CTAB Protocol for Obtaining High-Quality Total RNA from Hibiscus rosa-sinensis and Other Related Plants of the Family Malvaceae.

Malvaceae family, also known as the Mallow family, is a family of flowering plants containing Hibiscus rosa-sinensis and other plants of high medicinal value. This study focuses on the challenges associated with high-quality RNA extraction from Hibiscus rosa-sinensis and its related plants characterized by high levels of mucilage and phenolic compounds in their tissues. High mucilage and secondary metabolite content pose obstacles in obtaining high-quality RNA, negatively impacting downstream applications, such as gene expression analysis. Our research aimed to develop an efficient RNA extraction method tailored to the unique characteristics of Malvaceae family plants especially Hibiscus rosa-sinensis. Through the substitution of NaCl with KCl, a crucial component of the CTAB buffer, our methodology successfully addressed the challenges posed by high mucilage and phenolic compound levels. This modification led to a significant reduction in sample viscosity, which is because of the high mucilage in these plants. Our modified CTAB extraction method yielded significantly more RNA with higher purity than the conventional CTAB methods alone. The extracted RNA was largely intact, as indicated by 28S/18S ratios and RIN values, yielding high-quality RNA with improved purity suggested by the 260/280 and 260/230 ratios. The proposed approach not only serves as a solution to the specific challenges encountered in Hibiscus rosa-sinensis but also holds promise for broader applications across different plants within the family.

Unveiling the dynamic relationship of viruses and/or symbiotic bacteria with plant resilience in abiotic stress.

In the ecosphere, plants interact with environmental biotic and abiotic partners, where unbalanced interactions can induce unfavourable stress conditions. Abiotic factors (temperature, water, and salt) are primarily required for plants healthy survival, and any change in their availability is reflected as a stress signal. In certain cases, the presence of infectious pathogens such as viruses, bacteria, fungi, protozoa, nematodes, and insects can also create stress conditions in plants, leading to the emergence of disease or deficiency symptoms. While these symptoms are often typical of abiotic or biotic stress, however, there are instances where they can intensify under specific conditions. Here, we primarily summarize the viral interactions with plants during abiotic stress to understand how these associations are linked together during viral pathogenesis. Secondly, focus is given to the beneficial effects of root-associated symbiotic bacteria in fulfilling the basic needs of plants during normal as well as abiotic stress conditions. The modulations of plant functional proteins, and their occurrence/cross-talk, with pathogen (virus) and symbiont (bacteria) molecules are also discussed. Furthermore, we have highlighted the biochemical and systematic adaptations that develop in plants due to bacterial symbiosis to encounter stress hallmarks. Lastly, directions are provided towards exploring potential rhizospheric bacteria to maintain plant-microbes ecosystem and manage abiotic stress in plants to achieve better trait health in the horticulture crops.

Vaccine development: Current trends and technologies.

Despite the effectiveness of vaccination in reducing or eradicating diseases caused by pathogens, there remain certain diseases and emerging infections for which developing effective vaccines is inherently challenging. Additionally, developing vaccines for individuals with compromised immune systems or underlying medical conditions presents significant difficulties. As well as traditional vaccine different methods such as inactivated or live attenuated vaccines, viral vector vaccines, and subunit vaccines, emerging non-viral vaccine technologies, including viral-like particle and nanoparticle vaccines, DNA/RNA vaccines, and rational vaccine design, offer new strategies to address the existing challenges in vaccine development. These advancements have also greatly enhanced our understanding of vaccine immunology, which will guide future vaccine development for a broad range of diseases, including rapidly emerging infectious diseases like COVID-19 and diseases that have historically proven resistant to vaccination. This review provides a comprehensive assessment of emerging non-viral vaccine production methods and their application in addressing the fundamental and current challenges in vaccine development.

Demonstration of seed inoculation of dimer infectious clones of mungbean yellow mosaic India virus-soybean isolate.

In this study, the full-length components of mungbean yellow mosaic India virus (MYMIV) DNA-A (MW590720 & MW600934) and DNA-B (MW659819 & MW659820) from a soybean isolate were cloned and sequenced. Nucleotide sequence analysis of both MYMIV components revealed > 96% identity and close ancestry with MYMIV isolates from legumes in Southeast Asia. Furthermore, dimeric infectious clones of MYMIV were generated in the pCAMBIA1302 vector, and a seed infiltration protocol was established for mungbean, soybean, and Nicotiana tabacum. Agroinfiltration induced yellow mosaic symptoms in mungbean and N. tabacum plants 3 weeks post-infiltration, which were further confirmed by PCR using MYMIV-specific DNA primers. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03778-7.

Molecular detection and characterization of Potato spindle tuber viroid (PSTVd) infecting Tomato (Solanum lycopersicum L.) in Karnataka State of India.

Potato Spindle Tuber Viroid (PSTVd) is a non-coding, infectious, small, circular RNA known to cause disease in agricultural and horticultural plants. In the present work, an investigation was conducted in the southern districts of Karnataka state to assess the possible pospiviroid infections on tomato plants that are considered natural hosts for viroids. A total of 83 tomato samples showing disease symptoms (virus or viroid-like) along with healthy ones were collected and subjected to viroid detection by conventional Reverse Transcriptase Polymer Chain Reaction (RT-PCR) using universal (Pospi1-RE/Pospi1-FW) and a specific set of primers (3H1/2H1). The study confirmed the presence of PSTVd in one of the samples of tomato collected from Banghatta village of Mandy District, with an expected amplicon of ~ 361 bp. The bioassay conducted on tomato plants (cv. Rutgers) proved the association of PSTVd, which was further confirmed by RT-PCR. The amplicons were cloned, sequenced, and the representative sequences were deposited in the NCBI GenBank. The sequence alignment and secondary structure analysis of the isolated viroid with other reference sequences revealed the variations in the pathogenicity, central conserved region, and Terminal right domains. The variations observed between the isolated PSTVd with that of other Indian isolates support that viroid may have been transmitted among the crop plants, possibly through seed or mechanical means.

First report of apple stem grooving virus infection in loquat from India.

Loquat, commonly known as Eriobotrya japonica, is a major subtropical fruit from the Rosaceae family that is native to China but also found in most of Europe and Asia, including India. Apple stem grooving virus (ASGV) infecting loquat was detected using leaf samples collected from Himachal Pradesh (India) through DAC-ELISA followed by RT-PCR assays targeting coat protein (CP), movement protein (MP) and replicase (Rep) regions of ASGV genome. Sequencing of RT-PCR amplicons and sequence analyses revealed that CP, MP and Rep sequences of ASGV loquat Indian isolate of the current study shared a maximum of 98-100% nucleotide sequence identities with the corresponding sequences of available ASGV Indian isolates [LN559078, HE978837, MZ127820, MN912568]. Phylogenetic tree based on each sequenced gene confirmed the genetic diversity of ASGV. To the best of our knowledge, and based on review of the literature, this is the first report of ASGV infection in loquat from India.

Development of a new Collateral Cleavage-independent CRISPR/Cas12a based easy detection system for plant viruses.

Plant virus spread through various means, from mechanically to the insect vectors and act as obligate parasite, therefore, are extremely challenging to eradicate. Geminiviruses are an important class of viruses which have reported extensively in last two decades on several new hosts. They infect wide range of annual crops and perineal shrubs, therefore, essentially required to detect them on field and dispose to check their vector transmission to healthy crops. In this study, we have chosen two important begomovirus viz. Mungbean yellow mosaic India virus which infect wide range of leguminous crops while Ageratum enation virus is reported to infect a wide range of crops from weed to opium poppy. Here, we have utilized the binding and cleaving ability of LbaCas12a protein with target to detect the virus infection on field. We proposed here a new Collateral Cleavage Independent CRISPR/Cas12a based detection system (CCI-CRISPR) for plant viruses.

Serological and molecular analysis indicates the presence of distinct viral genotypes of Apple stem pitting virus in India.

Recombination leads to the generation of new viral progeny which remain undetected by routine testing procedures and may be a threat to the infected host. Here, we have characterised the complete genome sequences of two isolates of Apple stem pitting virus from apple cv. Red Chief (Palampur) and cv. Gold Spur (N) with distinct serological reactivities. The viral genomes consisted of 9267 nucleotides for isolate Palampur and 9254 nucleotides for isolate N, excluding the poly (A) tail and contained 5five open reading frames (ORFs). Isolate N shared 80.8% sequence identity with ASPV apple isolate GA2 from China, while isolate Palampur shared 81.4% sequence identity with ASPV apple isolate PB66 from the United Kingdom. The serological difference of isolates N and Palampur along with their low sequence identity indicated the existence of two distinct virus genotypes which was corroborated by evolutionary and genetic differentiation analyses. Recombination events were detected in the RdRp and CP sequences of Palampur isolate thereby suggesting the role of recombination in the evolution of distinct virus genotypes. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02798-5.

Apple scar skin viroid naked RNA is actively transmitted by the whitefly Trialeurodes vaporariorum.

Nucleic acid transfer between plants is a phenomenon which is likely to occur in many ways in nature. We report here the active transmission of Apple scar skin viroid (ASSVd) naked ssRNA species by the whitefly Trialeurodes vaporariorum (Tv). Not only the viroid RNA, its DNA form was also identified from the insect. The viroid transfer efficiency was enhanced with the help of Cucumis sativus Phloem protein 2 (CsPP2), a plant protein known to translocate viroid RNAs. This PP2/ASSVd complex is stably present in the viroid infected cucumber plants, as was identified with the help of immunological reaction. As viroid-like secondary structures are found in some plant RNAs, and PP2 is known to bind and translocate several RNAs, the results have huge implications in transfer of these RNA species between plants visited by the whitefly.

A simplified strategy for studying the etiology of viral diseases: Apple stem grooving virus as a case study.

A simple method to amplify infective, complete genomes of single stranded RNA viruses by long distance PCR (LD PCR) from woody plant tissues is described in detail. The present protocol eliminates partial purification of viral particles and the amplification is achieved in three steps: (i) easy preparation of template RNA by incorporating a pre processing step before loading onto the column (ii) reverse transcription by AMV or Superscript reverse transcriptase and (iii) amplification of cDNA by LD PCR using LA or Protoscript Taq DNA polymerase. Incorporation of a preprocessing step helped to isolate consistent quality RNA from recalcitrant woody tissues such as apple, which was critical for efficient amplification of the complete genomes of Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV). Complete genome of ASGV was cloned under T7 RNA polymerase promoter and was confirmed to be infectious through transcript inoculation producing symptoms similar to the wild type virus. This is the first report for the largest RNA virus genome amplified by PCR from total nucleic acid extracts of woody plant tissues.