The phenylpropanoid pathway varies across species and environments, producing diverse metabolites. The transcriptional regulation governing precursor feeding into this pathway remains largely unexplored. Here, we characterize OscWRKY7 TF from Ocimum sanctum as a positive regulator of arogenate dehydratase-5, a key shikimate pathway gene catalyzing the conversion of arogenate into phenylalanine. Phenylalanine treatment in O. sanctum induced key genes and metabolites of the phenylpropanoid pathway, with OscWRKY7 showing early induction. Recombinant OscWRKY7 interacts with the canonical W-box cis element and transactivates the reporter gene in yeast. Ectopic expression of OscWRKY7 in Arabidopsis thaliana induced transcriptomic changes in the shikimate and phenylpropanoid pathways, leading to upregulation of arogenate dehydratase-5 transcripts and enhanced accumulation of phenylalanine and downstream metabolites. The biological function of OscWRKY7 was determined in Ocimum sanctum using gain- and loss-of-function approaches via hairy root-mediated overexpression and tobacco rattle virus (TRV)-based VIGS. Overexpression of OscWRKY7 upregulated OscADT5 and enhanced phenylalanine accumulation, along with downstream phenylpropanoid metabolites, whereas silencing reduced them, providing evidence of OscWRKY7-mediated regulation of phenylpropanoid metabolism in O. sanctum. OscWRKY7 exhibits dual cis-element specificity, binding the W-box and a noncanonical WT-box variant (TAACTTTT) in the OscADT5 promoter, suggesting a novel regulatory module. The Electrophoretic Mobility Shift Assay and the Yeast One-Hybrid Assay demonstrated binding, while dual luciferase suggests the transactivation of OscADT5 by OscWRKY7 through a noncanonical WT-box. OscWRKY7 further modulates phenylpropanoid pathway-associated genes to alter the accumulation of anthocyanin and salicylic acid, thereby enhancing plant defense response in Arabidopsis. Overall, our study suggests that OscWRKY7 promotes phenylpropanoid accumulation by facilitating the availability of phenylalanine, a precursor of the phenylpropanoid pathway.

Rooibos (Aspalathus linearis) is one of the few endemic South African plants that has achieved economic importance and international acclaim, mostly as a herbal tea. Plant production, limited to a small mountainous region in South Africa, is at risk as commercial rooibos longevity is in decline, mostly due to low stress tolerance. Transcriptome data can serve to identify molecular markers for improved stress response, which would speed up selection and facilitate the establishment of breeding programmes. Previously, rooibos leaf transcriptomes have been sequenced using Illumina, which yields short reads, hampering correct reassembly of full-length transcripts. Here, we established Oxford Nanopore-based, long-read transcriptome analysis for leaf and root samples from rooibos. We report on potential pitfalls in data pre-processing (PolyA tail trimming and rRNA removal), and compare two assemblers (RATTLE and RNA-Bloom2) and two clustering algorithms (VSEARCH and CD-HIT). The best assembly comprising 169,122 transcripts was generated using RNA-Bloom2 with short-read polishing, followed by CD-HIT clustering. Of the 95,054 predicted proteins, only 67% were also present in the Illumina dataset. The remainder comprised substantially shorter, mostly full-length sequences from a wide range of primary and secondary biosynthesis pathways. Functional annotation indicated that this transcriptome represents a high-quality, comprehensive resource for data mining. In the leaf fraction, comparative transcriptomics identified overexpressed rooibos transcripts potentially involved in photosynthesis, photorespiration and carbon fixation. In the roots, overexpressed transcripts encoded enzymes potentially involved in regulation of root growth and secondary metabolite biosynthesis. These transcripts may represent first targets for molecular marker development.

Potato virus Y (PVY), potato mop-top virus (PMTV), potato virus S (PVS), and tobacco rattle virus (TRV) can be difficult to identify based on visual foliar symptoms. Using tuber samples collected from seven locations and 12 cultivars during 2017 to 2019, we developed a molecular assay using customized Whatman Flinders Technology Associates Plantsaver cards (FTA cards) and a reverse-transcription PCR (RT-PCR) for efficient sample collection and nucleic acid extraction. PMTV and PVY were detected more frequently on the stem end, TRV on the rose end, and PVS was evenly detected across the stem-end to rose-end axis of the tubers. Differences were seen in virus species localization within a tuber, thereby a composite of samples taken from multiple locations on a tuber improved virus detection. Regardless of sampling location on the tuber, tissue excised from the surface to 0.5 cm deep provided the best detection for all four viruses. For PVY, PMTV, and TRV, the proportion of tubers with viruses detected from field samples was highest at 100, 150, and 175 days after harvest, respectively. However, the probability of detecting PMTV and PVY 25 days after harvest was not different than the probability of detection at their peak detection times after harvest. The probability of TRV detection was lower at 25 days after harvest than at 175 days after harvest (P < 0.05).

To reduce reliance on time consuming postharvest seed potato grow outs, a Whatman Flinders Technology Associates Plantsaver Cards (FTA)-based pathogen detection protocol based on dormant tubers was developed for four potato viruses. Viruses tested included potato virus Y (PVY), potato virus S (PVS), potato mop-top virus (PMTV), and tobacco rattle virus (TRV). Viruses were also detected from potato tubers, sprouts, and leaves using different experimental test methods and sample collection timings (harvest-FTA, storage-FTA, sprouting-FTA, sprout-enzyme-linked immunosorbent assay [ELISA], sprout-recombinase polymerase amplification [RPA], leaf-RPA, and leaf-ELISA). When compared with the leaf-ELISA, accuracy of the harvest-FTA was 92.9% (PVY) and 93.8% (PVS), and test results were available 90-days earlier than leaf-ELISA. Results from the leaf-ELISA for PMTV and leaf-FTA for TRV grossly underreport detection of these two viruses compared with results obtained using multiple tuber and sprout test methods (harvest-FTA, storage-FTA, sprouting-FTA, sprout-ELISA, and sprout-RPA). In 2020, an on-farm collaboration resulted in field implementation of the FTA card-based tuber test for detecting PVY, PMTV, and TRV from eight seed lots. Accuracy, sensitivity, and specificity for detecting PVY from these eight seed lots using the farm-FTA test method were 89, 92, and 87%, respectively, and the results were provided approximately 3 months earlier than the official results from the state seed potato certification programs. Cost analysis of the FTA card-based detection protocols showed that this approach reduced testing costs for multiple pathogens and allowed growers to use a single sampling pipeline to measure and manage multiple pathogen risks at lower costs.

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.

The stubby root nematode, Paratrichodorus allius, is one of the most important plant-parasitic nematodes. Besides root feeding, P. allius also transmits the Tobacco rattle virus in potatoes, which causes corky ringspot disease. Rapid detection of P. allius is key for efficient management. This study was conducted to develop a real-time recombinase polymerase amplification (RPA) assay that is capable of detecting P. allius directly in DNA extracts from soil using a simple portable device in real time. A fluorophore-attached probe was designed to target the internal transcribed spacer (ITS)-rDNA of P. allius and was used along with primers designed previously. The real-time RPA assay had the ability to detect P. allius DNA extracted directly from infested soil with a sensitivity of one-sixteenth portion of a single nematode. This RPA assay was specific, as it did not produce positive signals from non-target nematodes tested. The real-time RPA was found to be rapid as it could even detect P. allius in as little as 7 min. Testing with 15 field soil samples validated the RPA assay developed in this study. This is the first report of P. allius detection directly from soil DNA using real-time RPA and is the fastest method for P. allius detection in soil to date.

In the post-genomic era, virus-induced gene silencing (VIGS) has played an important role in research on reverse genetics in plants. Commonly used Agrobacterium-mediated VIGS inoculation methods include stem scratching, leaf infiltration, use of agrodrench, and air-brush spraying. In this study, we developed a root wounding-immersion method in which 1/3 of the plant root (length) was cut and immersed in a tobacco rattle virus (TRV)1:TRV2 mixed solution for 30 min. We optimized the procedure in Nicotiana benthamiana and successfully silenced N. benthamiana, tomato (Solanum lycopersicum), pepper (Capsicum annuum L.), eggplant (Solanum melongena), and Arabidopsis thaliana phytoene desaturase (PDS), and we observed the movement of green fluorescent protein (GFP) from the roots to the stem and leaves. The silencing rate of PDS in N. benthamiana and tomato was 95-100%. In addition, we successfully silenced two disease-resistance genes, SITL5 and SITL6, to decrease disease resistance in tomatoes (CLN2037E). The root wounding-immersion method can be used to inoculate large batches of plants in a short time and with high efficiency, and fresh bacterial infusions can be reused several times. The most important aspect of the root wounding-immersion method is its application to plant species susceptible to root inoculation, as well as its ability to inoculate seedlings from early growth stages. This method offers a means to conduct large-scale functional genome screening in plants.

Plant-derived volatiles mediate interactions among plants, pathogenic viruses, and viral vectors. These volatile-dependent mechanisms have not been previously demonstrated belowground, despite their likely significant role in soil ecology and agricultural pest impacts. We investigated how the plant virus, tobacco rattle virus (TRV), attracts soil nematode vectors to infected plants. We infected Nicotiana benthamiana with TRV and compared root growth relative to that of uninfected plants. We tested whether TRV-infected N. benthamiana was more attractive to nematodes 7 d post infection and identified a compound critical to attraction. We also infected N. benthamiana with mutated TRV strains to identify virus genes involved in vector nematode attraction. Virus titre and associated impacts on root morphology were greatest 7 d post infection. Tobacco rattle virus infection enhanced 2-ethyl-1-hexanol production. Nematode chemotaxis and 2-ethyl-1-hexanol production correlated strongly with viral load. Uninfected plants were more attractive to nematodes after the addition of 2-ethyl-1-hexanol than were untreated plants. Mutation of TRV RNA2-encoded genes reduced the production of 2-ethyl-1-hexanol and nematode attraction. For the first time, this demonstrates that virus-driven alterations in root volatile emissions lead to increased chemotaxis of the virus's nematode vector, a finding with implications for sustainable management of both nematodes and viral pathogens in agricultural systems.

The stubby root nematode Paratrichodorus allius is an important plant-parasitic nematode species within the Trichodoridae family. It can directly harm the plants by feeding on the roots or indirectly by transmitting Tobacco rattle virus. These nematodes are mostly diagnosed either by traditional microscopic methods or a polymerase chain reaction (PCR)-based method. Droplet digital PCR (ddPCR) is a novel PCR technique which is sensitive and precise in quantifying DNA templates of the test samples. In this study, we developed a ddPCR assay to detect and quantify P. allius in soil. The specificity and sensitivity of the assay was first determined using P. allius nematode DNA or DNA from sterilized soil artificially inoculated with P. allius, and the assay was used to quantify P. allius populations in field soils. The assay did not detect nematodes other than P. allius, thus showing high specificity. It was able to detect P. allius equivalent to a 0.01 and 0.02 portion of a single nematode in soil DNA and nematode DNA extracts, respectively. Highly linear relationships between DNA copy numbers from ddPCR and serial dilutions of known concentrations were observed with DNA from P. allius nematodes (R[2] = 0.9842) and from artificially infested soil (R[2] = 0.9464). The P. allius populations from field soils determined by ddPCR were highly correlated with traditional microscopic counts (R[2] = 0.7963). To our knowledge, this is the first report of applying ddPCR to detect and quantify stubby root nematode in soil. The results of this study support the potentiality of a ddPCR assay as a new research tool in diagnostics of plant-parasitic nematodes.

Verticillium wilt (VW) caused by Verticillium dahliae is a soil-borne vascular fungal disease that severely affects cotton yield and fiber quality. Sugar metabolism plays an important role in the growth and pathogenicity of V. dahliae. However, limited information is known about the sugar transporter genes and their roles in the growth and pathogenicity of V. dahliae.

Copper-based chalcogenides have emerged as promising thermoelectric materials due to their high thermoelectric performance, tunable transport properties, earth abundance and low toxicity. We have presented an overview of experimental results and first-principal calculations investigating the thermoelectric properties of various polymorphs of Cu2SnS3 (CTS), Cu2ZnSnS4 (CZTS), and Cu2ZnSnSe4 (CZTSe) synthesized by high-energy reactive mechanical alloying (ball milling). Of particular interest are the disordered polymorphs of these materials, which exhibit phonon-glass-electron-crystal behavior-a decoupling of electron and phonon transport properties. The interplay of cationic disorder and nanostructuring leads to ultra-low thermal conductivities while enhancing electronic transport. These beneficial transport properties are the consequence of a plethora of features, including trap states, anharmonicity, rattling, and conductive surface states, both topologically trivial and non-trivial. Based on experimental results and computational methods, this report aims to elucidate the details of the electronic and lattice transport properties, thereby confirming that the higher thermoelectric (TE) performance of disordered polymorphs is essentially due to their complex crystallographic structures. In addition, we have presented synchrotron X-ray diffraction (SR-XRD) measurements and ab initio molecular dynamics (AIMD) simulations of the root-mean-square displacement (RMSD) in these materials, confirming anharmonicity and bond inhomogeneity for disordered polymorphs.

Root parasitic weed Phelipanche aegyptiaca is an obligate plant parasite that causes severe damage to host crops. Agriculture crops mainly belong to the Brassicaceae, Leguminosae, Cruciferae, and Solanaceae plant families affected by this parasitic weed, leading to the devastating loss of crop yield and economic growth. This root-specific parasitic plant is not able to complete its life cycle without a suitable host and is dependent on the host plant for nutrient uptake and germination. Therefore, selected parasitic genes of P. aegyptiaca which were known to be upregulated upon interaction with the host were chosen. These genes are essential for parasitism, and reduced activity of these genes could affect host-parasitic interaction and provide resistance to the host against these parasitic weeds. To check and examine the role of these parasitic genes which can affect the development of host resistance, we silenced selected genes in the P. aegyptiaca using the tobacco rattle virus (TRV) based virus-induced gene silencing (VIGS) method. Our results demonstrated that the total number of P. aegyptiaca parasite tubercles attached to the root of the host plant Nicotiana benthamiana was substantially decreased in all the silenced plants. However, silencing of the P. aegyptiaca MNT1 gene which encodes the mannitol transporter showed a significantly reduced number of germinated shoots and tubercles. Thus, our study indicates that the mannitol transport gene of P. aegyptiaca plays a crucial role in parasitic germination, and silencing of the PaMNT1 gene abolishes the germination of parasites on the host roots.