Small Heat Shock Protein (sHsp22.98) from Trialeurodes vaporariorum Plays Important Role in Apple Scar Skin Viroid Transmission

Trialeurodes vaporariorum, commonly known as the greenhouse whitefly, severely infests important crops and serves as a vector for apple scar skin viroid (ASSVd). This vector-mediated transmission may cause the spread of infection to other herbaceous crops. For effective management of ASSVd, it is important to explore the whitefly’s proteins, which interact with ASSVd RNA and are thereby involved in its transmission. In this study, it was found that a small heat shock protein (sHsp) from T. vaporariorum, which is expressed under stress, binds to ASSVd RNA. The sHsp gene is 606 bp in length and encodes for 202 amino acids, with a molecular weight of 22.98 kDa and an isoelectric point of 8.95. Intermolecular interaction was confirmed through in silico analysis, using electrophoretic mobility shift assays (EMSAs) and northwestern assays. The sHsp22.98 protein was found to exist in both monomeric and dimeric forms, and both forms showed strong binding to ASSVd RNA. To investigate the role of sHsp22.98 during ASSVd infection, transient silencing of sHsp22.98 was conducted, using a tobacco rattle virus (TRV)-based virus-induced gene silencing system. The sHsp22.98-silenced whiteflies showed an approximate 50% decrease in ASSVd transmission. These results suggest that sHsp22.98 from T. vaporariorum is associated with viroid RNA and plays a significant role in transmission.


Introduction
Apple (Malus domestica) is a commercially important temperate fruit crop.It is commonly infected with more than 21 viruses and 8 viroids [1,2].Apple scar skin viroid (ASSVd), which belongs to the genus Apscaviroid and family Pospiviroidae, is a major viroid infecting apple [3][4][5].The ASSVd genome consists of circular, single-stranded RNA (ssRNA) with ~330 nucleotides, and it autonomously replicates in the host cell nucleus.ASSVd is known to cause apple scar skin and dappling diseases in apple, as well as rusty skin, fruit crinkling, and fruit dimple diseases in pear [6][7][8][9].ASSVd spreads from infected to healthy plants naturally through grafting and mechanically through pruning tools.The viroid is known to be transmitted through seeds, but at a very low rate [10].As the major symptoms of viroids appear on fruits, their infection decreases the quality of the fruits by producing deformation, rendering them unmarketable, which leads to direct economic loss for farmers.ASSVd disease inhibits the growth of apple saplings by altering leaf metabolism [11].ASSVd is geographically widely distributed and has been reported in Japan, India, China, Viruses 2023, 15, 2069 2 of 14 South Korea, Iran, the USA, Canada, Italy, France, Australia, and Argentina [12].ASSVd has been reported to infect apple, wild apple, pear, peach, apricot, cherry, and Himalayan wild cherry [13,14].ASSVd is also known to replicate in several herbaceous plants, such as cucumber, tomato, pea, eggplant, Nicotiana benthamiana, N. tabacum, N. gluinosa, Chenopodium quinoa, and C. amaranticolar, under experimental conditions.Infected plants show mild chlorosis and stunting symptoms [15].Recently, it was reported that the transmission of ASSVd from infected apple trees to tree-associated fungi also occurs under natural conditions [16].
Cucumber phloem protein 2 (CsPP2) and tomato viroid RNA-binding protein 1 (Virp1) have been found to interact with potato spindle tuber viroid (PSTVd) and hop stunt viroid (HSVd), respectively [25,26].CsPP2 was also found to interact with ASSVd RNA, and its addition to the artificial diet enhances the uptake and transmission of viroid RNA by the greenhouse whitefly [17].For replication, PSTVd binds to ribosomal protein L5 (RPL5), regulates the splicing of transcription factor IIIA (TFIIIA), and produces a splice variant named TFIIIA-7ZF, which, in turn, directs the host polymerase II (Pol II) for PSTVd replication [27][28][29].The TFIIIA-7ZF also binds to HSVd, citrus bark cracking viroid, and apple fruit crinkle viroid, increasing their replication [30,31].PSTVd and other members of the Pospiviroidae family reprogram host DNA ligase 1 to function as an RNA ligase, which helps in the circularization of viroid monomeric RNA forms [32].HSVd binds to histone deacetylase 6 (HDA6) and reprograms it to promote epigenetic changes that lead to the transcriptional modifications observed during viroid pathogenesis [33,34].The chloroplastic replicating Avsunviroidae family members utilize nuclear-encoded polymerase (NEP) for replication [35].Limited information is available regarding the interaction between viroids and insect proteins and their role in transmission.PSTVd is known to be transmitted by aphids, i.e., Myzus persicae; however, transmission occurs only when plants are coinfected with potato leafroll virus (PLRV).In this case, PSTVd RNA is encapsulated by the PLRV coat protein, which leads to its uptake and transmission by aphids [36].
In a previous study, the analysis of MALDI-TOF data on ASSVd-interacting T. vaporariorum proteins revealed that the small heat shock protein (sHsp) interacts with ASSVd RNA [37].Small heat shock proteins (sHsps) are molecular chaperones that contain alphacrystallin domain (ACD) and are produced under stress conditions.The molecular weights of these proteins range from 12 to 43 kDa and lack ATPase domain.In conjunction with other heat shock proteins (Hsps), they respond quickly under stress and associate with other proteins to prevent their misfolding, aggregation, and refolding.sHsps are also known to protect messenger RNAs (mRNAs) during stress-induced translational arrest, although it is unclear whether RNA interactions with sHsps occur directly or via intermediary proteins [38,39].sHsp15 from Escherichia coli is known for its binding to RNA and DNA.Its structure reveals an RNA-binding domain known as the αL motif, and it is structurally homologous to the ribosomal protein S4 and tRNA synthetase [40][41][42].
Virus-induced gene silencing (VIGS) has been widely used for functional studies of interaction, allowing for the transient silencing of target genes.The delivery of dsRNA and siRNA into insects for target gene silencing has been carried out using methods such as injection, artificial feeding, and plant delivery.Tomato plants transiently expressing truncated genes of Bemisia tabaci, Cyclophilin B (CypB), and Heat shock protein 70 (Hsp70) through a tobacco rattle virus (TRV)-mediated VIGS system have been found to reduce tomato yellow leaf curl virus (TYLCV) transmission.However, the silencing of these genes had a deleterious effect on whitefly fitness [43].ASSVd is acquired and transmitted by T. vaporariorum as a ribonucleoprotein complex [17].However, the complex association of the viroid RNA with T. vaporariorum proteins, and its role in transmission, is unknown.The dynamic nature of sHsps in nucleotide binding and chaperone function prompted this study of its role in the transmission of ASSVd.In the present study, it is reported that T. vaporariorum sHsp22.98interacts with ASSVd RNA, and its silencing reduces ASSVd transmission.

Maintenance of Whitefly Colony and Pathogen
A pure colony of T. vaporariorum was raised from a single fertilized female on cucumber plants.The mitochondrial cytochrome oxidase I (mtCOI) gene sequence was used to confirm whitefly identification [44].Whiteflies were routinely checked for potential ASSVd and other viral infections in order to ensure a pathogen-free colony since our lab also conducts research on cucumber mosaic virus (CMV) and tomato leaf curl Palampur (ToLCPalV) virus.The plants and greenhouse whitefly colonies were maintained in insect-proof cages, and the glasshouse was under controlled conditions at 22 ± 1 • C, 60% relative humidity, and a 12h light/12h dark photoperiod.

Characterization of sHsp Gene
Based on MALDI-TOF hits, primers for amplification of the complete sHsp gene were designed.Total RNA of T. vaporariorum was extracted using TriZol reagent (Invitrogen, Carlsbad, CA, USA), and the first strand cDNA was synthesized using Verso cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions.The sHsp-gene-specific primers VH23F/VH23R were used to amplify the complete gene (Table 1).For amplification, proofreading TaKaRa LA Taq ® DNA polymerase (TakaraBio, Otsu, Shiga, Japan) was used.The reaction was set up as per manufacturer instructions, and for amplification, the reaction conditions used were as follows: initial denaturation for 1 min at 95 • C; 30 cycles at 98 • C/10 s; annealing at 55 • C/30 s; elongation at 68 • C/1 min.; and final elongation at 72 • C/10 min.The amplified gene was cloned into pGME ® -T easy vector (Promega, Madison, WI, USA) and transformed into chemically competent E. coli cells.Positive colonies were identified using colony PCR and a combination of vector (M13 forward) and gene-specific reverse primer (Table 1).SapphireAmp Fast PCR master mix (TakaraBio) was used for amplification (initial denaturation for 1 min at 95 • C; 30 cycles consisting of 98 • C/10 s, 55 • C/20 s; and elongation at 68 • C/15 s).Two individual positive recombinant colonies were sequenced using M13 forward and reverse primers in Sanger sequencing.Details of primers used are given in Table 1.

Heterologous Expression and Purification of Small Heat Shock Protein
For heterologous expression, the complete sHsp gene was amplified by using VH42F/ VH42R primers.For amplification proofreading, enzyme TaKaRa LA Taq ® DNA polymerase was used.The reaction conditions used are detailed in Section 2.2.The amplified product was cloned in pGEM ® -T easy vector (Promega) and mobilized to pET28a (+) vector (Novagen, Darmstadt, Germany) using BamHI and XhoI sites (introduced through primers in gene for cloning).The recombinant pET28a-sHsp plasmid was transformed into E. coli BL21(DE3) strain and protein expression was induced with 0.3 mM IPTG at 37 • C for 4 h.After sonication, the insoluble pellet containing the expressed protein was dissolved in binding buffer (0.5 M NaCl, 5 mM imidazole, 20 mM Tris HCl, pH 7.9, 6 M urea).The protein was purified using Ni-NTA column chromatography (Novagen).The purified protein was dialyzed for removal of urea and run on 12% SDS-PAGE gel.Details of primers used are given in Table 1.

Western Blotting
Heterologously expressed purified sHsp protein was fractioned on SDS-PAGE (12%) gel, and proteins were transferred to polyvinylidene diflouride (PVDF) membrane.The membrane was incubated for 1 h in 3% blocking solution (3% w/v skim milk in PBST: 137 mM NaCl, 2.7 mM KCl, 8 mM Na 2 HPO 4 , and 2 mM KH 2 PO4 with 0.5% Tween 20) at room temperature (RT).The protein-bound membrane was further incubated for one hour with polyclonal antiserum raised in rabbit against sHsp (diluted 1:5000 in PBST) at RT.The membrane was washed with PBST three times at RT/5min and then incubated with horseradish-peroxidase-conjugated anti-rabbit immunoglobulin G antibody (1:10,000; Sigma-Aldrich, St. Louis, MO, USA) at RT/1 h.For signal development, the membrane was incubated with HRP substrate (Immobilon ® Forte) in the dark for 1 min/RT, and signals were captured using a chemiluminescent detector (Azure Biosystems C300, Dublin, CA, USA).

In Vitro Binding Assay
The in vitro binding of heterologously expressed sHsp protein to ASSVd transcript RNA was carried out using an electrophoretic mobility shift assay (EMSA) and northwestern blot.A MEGAscript ® T7 kit (Thermo Fisher Scientific) was used for synthesis of ASSVd transcript using SpeI linearized recombinant ASSVd plasmid DNA as a template [15].The EMSA experiment was carried out by incubating different concentrations of sHsp proteins (50 to 800 ng) with 1 µg of ASSVd transcript.The RNA-protein complex was incubated at room temperature for 30 min in binding buffer (150 mM KCl, 0.1 mM dithiothreitol, 1 mM EDTA, 10 mM Tris, pH 7.4).The purified sHsp protein and ASSVd transcript were used as controls.RNA-protein complexes were separated using agarose gel electrophoresis in 2.5% agarose gel at 250 V in 0.5X TB buffer.The complex was detected by staining with ethidium bromide 0.2 µg/mL [45].
Northwestern assay was performed according to Marcos (1999), with minor modifications [46].Briefly, the purified protein was separated on 12% SDS-PAGE and electroblotted to nitrocellulose membrane.The protein-bound membrane was incubated for 2 h at 4 • C in RN buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM NaCl, 0.05% triton X-100, 1X Denhardt's reagent), followed by 3 h incubation with digoxigenin-labeled ASSVd probe.The membrane was then washed twice with 2X wash buffer (2X SSC and 0.1% SDS) at RT for 5 min and once with 1X wash buffer (0.1X SSC, 0.1% SDS) for 15 min./65• C. The membrane was further incubated in blocking solution (1 g blocking powder in 100 mL of 1X maleic acid buffer: 0.1 M maleic acid, 0.15 M NaCl) at 37 • C /1 h and followed by incubation with anti-digoxigenin-AP Fab fragment antibody (500 mU/mL) at 37 • C/1 h.The ASSVd interacting protein band was visualized by using one-step NBT/BCIP substrate.

Bioinformatic Analysis of ASSVd Association with Small Heat Shock Protein
The three-dimensional structural and dynamic association of ASSVd with T. vaporariorum small heat shock proteins was carried out using HADDOCK program and BIOVIA discovery studio software package.The ASSVd 330 base nucleotides (accession no.FM208138) and sHsp were used for modeling.The ASSVd viroid RNA and sHsp protein were modeled using Discovery Studio software package and then used for interaction analysis.The RNA-binding site on protein and protein-binding site on RNA were evaluated using the same package.This interaction was carried out by adopting HADDOCK program, which is governed by ambiguous interaction restraints (AIRs).

Transient Silencing of Small Heat Shock Protein in T. vaporariorum
Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) vectors (pTRV1 and pTRV2) were used for the generation of double-stranded RNA (dsRNA)/siRNA in tomato plants.The truncated sHsp gene (253 bp) was amplified by using VH65F/ VH65R primer pair (PCR conditions, as detailed in Section 2.2) and cloned in pTRV2 vector at SacI and XhoI sites, named TRV2-TvsHsp.Briefly, the TRV vector containing the truncated TvsHsp22.98region was designed to target the N-terminal region of the gene.Similarly, the truncated tomato PDS (Topds) gene (409 bp) was amplified by using primer pair To-PDS-F/To-PDS-R, cloned in TRV2 vector at XbaI and BamHI sites, and named TRV2-Topds.The TRV1, TRV2-TvsHsp, and TRV2-Topds constructs were transformed individually into Agrobacterium tumefaciens GV3101 strain.The bacterial suspensions were mixed at a 1:1 ratio of TRV2-TvsHsp, TRV2-Topds, and TRV2 with TRV1 and agroinfiltrated in tomato cotyledons.The TRV1:TRV2-Topds-inoculated plants were used as a visual marker for silencing, and TRV1:TRV2-mock-inoculated plants were used as mock controls.The agroinoculated plants were maintained in insect-proof cages under controlled conditions.The transient expression of TRV1 and TRV2 constructs were analyzed by employing RT-PCR using vector (TRV1-VH31F/VH31R; TRV2-VH30F/VH30R) and target-gene-specific primers.Details of primers are given in Table 1.
The non-viruliferous T. vaporariorum maintained on cucumber plants were starved for 2 h and then allowed to feed on tomato plants expressing TRV2-TvsHsp after 10 days post-agroinfiltration (dpa).Silencing of sHsp gene in whiteflies fed TRV2-TvsHsp plants was analyzed after 7 and 10 dpa.To confirm silencing, full-length primers of sHsp gene were used, along with cox gene as the internal control.Amplicons were quantified using Uvitech imaging software.Whiteflies were closely monitored for mortality, nymphal development, abnormalities, changes in behavior, growth, and development compared to non-silenced whiteflies maintained on healthy plants for three weeks.The primers used to quantify sHsp (VH42F/VH42R) and cox gene (VH25F/VH25R) are listed in Table 1.Experiments were repeated three times.

Transmission of ASSVd by T. vaporariorum
The sHsp-silenced whiteflies along with healthy controls were starved for 2 h, followed by artificial feeding with ASSVd transcript (5 µg) in 50 mL falcon tubes for viroid acquisition (6 h).The artificial diet contained 5 µg ASSVd transcript, 20% sugar solution, and 10 µg purified CsPP2.These whiteflies (viruliferous and non-viruliferous) were released on healthy cucumber plants at two leaf stages for 24 h.Later, the plants were sprayed with insecticide to kill whiteflies and kept in insect-proof cages.Whiteflies fed with 20% sugar solution and 10 µg purified CsPP2 were used as healthy controls.The whiteflies were later released on healthy cucumber plants and tested for transmission of ASSVd at 14 dpi using RT-PCR with viroid-specific primers (VH40F/VH40R) (Table 1).Statistical analysis was conducted using an unpaired t-test to assess the significance of the transmission efficiency of healthy WFs and silenced WFs.Experiments were repeated three times.

Molecular Characterization of T. vaporariorum Small Heat Shock Protein (sHsp22.98)
The full-length T. vaporariorum sHsp gene that was amplified from whitefly cDNA resulted in a specific ~600 base pair (bp) amplicon.The sequence analysis showed the sHsp gene is 606 bp in length and encodes for 202 amino acids, with a molecular weight of 22.98 kDa.ProtParam (incorporated in the Expasy translate tool) predicted the isoelectric point of sHsp is 8.95.The molecular weight of the identified sHsp protein was 22.98, and based on this, it was named sHsp22.98.Blastn and Blastx analysis showed the highest homology at 96.67% and 99.41% with the T. vaporariorum small heat shock protein (FJ183792.1),respectively.sHsp was found to contain the conserved alpha-crystallin domain (77-153 aa), identified using a conserved domain database search in NCBI.The sHsp22.98 protein contains only one cysteine residue (42 aa) and is present toward the N-terminal of the alpha-crystallin domain.This residue is considered important for the dimerization of protein.The sequence was submitted to the NCBI database under the following accession number: MT557571.1.

Recombinant Expression of sHsp22.98
The 606 bp sHsp22.98gene was cloned in a pET28a (+) vector and in-frame insertion was confirmed by sequencing.Sequence analysis of the pET28a-sHsp22.98 recombinant plasmid showed that the sHsp22.98gene was in frame with the His-tag.The E. coli BL21 (DE3) cells containing the sHsp22.98-pET28a(+) plasmid exhibited the expression of a ~25.6 kDa protein after induction with 0.3 mM IPTG at 37 • C, whereas no expression was observed in uninduced cells.The expressed recombinant protein was purified by using a Ni-NTA His-binding resin column (Figure 1A).The purified protein was used as an antigen for antisera production in rabbits.The purified protein was checked with sHsp22.98specific antibody, which showed signals specific to both sHsp22.98monomeric and dimeric forms (Figure 1B).The dimeric form was more pronounced after storage of protein over time (~1 week at −20 • C) when separated on 12% SDS-PAGE (Figure 1C).

In Vitro Binding of Recombinant sHsp22.98 to ASSVd RNA
The binding properties of sHsp22.98 protein to the ASSVd transcript were confirmed by in vitro RNA-protein interaction detection assays viz., EMSA and northwestern blot.In EMSA, the migration of the sHsp22.98-ASSVdRNA-bound complex was checked with varying concentrations of sHsp22.98 protein (50 ng to 800 ng: lane 3-lane 7) (Figure 2A).At all tested concentrations, the migration of the sHsp22.98-ASSVdRNA complex was retarded in agarose gel electrophoresis.Significant retardation in migration was observed at 400 ng and 800 ng of protein (lanes 6 and 7; Figure 2A).In northwestern assays, heterologously expressed recombinant sHsp22.98 protein showed binding to digoxigenin-labeled ASSVd RNA, and the interaction was found to both monomeric and dimeric forms (Figure 2B).The results obtained in EMSA and northwestern assays confirm the binding properties of sHsp22.98 to ASSVd RNA.

In vitro Binding of Recombinant sHsp22.98 to ASSVd RNA
The binding properties of sHsp22.98 protein to the ASSVd transcript were confirmed by in vitro RNA-protein interaction detection assays viz., EMSA and northwestern blot.In EMSA, the migration of the sHsp22.98-ASSVdRNA-bound complex was checked with varying concentrations of sHsp22.98 protein (50 ng to 800 ng: lane 3-lane 7) (Figure 2A).At all tested concentrations, the migration of the sHsp22.98-ASSVdRNA complex was retarded in agarose gel electrophoresis.Significant retardation in migration was observed at 400 ng and 800 ng of protein (lanes 6 and 7; Figure 2A).In northwestern assays, heterologously expressed recombinant sHsp22.98 protein showed binding to digoxigenin-labeled ASSVd RNA, and the interaction was found to both monomeric and dimeric forms (Figure 2B).The results obtained in EMSA and northwestern assays confirm the binding properties of sHsp22.98 to ASSVd RNA.

In vitro Binding of Recombinant sHsp22.98 to ASSVd RNA
The binding properties of sHsp22.98 protein to the ASSVd transcript were confirmed by in vitro RNA-protein interaction detection assays viz., EMSA and northwestern blot.In EMSA, the migration of the sHsp22.98-ASSVdRNA-bound complex was checked with varying concentrations of sHsp22.98 protein (50 ng to 800 ng: lane 3-lane 7) (Figure 2A).At all tested concentrations, the migration of the sHsp22.98-ASSVdRNA complex was retarded in agarose gel electrophoresis.Significant retardation in migration was observed at 400 ng and 800 ng of protein (lanes 6 and 7; Figure 2A).In northwestern assays, heterologously expressed recombinant sHsp22.98 protein showed binding to digoxigenin-labeled ASSVd RNA, and the interaction was found to both monomeric and dimeric forms (Figure 2B).The results obtained in EMSA and northwestern assays confirm the binding properties of sHsp22.98 to ASSVd RNA.In vitro interaction analysis of sHsp22.98 protein with digoxigenin-labeled ASSVd probe using northwestern blotting.Lane M: prestained protein ladder (Genetix).Lane 1: 10 µg of purified protein, lane 2: 5 µg of purified protein, lane 3: BSA as negative control.

Plant-Mediated Silencing of sHsp22.98 in T. vaporariorum
The mtCOI gene was used to determine the identity of the greenhouse whiteflies.In sequencing, the amplicon shows 100% identity with the T. vaporarium gene, confirming the identity of the colony.The healthy greenhouse whitefly colonies were also checked for cross-contamination of viroid, CMV, and ToLCPalV, and the colony was found to be free from these contaminants.TRV-mediated transient expression of Tv-sHsp22.98 in tomato was confirmed using RT-PCR at 10 and 14 dpi.The amplification of the desired bands from the upper uninoculated leaves confirmed the systemic movement and replication of the TRV-TvsHsp22.98 in tomato plants.The tomato PDS gene (phytoene desaturase) was used as a visual marker for the observation of silencing, as silencing of this gene leads to photobleaching in leaves (Figure S1).The targeted dsRNA forms were delivered to healthy whiteflies by feeding them TRV-TvsHsp22.98plants after 14 days post-agroinfiltration, aiming to achieve silencing of the target gene (sHsp22.98).Whiteflies were allowed to feed on these plants for 10 days.The whiteflies were checked at 7 and 10 dpi for silencing of the sHsp22.98gene.On day 10, no amplification for the sHsp22.98gene was observed (lanes 3 and 4, Figure 6A).Plant-mediated silencing led to a reduction in the expression of the sHsp22.98transcript by ~94 to 98%.The cox gene was used as an internal control for expression comparison.The sHsp22.98-silencedwhitefly did not show any significant morphological changes based on visual and microscopic observations.The reproduction of silenced whiteflies was analogous to control whiteflies.These results showed that, at 10 dpi, effective silencing of the target gene in T. vaporariorum was observed and silencing of sHsp22.98 had no deleterious effect on whiteflies.The ASSVd-sHsp22.98 interaction was carried out by adopting RNA homology modeling protoco built in Discovery Studio software package.

Plant-Mediated Silencing of sHsp22.98 in T. vaporariorum
The mtCOI gene was used to determine the identity of the greenhouse whiteflies.In sequencing, the amplicon shows 100% identity with the T. vaporarium gene, confirming the identity of the colony.The healthy greenhouse whitefly colonies were also checked for cross-contamination of viroid, CMV, and ToLCPalV, and the colony was found to be free from these contaminants.TRV-mediated transient expression of Tv-sHsp22.98 in tomato was confirmed using RT-PCR at 10 and 14 dpi.The amplification of the desired bands from the upper uninoculated leaves confirmed the systemic movement and replication of the TRV-TvsHsp22.98 in tomato plants.The tomato PDS gene (phytoene desaturase) was used as a visual marker for the observation of silencing, as silencing of this gene leads to photobleaching in leaves (Figure S1).The targeted dsRNA forms were delivered to healthy whiteflies by feeding them TRV-TvsHsp22.98plants after 14 days post-agroinfiltration, aiming to achieve silencing of the target gene (sHsp22.98).Whiteflies were allowed to feed on these plants for 10 days.The whiteflies were checked at 7 and 10 dpi for silencing of the sHsp22.98gene.On day 10, no amplification for the sHsp22.98gene was observed (lanes 3 and 4, Figure 6A).Plant-mediated silencing led to a reduction in the expression of the sHsp22.98transcript by ~94 to 98%.The cox gene was used as an internal control for expression comparison.The sHsp22.98-silencedwhitefly did not show any significant morphological changes based on visual and microscopic observations.The reproduction of silenced whiteflies was analogous to control whiteflies.These results showed that, at 10 dpi, effective silencing of the target gene in T. vaporariorum was observed and silencing of sHsp22.98 had no deleterious effect on whiteflies.

Transmission of ASSVd through sHsp22.98-Silenced Whiteflies
The sHsp22.98-silencedwhiteflies and non-silenced whiteflies (used as the positive control) were artificially fed with dimeric ASSVd RNA transcripts for 6 h and released on healthy cucumber plants to study the role of sHsp22.98 in viroid transmission.Both silenced and non-silenced whiteflies were allowed to feed on plants for 24 h (20 plants each) and 7−10 whiteflies were released per plant.After 24 h, whiteflies were killed via the spraying of insecticide.Out of the 20 plants, 7 plants were found positive for ASSVd infection when the viroid was transmitted through sHsp22.98-silencedwhiteflies, whereas the ASSVd transmission rate by up to 50% (Figure 6B).These results show that sHsp22.98 of T. vaporariorum plays an important role in the transmission of ASSVd.

Discussion
Viroids are the smallest known infectious non-coding, circular, single-stranded RNA molecules (234-401 nt) that infect many plant hosts, replicate autonomously, and cause important diseases [47][48][49].Viroid RNAs display extensive internal base pairing, which leads to the formation of metastable rod-like or quasi-rod-like conformation [50].Long- sHsp with an isoelectric point of more than 8 is known to bind to nucleotides [59].The sHsp22.98 identified in this study also has an isoelectric point of 8.9; based on this, it can be assumed as another feature for RNA-binding.These studies showed that sHsps are important RNA-binding proteins and regulate their stability, expression, and translation.
sHsp22.98 was found to exist in both monomer and dimer forms, with both interacting with ASSVd RNA.sHspB1 is known to form monomer and dimer forms and is present in the cytosol and mitochondrial membrane of HeLa cells.Although the ratio of monomer/dimer forms at cystol is almost equal, in the mitochondrial membrane, it is up to five-fold higher [60].Also, sHsps are well known to form hetero-oligomers. Based on this evidence, it seems that both dimeric and hetero-oligomer forms are important for their functioning.Apart from this, CsPP2 was shown to interact with ASSVd and HSVd RNA, and it also existed in monomer and dimer form [17,26,51]. VirP1, also known as bromodomain-containing host protein (BRP1), of tomato, binds to PSTVd at the TR domain.The VirP1 protein contains a nuclear localization signal (NLS) and bromodomain and is predicted to be involved in the transportation of PSTVd to the nucleus [25].Later, it was found that VirP1 also binds to both PSTVd C-loop and importin alpha protein for its transportation to the nucleus [61].VirP1 has also been reported in the transportation of citrus exocortis viroid (CEVd) to the nucleus [25,62].BRPI/VirP1 was also found to be involved in the transportation of cucumber mosaic virus Q-satRNA to the nucleus and aids in its replication [63].Co-infection of CMV and its satellite RNA results in its resistance against PSTVd [64,65], which is probably due to the sequestration of VirP1 by both the virus and its satellite RNA.Based on these studies, it can be concluded that both monomeric and dimeric forms of proteins show RNA-binding properties.Also, the viroid RNA depends on multiple host and vector factors for the establishment and spread of infection.
A number of insect proteins are known to play a role in the transmission of viruses.For example, silencing of cyclophilin B and heat shock protein 70 (Hsp70) of B. tabaci resulted in 43% and 12% reductions, respectively, in tomato yellow leaf curl virus transmission [43].In the case of cucumber vein yellowing virus (Family Potyviridae), amino acids 93-105 of the CP play an important role in B. tabaci-mediated transmission.Deletion of this region leads to the abolishment of B. tabaci-mediated virus transmission [66].In our study of the role of sHsp22.98 in viroid transmission, we found that the sHsp22.98gene was transiently silenced by feeding greenhouse whiteflies tomato plants inoculated with TRV-TvsHsp at 10 dpi.Silenced whiteflies showed a significant reduction (50%) in the transmission of ASSVd.These results show that sHsp22.98plays an important role in ASSVd transmission.
In summary, this study showed that viroid RNA not only interacts with host factors but is also capable of interacting with its vector proteins.Here, it was found that sHsp22.98associates with ASSVd RNA.sHsps is a multifunctional protein that plays an important role in biotic and abiotic stress.Transient silencing of sHsp22.98leads to a reduction in ASSVd transmission.

Figure 3 .
Figure 3. Molecular modeling and prediction of binding sites in ASSVd and T. vaporariorum small heat shock protein22.98(sHsp22.98).(A) Three-dimensional structure of ASSVd RNA showing three protein-binding sites.Purple: BS1; magenta: BS2; red: BS3.(B) Structure of sHsp22.98monomer showing the presence of alpha-crystalline domain in yellow.(C) Three-dimensional structure of sHsp22.98monomer protein amino acids showing predicted RNA-binding sites in circular dots.The ASSVd RNA and sHsp protein were modeled using Discovery Studio software package.

Figure 4 .
Figure 4. Dynamic association of sHsp22.98predicted protein-binding sites to ASSVd.The predicted interacting position of ASSVd and sHsp22.98 are given in brackets.The free energy of the interaction between ASSVd and sHsp22.98 is listed as a score.The ASSVd-sHsp22.98 interaction was carried out by adopting RNA homology modeling protocol built in Discovery Studio software package.(A) sHsp22.98-site3and RNA-BS1 complex (51-97), with a score of −159.30.(B) sHsp22.98-site3and

Figure 3 .
Figure 3. Molecular modeling and prediction of binding sites in ASSVd and T. vaporariorum small heat shock protein22.98(sHsp22.98).(A) Three-dimensional structure of ASSVd RNA showing three protein-binding sites.Purple: BS1; magenta: BS2; red: BS3.(B) Structure of sHsp22.98monomer showing the presence of alpha-crystalline domain in yellow.(C) Three-dimensional structure of sHsp22.98monomer protein amino acids showing predicted RNA-binding sites in circular dots.The ASSVd RNA and sHsp protein were modeled using Discovery Studio software package.

Figure 3 .
Figure 3. Molecular modeling and prediction of binding sites in ASSVd and T. vaporariorum small heat shock protein22.98(sHsp22.98).(A) Three-dimensional structure of ASSVd RNA showing three protein-binding sites.Purple: BS1; magenta: BS2; red: BS3.(B) Structure of sHsp22.98monomer showing the presence of alpha-crystalline domain in yellow.(C) Three-dimensional structure of sHsp22.98monomer protein amino acids showing predicted RNA-binding sites in circular dots.The ASSVd RNA and sHsp protein were modeled using Discovery Studio software package.

Figure 4 .
Figure 4. Dynamic association of sHsp22.98predicted protein-binding sites to ASSVd.The predicted interacting position of ASSVd and sHsp22.98 are given in brackets.The free energy of the interaction between ASSVd and sHsp22.98 is listed as a score.The ASSVd-sHsp22.98 interaction was carried out by adopting RNA homology modeling protocol built in Discovery Studio software package.(A) sHsp22.98-site3and RNA-BS1 complex (51-97), with a score of −159.30.(B) sHsp22.98-site3and

Figure 5 .
Figure 5. Dynamic association of sHsp22.98dimer with ASSVd RNA.(A) The predicted dimeric form of sHsp22.98.(B) The predicted interacting position of ASSVd and dimeric form of sHsp22.98The ASSVd-sHsp22.98 interaction was carried out by adopting RNA homology modeling protoco built in Discovery Studio software package.

Figure 5 .
Figure 5. Dynamic association of sHsp22.98dimer with ASSVd RNA.(A) The predicted dimeric form of sHsp22.98.(B) The predicted interacting position of ASSVd and dimeric form of sHsp22.98.The ASSVd-sHsp22.98 interaction was carried out by adopting RNA homology modeling protocol built in Discovery Studio software package.

Table 1 .
Details of primers used for amplification and their target region.