Plant Biotechnology Journal

Plant diseases pose a significant threat to global crop production. Most disease resistance genes used in crop breeding programs encode nucleotide-binding leucine-rich repeat receptors (NLRs) that are limited in pathogen specificity and durability. In this study, we leveraged synthetic biology to develop an inducible broad-spectrum resistance in tomatoes. Constitutive expression of autoactive NLRs in plants leads to robust resistance against multiple pathogens but significantly stunts growth. We expressed autoactive NLRs under the control of pathogen-inducible (PI) promoters to mitigate the fitness costs. Taking advantage of extensive, new genomic and transcriptomic resources, we identified PI promoters that responded to multiple pathogens but not abiotic stress. We further validated functionality of predicted elements through a promoter luciferase assay. We generated significant resistance in transgenic tomatoes but we also encountered unwanted expression induction of the native promoter regions in flowers which led to lethal fruit development. Thus, we pursued promoter engineering for fine-tuning the induction. We identified cis-regulatory regions responsible for pathogen-inducibility through promoter bashing experiments and recombined the native promoter with the inducible part and the core promoter. Furthermore, we rationally created synthetic promoters showing a gradient of expression levels, which will allow for selection for transgenic tomatoes with the best performance. We found that the spacing between functional sequences, repeat number of inducible sequences, and core promoters all influence the outcome of engineering. Our study outlines a framework for developing broad-spectrum synthetic immune constructs with reduced fitness cost and provides examples of pathogen-inducible promoter engineering.

Chloroplast synthetic biology holds promise for developing improved crops through improving the function of plastids. However, chloroplast engineering efforts face limitations due to the scarcity of genetic tools and the low throughput of plant-based systems. To address these challenges, we here established Chlamydomonas reinhardtii as a prototyping chassis for chloroplast synthetic biology. We developed an automation workflow that enables the generation, handling, and analysis of thousands of transplastomic strains in parallel, expanded the repertoire of selection markers for chloroplast transformation, established new reporter genes, and characterized over 140 regulatory parts, including native and synthetic promoters, UTRs, and intercistronic expression elements. We integrated the system within the Phytobrick cloning standard and demonstrate several applications, including a library-based approach to develop synthetic promoter designs in plastids. Finally, we provide a proof-of-concept for prototyping novel traits in plastids by introducing a chloroplast-based synthetic photorespiration pathway and demonstrating a twofold increase in biomass production. Overall, our study advances chloroplast engineering, and provides a promising platform to rapidly prototype chloroplast manipulations before their transfer into higher plants and crops.

Bread wheat (Triticum aestivum L.) plays a vital role in global food security, and its continuous genetic improvement is essential to meet the demands of a rapidly growing world population. Advances in genome sequencing and assembly have positioned wheat as a model crop for functional genomics and have increased the demand for highly efficient, genotype-independent transformation systems. A fusion technology involving a GROWTH-REGULATING FACTOR (GRF) and a GRF-INTERACTING FACTOR (GIF) has emerged as a powerful tool to enhance regeneration efficiency and expand the range of transformable genotypes. In this study, we present an optimized and robust Agrobacterium-mediated wheat transformation protocol incorporating GRF4-GIF1, tested across multiple wheat varieties. Across all tested wheat cultivars, GRF4-GIF1 containing constructs consistently enabled successful transformation, with varied efficiencies depending on the genotype and promotors used to drive the gene fusion. Our method significantly improves transformation efficiency while minimizing GRF4-GIF1 pleiotropic effects, providing a versatile platform for gene function analysis and gene editing. This work represents a critical step toward efficient, genotype-independent transformation in wheat, supporting both research and breeding applications aimed at improving crop resilience, nutritional value, and productivity.

Phtheirospermum japonicum is a genetic model for parasitic Orobanchaceae, a plant family that includes noxious parasitic weeds from the genera Striga, Orobanche, and Phelipanche (Ishida et al., 2011). Striga species alone cause billions of dollars in annual losses by reducing yields of major crops (Pennisi, 2010). The lack of stable transgenesis protocols often hinders heritable CRISPR/Cas9 genome editing for gene function analysis in crops and species beyond standard model plants, including parasitic Orobanchaceae (Steinberger and Voytas, 2025). Here, we adapted a virus-mediated delivery system for ultracompact TnpB nucleases, enabling genome editing independently of tissue regeneration or floral dip transformation in the parasitic plant P. japonicum (Nagalakshmi et al., 2025; Weiss et al., 2025).

Vascular plant pathogens transmitted by insects inflict devastating economic losses on crops worldwide. By obstructing and usurping the natural flow of nutrients, plant infection by these pathogens drastically reduces yields, vigor and productivity. Treating fruit-bearing trees against persistent vascular pathogens poses a unique challenge, as systemic delivery of therapeutics must navigate the compartmentalized architecture of the trees vascular system under changing environmental conditions without disrupting fruit production or long-term tree health. Plant viruses have gained traction as a novel approach to deliver therapies to crop plants, including fruit trees, but delivery of viral vectors to orchards at scale remains a significant challenge. We tested whether transgenic galls can be used to systemically infect plants with a plant virus infectious clone. We combined the plant growth regulator gene cassette from Agrobacterium tumefaciens strain C58 with the wild-type strain of citrus yellow vein associated virus-1 (CY1) on a single plasmid within the T-DNA for plant cell transformation. Using EHA105, a disarmed strain of A. tumefaciens, we inoculated stems with these gall-forming plasmids and initiated systemic CY1 infection in citrus and Arabidopsis thaliana over three independent experiments. We provide proof-of-concept that transgenic galls, referred to as symbionts, can launch the systemic infection of CY1 in economically important and model plants. Symbiont delivery of therapeutic viral vectors is theoretically scalable from inoculation of mother trees within the nursery to millions of trees in the field and may be a valuable tool for the commercial delivery of therapeutic plant viral vectors.

Leaves host remarkably diverse microbes, collectively referred to as the leaf microbiota. While many beneficial functions have been attributed to the plant microbiota, the individual contributions of leaf-colonising bacteria range from pathogenic to mutualistic interactions. Omics approaches demonstrated that some leaf-colonising bacteria evoke substantial changes in gene expression and metabolic profiles in the plant host, including plant immunity. While omic approaches provide a system level view on cellular functions, they are costly and laborious, thereby severely limiting the throughput of the number of bacterial strains that can be tested in planta. To enable cost-effective high-throughput screens, we have developed a plant protoplast-based assay to measure real-time target gene expression changes following bacterial inoculation. Here, protoplasts were isolated from leaves of stable transgenic plants containing a pPR1:eYFP-nls construct. Changes in yellow fluorescence were captured for up to 96 treatments using a plate reader. This allowed the monitoring of changes in the salicylic acid-dependent plant immune response over time. Protoplast isolation per se evoked mild fluorescence responses, likely linked to endogenous salicylic acid production. This is advantageous in a bacterial assay, as bidirectional changes in PR1 expression can be measured. Plate reader-generated data were validated via fluorescence microscopy and RT-qPCR. Fluorescence microscopy further demonstrated heterogeneity in the response of individual protoplasts, which is potentially linked to differences in cell-type. In summary, the protoplast assay is an affordable and easily up-scalable way of measuring changes in target gene expression to bacterial colonisation.

Banana Xanthomonas wilt (BXW) disease, caused by Xanthomonas vasicola pv. musacearum, is a major constraint to banana production in East and Central Africa. All cultivated banana varieties are susceptible, with the wild progenitor Musa balbisiana being the only known source of complete resistance. Limitations in classical breeding have prompted the exploration of molecular genetic tools, such as genetic modification, to develop resistant cultivars. Comparative transcriptomic analyses revealed a five-fold upregulation of MusaVicilin gene in M. balbisiana (BB genome) compared to the BXW- susceptible Pisang Awak when challenged with the pathogen, suggesting its role in defense. This study investigated whether constitutive overexpression of the MusaVicilin gene cloned from M. balbisiana could enhance resistance to BXW in the susceptible Sukali Ndiizi cultivar (AAB genome). Transgenic events were developed with the MusaVicilin gene under the control of the constitutive CaMV 35S promoter. These events exhibited enhanced disease resistance compared with non-transgenic control plants. The overexpression of MusaVicilin highlights its potential as a candidate gene for engineering resistance to BXW in susceptible cultivars. Moreover, MusaVicilin could serve as a valuable component in gene stacking strategies aimed at developing durable, disease-resistant banana varieties.

Efficient transformation remains a major constraint to functional genomics and genome editing in Vaccinium, where stable transformation systems are often genotype-dependent and inefficient. Here, we establish a rapid and high-efficiency Rhizobium rhizogenes-mediated hairy root transformation platform optimized for the genus. Using the RUBY visual reporter, transformation efficiency reached 46.7% in leaf explants infected with strain Ar. A4 and cultured on half-strength Woody Plant Medium, with transgenic roots visible within 16 days post-co-cultivation. Comparative evaluation of six R. rhizogenes strains identified Ar. A4 and ATCC15834 as consistently superior across diverse Vaccinium germplasm representing different taxonomic sections, achieving up to 80% efficiency in selected genotypes. While conventional regeneration from transgenic roots was not successful, overexpression of developmental regulators enabled shoot formation with 7% efficiency, demonstrating a path toward stable plant recovery. This platform delivers a rapid, genotype-flexible system for gene validation, metabolic pathway analysis, and genome editing in Vaccinium, substantially expanding the molecular toolkit available for perennial fruit crop research and translational breeding.

Recent advances in genome sequencing and assembly techniques have made it possible to achieve chromosome level reference genomes for citrus. Relatively few genomes have been anchored at the chromosome level and/or are haplotype phased, with the available genomes of varying accuracy and completeness. We now report a phased high-quality chromosome level genome assembly for an Australian native citrus species; Citrus australis (round lime) using highly accurate PacBio HiFi long reads, complemented with Hi-C scaffolding. Hifiasm with Hi-C integrated assembly resulted in a 331 Mb genome of C. australis with two haplotypes of nine pseudochromosomes with an N50 of 36.3 Mb and 98.8% genome assembly completeness (BUSCO). Repeat analysis showed that more than 50% of the genome contained interspersed repeats. Among them, LTR elements were the predominant type (21.0%), of which LTR Gypsy (9.8 %) and LTR copia (7.7 %) elements were the most abundant repeats. A total of 29,464 genes and 32,009 transcripts were identified in the genome. Of these, 28,222 CDS (25,753 genes) had BLAST hits and 21,401 CDS (75.8%) were annotated with at least one GO term. Citrus specific genes for antimicrobial peptides, defense, volatile compounds and acidity regulation were identified. This chromosome scale, and haplotype resolved C. australis genome will facilitate the study of important genes for citrus breeding and will also allow the enhanced definition of the evolutionary relationships between wild and domesticated citrus species.

Wild tomatoes are important genomic resources for tomato research and breeding. Development of a foreign DNA-free CRISPR-Cas delivery system has potential to mitigate public concern about genetically modified organisms. Here, we established a DNA-free protoplast regeneration and CRISPR-Cas9 genome editing system for Solanum peruvianum, an important resource for tomato introgression breeding. We generated mutants for genes involved in small interfering RNAs (siRNA) biogenesis, RNA-DEPENDENT RNA POLYMERASE 6 (SpRDR6) and SUPPRESSOR OF GENE SILENCING 3 (SpSGS3); pathogen-related peptide precursors, PATHOGENESIS-RELATED PROTEIN-1 (SpPR-1) and PROSYSTEMIN (SpProsys); and fungal resistance (MILDEW RESISTANT LOCUS O, SpMlo1) using diploid or tetraploid protoplasts derived from in vitro-grown shoots. The ploidy level of these regenerants was not affected by PEG-calcium-mediated transfection, CRISPR reagents, or the target genes. By karyotyping and whole genome sequencing analysis, we confirmed that CRISPR-Cas9 editing did not introduce chromosomal changes or unintended genome editing sites. All mutated genes in both diploid and tetraploid regenerants were heritable in the next generation. spsgs3 null T0 regenerants and sprdr6 null T1 progeny had wiry, sterile phenotypes in both diploid and tetraploid lines. The sterility of the spsgs3 null mutant was partially rescued, and fruits were obtained by grafting to wild-type stock and pollination with wild-type pollen. The resulting seeds contained the mutated alleles. Tomato yellow leaf curl virus proliferated at higher levels in spsgs3 and sprdr6 mutants than in the wild type. Therefore, this protoplast regeneration technique should greatly facilitate tomato polyploidization and enable the use of CRISPR-Cas for S. peruvianum domestication and tomato breeding. One-sentence summaryDNA-free CRISPR-Cas9 genome editing in wild tomatoes creates stable and inheritable diploid and tetraploid regenerants.

Wheat (Triticum aestivum) is one of the most important food crops with an urgent need for increase in its production to feed the growing world. Triticum timopheevii (2n = 4x = 28) is an allotetraploid wheat wild relative species containing the At and G genomes that has been exploited in many pre-breeding programmes for wheat improvement. In this study, we report the generation of a chromosome-scale reference genome assembly of T. timopheevii accession PI 94760 based on PacBio HiFi reads and chromosome conformation capture (Hi-C). The assembly comprised a total size of 9.35 Gb, featuring a contig N50 of 42.4 Mb, and 166,325 predicted gene models. DNA methylation analysis showed that the G genome had on average more methylated bases than the At genome. The G genome was also more closely related to the S genome of Aegilops speltoides than to the B genome of hexaploid or tetraploid wheat. In summary, the T. timopheevii genome assembly provides a valuable resource for genome-informed discovery of agronomically important genes for food security.

Fusarium oxysporum is a significant threat to agriculture and One Health, requiring advanced molecular tools for functional genomics analyses and biological control agent development. Existing gene-editing methods are hampered by costly protoplast preparation protocols and by CRISPR/Cas9 limitations, such as restricted PAM sequences and complex guide RNA requirements. We engineered an efficient CRISPR/Cpf1 system that overcomes these issues through three main innovations: small-scale protoplast generation using novel filter columns that greatly reduces enzyme consumption while simplifying workflows, a CRISPR/Cpf1 system with flexible PAM recognition and staggered DNA cleavage to promote homologous recombination, and minimal homology arm strategies that significantly decrease cloning complexity. Extensive validation confirms successful gene targeting with molecular verification and functional analysis via standardized pathogenicity assays. This integrated platform offers affordable, accessible tools for systematic F. oxysporum research, enhancing fundamental understanding of plant-pathogen interactions and supporting high-throughput screening vital for agricultural biotechnology and biological agent development. MOTIVATIONFusarium oxysporum plays crucial roles as both a damaging plant pathogen and a model for studying host-pathogen interactions. It is well established that systematic functional genomics approaches are vital for advancing agricultural biotechnology and developing biological agents. Creating efficient gene-editing systems can help elucidate virulence mechanisms and enable rapid production of modified strains for practical use. However, current transformation methods face major challenges, such as high enzyme costs, and CRISPR/Cas9 systems are limited by PAM sequence availability and by blunt-end DNA cleavage, which hampers homologous recombination. Furthermore, complex guide RNA scaffolds complicate large-scale functional studies with traditional methods. To address these challenges, we have developed a streamlined CRISPR/Cpf1 (Cas12a) platform that combines small-scale protoplast preparation with significantly reduced enzyme use, exploiting Cpf1s unique features, such as flexible PAM recognition, staggered DNA cuts that promote recombination, improved target specificity, and simpler guide RNA design. This platform can also accelerate the development of biological agents and support high-throughput screening applications essential to the progress of agricultural biotechnology. HIGH LIGHTSO_LIReduced enzyme costs by 95% through small-scale protoplast preparation C_LIO_LICRISPR/Cpf1 system established for efficient gene editing in Fusarium oxysporum C_LIO_LIStreamlined workflow enables routine gene targeting and rapid mutant screening C_LIO_LIComplete workflow validated with EGFP-marked pathogenicity C_LI

Sorghum bicolor, one of the most important grass crops around the world, harbors a high degree of genetic diversity. We constructed chromosome-level genome assemblies for two important sorghum inbred lines, Tx2783 and RTx436. The final high-quality reference assemblies consist of 19 and 18 scaffolds, respectively, with contig N50 values of 25.6 and 20.3 Mb. Genes were annotated using evidence-based and de novo gene predictors, and RAMPAGE data demonstrate that transcription start sites were effectively captured. Together with other public sorghum genomes, BTx623, RTx430, and Rio, extensive structural variations (SVs) of various sizes were characterized using Tx2783 as a reference. Genome-wide scanning for disease resistance (R) genes revealed high levels of diversity among these five sorghum accessions. To characterize sugarcane aphid (SCA) resistance in Tx2783, we mapped the resistance region on chromosome 6 using a recombinant inbred line (RIL) population and found a SV of 191 kb containing a cluster of R genes in Tx2783. Using Tx2783 as a backbone, along with the SVs, we constructed a pan-genome to support alignment of resequencing data from 62 sorghum accessions, and then identified core and dispensable genes using this population. This study provides the first overview of the extent of genomic structural variations and R genes in the sorghum population, and reveals potential targets for breeding of SCA resistance.

Plant bioengineering is a time-consuming and labor-intensive process, with no guarantee of achieving the desired trait. Here we report a fast, automated, scalable, high-throughput pipeline for plant bioengineering (FAST-PB). FAST-PB achieves gene cloning, genome editing, and product characterization by integrating automated biofoundry engineering of callus and protoplast cells with single cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We first demonstrate that FAST-PB can streamline the Golden Gate cloning process, with the capacity to construct 96 vectors in parallel. To prove the concept, using FAST-PB, we first found that PEG2050 significantly increases transfection efficiency by over 45%. To validate the pipeline, we established a reporter-gene-free method for CRISPR editing via mutation of HCF136, affecting cellular chlorophyll fluorescence. Next, we applied this pipeline for lipid production and found that diverse lipids were significantly enhanced up to sixfold through introducing multi-gene cassettes via CRISPR activation, and regenerated plant using this platform. Lastly, we harnessed FAST-PB to achieve high-throughput single-cell lipid profiling through the integration of MALDI-MS with the biofoundry, and differentiated engineered and unengineered cells using the single-cell lipidomics. These innovations massively increase the throughput of synthetic biology, genome editing, and metabolic engineering, and change what is possibly using single-cell metabolomics in plants.

Doubled haploid (DH) technology is used to obtain homozygous lines in a single generation, which significantly accelerates the crop breeding trajectory. Traditionally, in vitro culture is used to generate DHs, but is limited by species and genotype recalcitrance. In vivo haploid induction (HI) through seed is been widely and efficiently used in maize and was recently extended to several monocot crops. However, a similar generic and efficient HI system is still lacking in dicot crops. Here we show that genotype-independent in vivo HI can be triggered by mutation of DMP genes in tomato, rapeseed and tobacco with HI rates of ~1.9%, 2.4% and 1.2%, respectively. The DMP-HI system offers a robust DH technology to facilitate variety improvement in these crops. The success of this approach and the conservation of DMP genes paves the way for a generic and efficient genotype-independent HI system in other dicot crops.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a significant threat to global wheat production. Genetic resistance plays a crucial role in controlling this disease. Among wheat breeding innovations, the wheat-rye 1BL.1RS translocations are notable for introducing alien genetic diversity, partly due to the presence of the stripe rust resistance gene Yr9 on 1RS. To clone Yr9, we first identified four Pst-susceptible mutants from Lumai 15, which carries the 1BL.1RS translocation and Yr9. Using these mutants, we performed Sequencing Trait-Associated Mutations (STAM). A single candidate gene, YrChr1B, was identified within the Yr9 locus and later confirmed as Yr9 through genetic complementation and gene editing. The Yr9 gene encodes a coiled-coil nucleotide-binding site leucine-rich repeat (CC-NBS-LRR or NLR) protein and is part of a 14-member NLR gene cluster. This cluster is conserved among Triticeae species and is an ortholog of the barley Mla locus. Cloning Yr9 expands the genetic resources available for molecular wheat breeding aimed at durable and broad-spectrum disease resistance.

Genetic engineering of wheat is complex due to its large genome size, the presence of numerous genes with high sequence similarities, and a multitude of repetitive elements. In addition, genetic transformation of wheat has been difficult, mainly due to poor regeneration in tissue cultures. Recent advances in plant biotechnology, particularly the use of the regenerative genes GROWTH-REGULATING FACTOR (GRF) and GRF-INTERACTING FACTOR (GIF), have provided new tools for wheat transformation and regeneration. Another transformative tool is the RUBY system, that involves genetic engineering of three betalain biosynthesis genes, providing a noninvasive, visually detectable red pigment. In this study, we used the GRF4-GIF1 chimera along with the RUBY system to advance transformation and gene editing in wheat and barley. The GRF4-GIF1 chimera significantly aided wheat regeneration; however, it had an opposite effect in barley, where it inhibited the regeneration process. Therefore, we primarily generated RUBY transgenic barley lines using constructs that did not include the GRF4-GIF1 chimera. Additionally, we used the RUBY cassette for fast assessment of gene editing by knockingout the first betalain biosynthetic gene in RUBY-positive transgenic wheat plants, resulting in a change of leaf color from red to green. The edited RUBY wheat lines lost more than just the red color. They also lost betalain-related traits, such as being less likely to get leaf rust (Puccinia triticina) and salt stress. Importantly, the loss of RUBY did not affect plant viability, making it a useful tool for genome editing and a viable alternative to destructive methods.

Bioengineering aimed at producing complex and valuable plant specialized metabolites in microbial hosts requires efficient uptake of precursor molecules and export of final products to alleviate toxicity and feedback inhibition. Plant genomes encode a vast repository of transporters of specialized metabolites that-- due to lack of molecular knowledge--remains largely unexplored in bioengineering. Using phlorizin as a case study--an anti-diabetic and anti-cancerous flavonoid from apple--we demonstrate that brute-force functional screening of plant transporter libraries in Xenopus oocytes is a viable approach to identify transporters for bioengineering. By screening 600 Arabidopsis transporters, we identified and characterized purine permease 8 (AtPUP8) as a bidirectional phlorizin transporter. Functional expression in the plasma membrane of a phlorizin-producing yeast strain increased phlorizin titer by more than 80 %. This study provides a generic approach for identifying plant exporters of specialized metabolites and demonstrates the potential of transport-engineering for improving yield in bioengineering approaches.

Breeding for less digestible starch in wheat can improve the health impact of bread and other wheat foods. Based on an established in vitro starch digestibility assay by Edwards et al. (2019) we developed a high-throughput assay to measure starch digestibility in hydrothermally processed samples for use in forward genetic approaches. Digestibility of purified starch from maize and wheat was measured using both methods and produced comparable results. Using the high-throughput assay, we estimated starch digestibility of 118 wheat landraces from the core Watkins collection and found wide variation across lines and elite UK varieties, (20% to 40% and 31% to 44% starch digested after 90 minutes respectively). Sieved flour fractions and purified starch for selected lines showed altered starch digestibility profiles compared with wholemeal flour, suggesting that matrix properties of flour rather than intrinsic properties of starch granules conferred the low starch digestibility observed.

Hemicelluloses are important dietary fibers and a key component of lignocellulosic biomass. Despite numerous observations for fluorescently tagged cellulose synthases, the subcellular journeys and biochemical activities of intracellular cellulose synthase-like enzymes such as {beta}-mannan synthases (ManS) remain largely unexplored. This study identifies C-terminal fluorescent protein tags that maintain ManS activity in the yeast to accelerate the Design, Build, Test, Learn cycles for polysaccharide biosynthesis. Using the Amorphophallus konjac ManS as a case study, we demonstrate that the enzyme co-localizes with a known yeast marker for the Golgi apparatus despite the toxic effects of plant glucomannan accumulation in Pichia pastoris. The ManS first transmembrane domain was found to be critical for the punctate localization of the enzyme, its overall expression level and its function. Additionally, we explored how fluorescently tagged ManS is influenced by genetic or chemical perturbations of native yeast cell wall components, such as reducing protein mannosylation and severely disrupting {beta}-1,3-glucans. Finally, we identified alternative feeding strategies and episomal vectors for Pichia, which were extended to Saccharomyces cerevisiae, to accelerate hemicellulose research. We propose that expanding the Plant MoClo-compatible plasmid repertoire is essential to swiftly prototype carbohydrate-active enzymes in yeast before proceeding with more time-intensive analyses in plants. Requiring only hours or days instead of weeks or months for plant transformation/regeneration, our yeast prototyping strategies can de-risk the bioengineering of carbohydrate-active enzymes.