Protoplasts are plant cells that have been stripped of their rigid cell walls, leaving behind a fragile, membrane-bound sphere of living cytoplasm. Because the wall is removed, protoplasts readily take up foreign DNA, making them a workhorse system for plant research. In a typical protoplast experiment, scientists can introduce reporter constructs to watch gene expression unfold in real time, map protein localization inside the cell, dissect gene regulatory networks, test gene-editing reagents, and probe protein function, all within a matter of days and without the months required to generate a stable transgenic plant. The technique has been refined for major crops and model species such as Arabidopsis, tobacco, and rice, where leaves provide an abundant and convenient source of cells. But the standard recipe assumes a plant with expanded leaves and roots, and dodder has neither.

That anatomical peculiarity posed the central challenge for the research team at Virginia Tech, led by Hope Gruszewski and Matilda Cashman, who contributed equally to the work, alongside Joseph S. Taylor, Bastiaan O. R. Bargmann, and James H. Westwood. As an obligate parasite, C. campestris has evolutionarily abandoned most of the structures that define a conventional plant. It produces slender, leafless, yellow-orange stems that twine around hosts, and it lacks true roots, relying entirely on haustoria for sustenance. The team therefore had to rethink the very first step of the protocol: which tissue to use as the starting material. The options available were limited. They tested shoots harvested from plants that were actively growing on a host, young seedlings, and undifferentiated callus tissue grown in culture, systematically evaluating how well each source yielded intact, viable protoplasts after enzymatic digestion of the cell walls.

The answer, somewhat unexpectedly, came from the tissue least connected to the parasite’s natural lifestyle. Shoots that had developed from callus culture, essentially laboratory-grown stem tissue coaxed into existence on nutrient media, proved to be the best starting material, outperforming both host-grown shoots and seedlings. The researchers grew the callus on modified Murashige and Skoog media supplemented with the plant hormones 6-benzylaminopurine and naphthalene acetic acid, which promote the proliferation of the soft, actively dividing tissue that digests cleanly into single cells. Once the optimal tissue source was identified, the team turned to optimizing the remaining parameters of the isolation procedure, including the enzyme cocktail, digestion conditions, and purification steps, which involve filtering and washing the fragile protoplasts in solutions buffered with MES and protected with bovine serum albumin to preserve membrane integrity.

The optimized protocol delivers numbers that make real experiments possible. From just three grams of starting material, the researchers can routinely isolate approximately one million protoplasts, with more than 85 percent of them remaining viable after the isolation process. That viability figure is critical, because protoplasts are notoriously delicate; even in well-established systems, the stresses of enzymatic digestion and purification can rupture a large fraction of the cells. Achieving consistently high yields and viability from a parasitic plant with no conventional leaves means that downstream applications, from fluorescence microscopy to flow cytometry, now have enough material to work with. The method transforms what was once a bespoke, uncertain exercise into a repeatable routine that any competent plant molecular biology lab could adopt.

Isolation, however, was only half the battle. A protoplast is only useful if foreign DNA can be delivered into it and expressed. The team tackled this with polyethylene glycol-mediated transformation, the most widely used chemical method for shuttling DNA across protoplast membranes. In this approach, protoplasts are suspended in a mannitol-magnesium transformation buffer, mixed with plasmid DNA, and exposed to PEG, which transiently disrupts the membrane and permits DNA uptake before the membrane reseals. The researchers transformed the C. campestris protoplasts with multiple different constructs, and the technique reliably produced an approximate transformation rate of 10 percent. That robustness across constructs matters, because it demonstrates that the method is not a one-off success tuned to a single plasmid but a general platform for genetic manipulation of the parasite’s cells.

To showcase the research value of the new system, the team put the transformed protoplasts to work on two fronts. First, they expressed subcellular-targeted fluorescent proteins, confirming that the parasite’s cells can correctly sort tagged proteins to their intended destinations, a prerequisite for studying protein localization and trafficking in Cuscuta. Second, they used flow cytometry to quantify the activation of an exogenous auxin-responsive reporter gene, treating the protoplasts with the plant hormone auxin, indole-3-acetic acid, and measuring the fluorescent output across large cell populations. Auxin signaling is a master regulator of plant growth and development, and being able to measure its response quantitatively in dodder cells opens the door to comparing hormone signaling between the parasite and its autotrophic relatives at single-cell resolution.

The significance of the work extends well beyond the technical achievement. Parasitic plants such as Cuscuta, Striga, and Orobanche inflict enormous crop losses worldwide, and understanding how their cells sense hosts, form haustoria, and negotiate molecular exchanges with their victims is a major goal of plant science. Cuscuta is particularly intriguing because the haustorial connection allows the movement of RNAs and proteins between parasite and host, raising questions about cross-species signaling that cannot be answered with conventional model systems. Until now, however, progress has been hampered by the scarcity of genetic and cellular tools for these organisms. A reliable protoplast platform fills a crucial gap, offering a fast, flexible system for testing gene function without waiting for slow and difficult stable transformation protocols that have yet to be perfected for obligate parasites.

The Virginia Tech team, whose work was supported by the United States National Science Foundation and the United States Department of Agriculture, emphasizes that protoplasts are efficient systems for analyzing cell-level events, including gene expression, gene regulatory networks, gene editing, protein localization, and protein function. With the new protocol in hand, researchers can begin asking how the fundamental machinery of a parasitic plant cell differs from that of an autotrophic one, whether hormone signaling pathways have been rewired to serve the parasite’s needs, and which cellular programs underpin the formation of the haustorium. The method has the potential to accelerate research into the basic biology of parasitic plants, and in doing so, it may eventually inform new strategies for controlling one of agriculture’s most persistent enemies. For a plant that has spent millions of years perfecting the art of stealth, Cuscuta campestris has just lost a significant measure of its anonymity.

Subject of Research: Protoplast isolation and transient genetic transformation of the obligate parasitic plant Cuscuta campestris

Article Title: Isolation and transient transformation of protoplasts from Cuscuta campestris, an obligate parasitic plant

Article References: Gruszewski, H., Cashman, M., Taylor, J. S., Bargmann, B. O. R., & Westwood, J. H. (2026). Isolation and transient transformation of protoplasts from Cuscuta campestris, an obligate parasitic plant. Plant Methods. https://doi.org/10.1186/s13007-026-01600-y

Image Credits: AI Generated

DOI: 10.1186/s13007-026-01600-y

Keywords: Cuscuta campestris, protoplasts, parasitic plant, plant genetic transformation, haustoria, auxin signaling, flow cytometry, fluorescent proteins, plant cell biology, Plant Methods, callus culture, gene expression

The two most intensively studied feeding structures are giant cells and syncytia, and their developmental strategies could hardly be more different. Root-knot nematodes induce giant cells through rounds of mitosis that lack cytokinesis, combined with repeated endoreduplication, producing enlarged, multinucleate cells stuffed with dense cytoplasm. These cells develop wall ingrowths resembling xylem transfer cells, dramatically increasing the plasma membrane surface area available for solute exchange. Cyst nematodes, by contrast, dissolve the cell walls of neighboring root cells and fuse their protoplasts into a syncytium, a single continuous multinucleate compartment that can eventually encompass more than two hundred cells. Remarkably, three-dimensional reconstructions have shown that individual giant cells can expand roughly sixty-fold in volume between three and forty days after infection, a transformation driven by cell wall loosening and continuous cytoskeletal rearrangement.

None of this happens by accident. The engine of the entire process is the nematode’s stylet, a hollow, protrusible spear connected to esophageal gland cells that secrete a cocktail of effector proteins directly into host tissue. These effectors include cell wall-degrading enzymes such as cellulases, hemicellulases, and pectinases that ease penetration and migration, but the more intriguing molecules are those that manipulate host physiology from within. Once inside plant cells, effectors travel to the nucleus, the cytoplasm, or the apoplast, where they bind host proteins and bend gene expression to the parasite’s advantage. In compatible interactions, the host’s normal developmental pathways are subverted: the cell cycle is reactivated, cell wall architecture is remodeled, and metabolism is redirected toward feeding site construction.

Plant hormones sit at the center of this manipulation. Local auxin accumulation is a master switch for feeding site formation, and nematodes have evolved multiple ways to boost it. The cyst nematode effector 19C07 interacts with the auxin influx transporter LAX3 to enhance auxin flow into selected cells and upregulate cell wall-degrading enzymes, while the effector 10A07 binds the auxin response repressor IAA16, destabilizing its suppressive grip on auxin response factors and even recruiting a host kinase, IPK, to shepherd it into the nucleus. Cytokinin plays an equally critical role in driving cell cycle reactivation at the G1/S transition. Strikingly, Heterodera schachtii carries its own isopentenyltransferase gene, effectively synthesizing cytokinin from the parasite’s side of the interface; silencing that gene suppresses feeding site expansion, and Arabidopsis mutants lacking the cytokinin receptors AHK3 and AHK4 show markedly reduced susceptibility. Ethylene plays a double game, sometimes attracting nematodes to roots and promoting syncytial cell wall expansion, yet also capable of reducing infection through interactions with the receptor ETR1, a contradiction the review flags as an open question.

At the same time, nematodes must dismantle the plant’s immune system, which operates in two layers. Pattern-triggered immunity begins when cell surface receptors detect pathogen-associated molecular patterns, triggering reactive oxygen species production, MAP kinase cascades, and cell wall reinforcement. A deeper layer, effector-triggered immunity, mediated by intracellular resistance proteins, produces localized cell death that starves the parasite. Nematode effectors attack both layers with surgical precision. In rice, the Meloidogyne graminicola effector MgMO237 suppresses pattern-triggered immunity by binding multiple defense-related host proteins, while MgMO289 manipulates the host’s reactive oxygen system through the copper metallochaperone OsHPP04. The M. javanica effector MjTTL5 recruits the thioredoxin reductase subunit AtFTRc to scavenge ROS during the oxidative burst, and the sugar beet cyst nematode effector 10A06 targets spermidine synthase 2 to bolster antioxidant capacity at the infection site. In Globodera rostochiensis, the ubiquitin carboxyl extension protein GrUBCEP12 suppresses flg22-induced immunity, and the effector Gr29D09 disables the potato hexokinase StHXK1.

The review devotes particular attention to transcription factors, the master regulators that nematodes either hijack or exploit as part of the plant’s own response. Functional studies in Arabidopsis and tomato have identified fifteen co-regulated transcription factor hubs during infection, spanning the MYB, WRKY, and ARF families. One especially revealing case is DEL1, an atypical E2F factor that suppresses salicylic acid accumulation and lignin biosynthesis during root-knot infection by repressing EDS5, a gene encoding a salicylic acid transporter. del1 mutants accumulate more salicylic acid, lignify their galls more heavily, and resist infection, while the nematode’s own effector Mi-ISC-1 independently attacks the isochorismate pathway that feeds salicylic acid synthesis. The convergence of host and parasite manipulation on the same pathway underscores how central it is to the outcome of infection.

The WRKY family illustrates the bewildering duality of transcriptional control in these interactions. WRKY11 and WRKY17 act as positive defenders in Arabidopsis, with mutants of either gene showing heightened susceptibility to both cyst and root-knot nematodes. Yet in tomato, SlWRKY16 and SlWRKY31 behave as negative regulators, and their overexpression enhances infection while suppressing defense-related genes. SlWRKY45 goes further: it interacts with JAZ repressor proteins and, once released, binds the promoter of the jasmonic acid biosynthesis gene AOC, throttling jasmonate production and opening the door to the parasite. Meanwhile, WRKY23 promotes feeding site formation by inducing auxin-responsive genes, and bHLH25 and bHLH27 act synergistically to support syncytium development in cyst nematode infection. The same family can thus contain both shields and traitors, and which role a given member plays depends on species, tissue, and developmental stage.

Effectors also reach deep into the plant’s information-processing machinery. The M. incognita effector Mi16D10 binds SCARECROW-like GRAS transcription factors to accelerate giant cell formation, and Mi2G02 hijacks the trihelix factor GT-3a, converting it into a transcriptional inhibitor that silences cell development genes through the 26S proteasome pathway. The cyst nematode effector 30D08 targets AtSMU2, a spliceosome component, perturbing pre-mRNA splicing across thousands of transcripts, and MiEFF18 attacks the core spliceosomal protein SmD1 to fine-tune transcripts governing the cell cycle and cytoskeleton. Indeed, root-knot nematodes induce splicing reprogramming of roughly 2,898 genes involving more than 9,065 transcripts during gall formation. Beyond transcription, epigenetic mechanisms add another layer of control: DNA hypomethylation occurs early in infection, potentially activating stress genes, while the effector Hs32E03 interacts with the histone deacetylase HDT1 to alter acetylation patterns in ribosomal DNA regions, reshaping chromatin to favor parasitism.

What makes the review compelling is its translational ambition. The authors argue that understanding these molecular vulnerabilities opens concrete routes to resistance: CRISPR/Cas editing of transcription factors that mediate feeding site formation, breeding informed by epigenetic marks associated with defense, and host-induced gene silencing that knocks out essential nematode effectors. They also champion single-cell and single-nucleus RNA sequencing as the next frontier, since bulk transcriptomics averages away the cellular heterogeneity that defines galls and syncytia, and functional redundancy among transcription factors often masks the effect of silencing any single gene. Gland-cell-specific transcriptomics is meanwhile expanding the catalog of known effectors, offering a moving target that breeders and biotechnologists must track.

The bigger picture is one of an evolutionary arms race fought at the level of gene regulation. Nematodes exploit conserved host hubs, from the COP9 signalosome to JAZ proteins and translation initiation factors, that other pathogens also target, suggesting shared vulnerabilities that could yield broadly durable resistance. Plants, for their part, retain the capacity to resist when their immune signaling outpaces the parasite’s reprogramming, as resistant interactions shift the sex ratio of developing nematodes toward males and curtail reproduction. Decoding the precise choreography of effectors, hormones, transcription factors, and epigenetic marks, the authors conclude, is not merely an academic exercise but a prerequisite for engineering crops that refuse to build the parasite’s dining room, a goal with enormous implications for global food security as biotic pressures on agriculture intensify.

Subject of Research: Molecular mechanisms of feeding site formation and transcriptional reprogramming in plant-parasitic nematode interactions

Article Title: Cellular plasticity and transcriptional reprogramming in plant-nematode interactions: insights into feeding site formation and plant defense

Article References: Yusuf, A. G., & Bello, T. T. (2025). Cellular plasticity and transcriptional reprogramming in plant-nematode interactions: insights into feeding site formation and plant defense. Crop Health, 3(1), Article 16. https://doi.org/10.1007/s44297-025-00056-1

Image Credits: AI Generated

DOI: 10.1007/s44297-025-00056-1

Keywords: plant-parasitic nematodes, root-knot nematode, cyst nematode, giant cells, syncytium, transcription factors, effector proteins, auxin signaling, cytokinin, plant immunity, CRISPR, RNA interference