In silico Prediction and Validations of Domains Involved in Gossypium hirsutum SnRK1 Protein Interaction With Cotton Leaf Curl Multan Betasatellite Encoded βC1

Cotton leaf curl disease (CLCuD) caused by viruses of genus Begomovirus is a major constraint to cotton (Gossypium hirsutum) production in many cotton-growing regions of the world. Symptoms of the disease are caused by Cotton leaf curl Multan betasatellite (CLCuMB) that encodes a pathogenicity determinant protein, βC1. Here, we report the identification of interacting regions in βC1 protein by using computational approaches including sequence recognition, and binding site and interface prediction methods. We show the domain-level interactions based on the structural analysis of G. hirsutum SnRK1 protein and its domains with CLCuMB-βC1. To verify and validate the in silico predictions, three different experimental approaches, yeast two hybrid, bimolecular fluorescence complementation and pull down assay were used. Our results showed that ubiquitin-associated domain (UBA) and autoinhibitory sequence (AIS) domains of G. hirsutum-encoded SnRK1 are involved in CLCuMB-βC1 interaction. This is the first comprehensive investigation that combined in silico interaction prediction followed by experimental validation of interaction between CLCuMB-βC1 and a host protein. We demonstrated that data from computational biology could provide binding site information between CLCuD-associated viruses/satellites and new hosts that lack known binding site information for protein–protein interaction studies. Implications of these findings are discussed

A plant virus protein, NIa-pro, interacts with Indole-3-acetic acid-amido synthetase, whose levels positively correlate with disease severity

Potato virus Y (PVY) is an economically important plant pathogen that reduces the productivity of several host plants. To develop PVY-resistant cultivars, it is essential to identify the plant-PVY interactome and decipher the biological significance of those molecular interactions. We performed a yeast two-hybrid (Y2H) screen of Nicotiana benthamiana cDNA library using PVY-encoded NIa-pro as the bait. The N. benthamiana Indole-3-acetic acid-amido synthetase (IAAS) was identified as an interactor of NIa-pro protein. The interaction was confirmed via targeted Y2H and bimolecular fluorescence complementation (BiFC) assays. NIa-pro interacts with IAAS protein and consequently increasing the stability of IAAS protein. Also, the subcellular localization of both NIa-pro and IAAS protein in the nucleus and cytosol was demonstrated. By converting free IAA (active form) to conjugated IAA (inactive form), IAAS plays a crucial regulatory role in auxin signaling. Transient silencing of IAAS in N. benthamiana plants reduced the PVY-mediated symptom induction and virus accumulation. Conversely, overexpression of IAAS enhanced symptom induction and virus accumulation in infected plants. In addition, the expression of auxin-responsive genes was found to be downregulated during PVY infection. Our findings demonstrate that PVY NIa-pro protein potentially promotes disease development via modulating auxin homeostasis

Investigating the Roles of Coat Protein and Triple Gene Block Proteins of Potato Mop-Top Virus Using a Heterologous Expression System

Potato mop-top virus (PMTV) is an emerging viral pathogen that causes tuber necrosis in potatoes. PMTV is composed of three single-stranded RNA segments: RNA1 encodes RNA-dependent RNA polymerase, RNA2 contains the coat protein (CP), and RNA3 harbors a triple gene block (TGB 1, TGB2, and TGB3). CP plays a role in viral transmission, while TGB is known to facilitate cell-to-cell and long-distance systemic movement. The role of CP in symptom development, specifically in the presence of TGB genes, was investigated using potato virus X (PVX) as a delivery vehicle to express PMTV genes in the model plant Nicotiana benthamiana. Plants expressing individual genes showed mild symptoms that included leaf curling and crumpling. Interestingly, symptom severity varied among plants infected with three different combinations: CP with TGB1, CP with TGB2, and CP with TGB3. Notably, the combination of CP and TGB3 induced a hypersensitive response, accompanied by stunted growth and downward curling and crumpling. These results suggest the potential role of TGB co-expressed with CP in symptom development during PMTV infection. Additionally, this study demonstrates the use of the PVX-based expression system as a valuable platform for assessing the role of unknown genes in viral pathogenicity

DataSheet_1_A plant virus protein, NIa-pro, interacts with Indole-3-acetic acid-amido synthetase, whose levels positively correlate with disease severity.docx

Potato virus Y (PVY) is an economically important plant pathogen that reduces the productivity of several host plants. To develop PVY-resistant cultivars, it is essential to identify the plant-PVY interactome and decipher the biological significance of those molecular interactions. We performed a yeast two-hybrid (Y2H) screen of Nicotiana benthamiana cDNA library using PVY-encoded NIa-pro as the bait. The N. benthamiana Indole-3-acetic acid-amido synthetase (IAAS) was identified as an interactor of NIa-pro protein. The interaction was confirmed via targeted Y2H and bimolecular fluorescence complementation (BiFC) assays. NIa-pro interacts with IAAS protein and consequently increasing the stability of IAAS protein. Also, the subcellular localization of both NIa-pro and IAAS protein in the nucleus and cytosol was demonstrated. By converting free IAA (active form) to conjugated IAA (inactive form), IAAS plays a crucial regulatory role in auxin signaling. Transient silencing of IAAS in N. benthamiana plants reduced the PVY-mediated symptom induction and virus accumulation. Conversely, overexpression of IAAS enhanced symptom induction and virus accumulation in infected plants. In addition, the expression of auxin-responsive genes was found to be downregulated during PVY infection. Our findings demonstrate that PVY NIa-pro protein potentially promotes disease development via modulating auxin homeostasis.</p

Phytoextracts as Crop Biostimulants and Natural Protective Agents—A Critical Review

Excessive application of synthetic chemicals to crops is a serious environmental concern. This review suggests that some potential natural compounds can be used as alternatives and could be applied directly to plants to improve crop growth and productivity. These phytoextracts can serve as biostimulants to induce abiotic and biotic stress tolerance in different crops growing under diverse environmental conditions. The biosynthesis and accumulation of a variety of chemical compounds such as glycinebetaine, vitamins, nutrients, and secondary metabolites in some plants are of great value and an environmentally friendly cheaper source than several synthetic substances of a similar nature. The review summarizes the information regarding the potential role of different plant phytoextracts and suggests subsequent applications to modulate crop stress tolerance. Future studies should focus on the relative effectiveness of these plant-based extracts compared with their synthetic counterparts and focus on practical applications to signify sustainable practices linked with the use of natural products

βC1, pathogenicity determinant encoded by Cotton leaf curl Multan betasatellite, interacts with calmodulin-like protein 11 (Gh-CML11) in Gossypium hirsutum.

Begomoviruses interfere with host plant machinery to evade host defense mechanism by interacting with plant proteins. In the old world, this group of viruses are usually associated with betasatellite that induces severe disease symptoms by encoding a protein, βC1, which is a pathogenicity determinant. Here, we show that βC1 encoded by Cotton leaf curl Multan betasatellite (CLCuMB) requires Gossypium hirsutum calmodulin-like protein 11 (Gh-CML11) to infect cotton. First, we used the in silico approach to predict the interaction of CLCuMB-βC1 with Gh-CML11. A number of sequence- and structure-based in-silico interaction prediction techniques suggested a strong putative binding of CLCuMB-βC1 with Gh-CML11 in a Ca+2-dependent manner. In-silico interaction prediction was then confirmed by three different experimental approaches: The Gh-CML11 interaction was confirmed using CLCuMB-βC1 in a yeast two hybrid system and pull down assay. These results were further validated using bimolecular fluorescence complementation system showing the interaction in cytoplasmic veins of Nicotiana benthamiana. Bioinformatics and molecular studies suggested that CLCuMB-βC1 induces the overexpression of Gh-CML11 protein and ultimately provides calcium as a nutrient source for virus movement and transmission. This is the first comprehensive study on the interaction between CLCuMB-βC1 and Gh-CML11 proteins which provided insights into our understating of the role of βC1 in cotton leaf curl disease

Ipomoea carnea associated phytochemicals and their in silico investigation towards Meloidogyne incognita

Root-knot nematodes (Meloidogyne spp.) are sedentary endo-parasite that causes severe yield loss in carrot. Chemical nematicides currently used to manage Meloidogyne incognita are being phased out because of rising health and environmental issues. This study aimed to evaluate nematicidal effect of various concentrations, viz., 250, 500, 750, 1000ppm of leaf extract of Ipomoea carnea against M. incognita infecting carrot under in vitro and in pots assays. In our result, all tested concentrations displayed J2s mortality and egg hatching inhibition along with improving growth of carrot and reduced J2s population and root-knot index. Molecular docking performed predicts binding interactions of two major compounds, viz., neophytadiene and 2-amino-2-methyl-1-propanol as shown by GC-MS analysis with targeted protein, odorant response gene-1 of M. incognita, to confirm nematicidal action of I. carnea leaf extract. The obtained results also suggested that neophytadiene interacted more and strongly bound with odorant response gene-3 than 2-amino-2-methyl-1-propanol. The biochemical ligand-target protein interaction described in the present work will be helpful in the logical selection of biomolecules and essential proteins. Therefore, plant extract may be used the best alternative to chemical nematicides to control root-knot nematodes and caused longitudinal growth of the plant as well as reduce environmental risks

The Past, Present, and Future of Monkeypox: A Rapid Review Regarding Prevalence and Prevention

While monkeypox virus (MPXV) remained endemic in central and western African countries, a sudden unusual spike of global cases among non-endemic countries is an enigma for scientists. With 257 cases reported as of 26th May 2021, a multi-country outbreak of monkeypox has been declared in countries including the UK, EU/EEA states, and North America. Even though the likelihood of transmissibility of MPXV is limited compared to COVID-19, yet a coordinated multidisciplinary effort is required to prevent any further global expansion. Few appropriate responsive approaches to contain the infection could be; limiting the contact with potential animal reservoirs, isolation of confirmed cases, using PPEs to prevent human-human transmission, awareness activities, and administration of pre and post prophylactic vaccination. In this review, we have discussed the previous and current outbreaks of MPXV along with the abrupt actions that are needed to address the situation