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SummaryThe shared diseases between animals and humans are known as zoonotic diseases and spread infectious diseases among humans. Zoonotic diseases are not only a major burden to livestock industry but also threaten humans accounting for >60% cases of human illness. About 75% of emerging infectious diseases in humans have been reported to originate from zoonotic pathogens. Because antibiotics are frequently used to protect livestock from bacterial diseases, the development of antibiotic‐resistant strains of epidemic and zoonotic pathogens is now a major concern. Live attenuated and killed vaccines are the only option to control these infectious diseases and this approach has been used since 1890. However, major problems with this approach include high cost and injectable vaccines is impractical for >20 billion poultry animals or fish in aquaculture. Plants offer an attractive and affordable platform for vaccines against animal diseases because of their low cost, and they are free of attenuated pathogens and cold chain requirement. Therefore, several plant‐based vaccines against human and animals diseases have been developed recently that undergo clinical and regulatory approval. Plant‐based vaccines serve as ideal booster vaccines that could eliminate multiple boosters of attenuated bacteria or viruses, but requirement of injectable priming with adjuvant is a current limitation. So, new approaches like oral vaccines are needed to overcome this challenge. In this review, we discuss the progress made in plant‐based vaccines against zoonotic or other animal diseases and future challenges in advancing this field.

To advance a novel concept of debulking virus in the oral cavity, the primary site of viral replication, virus-trapping proteins CTB-ACE2 were expressed in chloroplasts and clinical-grade plant material was developed to meet FDA requirements. Chewing gum (2 g) containing plant cells expressed CTB-ACE2 up to 17.2 mg ACE2/g dry weight (11.7% leaf protein), have physical characteristics and taste/flavor like conventional gums, and no protein was lost during gum compression. CTB-ACE2 gum efficiently (>95%) inhibited entry of lentivirus spike or VSV-spike pseudovirus into Vero/CHO cells when quantified by luciferase or red fluorescence. Incubation of CTB-ACE2 microparticles reduced SARS-CoV-2 virus count in COVID-19 swab/saliva samples by >95% when evaluated by microbubbles (femtomolar concentration) or qPCR, demonstrating both virus trapping and blocking of cellular entry. COVID-19 saliva samples showed low or undetectable ACE2 activity when compared with healthy individuals (2,582 versus 50,126 DRFU; 27 versus 225 enzyme units), confirming greater susceptibility of infected patients for viral entry. CTB-ACE2 activity was completely inhibited by pre-incubation with SARS-CoV-2 receptor-binding domain, offering an explanation for reduced saliva ACE2 activity among COVID-19 patients. Chewing gum with virus-trapping proteins offers a general affordable strategy to protect patients from most oral virus re-infections through debulking or minimizing transmission to others.

BackgroundCotton yield has been badly affected by different insects and weed competition. In Past Application of multiple chemicals is required to manage insects and weed control was achieved by different conventional means, such as hand weeding, crop rotation and polyculture, because no synthetic chemicals were available. The control methods shifted towards high input and target-oriented methods after the discovery of synthetic herbicide in the 1930s. To utilise the transgenic approach, cotton plants expressing the codon-optimised CEMB GTGene were produced in the present study.ResultsLocal cotton variety CEMB-02 containing Cry1Ac and Cry2A in single cassette was transformed by synthetic codon-optimised 5-enolpyruvylshikimate-3-phosphate synthase gene cloned into pCAMBIA 1301 vector under 35S promoter with Agrobacterium tumifaciens. Putative transgenic plants were screened in MS medium containing 120 µmol/L glyphosate. Integration and expression of the gene were evaluated by PCR from genomic DNA and ELISA from protein. A 1.4-kb PCR product for Glyphosate and 167-bp product for Cry2A were obtained by amplification through gene specific primers. Expression level of Glyphosate and Bt proteins in two transgenic lines were recorded to be 0.362, 0.325 µg/g leaf and 0.390, 0.300 µg/g leaf respectively. FISH analysis of transgenic lines demonstrates the presence of one and two copy no. of Cp4 EPSPS transgene respectively. Efficacy of the transgene Cp4 EPSPS was further evaluated by Glyphosate spray (41 %) assay at 1900 ml/acre and insect bioassay which shows 100 %mortality of insect feeding on transgenic lines as compared to control.ConclusionThe present study shows that the transgenic lines produced in this study were resistant not only to insects but also equally good against 1900 ml/acre field spray concentration of glyphosate.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1397-0) contains supplementary material, which is available to authorized users.

Agricultural crops suffer many diseases, including fungal and bacterial infections, causing significant yield losses. The identification and characterisation of pathogenesis-related protein genes, such as chitinases, can lead to reduction in pathogen growth, thereby increasing tolerance against fungal pathogens. In the present study, the chitinase I gene was isolated from the genomic DNA of Barley (Hordeum vulgare L.) cultivar, Haider-93. The isolated DNA was used as template for the amplification of the ∼935 bp full-length chitinase I gene. Based on the sequence of the amplified gene fragment, class I barley chitinase shares 93% amino acid sequence homology with class II wheat chitinase. Interestingly, barley class I chitinase and class II chitinase do not share sequence homology. Furthermore, the amplified fragment was expressed in Escherichia coli Rosetta strain under the control of T7 promoter in pET 30a vector. Recombinant chitinase protein of 35 kDa exhibited highest expression at 0.5 mM concentration of IPTG. Expressed recombinant protein of 35 kDa was purified to homogeneity with affinity chromatography. Following purification, a Western blot assay for recombinant chitinase protein measuring 35 kDa was developed with His-tag specific antibodies. The purified recombinant chitinase protein was demonstrated to inhibit significantly the important phytopathogenic fungi Alternaria solani, Fusarium spp, Rhizoctonia solani and Verticillium dahliae compared to the control at concentrations of 80 μg and 200 μg.

Newcastle disease (ND) is a viral disease that causes labored breathing, periorbital oedema, and ataxia in the majority of avian species. The available vaccines against Newcastle disease virus (NDV) are limited, owing to their low reactivity and multiple dosage requirements. Plant-based machinery provides an attractive and safe system for vaccine production. In the current study, we attempted to express fusion (F) and hemagglutinin-neuraminidase (HN) proteins (the protective antigens against NDV) under constitutive 35S and seed-specific Zein promoters, respectively. Almost 2-7.1-fold higher expression of F gene mRNA in transgenic corn leaves and 8-28-fold higher expression of HN gene mRNA in transgenic corn seeds were observed, when the expression was analyzed by real-time PCR on a relative basis as compared to non-transgenic control plant material (Leaves and seeds). Similarly, 1.66 µg/ml of F protein in corn leaves, i.e., 0.5% of total soluble protein, and 2.4 µg/ml of HN protein in corn seed, i.e., 0.8% of total seed protein, were found when calculated through ELISA. Similar levels of immunological response were generated in chicks immunized through injection of E. coli-produced pET F and pET HN protein as in chickens orally fed leaves and seeds of maize with expressed immunogenic protein. Moreover, the detection of anti-NDV antibodies in the sera of chickens that were fed maize with immunogenic protein, and the absence of these antibodies in chickens fed a normal diet, confirmed the specificity of the antibodies generated through feeding, and demonstrated the potential of utilizing plants for producing more vaccine doses, vaccine generation at higher levels and against other infectious diseases.

Zinc finger proteins are encoded by the genes chiefly involved in stress resistance hormone signal transduction of plants. In this study, a cDNA encoding a zinc finger transcription factor was isolated by the yeast one-hybrid system from Gossypium arboreum using the MYB-box element of the universal stress gene (GUSP1) promoter as bait. The corresponding protein (named GaZnF) can bind specifically to a 13 bp MYB-box region. The GaZnF cDNA is 1093 bp in length, including a 510 bp open reading frame. The predicted GaZnF protein contains ANI-A20 motifs and shares a high sequence similarity with zinc finger proteins from other plants. Spatial expression pattern of GaZnF was studied under drought, heavy metals and salt stresses through real-time PCR. The gene showed enhanced expression under each stress treatment with maximum transcript abundance in root tissues. The results support the hypothesis that G. arboreum zinc finger proteins are involved in plant response to drought, salt and heavy metal stresses.

Biological control is a novel approach in crop protection. Bacteria, such as Bacillus spp. and Pseudomonas spp., are reported for this purpose and some of their products are already commercially available. In this study, the rhizosphere and phyllosphere of healthy cotton plants were used as a source of bacterial isolates with properties of potential biocontrol agents. The isolates were screened for phosphate solubilization activity, indole acetic acid (IAA) production and antifungal activity. Two isolates, S 1 HL 3 and S 1 HL 4 , showed phosphate solubilization and IAA production simultaneously, while another two, JS 2 HR 4 and JS 3 HR 2 , demonstrated potential to inhibit fungal pathogens. These bacteria were identified as Pseudomonas aeruginosa (S 1 HL 3), Burkholderia sp. (S 1 HL 4) and Bacillus sp. (JS 2 HR 4 and JS 3 HR 2) based on biochemical and molecular characteristics. The isolates were tested against Cotton leaf curl virus (CLCuV) in greenhouse conditions, both as individual bacterial isolates and consortia. Treated plants were healthy as compared to control plants, where up to 74% of the plants were symptomatic for CLCuV infection. Maximum inhibition of CLCuV was observed in the plants treated with a mixture of bacterial isolates: the viral load in the treated plants was only 0.4% vs. up to 74% in controls. This treatment consortium included P. aeruginosa S 1 HL 3 , Burkholderia sp. S 1 HL 4 and Bacillus spp. isolates, JS 2 HR 4 and JS 3 HR 2. The principal-component biplot showed a highly significant correlation between the viral load percentage and the disease incidence.