Multiplex Real-Time Loop-Mediated Isothermal Amplification (LAMP) Based on the Annealing Curve Analysis: Toward an On-Site Multiplex Detection of Transgenic Sequences in Seeds and Food Products.

Monitoring of the introduction of unapproved genetically modified (GM) events is required because the approval status of a GM event may differ from country to country. The on-site methods such as loop-mediated isothermal amplification (LAMP) offer a technological answer for the rapid GM detection beyond the laboratories. Real-time LAMP assays detecting one GM target were reported earlier. To increase the efficiency of the assay, a multiplex real-time LAMP simultaneously targeting Figwort Mosaic Virus promoter (P-FMV) that constructs region between the Cauliflower Mosaic Virus 35S promoter and cry1Ac gene (p35S-cry1Ac) and neomycin phosphotransferase II (nptII) marker gene was developed. The assay could detect as low as 0.1% for each GM target within 45 min. To the best of our knowledge, multiplexing in real-time LAMP using the Genie II system with applicability in GM detection has been reported herein for the first time. The developed method provides rapid, on-site, and real-time GM detection in seeds and food products.

Comparative analysis of herbivory responsive miRNAs to delineate pod borer (Helicoverpa armigera) resistance mechanisms in Cajanus cajan and its wild relative Cajanus scarabaeoides.

KEY MESSAGE: Comparative analysis of herbivory responsive miRNAs between pod borer susceptible C. cajan and its resistant Crop Wild Relative (CWR) C. scarabaeoides revealed miRNA-based regulation of defense genes and plant-insect interactions. Gram pod borer (Helicoverpa armigera) is one of most devastating pests of pigeon pea (Cajanus cajan) worldwide, responsible for huge losses in crop productivity. The lack of genes conferring resistance to pod borer in pigeon pea has proven to be a bottleneck for its improvement. One of its CWR, C. scarabaeoides has demonstrated resistance to this pest and can be exploited for developing pest resistant crop varieties. Differences in expression patterns of herbivory responsive microRNAs in the susceptible C. cajan and resistant C. scarabaeoides after different time duration of pod borer infestation (2 h, 8 h and 18 h) were identified, characterized and functionally validated to understand their role in insect defense response. A total of 462 conserved and 449 novel miRNAs and 273 conserved and 185 novel miRNAs, were identified in C. cajan and C. scarabaeoides, respectively. Among the identified miRNAs, 65, 68 and 65 miRNAs were found to be differentially expressing between the C. scarabaeoides and C. cajan libraries 2 h, 8 h and 18 h post infestation, respectively. These miRNAs were found to target genes involved in a number of pathways contributing to defense and acquired resistance in C. scarabaeoides against pod borer, indicating miRNA-based regulation of defense pathways. Expression patterns of eight of these miRNAs were validated by qRT-PCR. This study provides novel insights into the miRNA-mediated plant-insect interactions and the mechanisms of regulatory pathways involved in insect defense. These findings can be utilized for further exploring the mechanism of herbivore defense in plant systems.

Host-mediated attenuation of gut sucrase in mustard aphid Lipaphis erysimi impaired its parthenogenetic reproduction on Indian mustard Brassica juncea.

BACKGROUND: The nefarious hemipteran mustard aphid (Lipaphis erysimi) inflicts colossal yield losses in Brassica crops including Indian mustard (Brassica juncea). Lack of an accessible resistance source has been the primary impediment in breeding varietal resistance against aphids. In recent years, in planta RNAi-mediated resistance has been demonstrated in model plants as a potential tool for protection against insect pests. However, translational application in crop species is imperative for critical assessment of this technology in breeding effective resistance. RESULTS: The essential role of sucrase 1 (SUC1) in mitigating osmotic pressure imposed by sucrose-rich phloem sap inside the insect gut is corroborated by its expression pattern in L. erysimi. Transgenic lines of Indian mustard were developed expressing SUC1 hairpin RNA for its host-mediated delivery into the infesting aphids. The expression of the dsRNA encoding cassette, and generation of siRNA molecules in transgenic B. juncea lines were verified by quantitative reverse transcription (RT)-PCR, stem-loop RT-PCR and Northern hybridization. Rearing of L. erysimi on the transgenic lines resulted in 22-40% reduction in aphid fecundity. The observed retardation in aphid reproduction was coherent with the detection of SUC1-specific siRNA molecules and attenuation of the SUC1 transcript level in L. erysimi fed on the transgenic lines. CONCLUSION: Augmenting varietal resistance can substantially reduce usage of toxic agrochemicals in crop protection. This attempt was the first successful demonstration of host-mediated RNAi of an aphid gene in any Brassica crop. It paves the way for more rigorous attempt of engineering RNAi-based resistance against aphids in Brassica crops. © 2021 Society of Chemical Industry.

Concepts and considerations for enhancing RNAi efficiency in phytopathogenic fungi for RNAi-based crop protection using nanocarrier-mediated dsRNA delivery systems.

Existing, emerging, and reemerging strains of phytopathogenic fungi pose a significant threat to agricultural productivity globally. This risk is further exacerbated by the lack of resistance source(s) in plants or a breakdown of resistance by pathogens through co-evolution. In recent years, attenuation of essential pathogen gene(s) via double-stranded (ds) RNA-mediated RNA interference (RNAi) in host plants, a phenomenon known as host-induced gene silencing, has gained significant attention as a way to combat pathogen attack. Yet, due to biosafety concerns regarding transgenics, country-specific GMO legislation has limited the practical application of desirable attributes in plants. The topical application of dsRNA/siRNA targeting essential fungal gene(s) through spray-induced gene silencing (SIGS) on host plants has opened up a transgene-free avenue for crop protection. However, several factors influence the outcome of RNAi, including but not limited to RNAi mechanism in plant/fungi, dsRNA/siRNA uptake efficiency, dsRNA/siRNA design parameters, dsRNA stability and delivery strategy, off-target effects, etc. This review emphasizes the significance of these factors and suggests appropriate measures to consider while designing in silico and in vitro experiments for successful RNAi in open-field conditions. We also highlight prospective nanoparticles as smart delivery vehicles for deploying RNAi molecules in plant systems for long-term crop protection and ecosystem compatibility. Lastly, we provide specific directions for future investigations that focus on blending nanotechnology and RNAi-based fungal control for practical applications.

Effector mining from the Erysiphe pisi haustorial transcriptome identifies novel candidates involved in pea powdery mildew pathogenesis.

Pea powdery mildew (PM) is an important fungal disease caused by an obligate biotroph, Erysiphe pisi (Ep), which significantly impacts pea production worldwide. The phytopathogen secretes a plethora of effectors, primarily through specialized infection structures termed haustoria, to establish a dynamic relationship with its host. To identify Ep effector candidates, a cDNA library of enriched haustoria from Ep-infected pea leaves was sequenced. The Ep transcriptome encodes 622 Ep candidate secreted proteins (CSPs), of which 167 were predicted to be candidate secreted effector proteins (CSEPs). Phylogenetic analysis indicates that Ep CSEPs are highly diverse, but, unlike cereal PM CSEPs, exhibit extensive sequence similarity with effectors from other PMs. Quantitative real-time PCR of a subset of EpCSEP/CSPs revealed that the majority are preferentially expressed in haustoria and exhibit infection stage-specific expression patterns. The functional roles of EpCSEP001, EpCSEP009 and EpCSP083 were probed by host-induced gene silencing (HIGS) via a double-stranded (ds) RNA-mediated RNAi approach. Foliar application of individual EpCSEP/CSP dsRNAs resulted in a marked reduction in PM disease symptoms. These findings were consistent with microscopic and molecular studies, suggesting that these Ep CSEP/CSPs play important roles in pea PM pathogenesis. Homology modelling revealed that EpCSEP001 and EpCSEP009 are analogous to fungal ribonucleases and belong to the RALPH family of effectors. This is the first study to identify and functionally validate candidate effectors from the agriculturally relevant pea PM, and highlights the utility of transcriptomics and HIGS to elucidate the key proteins associated with Ep pathogenesis.

Comprehensive evaluation of candidate reference genes for qRT-PCR studies of gene expression in mustard aphid, Lipaphis erysimi (Kalt).

Mustard aphid, also known as turnip aphid (Lipaphis erysimi) is a major insect pest of rapeseed-mustard group of crops. Tremendous economic significance has led to substantial basic research involving gene-expression studies in this insect species. In qRT-PCR analysis of gene-expression, normalization of data against RNA variation by using appropriate reference gene is fundamental. However, appropriate reference genes are not known in case of L. erysimi. We evaluated 11 candidate reference genes for their expression stability in 21 samples of L. erysimi subjected to various regimes of experimental treatments. Unlike other studies, we validated true effects of the treatments on the samples either by gene-expression study of an associated marker gene or by biochemical tests. In the validated samples, expression stability of the reference genes was analysed by employing four different statistical softwares geNorm, NormFinder, BestKeeper and deltaCt. Drawing consensus on the results from different softwares, we recommend three best reference genes 16S, RPS18 and RPL13 for normalization of qRT-PCR data in L. erysimi. This study provides for the first time a comprehensive list of suitable reference genes for mustard aphid and demonstrates the advantage of using more than one reference gene in combination for certain experimental conditions.

Shape-dependent bactericidal activity of TiO2 for the killing of Gram-negative bacteria Agrobacterium tumefaciens under UV torch irradiation.

This paper demonstrated the relative bactericidal activity of photoirradiated (6W-UV Torch, λ > 340 nm and intensity = 0.64 mW/cm(2)) P25-TiO2 nanoparticles, nanorods, and nanotubes for the killing of Gram-negative bacterium Agrobacterium tumefaciens LBA4404 for the first time. TiO2 nanorod (anatase) with length of 70-100 nm and diameter of 10-12 nm, and TiO2 nanotube with length of 90-110 nm and diameter of 9-11 nm were prepared from P-25 Degussa TiO2 (size, 30-50 nm) by hydrothermal method and compared their biocidal activity both in aqueous slurry and thin films. The mode of bacterial cell decomposition was analyzed through transmission electron microscopy (TEM), Fourier transform-infrared (FT-IR), and K(+) ion leakage. The antimicrobial activity of photoirradiated TiO2 of different shapes was found to be in the order P25-TiO2 > nanorod > nanotube which is reverse to their specific surface area as 54 < 79 < 176 m(2) g(-1), evidencing that the highest activity of P25-TiO2 nanoparticles is not due to surface area as their crystal structure and surface morphology are entirely different. TiO2 thin films always exhibited less photoactivity as compared to its aqueous suspension under similar conditions of cell viability test. The changes in the bacterial surface morphology by UV-irradiated P25-TiO2 nanoparticles was examined by TEM, oxidative degradation of cell components such as proteins, carbohydrates, phospholipids, nucleic acids by FT-IR spectral analysis, and K(+) ion leakage (2.5 ppm as compared to 0.4 ppm for control culture) as a measure of loss in cell membrane permeability.

Synthesis and spectral characterization of new bis(2-(pyrimidin-2-yl)ethoxy)alkanes and their pharmacological activity.

The pyrimidine nucleus is an important component of nucleic acids (DNA and RNA) and vitamins (B(2) and folic acid). It is evident from the literature that pyrimidine derivatives possess a wide spectrum of biological activities such as antioxidant, anticancer, antibacterial, and anti-inflammatory activities. On the basis of diverse biological activities, we attempted to synthesize a series of novel bis(2-(pyrimidin-2-yl)ethoxy)alkanes 5a-j in four steps with good yields. 2-Chloropyrimidine (1) was reacted with diethyl malonate in the presence of sodium hydride in dry dimethyl formamide to yield the intermediate diethyl 2-(pyrimidin-2-yl)malonate (2), which on further reaction with sodium chloride and dimethyl sulfoxide yielded ethyl 2-(pyrimidin-2-yl)ethanoate (3). Reduction with sodium borohydride (NaBH(4) ) resulted in the formation of 2-(pyrimidin-2-yl)ethanol (4). This was further reacted with various dibromoalkanes to obtain the title compounds 5a-j. In this current study, we evaluated the antioxidant properties of the title compounds using four in vitro test systems: the 2,2-diphenyl-2-picrylhydrazyl radical-, superoxide radical-, and hydroxyl radical-scavenging assays, and the anti-lipid peroxidation activity test. The title compounds showed promising antioxidant activity when compared to butylated hydroxytoluene. The potency of their antioxidant activity was mainly influenced by the alkyl fragment attached to 2-(pyrimidin-2-yl)ethanol. The ethyl and butyl fragments linked to oxygen led to increased antioxidant activity of the title compounds (i.e., 5b and 5d) in all our in vitro assays.