Structural and Functional Analysis of Groundnut bud necrosis virus (GBNV) Using Computational and Biochemical Approaches.

Groundnut bud necrosis virus (GBNV) belonging to the genus Orthotospovirus is transmitted by its vector Thrips palmi. It is a tri-segmented RNA virus that consists of L, M, and S RNA segments. We analysed the secondary structure features of GBNV proteins through various software and predicted the transmembrane helix, glycosylation, and signal peptidase sites within the GBNV protein sequences (GN, GC, N, NSm, and NSs). In glycoprotein sequence, extended strands are predominant (52.87%) whereas the N protein sequence mostly contains alpha helices (47.46%). The random coils are present in movement protein (43.97%) and structural protein (39.41%). We generated the 3D structure of GN and N protein using SWISS MODEL software and quality is validated through PROCHECK and PDBsum software. We also expressed the GBNV proteins (GN, GC, N, NSm, and NSs) in bacterial expression system. The recombinant proteins were used to raise polyclonal antibodies in mice. Our study will be useful in understanding GBNV protein structures in further detail by analysing the important domains that interact with the thrips proteins. This will further aid us in understanding virus-vector relationship through the application of protein-protein interaction and other immunodiagnostic techniques.

A thioredoxin-like protein of Bemisia tabaci interacts with coat protein of begomoviruses.

Bemisia tabaci (whitefly) is the sole vector of begomoviruses, which transmits them in a persistent and circulative manner from infected to healthy plants. During this process, begomoviruses interact with various proteins in the insect vector B. tabaci that would play a specific role in the virus transmission. Identification and characterization of such proteins are important to understand the complete process of virus transmission. Coat protein (CP) of begomoviruses is the only protein which is reported to interact with proteins of the insect vector B. tabaci. In this study, we performed yeast two-hybrid assay using CP of cotton leaf curl Rajasthan virus (CLCuV) and Tomato leaf curl New Delhi virus (ToLCNDV) as bait in separate experiments and cDNA prepared from total RNA of B. tabaci was used as prey. Yeast two-hybrid assay resulted in identification of a thioredoxin-like protein (TLP) from CLCuV yeast two-hybrid library. Later TLP was also found to interact with CP of ToLCNDV. In vitro pull-down assay showed TLP interaction with CP of both CLCuV and ToLCNDV. TLP was found to interact with ToLCNDV virus particles isolated from tomato leaves.

Influence of Groundnut bud necrosis virus on the Life History Traits and Feeding Preference of Its Vector, Thrips palmi.

The effect of Groundnut bud necrosis virus (GBNV) infection on the life history traits of its vector, Thrips palmi, and its feeding preference on GBNV-infected plants were studied. A significant difference was observed in the developmental period (first instar to adult) between the GBNV-infected and healthy thrips, wherein the developmental period of GBNV-infected thrips was decreased. However, there was no effect on the other parameters such as preadult mortality, adult longevity, and fecundity. Further investigation on a settling and feeding choice assay of T. palmi to GBNV-infected and healthy plants showed that T. palmi preferred GBNV-infected cowpea plants more than the healthy cowpea plants. This preference was also noticed for leaf disks from GBNV-infected cowpea, groundnut, and tomato plants.

Detection and Localization of Wolbachia in Thrips palmi Karny (Thysanoptera: Thripidae).

Thrips palmi Karny is a globally distributed polyphagous agricultural pest. It causes huge economic loss by its biological behaviors like feeding, reproduction and transmission of tospoviruses. Since T. palmi shows close morphological similarities with other thrips species, we employed mitochondrial cytochrome oxidase 1 (mtCO1) gene as a molecular marker. BLAST analysis of this sequence helped us to identify the collected specimen as T. palmi. We observed the female to male ratio of about 3:1 from collected samples and suspected the presence of Wolbachia. The presence of Wolbachia was detected by PCR using genus specific primers of 16S rRNA gene. Further confirmation of Wolbachia strain was achieved by conducting PCR amplification of three ubiquitous genes ftsZ, gatB and groEL. A phylogenetic tree was constructed with concatenated sequences of ftsZ and gatB gene to assign supergroup to Wolbachia. Finally, we localized Wolbachia in abdominal region of the insect using fluorescent in situ hybridization with the help of confocal microscope. Our result confirmed the presence of Wolbachia supergroup B strain for the first time in T. palmi.

Elimination of Arsenophonus and decrease in the bacterial symbionts diversity by antibiotic treatment leads to increase in fitness of whitefly, Bemisia tabaci.

Bemisia tabaci is an invasive agricultural pest with more than 24 genetic groups harboring different bacterial endosymbionts categorized into obligatory and facultative endosymbionts. Arsenophonus is one of the facultative endosymbionts prevalent in B. tabaci of Indian sub-continent. Not much is known about the functional role of this endosymbiont in its host. Some studies have revealed its involvement in virus transmission by B. tabaci, but how it effects the biology of B. tabaci is unknown. In this study, tetracycline was used to eliminate Arsenophonus from B. tabaci to study its effects with regard to development and other fitness parameters. Bacteria specific 16S Polymerase chain reaction (PCR) was used to ascertain Arsenophonus absence with differential effects on other secondary endosymbionts present in B. tabaci. Our results revealed that Arsenophonus negative (A(-)) whiteflies had more fecundity, increased juvenile developmental time, increased nymphal survival and increased adult life span as compared to control (A(+)) whiteflies. Thus, our results demonstrate that A(+) whiteflies have lesser fitness as compared to A(-) whiteflies. These observations give a new insight about the probable role of Arsenophonus in B. tabaci, that need to be explored further.