogenesis process. CRISPR-based genome editing variants have been developed for a wide range of applications in eukaryotes and prokaryotes. However, the unique mechanisms of different hosts restrict the wide adaptation for specific applications. Here, CRISPR-dCas9 (dead Cas9) and nCas9 (Cas9 nickase) deaminase vectors were developed for a broad range of phytopathogenic bacteria. A gene for a dCas9 or nCas9, cytosine deaminase CDA1, and glycosylase inhibitor fusion protein (cytosine base editor, or CBE) was applied to base editing under the control of different promoters. Results showed that the RecA promoter led to nearly 100% modification of the target region. When residing on the broad host range plasmid pHM1, CBERecAp is efficient in creating base edits in strains of Xanthomonas, Pseudomonas, Erwinia and Agrobacterium. CBE based on nCas9 extended the editing window and produced a significantly higher editing rate in Pseudomonas. Strains with nonsynonymous mutations in test genes displayed expected phenotypes. By multiplexing guide RNA genes, the vectors can modify up to four genes in a single round of editing. Whole-genome sequencing of base-edited isolates of Xanthomonas oryzae pv. oryzae revealed guide RNA-independent off-target mutations. Further modifications of the CBE, using a CDA1 variant (CBERecAp-A) reduced off-target effects, providing an improved editing tool for a broad group of phytopathogenic bacteria.

The recalcitrance exhibited by many maize (Zea mays) genotypes to traditional genetic transformation protocols poses a significant challenge to the large-scale application of genome editing (GE) in this major crop species. Although a few maize genotypes are widely used for genetic transformation, they prove unsuitable for agronomic tests in field trials or commercial applications. This challenge is exacerbated by the predominance of transformable maize lines adapted to temperate geographies, despite a considerable proportion of maize production occurring in the tropics. Ectopic expression of morphogenic regulators (MRs) stands out as a promising approach to overcome low efficiency and genotype dependency, aiming to achieve ’universal’ transformation and GE capabilities in maize. Here, we report the successful GE of agronomically relevant tropical maize lines using a MR-based, Agrobacterium-mediated transformation protocol previously optimized for the B104 temperate inbred line. To this end, we used a CRISPR/Cas9-based construct aiming at the knockout of the VIRESCENT YELLOW-LIKE (VYL) gene, which results in an easily recognizable phenotype. Mutations at VYL were verified in protoplasts prepared from B104 and three tropical lines, regardless of the presence of a single nucleotide polymorphism (SNP) at the seed region of the VYL target site in two of the tropical lines. Three out of five tropical lines were amenable to transformation, with efficiencies reaching up to 6.63%. Remarkably, 97% of the recovered events presented indels at the target site, which were inherited by the next generation. We observed off-target activity of the CRISPR/Cas9-based construct towards the VYL paralog VYL-MODIFIER, which could be partly due to the expression of the WUSCHEL (WUS) MR. Our results demonstrate efficient GE of relevant tropical maize lines, expanding the current availability of GE-amenable genotypes of this major crop.