Activation of the native PHYTOENE SYNTHASE 1 promoter by modifying near-miss cis-acting elements induces carotenoid biosynthesis in embryogenic rice callus.

KEY MESSAGE: Modification of silent latent endosperm-enabled promoters (SLEEPERs) allows the ectopic activation of non-expressed metabolic genes in rice callus Metabolic engineering in plants typically involves transgene expression or the mutation of endogenous genes. An alternative is promoter modification, where small changes in the promoter sequence allow genes to be switched on or off in particular tissues. To activate silent genes in rice endosperm, we screened native promoters for near-miss cis-acting elements that can be converted to endosperm-active regulatory motifs. We chose rice PHYTOENE SYNTHASE 1 (PSY1), encoding the enzyme responsible for the first committed step in the carotenoid biosynthesis pathway, because it is not expressed in rice endosperm. We identified six motifs within a 120-bp region, upstream of the transcriptional start site, which differed from endosperm-active elements by up to four nucleotides. We mutated four motifs to match functional elements in the endosperm-active BCH2 promoter, and this promoter was able to drive GFP expression in callus and in seeds of regenerated plants. The 4 M promoter was not sufficient to drive PSY1 expression, so we mutated the remaining two elements and used the resulting 6 M promoter to drive PSY1 expression in combination with a PDS transgene. This resulted in deep orange callus tissue indicating the accumulation of carotenoids, which was subsequently confirmed by targeted metabolomics analysis. PSY1 expression driven by the uncorrected or 4 M variants of the promoter plus a PDS transgene produced callus that lacked carotenoids. These results confirm that the adjustment of promoter elements can facilitate the ectopic activation of endogenous plant promoters in rice callus and endosperm and most likely in other tissues and plant species.

Epigenetic variation in early and late flowering plants of the rubber-producing Russian dandelion Taraxacum koksaghyz provides insights into the regulation of flowering time.

The Russian dandelion (Taraxacum koksaghyz) grows in temperate zones and produces large amounts of poly(cis-1,4-isoprene) in its roots, making it an attractive alternative source of natural rubber. Most T. koksaghyz plants require vernalization to trigger flower development, whereas early flowering varieties that have lost their vernalization dependence are more suitable for breeding and domestication. To provide insight into the regulation of flowering time in T. koksaghyz, we induced epigenetic variation by in vitro cultivation and applied epigenomic and transcriptomic analysis to the resulting early flowering plants and late flowering controls, allowing us to identify differences in methylation patterns and gene expression that correlated with flowering. This led to the identification of candidate genes homologous to vernalization and photoperiodism response genes in other plants, as well as epigenetic modifications that may contribute to the control of flower development. Some of the candidate genes were homologous to known floral regulators, including those that directly or indirectly regulate the major flowering control gene FT. Our atlas of genes can be used as a starting point to investigate mechanisms that control flowering time in T. koksaghyz in greater detail and to develop new breeding varieties that are more suited to domestication.

Overexpression of a pseudo-etiolated-in-light-like protein in Taraxacum koksaghyz leads to a pale green phenotype and enables transcriptome-based network analysis of photomorphogenesis and isoprenoid biosynthesis.

Introduction: Plant growth and greening in response to light require the synthesis of photosynthetic pigments such as chlorophylls and carotenoids, which are derived from isoprenoid precursors. In Arabidopsis, the pseudo-etiolated-in-light phenotype is caused by the overexpression of repressor of photosynthetic genes 2 (RPGE2), which regulates chlorophyll synthesis and photosynthetic genes. Methods: We investigated a homologous protein in the Russian dandelion (Taraxacum koksaghyz) to determine its influence on the rich isoprenoid network in this species, using a combination of in silico analysis, gene overexpression, transcriptomics and metabolic profiling. Results: Homology-based screening revealed a gene designated pseudo-etiolated-in-light-like (TkPEL-like), and in silico analysis identified a light-responsive G-box element in its promoter. TkPEL-like overexpression in dandelion plants and other systems reduced the levels of chlorophylls and carotenoids, but this was ameliorated by the mutation of one or both conserved cysteine residues. Comparative transcriptomics in dandelions overexpressing TkPEL-like showed that genes responsible for the synthesis of isoprenoid precursors and chlorophyll were downregulated, probably explaining the observed pale green leaf phenotype. In contrast, genes responsible for carotenoid synthesis were upregulated, possibly in response to feedback signaling. The evaluation of additional differentially expressed genes revealed interactions between pathways. Discussion: We propose that TkPEL-like negatively regulates chlorophyll- and photosynthesis-related genes in a light-dependent manner, which appears to be conserved across species. Our data will inform future studies addressing the regulation of leaf isoprenoid biosynthesis and photomorphogenesis and could be used in future breeding strategies to optimize selected plant isoprenoid profiles and generate suitable plant-based production platforms.

Transcriptomic analysis of Chinese yam (Dioscorea polystachya Turcz.) variants indicates brassinosteroid involvement in tuber development.

Dioscorea is an important but underutilized genus of flowering plants that grows predominantly in tropical and subtropical regions. Several species, known as yam, develop large underground tubers and aerial bulbils that are used as food. The Chinese yam (D. polystachya Turcz.) is one of the few Dioscorea species that grows well in temperate regions and has been proposed as a climate-resilient crop to enhance food security in Europe. However, the fragile, club-like tubers are unsuitable for mechanical harvesting, which is facilitated by shorter and thicker storage organs. Brassinosteroids (BRs) play a key role in plant cell division, cell elongation and proliferation, as well as in the gravitropic response. We collected RNA-Seq data from the head, middle and tip of two tuber shape variants: F60 (long, thin) and F2000 (short, thick). Comparative transcriptome analysis of F60 vs. F2000 revealed 30,229 differentially expressed genes (DEGs), 1,393 of which were differentially expressed in the growing tip. Several DEGs are involved in steroid/BR biosynthesis or signaling, or may be regulated by BRs. The quantification of endogenous BRs revealed higher levels of castasterone (CS), 28-norCS, 28-homoCS and brassinolide in F2000 compared to F60 tubers. The highest BR levels were detected in the growing tip, and CS was the most abundant (439.6 ± 196.41 pmol/g in F2000 and 365.6 ± 112.78 pmol/g in F60). Exogenous 24-epi-brassinolide (epi-BL) treatment (20 nM) in an aeroponic system significantly increased the width-to-length ratio (0.045 ± 0.002) compared to the mock-treated plants (0.03 ± 0.002) after 7 weeks, indicating that exogenous epi-BL produces shorter and thicker tubers. In this study we demonstrate the role of BRs in D. polystachya tuber shape, providing insight into the role of plant hormones in yam storage organ development. We found that BRs can influence tuber shape in Chinese yam by regulating the expression of genes involved cell expansion. Our data can help to improve the efficiency of Chinese yam cultivation, which could provide an alternative food source and thus contribute to future food security in Europe.

Cyanovirin-N binds to select SARS-CoV-2 spike oligosaccharides outside of the receptor binding domain and blocks infection by SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped positive stranded RNA virus which has caused the recent deadly pandemic called COVID-19. The SARS-CoV-2 virion is coated with a heavily glycosylated Spike glycoprotein which is responsible for attachment and entry into target cells. One, as yet unexploited strategy for preventing SARS-CoV-2 infections, is the targeting of the glycans on Spike. Lectins are carbohydrate-binding proteins produced by plants, algae, and cyanobacteria. Some lectins can neutralize enveloped viruses displaying external glycoproteins, offering an alternative therapeutic approach for the prevention of infection with virulent ß-coronaviruses, such as SARS-CoV-2. Here we show that the cyanobacterial lectin cyanovirin-N (CV-N) can selectively target SARS-CoV-2 Spike oligosaccharides and inhibit SARS-CoV-2 infection in vitro and in vivo. CV-N neutralizes Delta and Omicron variants in vitro better than earlier circulating viral variants. CV-N binds selectively to Spike with a Kd as low as 15 nM and a stoichiometry of 2 CV-N: 1 Spike but does not bind to the receptor binding domain (RBD). Further mapping of CV-N binding sites on Spike shows that select high-mannose oligosaccharides in the S1 domain of Spike are targeted by CV-N. CV-N also reduced viral loads in the nares and lungs in vivo to protect hamsters against a lethal viral challenge. In summary, we present an anti-coronavirus agent that works by an unexploited mechanism and prevents infection by a broad range of SARS-CoV-2 strains.

Improving environmental stress resilience in crops by genome editing: insights from extremophile plants.

In basic and applied sciences, genome editing has become an indispensable tool, especially the versatile and adaptable CRISPR/Cas9 system. Using CRISPR/Cas9 in plants has enabled modifications of many valuable traits, including environmental stress tolerance, an essential aspect when it comes to ensuring food security under climate change pressure. The CRISPR toolbox enables faster and more precise plant breeding by facilitating: multiplex gene editing, gene pyramiding, and de novo domestication. In this paper, we discuss the most recent advances in CRISPR/Cas9 and alternative CRISPR-based systems, along with the technical challenges that remain to be overcome. A revision of the latest proof-of-concept and functional characterization studies has indeed provided more insight into the quantitative traits affecting crop yield and stress tolerance. Additionally, we focus on the applications of CRISPR/Cas9 technology in regard to extremophile plants, due to their significance on: industrial, ecological and economic levels. These still unexplored genetic resources could provide the means to harden our crops against the threat of climate change, thus ensuring food security over the next century.

COL2-dependent photoperiodic floral induction in Nicotiana sylvestris seems to be lost in the N. sylvestris × N. tomentosiformis hybrid N. tabacum.

Introduction: Plants are sessile organisms that maximize reproductive success by adapting to their environment. One of the key steps in the reproductive phase of angiosperms is flower development, requiring the perception of multiple endogenous and exogenous signals integrated via a complex regulatory network. Key floral regulators, including the main transcription factor of the photoperiodic pathway (CONSTANS, CO) and the central floral pathway integrator (FLOWERING LOCUS T, FT), are known in many species. Methods and results: We identified several CO-like (COL) proteins in tobacco (Nicotiana tabacum). The NtCOL2a/b proteins in the day-neutral plant N. tabacum were most closely related to Arabidopsis CO. We characterized the diurnal expression profiles of corresponding genes in leaves under short-day (SD) and long-day (LD) conditions and confirmed their expression in phloem companion cells. Furthermore, we analyzed the orthologs of NtCOL2a/b in the maternal LD ancestor (N. sylvestris) and paternal, facultative SD ancestor (N. tomentosiformis) of N. tabacum and found that they were expressed in the same diurnal manner. NtCOL2a/b overexpression or knock-out using the CRISPR/Cas9 system did not support a substantial role for the CO homologs in the control of floral transition in N. tabacum. However, NsCOL2 overexpression induced flowering in N. sylvestris under typically non-inductive SD conditions, correlating with the upregulation of the endogenous NsFTd gene. Discussion: Our results suggest that NsFTd is transcriptionally regulated by NsCOL2 and that this COL2-dependent photoperiodic floral induction seems to be lost in N. tabacum, providing insight into the diverse genetics of photoperiod-dependent flowering in different Nicotiana species.

Omics and Multi-Omics Analysis for the Early Identification and Improved Outcome of Patients with Psoriatic Arthritis.

The definitive diagnosis and early treatment of many immune-mediated inflammatory diseases (IMIDs) is hindered by variable and overlapping clinical manifestations. Psoriatic arthritis (PsA), which develops in ~30% of people with psoriasis, is a key example. This mixed-pattern IMID is apparent in entheseal and synovial musculoskeletal structures, but a definitive diagnosis often can only be made by clinical experts or when an extensive progressive disease state is apparent. As with other IMIDs, the detection of multimodal molecular biomarkers offers some hope for the early diagnosis of PsA and the initiation of effective management and treatment strategies. However, specific biomarkers are not yet available for PsA. The assessment of new markers by genomic and epigenomic profiling, or the analysis of blood and synovial fluid/tissue samples using proteomics, metabolomics and lipidomics, provides hope that complex molecular biomarker profiles could be developed to diagnose PsA. Importantly, the integration of these markers with high-throughput histology, imaging and standardized clinical assessment data provides an important opportunity to develop molecular profiles that could improve the diagnosis of PsA, predict its occurrence in cohorts of individuals with psoriasis, differentiate PsA from other IMIDs, and improve therapeutic responses. In this review, we consider the technologies that are currently deployed in the EU IMI2 project HIPPOCRATES to define biomarker profiles specific for PsA and discuss the advantages of combining multi-omics data to improve the outcome of PsA patients.

Regulation of Molecular Farming Products.

The regulation of molecular farming is a complex topic because plants and plant-based systems are relative newcomers among the many production platforms available for recombinant proteins. The regulations specific for different types of product (human/veterinary pharmaceuticals and medical devices, cosmetics, diagnostics, and research reagents) must therefore be overlaid with the regulations governing hitherto unfamiliar production platforms, and this must be achieved in different jurisdictions that handle genetically modified organisms (and genetically modified plants in particular) in very different ways. This chapter uses examples of different product types and production methods in three different jurisdictions (the USA, the EU, and Canada) to demonstrate some of the challenges facing the regulatory authorities.

The tobacco phosphatidylethanolamine-binding protein NtFT4 increases the lifespan of Drosophila melanogaster by interacting with the proteostasis network.

Proteostasis reflects the well-balanced synthesis, trafficking and degradation of cellular proteins. This is a fundamental aspect of the dynamic cellular proteome, which integrates multiple signaling pathways, but it becomes increasingly error-prone during aging. Phosphatidylethanolamine-binding proteins (PEBPs) are highly conserved regulators of signaling networks and could therefore affect aging-related processes. To test this hypothesis, we expressed PEPBs in a heterologous context to determine their ectopic activity. We found that heterologous expression of the tobacco (Nicotiana tabacum) PEBP NtFT4 in Drosophila melanogaster significantly increased the lifespan of adult flies and reduced age-related locomotor decline. Similarly, overexpression of the Drosophila ortholog CG7054 increased longevity, whereas its suppression by RNA interference had the opposite effect. In tobacco, NtFT4 acts as a floral regulator by integrating environmental and intrinsic stimuli to promote the transition to reproductive growth. In Drosophila, NtFT4 engaged distinct targets related to proteostasis, such as HSP26. In older flies, it also prolonged Hsp26 gene expression, which promotes longevity by maintaining protein integrity. In NtFT4-transgenic flies, we identified deregulated genes encoding proteases that may contribute to proteome stability at equilibrium. Our results demonstrate that the expression of NtFT4 influences multiple aspects of the proteome maintenance system via both physical interactions and transcriptional regulation, potentially explaining the aging-related phenotypes we observed.

So similar yet so different: The distinct contributions of extrafascicular and fascicular phloem to transport and exudation in cucumber plants.

Cucurbits have been used as phloem research models for many decades because their exudates can be accessed with ease. However, cucurbit plants possess two distinct phloem systems known as the fascicular phloem (FP) and extrafascicular phloem (EFP). Therefore, the molecular composition and function of certain exudates can be misinterpreted due to their unclear origin. To characterize the anatomy and function of the different phloem systems more clearly, we generated specific antibodies against marker proteins (PP1 homologs) allowing the clear identification of the EFP at the organ, tissue and cellular levels by immunological staining. We also used detailed microscopy to determine common and unique anatomical features of the FP and EFP sieve elements (SEs) in cucumber (Cucumis sativus). The comparison of exudation rates and the dynamic viscosity, density and sugar content of the exudates from plants grown in the light and dark revealed the consistent composition and behavior of the EFP exudate even when photosynthesis was prevented, thus differing from the properties of the FP exudate. Furthermore, the analysis of phloem transport using a fluorescein disodium salt showed only wound-induced exudation of dye from the EFP, indicating the absence of transport in this tissue. Our results show that it is important to distinguish between the EFP and FP in cucurbits, particularly their differing behaviors in response to wounding.

The Ca2+ response of a smart forisome protein is dependent on polymerization.

Forisomes are giant self-assembling mechanoproteins that undergo reversible structural changes in response to Ca2+ and various other stimuli. Artificial forisomes assembled from the monomer MtSEO-F1 can be used as smart biomaterials, but the molecular basis of their functionality is not understood. To determine the role of protein polymerization in forisome activity, we tested the Ca2+ association of MtSEO-F1 dimers (the basic polymerization unit) by circular dichroism spectroscopy and microscale thermophoresis. We found that soluble MtSEO-F1 dimers neither associate with Ca2+ nor undergo structural changes. However, polarization modulation infrared reflection absorption spectroscopy revealed that aggregated MtSEO-F1 dimers and fully-assembled forisomes associate with Ca2+ , allowing the hydration of poorly-hydrated protein areas. A change in the signal profile of complete forisomes indicated that Ca2+ interacts with negatively-charged regions in the protein complexes that only become available during aggregation. We conclude that aggregation is required to establish the Ca2+ response of forisome polymers.

The functionality of plant mechanoproteins (forisomes) is dependent on the dual role of conserved cysteine residues.

Forisomes are giant polyprotein complexes that undergo reversible conformational rearrangements from a spindle-like to a plug-like state in response to Ca2+ or changes in pH. They act as valves in the plant vasculature, and reproduce this function in vitro to regulate flow in microfluidic capillaries controlled by electro-titration. Heterologous expression in yeast or plants allows the large-scale production of tailor-made artificial forisomes for technical applications. Here we investigated the unexpected disintegration of artificial forisomes in response to Ca2+ following the deletion of the M1 motif in the MtSEO-F1 protein or the replacement of all four conserved cysteine residues therein. This phenomenon could be mimicked in wild-type forisomes under reducing conditions by adding a thiol alkylating agent. We propose a model in which reversible changes in forisome structure depend on cysteine residues with ambiguous redox states, allowing the formation of intermolecular disulfide bridges (confirmed by mass spectrometry) as well as noncovalent thiol interactions to connect forisome substructures in the dispersed state. This is facilitated by the projection of the M1 motif from the MtSEO-F1 protein as part of an extended loop. Our findings support the rational engineering of disintegrating forisomes to control the release of peptides or enzymes in microfluidic systems.

The tobacco phosphatidylethanolamine-binding protein NtFT4 simultaneously improves vitality, growth, and protein yield in human cells.

The production of biopharmaceutical proteins in mammalian cells by transient expression or stable transformation requires robust and viable cells. Cell line engineering must therefore balance improved cell growth and viability with high productivity. We tested the ability of nonmammalian phosphatidylethanolamine-binding proteins to enhance cell proliferation in monolayers and suspension cultures. The tobacco protein NtFT4 improved the proliferation of multiple human cell lines. Viable cell density is usually impaired by efficient transfection, but we found that the number of HEK-293TNtFT4 cells at the peak of protein expression was twice that of standard HEK-293T cells, and the antibody yield increased by approximately one-third. Improved growth and viability were observed in different cell lines, in different culture media, and also after transient transfection, suggesting the beneficial trait is consistent and transferable. Additional modifications could boost the productivity of high-density HEK-293TNtFT4 cells even further as we showed for a fluorescent marker protein and recombinant antibody expressed in monolayer cultures. The HEK-293TNtFT4 cell line provides a new human model platform that increases cell proliferation, also achieving a fundamental improvement in recombinant protein expression.

Contributions of the international plant science community to the fight against infectious diseases in humans-part 2: Affordable drugs in edible plants for endemic and re-emerging diseases.

The fight against infectious diseases often focuses on epidemics and pandemics, which demand urgent resources and command attention from the health authorities and media. However, the vast majority of deaths caused by infectious diseases occur in endemic zones, particularly in developing countries, placing a disproportionate burden on underfunded health systems and often requiring international interventions. The provision of vaccines and other biologics is hampered not only by the high cost and limited scalability of traditional manufacturing platforms based on microbial and animal cells, but also by challenges caused by distribution and storage, particularly in regions without a complete cold chain. In this review article, we consider the potential of molecular farming to address the challenges of endemic and re-emerging diseases, focusing on edible plants for the development of oral drugs. Key recent developments in this field include successful clinical trials based on orally delivered dried leaves of Artemisia annua against malarial parasite strains resistant to artemisinin combination therapy, the ability to produce clinical-grade protein drugs in leaves to treat infectious diseases and the long-term storage of protein drugs in dried leaves at ambient temperatures. Recent FDA approval of the first orally delivered protein drug encapsulated in plant cells to treat peanut allergy has opened the door for the development of affordable oral drugs that can be manufactured and distributed in remote areas without cold storage infrastructure and that eliminate the need for expensive purification steps and sterile delivery by injection.

Contributions of the international plant science community to the fight against human infectious diseases - part 1: epidemic and pandemic diseases.

Infectious diseases, also known as transmissible or communicable diseases, are caused by pathogens or parasites that spread in communities by direct contact with infected individuals or contaminated materials, through droplets and aerosols, or via vectors such as insects. Such diseases cause ˜17% of all human deaths and their management and control places an immense burden on healthcare systems worldwide. Traditional approaches for the prevention and control of infectious diseases include vaccination programmes, hygiene measures and drugs that suppress the pathogen, treat the disease symptoms or attenuate aggressive reactions of the host immune system. The provision of vaccines and biologic drugs such as antibodies is hampered by the high cost and limited scalability of traditional manufacturing platforms based on microbial and animal cells, particularly in developing countries where infectious diseases are prevalent and poorly controlled. Molecular farming, which uses plants for protein expression, is a promising strategy to address the drawbacks of current manufacturing platforms. In this review article, we consider the potential of molecular farming to address healthcare demands for the most prevalent and important epidemic and pandemic diseases, focussing on recent outbreaks of high-mortality coronavirus infections and diseases that disproportionately affect the developing world.

Comparative Transcriptome Analysis in Taraxacum koksaghyz to Identify Genes that Determine Root Volume and Root Length.

The Russian dandelion (Taraxacum koksaghyz, family Asteraceae) produces large amounts of natural rubber in the laticifers of its roots. This species has been proposed as an alternative source of natural rubber to augment or partly replace the rubber tree (Hevea brasiliensis) but domestication would require genetic improvement to increase rubber yields and agronomic optimization to facilitate harvesting and processing. Optimization has focused thus far on the size and shape of the roots, the primary storage organ for natural rubber and inulin. However, the corresponding genetic factors are poorly understood. Here we describe the comparative transcriptomic analysis of root tissues from T. koksaghyz plant sets featuring different root sizes and shapes, aiming to identify differentially expressed genes correlating with root length or root diameter in the upper root and root tip. The resulting datasets revealed multiple candidate genes for each trait and root part, including a glucan endo-1,3-ß-d-glucosidase, an allene oxide synthase 3, and a TIFY10A/JAZ1 homolog. These three genes were tested by qRT-PCR in outdoor-grown plants with diverse root morphology, and the expression of two genes correlated with the appropriate root morphotype, confirming the effectiveness of our method. We evaluated the candidate genes to gain insight into their potential functions in root development. Such candidate genes could be suitable for marker-assisted breeding programs in the future.

Glyco-Engineering of Plant-Based Expression Systems.

Most secreted proteins in eukaryotes are glycosylated, and after a number of common biosynthesis steps the glycan structures mature in a species-dependent manner. Therefore, human therapeutic proteins produced in plants often carry plant-like rather than human-like glycans, which can affect protein stability, biological function, and immunogenicity. The glyco-engineering of plant-based expression systems began as a strategy to eliminate plant-like glycans and produce human proteins with authentic or at least compatible glycan structures. The precise replication of human glycans is challenging, owing to the absence of a pathway in plants for the synthesis of sialylated proteins and the necessary precursors, but this can now be achieved by the coordinated expression of multiple human enzymes. Although the research community has focused on the removal of plant glycans and their replacement with human counterparts, the presence of plant glycans on proteins can also provide benefits, such as boosting the immunogenicity of some vaccines, facilitating the interaction between therapeutic proteins and their receptors, and increasing the efficacy of antibody effector functions. Graphical Abstract Typical structures of native mammalian and plant glycans with symbols indicating sugar residues identified by their short form and single-letter codes. Both glycans contain fucose, albeit with different linkages.

Fast, Precise, and Reliable Multiplex Detection of Potato Viruses by Loop-Mediated Isothermal Amplification.

Potato is an important staple food crop in both developed and developing countries. However, potato plants are susceptible to several economically important viruses that reduce yields by up to 50% and affect tuber quality. One of the major threats is corky ringspot, which is a tuber necrosis caused by tobacco rattle virus (TRV). The appearance of corky ringspot symptoms on tubers prior to commercialization results in ≈ 45% of the tubers being downgraded in quality and value, while ≈ 55% are declared unsaleable. To improve current disease management practices, we have developed simple diagnostic methods for the reliable detection of TRV without RNA purification, involving minimalized sample handling (mini), subsequent improved colorimetric loop-mediated isothermal amplification (LAMP), and final verification by lateral-flow dipstick (LFD) analysis. Having optimized the mini-LAMP-LFD approach for the sensitive and specific detection of TRV, we confirmed the reliability and robustness of this approach by the simultaneous detection of TRV and other harmful viruses in duplex LAMP reactions. Therefore, our new approach offers breeders, producers, and farmers an inexpensive and efficient new platform for disease management in potato breeding and cultivation.