Nicotiana Benthamiana Research Articles

Betaine lipids overproduced in seed plants are excluded from plastid membranes and promote endomembrane expansion

Abstract Plants and algae have to adapt to environmental changes and face various stresses that negatively affect their growth and development. One common stress is phosphate (Pi) deficiency, which is often present in the environment at limiting levels. In response to Pi deficiency, these organisms increase Pi uptake and remobilize intracellular Pi. Phospholipids are degraded to provide Pi and replaced by non-phosphorus lipids, such as glycolipids or betaine lipids. During evolution, seed plants lost the ability to synthesize betaine lipids. By expressing BTA1 genes, which are involved in the synthesis of diacylglyceryl-N,N,N-trimethyl-homoserine (DGTS), from different species, our work shows that DGTS can be produced in seed plants. In Arabidopsis, expression of BTA1 under a phosphate starvation-inducible promoter resulted in limited DGTS production without having any impact on plant growth or lipid remodeling. In transient expression systems in Nicotiana benthamiana, leaves were able to accumulate DGTS up to 30 % of their glycerolipid content at a slight expense of galactolipid and phospholipid production. At the subcellular level, we showed that DGTS is absent from the plastids and seems to be enriched in the endomembranes, driving an ER membrane proliferation. Finally, the DGTS synthesis pathway seems to compete with PC synthesis via the Kennedy pathway but does not seem to be derived from the PC diacylglycerol backbone and therefore does not interfere with the eukaryotic pathway involved in galactolipid synthesis.

Turnip mosaic virus selectively subverts a PR‐5 thaumatin‐like, plasmodesmal protein to promote viral infection

Summary Pathogenesis‐related (PR) proteins are induced by abiotic and biotic stresses and generally considered as part of the plant defense mechanism. However, it remains yet largely unclear if and how they are involved in virus infection. Our recent quantitative, comparative proteomic study identified three PR‐5 family proteins that are significantly differentially accumulated in the plasmodesmata (PD)‐enriched fraction isolated from Nicotiana benthamiana leaves infected by turnip mosaic virus (TuMV). In this study, we employed the TuMV‐Arabidopsis pathosystem to characterize the involvement of two Arabidopsis orthologs, AtOSM34 and AtOLP of the three N. benthamiana PR‐5‐like proteins. We show that AtOSM34 and AtOLP are PD‐localized proteins and their expression is up‐ and downregulated in response to TuMV infection, respectively. Deficiency or overexpression of AtOLP does not affect viral RNA accumulation. Knockdown of AtOSM34 inhibits TuMV infection, whereas its overexpression promotes viral infection. We further demonstrate that AtOSM34 functions as a proviral factor through diminishing PD callose deposition to promote viral intercellular movement, targeting the viral replication complex to enhance viral replication, and suppressing the ROS‐mediated antiviral response. Taken together, these data suggest that TuMV has evolved the ability to selectively upregulate and subvert AtOSM34, a PR‐5 family protein to assist its infection.

First Report of Pepper Chlorosis-Associated Virus in Burdock (Arctium lappa) in Korea

Burdock (Arctium lappa) is a fiber-rich root vegetable widely used in Asian cuisine with many health benefits. In July 2023, two burdock leaf samples showing viral symptoms, such as yellowing and interveinal chlorosis, were collected from a field in Daegu, South Korea, with an incidence rate of about 10%. The samples were pooled, ground with liquid nitrogen, and total RNA was extracted using a Maxwell® 16 LEV Plant RNA Kit (Promega, Madison, USA). A cDNA library was constructed using TruSeq Standard Total RNA with Ribo-Zero (Illumina, San Diego, USA) and sequenced on an Illumina NovaSeq 6000 (BIOTO, Daejeon, Korea) with 100 bp paired-end reads. Of the approximately 84 million reads generated, about 61 million clean reads were identified using Trimmomatic 0.39 with default parameters. Subsequently, Trinity 2.13.0 was used for de novo assembly, resulting in 179,910 contigs, which, when annotated as previously described (Bak et al., 2024), revealed the presence of tomato spotted wilt virus (TSWV) and pepper chlorosis-associated virus (PepCaV). Three PepCaV-related contigs, representing a near-complete genome, were submitted to NCBI GenBank (PP502423-PP502425). BLASTn analysis showed 95.21% (7275/7641 bp), 95.89% (1680/1752 bp), and 96.18% (1461/1519 bp) identities with the L, M, and S segments of the PepCaV isolate Higashitsuno_2021 (LC719619-LC719621), respectively. RT-PCR was performed to confirm the presence of viruses using specific primers for PepCaV (5’-CACCTTGATTATAGACATGACAG/GTTCTCAAATCTTCCCAATCG-3’) targeting the coat protein region, and TSWV (5’-GCAACAACTTGCAAGAAGATG/CATGACCTTCAGAAGGCTTG-3’; Kim et al., 2021). Initial results showed both samples were positive for TSWV, and one was positive for PepCaV. Additionally, 26 symptomatic and 8 asymptomatic samples were collected from the same field. PepCaV was positive in 3 samples, TSWV in 12, and TSWV+PepCaV in 11 by RT-PCR. All asymptomatic samples were negative. Leaves infected with PepCaV, whether solely or co-infected with TSWV, initially exhibited interveinal chlorosis and vein contraction on one side of the main vein (Fig. S1). In plants infected with PepCaV alone, these symptoms were mild or less noticeable in the initial phases. The majority of PepCaV-infected samples were co-infected with TSWV, during which symptoms appeared more severe. Following this, an amplicon, derived from the sample that initially tested positive for PepCaV in the first RT-PCR was cloned using the All in OneTM Cloning Kit (BioFact, Daejeon, Korea) and sequenced by Bionner (Daejeon, Korea). The resulting sequence, submitted to NCBI GenBank (PP928956), demonstrated 96.87% (805/831 bp) identity with the PepCaV isolate Higashitsuno_2021 (LC719600). Moreover, sap inoculation was performed on A. lappa, Nicotiana benthamiana, N. clevelandii, N. debneyi, N. rustica, N. tabacum, Chenopodium amaranticolor, and C. quinoa, but no transmission was identified in any case. PepCaV, recently identified and likely belonging to the genus Ophiovirus within the family Ophioviridae, is known to infect pepper and tobacco (Shimomoto et al., 2023; Wang et al., 2024). This is the first report of PepCaV in Korea and the first identification of burdock as a natural host. Although studies on its vectors and pathogenicity are lacking, PepCaV’s presence suggests potential risks. Given the widespread cultivation of burdock in Korea, PepCaV could significantly impact local agriculture by affecting crop yield and health.

Fusarium sacchari Effector FsMEP1 Contributes to Virulence by Disturbing Localization of Thiamine Thiazole Synthase ScTHI2 from Sugarcane

Fusarium sacchari is a significant pathogenic fungus that causes sugarcane Pokkah Boeng. Proteins secreted by pathogenic fungi can be delivered into hosts to suppress plant immunity and establish infection. However, there is still much to be discovered regarding F. sacchari’s secreted effectors in overcoming plant immunity. In this paper, we characterize a novel effector called FsMEP1, which is essential for the virulence of F. sacchari. FsMEP1 contains a conserved zinc-binding motif sequence, HEXXH, and is highly expressed during host infection. Using the Agrobacterium tumefaciens-mediated transient expression system, it was confirmed that FsMEP1 could suppress Bcl-2-associated X protein (BAX)-triggered cell death, callose deposition, and ROS explosion in Nicotiana benthamiana. Furthermore, the deletion of FsMEP1 demonstrated its requirement for contributing to the pathogenicity of F. sacchari in sugarcane. Further analysis revealed that FsMEP1 could interact with the sugarcane thiamine thiazole synthase ScTHI2 and disrupt its normal localization, thereby inhibiting the synthesis of thiamine and the defense responses mediated by ScTHI2. Based on these findings, we propose that ScTHI2 represents a potential molecular target for improving sugarcane resistance to Pokkah Boeng disease.

MicroRNA858 represses the transcription factor gene SbMYB47 and regulates flavonoid biosynthesis in Scutellaria baicalensis

Abstract MicroRNAs (miRNAs) are non-coding endogenous single-stranded RNAs that regulate target gene expression by reducing their transcription and translation. Several miRNAs in plants function in secondary metabolism. The dried root of Scutellaria baicalensis Georgi is a traditional Chinese medicine that contains flavonoids (baicalin, wogonoside, and baicalein) as its main active ingredients. Although the S. baicalensis genome sequence has been published, information regarding its miRNAs is lacking. In this study, 12 small RNA libraries of different S. baicalensis tissues were compiled, including roots, stems, leaves, and flowers. A total of 129 miRNAs were identified, including 99 miRNAs from 27 miRNA families and 30 predicted miRNAs. Furthermore, 46 reliable target genes of 15 miRNA families were revealed using psRNAtarget and confirmed by degradome sequencing. It was speculated that the microRNA858 (miR858)–SbMYB47 module might be involved in flavonoid biosynthesis. Transient assays in Nicotiana benthamiana leaves indicated that miR858 targets SbMYB47 and suppresses its expression. Artificial miRNA-mediated knockdown of miR858 and overexpression of SbMYB47 significantly increased the flavonoid content in S. baicalensis hairy roots, while SbMYB47 knockdown inhibited flavonoid accumulation. Yeast one-hybrid and dual-luciferase assays indicated that SbMYB47 directly binds to and activates the S. baicalensis phenylalanine ammonia-lyase 3 (SbPAL-3) and flavone synthase II (SbFNSⅡ-2) promoters. Our findings reveal the link between the miR858–SbMYB47 module and flavonoid biosynthesis, providing a potential strategy for the production of flavonoids with important pharmacological activities through metabolic engineering.

Use of the Puccinia sorghi haustorial transcriptome to identify and characterize AvrRp1-D recognized by the maize Rp1-D resistance protein.

The common rust disease of maize is caused by the obligate biotrophic fungus Puccinia sorghi. The maize Rp1-D allele imparts resistance against the P. sorghi IN2 isolate by initiating a defense response that includes a rapid localized programmed cell death process, the hypersensitive response (HR). In this study, to identify AvrRp1-D from P. sorghi IN2, we employed the isolation of haustoria, facilitated by a biotin-streptavidin interaction, as a powerful approach. This method proves particularly advantageous in cases where the genome information for the fungal pathogen is unavailable, enhancing our ability to explore and understand the molecular interactions between maize and P. sorghi. The haustorial transcriptome generated through this technique, in combination with bioinformatic analyses such as SignalP and TMHMM, enabled the identification of 251 candidate effectors. We ultimately identified two closely related genes, AvrRp1-D.1 and AvrRp1-D.2, which triggered an Rp1-D-dependent defense response in Nicotiana benthamiana. AvrRp1-D-induced Rp1-D-dependent HR was further confirmed in maize protoplasts. We demonstrated that AvrRp1-D.1 interacts directly and specifically with the leucine-rich repeat (LRR) domain of Rp1-D through yeast two-hybrid assay. We also provide evidence that, in the absence of Rp1-D, AvrRp1-D.1 plays a role in suppressing the plant immune response. Our research provides valuable insights into the molecular interactions driving resistance against common rust in maize.

Virulence effectors encoded in the rice yellow dwarf phytoplasma genome participate in pathogenesis.

Bacteria-like phytoplasmas alternate between plant and insect hosts, secreting proteins that disrupt host development. In this study, we sequenced the complete genome of 'Candidatus Phytoplasma oryzae' strain HN2022, associated with rice yellow dwarf (RYD) disease, using PacBio HiFi technology. The strain was classified within the 16Sr XI-B subgroup. Through SignalP v5.0 for prediction and subsequent expression analysis of secreted proteins in Nicotiana benthamiana and rice (Oryza sativa L.), we identified the key virulence effector proteins RY348 and RY378. RY348, a homologue of Secreted Aster Yellows Phytoplasma Effector 54 (SAP54), targets and degrades the MADS-box transcription factors MADS1 and MADS15, causing pollen sterility. Meanwhile, RY378 impacts the strigolactone and auxin signaling pathways, substantially increasing tillering. These findings offer insights into the interactions between plants and phytoplasmas.

Biosynthetic Origin of the Methoxy Group in Quinine and RelatedAlkaloids.

a methoxy group that has been assumed to be incorporated at a late pathway stage. Here we show that the methoxy group in quinine and related alkaloids is introduced onto the starting substrate tryptamine. Feeding studies definitively show that 5-methoxytryptamine is utilized as a quinine biosynthetic intermediate in planta. We discover the biosynthetic genes that encode the responsible oxidase and methyltransferase, and we use these genes to reconstitute the early steps of the alkaloid biosynthetic pathway in Nicotiana benthamiana to produce a mixture of methoxylated and non-methoxylated alkaloid intermediates. Importantly, we show that the co-occurrence of both tryptamine and 5-methoxytryptamine substrates, along with the substrate promiscuity of downstream pathway enzymes, enable parallel formation of both methoxylated and non-methoxylated alkaloids.

Application of an Efficient Enhancer in Gene Function Research

Although great progress has been made in transgenic technology, increasing the expression level and thus promising the expected phenotypes of exogenous genes in transgenic plants is still a crucial task for genetic transformation and crop engineering. Here, we conducted a comparative study of the enhancing efficiency of three putative translational enhancers, including Ω (natural leader from a plant virus), OsADH 5′ (natural leader from a plant gene), and ARC (active ribosomal RNA complementary), using the transient gene expression systems of Nicotiana benthamiana and Chirita pumila. We demonstrate that three tandem repeats of ARC (3 × ARC) are more efficient than other enhancers in expression. The enhancing efficiency of 6 × ARC is further increased, up to 130 times the expression level without the insertion of enhancers. We further evaluated the enhancing efficiency of 6 × ARC under agrobacterium-mediated transformation systems. In C. pumila, 6 × ARC significantly amplifies the phenotypic effect of CpCYC1 and CpCYC2 in repressing stamen development and yellow pigmentation. In Arabidopsis thaliana, 6 × ARC and the AtAP1 promoter work together to promote the accumulation of anthocyanin pigments in vegetative and reproductive organs. Most significantly, the fusion of 6 × ARC in a CpCYC1/2 transgenic system in C. pumila fully reveals that these genes have the complete function of repressing the yellow spots, displaying an advantage in manifesting the function of exogenous genes. This study highlights the application potential of the enhancer 6 × ARC in gene function research in plants.

MYB168 and WRKY20 transcription factors synergistically regulate lignin monomer synthesis during potato tuber wound healing.

Lignin is a critical component of the closing layer of the potato (Solanum tuberosum L.) tuber during healing; however, the molecular mechanism of its formation remains poorly understood. To elucidate the molecular mechanism of tuber healing, we screened the genes encoding transcription factors that regulate lignin synthesis(StMYB24/49/105/144/168, StWRKY19/20/22/23/34) and the key genes involved in lignin monomer synthesis (PHENYLALANINE AMMONIA LYASE 5 (StPAL5) and CINNAMYL ALCOHOL DEHYDROGENASE 14 (StCAD14)) for induced expression after wounding using transcriptome data. DLR, Y1H, EMSA, and ChIP-qPCR assays revealed that StMYB168 could bind directly to the StPAL5 and StCAD14 promoters to activate their expression and that StWRKY20 enhanced this regulation with a synergistic effect. Y2H, BiFC, and Co-IP assays showed that StMYB168 interacted with StWRKY20 to form a MYB-WRKY complex. Furthermore, transient overexpression of StMYB168 and StWRKY20 in Nicotiana benthamiana leaves upregulated the expression of NbPAL and NbCAD10 and promoted lignin accumulation in the leaves. In addition, overexpression of StWRKY20 and StMYB168 together resulted in higher expression levels of NbPAL and NbCAD10 and higher levels of lignin monomer and total lignin. In contrast, silencing of StMYB168 and StWRKY20 in potato significantly reduced the lignin content of wounded tubers. In conclusion, StMYB168 and StWRKY20 are important regulators of lignin biosynthesis in potato tubers during healing and can positively regulate lignin biosynthesis by forming a complex. The elucidation of this regulatory module provides information on the regulatory mechanism of lignin monomer synthesis in wounded tubers at the transcriptional level.

Binary vector copy number engineering improves Agrobacterium-mediated transformation.

The copy number of a plasmid is linked to its functionality, yet there have been few attempts to optimize higher-copy-number mutants for use across diverse origins of replication in different hosts. We use a high-throughput growth-coupled selection assay and a directed evolution approach to rapidly identify origin of replication mutations that influence copy number and screen for mutants that improve Agrobacterium-mediated transformation (AMT) efficiency. By introducing these mutations into binary vectors within the plasmid backbone used for AMT, we observe improved transient transformation of Nicotiana benthamiana in four diverse tested origins (pVS1, RK2, pSa and BBR1). For the best-performing origin, pVS1, we isolate higher-copy-number variants that increase stable transformation efficiencies by 60-100% in Arabidopsis thaliana and 390% in the oleaginous yeast Rhodosporidium toruloides. Our work provides an easily deployable framework to generate plasmid copy number variants that will enable greater precision in prokaryotic genetic engineering, in addition to improving AMT efficiency.

Recognition of a Fungal Effector Potentiates Pathogen-Associated Molecular Pattern-Triggered Immunity in Cotton.

Plants are equipped with multi-layered immune systems that recognize pathogen-derived elicitors to activate immunity. Verticillium dahliae is a soil-borne fungus that infects a broad range of plants and causes devastating wilt disease. The mechanisms underlying immune recognition between plants and V. dahliae remain elusive. Here, a V. dahliae secretory protein, elicitor of plant defense gene (VdEPD1), acts as an elicitor that triggers defense responses in both Nicotiana benthamiana and cotton plants is identified. Targeted gene deletion of VdEPD1 enhances V. dahliae virulence in plants. Expression of VdEPD1 triggers the accumulation of reactive oxygen species (ROS) and the activation of cell death in cotton plants. Gossypium barbadense EPD1-interacting receptor-like cytoplasmic kinase (GbEIR5A) and GbEIR5D interact with VdEPD1. Silencing of GbEIR5A/D significantly impairs VdEPD1-triggered cell death in cotton plants, indicating the contribution of GbEIR5A/D to VdEPD1-activated effector-triggered immunity (ETI). VdEPD1 stimulates the expression of GbEIR5A and GbEIR5D in cotton plants. Interestingly, cotton plants with silenced GbEIR5A/D genes exhibit compromised pathogen-associated molecular patterns (PAMPs)-triggered ROS accumulation, whereas overexpression of GbEIR5A or GbEIR5D enhances PAMP-induced ROS. These findings indicate that recognition of VdEPD1 potentiates GbEIRs to enhance cotton PAMP-triggered immunity (PTI), uncovering a cooperative interplay of PTI and ETI in cotton.

Viral condensates formed by Pea enation mosaic virus 2 sequester ribosomal components and suppress translation

Viral proteins with intrinsic disorder, such as the p26 movement protein from Pea enation mosaic virus 2 (PEMV2), can phase separate and form condensates that aid specific stages of virus replication. However, little is known about the impact of viral condensate formation on essential cellular processes, like translation. In this study, we performed mass spectrometry on affinity-purified p26 condensates and found an enrichment of RNA-binding proteins involved in translation and ribosome biogenesis. Puromycin assays and polysome profiling show that ectopic p26 expression suppresses ribosome assembly and translation in Nicotiana benthamiana, mirroring defects in late-stage PEMV2 infection. Despite interactions with the 2'-O-methyltransferase fibrillarin, p26 does not inhibit translation by altering rRNA methylation but instead binds directly to rRNAs and decreases their solubility. Disruption of ribosome assembly and translation by p26 during late PEMV2 infection may promote stages of the virus replication cycle that are incompatible with translation, including systemic movement.

A novel protein elicitor (Cs08297) from Ciboria shiraiana enhances plant disease resistance.

Ciboria shiraiana is a necrotrophic fungus that causes mulberry sclerotinia disease resulting in huge economic losses in agriculture. During infection, the fungus uses immunity elicitors to induce plant tissue necrosis that could facilitate its colonization on plants. However, the key elicitors and immune mechanisms remain unclear in C. shiraiana. Herein, a novel elicitor Cs08297 secreted by C. shiraiana was identified, and it was found to target the apoplast in plants to induce cell death. Cs08297 is a cysteine-rich protein unique to C. shiraiana, and cysteine residues in Cs08297 were crucial for its ability to induce cell death. Cs08297 induced a series of defence responses in Nicotiana benthamiana, including the burst of reactive oxygen species (ROS), callose deposition, and activation of defence-related genes. Cs08297 induced-cell death was mediated by leucine-rich repeat (LRR) receptor-like kinases BAK1 and SOBIR1. Purified His-tagged Cs08297-thioredoxin fusion protein triggered cell death in different plants and enhanced plant resistance to diseases. Cs08297 was necessary for sclerotial development, oxidative-stress adaptation, and cell wall integrity but negatively regulated virulence of C. shiraiana. In conclusion, our results revealed that Cs08297 is a novel fungal elicitor in fungi inducing plant immunity. Furthermore, its potential to enhance plant resistance provides a new target to control agricultural diseases biologically.

Functional Identification of Two Glycerol-3-phosphate Acyltransferase5 Homologs from Chenopodium quinoa

Glycerol-3-phosphate acyltransferase5 (GPAT5) is the key enzyme in suberin biosynthesis in Arabidopsis, tomato and Sarracenia purpurea. However, little is known about whether GPAT5 function is conserved in halophytes. In this study, we identified two GPAT5 homologs, CqGPAT5a and CqGPAT5b, in Chenopodium quinoa, the typical halophyte. Using RT-qPCR, we found that CqGPAT5a and CqGPAT5b were highly expressed in quinoa roots and rapidly induced by high salt stress. CqGPAT5a and CqGPAT5b were localized to the endoplasmic reticulum and found to have glycerol-3-phosphate acyltransferase activity using yeast complementation assays. Compared with CqGPAT5b, CqGPAT5a showed relatively weaker function and less protein abundance when expressed in yeast, Arabidopsis or Nicotiana benthamiana. Subsequently, we identified a serine (S) to leucine (L) variation in the CqGPAT5a protein sequence (S251L) compared with CqGPAT5b, located in the connecting region between the second and third transmembrane domains. Site-directed mutagenesis together with yeast mutant complementation and transient expression in tobacco demonstrated that this variation significantly affected CqGPAT5a activity and protein abundance. These findings expand our understanding of GPAT5 and provide new evidence that GPAT5 may be functionally conserved in halophytes.

Saccharomyces cerevisiae whole cell biotransformation for the production of aldehyde flavors and fragrances

9-Carbon aldehydes such as (2E)-nonenal, (3Z)-nonenal, and (2E,6Z)-nonadienal are important melon and cucumber fragrance compounds. Currently, these molecules are produced either synthetically, which faces consumer aversion, or through biotransformation using plant-extracted enzymes, which is costly and inefficient. In this study, we constructed a Saccharomyces cerevisiae platform for the whole cell biotransformation of polyunsaturated fatty acids (PUFAs) to 9-carbon aldehydes. Heterologous expression of lipoxygenase (LOX) from Nicotiana benthamiana and hydroperoxide lyase (HPL) from Cucumis melo (melon) in S. cerevisiae enabled the production of (2E)-nonenal from readily available polyunsaturated fatty acid substrates. A 5.5-fold increase in (2E)-nonenal titer was then achieved utilizing genetic and reaction condition enhancement strategies. The highest titer of (2E)-nonenal was more than 0.11 mM, with about 9% yield. This platform can potentially be used to produce a variety of other aldehyde products by customizing with LOX and HPL enzymes of different regio-selectivities.Key points• Establishment of a S. cerevisiae whole-cell biotransformation platform for cost-efficient, high-yield conversion of PUFAs into high value 9-carbon aldehyde compounds• 5.5-Fold improvement of (2E)-nonenal titer to > 0.11 mM achieved by enhancing reaction conditions and gene expression levels of LOX and HPL

Secretory Proteins Are Involved in the Parasitism of Melon by Phelipanche aegyptiaca During the Attachment Stage

Parasitic plants represent a significant challenge in global agriculture, with Broomrape (Orobanche/Phelipanche spp.) being a notable example of a holoparasitic species that targets the roots of host plants. This study employed comparative transcriptomics to investigate the mechanisms underlying the parasitism of P. aegyptiaca on melon, focusing on both resistant and susceptible interactions. The findings indicate that the critical phase of P. aegyptiaca parasitism occurs during the post-attachment stage. It is suggested that peptidases may play a role in the development of invasive cells, while cell wall-degrading enzymes (CWDEs) are likely involved in cell wall modification and degradation, and transferases, elicitors, and effectors may play a role in immune regulation. In this study, 25 tobacco rattle virus (TRV) recombinant vectors were successfully constructed and functionally validated using a host-induced gene silencing assay to explore the functions of candidate-secreted effector proteins. The results revealed that silencing Cluster-107894.0, Cluster-11592.0, and Cluster-12482.0 significantly decreased the parasitism rate of P. aegyptiaca on Nicotiana benthamiana. Notably, Cluster-107849.0 encodes a cellulase with hydrolase activity, Cluster-11592.0 encodes a periodic-dependent kinase inhibitor with phosphoprotein activity, and Cluster-12482.0 encodes a glucan 1,3-β-glucosidase with hydrolase activity. These findings potentially offer a novel theoretical framework and justification for understanding host–parasite plant interactions, and suggest new avenues for developing crop varieties resistant to parasitic infestation.

Isolation of Small Extracellular Vesicles (sEVs) from the Apoplastic Wash Fluid of Nicotiana benthamiana Leaves.

Extracellular vesicles (EVs) are small membranous vesicles secreted by cells into their surrounding extracellular environment. Similar to mammalian EVs, plant EVs have emerged as essential mediators of intercellular communication in plants that facilitate the transfer of biological material between cells. They also play essential roles in diverse physiological processes including stress responses, developmental regulation, and defense mechanisms against pathogens. In addition, plant EVs have demonstrated promising health benefits as well as potential therapeutic effects in mammalian health. Despite the plethora of potential applications using plant EVs, their isolation and characterization remains challenging. In contrast to mammalian EVs, which benefit from more standardized isolation protocols, methods for isolating plant EVs can vary depending on the starting material used, resulting in diverse levels of purity and composition. Additionally, the field suffers from the lack of plant EV markers. Nevertheless, three main EV subclasses have been described from leaf apoplasts: tetraspanin 8 positive (TET8), penetration-1-positive (PEN1), and EXPO vesicles derived from exocyst-positive organelles (EXPO). Here, we present an optimized protocol for the isolation and enrichment of small EVs (sEVs; <200 nm) from the apoplastic fluid from Nicotiana benthamiana leaves by ultracentrifugation. We analyze the preparation through transmitted electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting. We believe this method will establish a basic protocol for the isolation of EVs from N. benthamiana leaves, and we discuss technical considerations to be evaluated by each researcher working towards improving their plant sEV preparations. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Isolation and enrichment of small extracellular vesicles (sEVs) from the apoplastic fluid of Nicotiana benthamiana leaves.

The Phytophthora infestans effector Pi05910 suppresses and destabilizes host glycolate oxidase StGOX4 to promote plant susceptibility.

Phytophthora infestans is a notorious oomycete pathogen that causes potato late blight. It secretes numerous effector proteins to manipulate host immunity. Understanding mechanisms underlying their host cell manipulation is crucial for developing disease resistance strategies. Here, we report that the conserved RXLR effector Pi05910 of P. infestans is a genotype-specific avirulence elicitor on potato variety Longshu 12 and contributes virulence by suppressing and destabilizing host glycolate oxidase StGOX4. By performing co-immunoprecipitation, yeast-two-hybrid assays, luciferase complementation imaging, bimolecular fluorescence complementation and isothermal titration calorimetry assays, we identified and confirmed potato StGOX4 as a target of Pi05910. Further analysis revealed that StGOX4 and its homologue NbGOX4 are positive immune regulators against P. infestans, as indicated by infection assays on potato and Nicotiana benthamiana overexpressing StGOX4 and TRV-NbGOX4 plants. StGOX4-mediated disease resistance involves enhanced reactive oxygen species accumulation and activated the salicylic acid signalling pathway. Pi05910 binding inhibited enzymatic activity and destabilized StGOX4. Furthermore, mutagenesis analyses indicated that the 25th residue (tyrosine, Y25) of StGOX4 mediates Pi05910 binding and is required for its immune function. Our results revealed that the core RXLR effector of P. infestans Pi05910 suppresses plant immunity by targeting StGOX4, which results in decreased enzymatic activity and protein accumulation, leading to enhanced plant susceptibility.

Discovery and characterization of a novel carlavirus in Ligularia jaluensis plants

Ligularia jaluensis is an important medicinal and ornamental plant in China. However, the viruses capable of infecting Ligularia jaluensis remains unknown. Here, we identified a novel carlavirus, tentatively named ligularia jaluensis carlavirus (LJCV), as well as a known iris severe mosaic virus (ISMV), in L. jaluensis plants displaying chlorosis and yellow ring spot symptoms, using RNA-seq analysis. The LJCV genome consists of an 8497 nt positive-sense, single-stranded RNA [excluding the poly(A) tail], and contains six open reading frames (ORFs). Phylogenetic analyses based on the full-length genome and RNA-dependent RNA polymerase (RdRp) amino acid (aa) sequences revealed that LJCV clusters within an evolutionary branch alongside known viruses in the Carlavirus genus. The RdRp protein encoded by ORF1 of LJCV shared 45.38%–67.41% identity with the corresponding proteins of eight closely related carlaviruses. ORFs 2–4 constitute the triple gene block (TGB), with TGBp1 and TGBp3 localized in the endoplasmic reticulum (ER), while TGBp2 is localized at plasmodesmata (PD) and facilitates viral intercellular movement, as demonstrated by its ability to complement the potato virus X with movement-deficient mutant (PVX-Δp25-GFP). Additionally, ORF6 encodes a cysteine-rich protein (CRP) that is localized in the chloroplast and functions as a viral pathogenicity factor, inducing severe viral symptoms in the heterologous PVX expression system. Furthermore, we successfully constructed an infectious cDNA clone of LJCV, and found that it can infect Nicotiana benthamiana plants through mechanical inoculation or agrobacterium-mediated infiltration of the LJCV infectious clone. These findings enhance our understanding of the characteristics and host range of carlaviruses, as well as the viruses capable of infecting L. jaluensis.