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. 2022 Feb 10;12(3):66. doi: 10.1007/s13205-021-03085-z

Knockdown of capsid protein encoding novel ATPase domain inhibits genome packaging in potato leafroll virus

Jitesh Kumar 1,#, Ravi Ranjan Kumar 1,#, Dilip Kumar Das 2, Auroshikha Mohanty 1, Kumari Rajani 3, Namaste Kumari 4, Vinod Kumar 5,6, Sunil Kumar 7, Bajarang Vasant Kumbhar 8, Tushar Ranjan 1,✉
PMCID: PMC8831665  PMID: 35186663

Abstract

Potato leafroll virus (PLRV) uses powerful molecular machines to package its genome into a viral capsid employing ATP as fuel. Although, recent bioinformatics and structural studies have revealed detailed mechanism of DNA packaging, little is known about the mechanochemistry of genome packaging in small plant viruses such as PLRV. We have identified a novel P-loop-containing ATPase domain with two Walker A-like motifs, two arginine fingers, and two sensor motifs distributed throughout the polypeptide chain of PLRV capsid protein (CP). The composition and arrangement of the ATP binding and hydrolysis domain of PLRV CP is unique and rarely reported. The discovery of the system sheds new light on the mechanism of viral genome packaging, regulation of viral assembly process, and evolution of plant viruses. Here, we used the RNAi approach to suppress CP gene expression, which in turn prevented PLRV genome packaging and assembly in Solanum tuberosum cv. Khufri Ashoka. Potato plants agroinfiltrated with siRNA constructs against the CP with ATPase domain exhibited no rolling symptoms upon PLRV infection, indicating that the silencing of CP gene expression is an efficient method for generating PLRV-resistant potato plants. In addition, molecular docking study reveals that the PLRV CP protein has ATP-binding pocket at the interface of each monomer. This further confirms that knockdown of the CP harboring ATP-binding domain could hamper the process of viral genome packaging and assembly. Moreover, our findings provide a robust approach to generate PLRV-resistant potato plants, which can be further extended to other species. Finally, we propose a new mechanism of genome packaging and assembly in plant viruses.

Keywords: Plant virus, Genome packaging, P-loop-containing ATPase domain, Packaging motor, Capsid protein, RNA interference

Introduction

Plant viruses cause diverse diseases in crops and are responsible for huge economic losses. The potato crop is severely affected by various biotic stresses. Among these stresses, viruses have a significant contribution to yield losses worldwide (Palukaitis 2012). Due to vegetative reproduction of potato, viruses propagate through seed tubers and maintain their life cycle (Kumar et al. 2020). Tubers used for planting in the next season can harbor latent viruses, which subsequently reduce emergence, plant vigor, and yield (Kumar et al. 2020). More than 40 different viruses affect the cultivation of potato crop across the globe (Kumar et al. 2020; Palukaitis 2012). Potato leafroll virus (PLRV), which belongs to the genus Polerovirus and family Luteoviridae, is a ubiquitous potato virus worldwide and is responsible for more than 20 million tons yield loss every year (Kreuze et al. 2020; Kumari et al. 2020). Thus, understanding the mode of infection, genome packaging, and assembly of PLRV is crucial for developing control strategies (Kumari et al. 2020; Ranjan et al. 2021).

Genome packaging is an important step in the process of viral maturation. During virus life cycle, genetic information needs to be incorporated into newly produced virus particles (Chelikani et al. 2014a, b). Three types of genome packaging system have been reported in viruses so far (Chelikani et al. 2014a; Ranjan et al. 2021). In the passive type I system, which exists in majority of plant viruses, the capsid proteins (CPs) nucleate over the genome and form a mature virus particle without utilizing any energy. On the contrary, both type II and III systems, which are found in bacteriophages and large eukaryotic DNA viruses, are active, ATP-dependent packaging systems (Chelikani et al. 2014a, 2014b; Ranjan et al. 2021). Viruses from type II and III system use powerful ATPase motors to achieve genome packaging (Chelikani et al., 2014a). These motors generate forces as high as 100 pN and translocate DNA into a preformed prohead until DNA condenses into crystalline form (Vafabakhsh et al. 2014). These viral packaging motors share a common architecture with P-loop superfamily of multimeric ring ATPases that perform diverse functions such as chromosome segregation (helicases), protein remodeling (chaperones), and cargo transport (dyneins) (Thomsen and Berger 2008).

Recently, we proposed a novel, expanded sub-classification system for the type I genome packaging system and placed many of the plant viruses, including PLRV, under the ATP-dependent sub-type IA system based on the ATPase domain present in CP (Ranjan et al. 2021). The mechanochemistry of the ATPase motor encoded by plant viruses is hardly explored. How a functional CP ATPase oligomerizes and nucleates on the genomic end and initiates its activity is not yet known. The ATPase motors drive the difficult task of precisely inserting the viral genome into the capsid from the cytoplasmic pool of RNA inside the host cell. Presence of a powerful motor across viruses infecting different domains of life might represent a unique variation of the genome packaging apparatus acquired and contrived by plant viruses (Chelikani et al. 2014b).

As CP is known to play a crucial role in genome packaging in PLRV (Ranjan et al. 2021), we sought to analyze its amino acid sequence to identify conserved motifs and use them to develop a potential method of developing virus-resistant plants. Our comprehensive bioinformatic analysis of CP amino acid sequence revealed a unique P-loop containing ATPase domain with two Walker A-like motifs, two arginine fingers, two sensor-like motifs, and one Walker B motif, which possess ATP-binding and hydrolysis activities and are spread throughout the polypeptide chain of CP. To support the above juncture, blind docking was performed to check the interaction of CP with ATP molecules. Our structural and docking analysis revealed that these critical motifs are either part of the loop or present at the tip of strand and make a flexible active site for ATP binding and hydrolysis. The P-loop plays a key role in coordinating ATP hydrolysis with DNA translocation. We believe that the ATPase domain of CP has a direct role in genome packaging of PLRV. To validate this hypothesis, we conducted RNAi experiments on the CP gene to check its effects on PLRV infection. Knockdown of the CP gene encoding ATPase domain resulted in the generation of potato leaves free from PLRV, indicating its role in genome packaging and assembly. Furthermore, northern blotting, RT-PCR, and ELISA analyses confirmed that the PLRV CP mRNA and protein were not detected in the leaves of plants agroinfiltrated with CP siRNA. However, the same mRNA was detected at high levels in the tertiary leaves of control plants (naturally infected) and those agroinfiltrated with the empty vector. As CP homologs are present in almost every plant virus, their inhibition via RNAi can be a potential method for generating virus-resistant plants.

Material and methods

Sequence analysis and motif identification

The sequences of CP from different strains of PLRV were retrieved from the NCBI database (http://www.ncbi.nlm.nih.gov/). Multiple sequence alignments were generated using ClustalW (Larkin 2007) and were manually corrected for domain superimpositions.

Molecular docking

To explore the binding mode of CP with ATP, a molecular docking tool AutoDock4.2 was employed (Morris et al. 2009). Crystal structure of PLRV CP was taken from the protein database (source code: 6SCO.pdb), where it was crystallized in the trimeric form. In this study, we used the monomer of CP to investigate their binding pocket and mode of interaction with ATP. The atomic coordinates of the ATP were generated using the Discovery Studio Visualizer (BIOVIA 2016). Blind and local docking approaches were explored to investigate the binding mode of CP with the ATP molecules.

Preparation of siRNA constructs

To generate target-specific siRNAs against the CP gene, the CP-encoding gene fragments were amplified using the cDNA of PLRV as template. The amplified fragments were digested with Xho I and Kpn I and cloned into the pHANNIBAL vector at Xho I–Kpn I sites in sense orientation (pHANNIBAL-sense CP). The primer sequence for cloning of the CP-encoding gene fragment in sense orientation was designed manually and synthesized at IDT, USA. These sequences are as follows: CPs FP: 5′-CCGGCTCGAGATGAGTACGGTCGTGGTTAGAGG.

A-3′; CPs RP: 5′-GCGCGGTACCCTATCTGGGGTTCTGCAAAGCCAC-3′. The pHANNIBAL vector contains the CaMV 35S promoter and the NOS terminator in sense orientation. Subsequently, the amplified and digested antisense CP gene fragments were cloned into the same vector at Hind III–Xba I sites to generate the antisense construct (pHANNIBAL-antisense CP). The sequences of these primers are as follows: CPas FP: 5′- GGGTAAGCTTCTATCTGGGGTTCTGCAAAGCCAC-3′; CPas RP: 5′- GCGCTCTAGAAT.

GAGTACGGTCGTGGTTAGAGG-3′. The resulting siRNA construct containing the sense and antisense fragments of the CP cDNA sequence was named as pHANNIBAL-CP. The siRNA cassettes were released from pHANNIBAL-CP by Not I digestion and introduced into the binary vector pART27 to generate the siRNA construct pART27-CP for plant transformation. Subsequently, the orientation of the cloned CP genes was confirmed through sequencing at each step.

Transient expression assay

The binary plasmid pART27-CP and the empty vector pART27 were extracted and purified from E. coli cultures, and were transformed into Agrobacterium tumefaciens EHA101 using the freeze–thaw transformation method (Jyothishwaran et al. 2006). The transformed cells of A. tumefaciens were plated on Luria–Bertani (LB) agar plates containing 50 μg/mL kanamycin, 100 μg/mL spectinomycin, and 100 μg/mL chloramphenicol for selecting successful transformants. Transformation of the binary plasmid was further confirmed using colony PCR for the CP gene. For agroinfiltration, A. tumefaciens cells harboring the pART27-CP siRNA constructs were grown overnight at 28 °C in the LB medium supplemented with appropriate antibiotics. The overnight cultures were diluted 1:10 in fresh media containing the above-mentioned antibiotics, 10 mM 2-(N-morpholino) ethanesulfonic acid (MES), and 200 μM acetosyringone to reach an OD600 of 0.3. The cells were collected by centrifugation at 5000 × g for 5 min, and were resuspended in the infiltration medium containing 10 mM MES, 10 mM MgCl2, and 200 μM acetosyringone. The cells were then incubated at room temperature for 2–3 h before agroinfiltration. Solanum tuberosum cv. Khufri Ashoka plants (35–40 days old) were agroinfiltrated twice at the interval of 24 h by injecting 2 mL of these cells directly into the phloem and leaves using a syringe. Whole plants were covered with a transparent plastic bag for 3–4 days. All experiments were repeated thrice with five plants in each experiment for each siRNA construct and control.

Viral infectivity assay

Symptomatic potato plants newly emerged from PLRV-infected tubers were used for agroinfiltration of pART27-CP siRNA constructs. PLRV-infected tubers were grown in a controlled environment of transgenic glass room. The presence of PLRV titer in tubers and newly emerged plants were confirmed through enzyme-linked immunosorbent assay (ELISA) and PCR using CP-specific primers (FP: 5ʹATGAGTACGGTCGTGGTTAGA-3ʹ; RP: 5ʹ CTATCTGGGGTTCTGCAAAGCC-3’). The plants were grown in a transgenic glass house at 25 °C with 16 h of light and 8 h of darkness after agroinfiltration. Ten days post-agroinfiltration (dpi), newly emerged tertiary leaves were harvested for RNAi analysis. The plants harboring RNAi constructs were analyzed using Northern blotting, RT-PCR, and ELISA.

Northern blotting analysis of siRNA

Leaves of 5–10 dpi agroinfiltrated potato plants and healthy control plants were used for small RNA isolation. Small RNA was extracted using PAX gene Tissue RNA/miRNA Kit (Qiagen, Germany) following the manufacturer’s protocol. Denaturing polyacrylamide gels (15%) containing 7 M urea were run at 40 mA (600 V) for approximately 2 h to resolve the total RNA, which contained the siRNA pool. The gels were stained for 10 min with 0.5 μg/ml ethidium bromide (EtBr) in DEPC-treated TBE buffer. A Bio-Rad transblot apparatus (Bio-Rad, USA) was used to transfer the RNA onto a Hybond-N + positively charged nylon membrane (GE healthcare, USA) at 200 mA (9–10 V) for 3 h followed by crosslinking for 20 min at 1200 μJ and drying for 30 min at 50 °C to improve sensitivity. Furthermore, the membrane was pre-hybridized in pre-hybridization buffer [7% SDS, 200 mM Na2HPO4 (pH 7.0), 5 μg/ml salmon sperm DNA (SSDNA)] at 40 °C for 30 min. The hybridization buffer was replaced with hybridization buffer containing biotin-labeled probes at 50 pmol/mL concentration. The membrane was subsequently hybridized at 40 °C for 12–16 h with continuous gentle shaking followed by rinsing with washing buffer (1 × SSC, 0.1% SDS) thrice for a total of 15 min at room temperature. The membrane was then blocked for 15 min with blocking buffer using gentle shaking at room temperature followed by incubation with hybridization buffer containing stabilized streptavidin–HRP conjugate for an additional 15 min. The blot was visualized using Amersham typhoon blot imaging systems (GE, Healthcare) after developing by ECL (GE Healthcare).

RT-PCR analysis of viral RNA in potato plants

Total RNA was extracted from the newly emerged tertiary leaves (100 mg) of agroinfiltrated potato plants and healthy control plants using RNeasy Plant Mini kit (Thermo Scientific, USA) following the manufacturer’s instructions. RT-PCR was conducted for CP-infiltrated plants individually using the PLRV-specific primers. cDNA was synthesized using an RT-PCR kit (Thermo Scientific, USA).

ELISA

Indirect ELISA was performed for the detection of PLRV infection in tertiary leaves of agroinfiltrated plants and healthy control plants (both positive and negative) using modified protocol developed by our lab (Kumari et al. 2020). Healthy uninfected plants and PLRV-infected plants were considered as negative and positive control, respectively. The absorbances of different samples and controls at 405 nm were normalized with the positive control.

Results

Insights on the structural details of the PLRV CP ATPase domain

CPs are known as plant virus-encoded factors that nucleate over viral genomes and encapsidate them by deriving energy from ATP (Ranjan et al. 2021). The motifs necessary for ATP binding and hydrolysis are present throughout the polypeptide chain of PLRV CP (Fig. 1). Figure 1 presents a multiple sequence alignment of CP sequences from different PLRV variants. Role of the ATPase motifs toward ATP binding and hydrolysis in PLRV movement protein has already been confirmed in our previous report (Kumari et al. 2020). Intriguingly, in the present research, our sequence analysis revealed a unique pattern of ATPase domain, which contained two sets of Walker A, arginine finger, and sensor-like motifs, suggesting divergent evolution within the classical P-loop-containing ATPase superfamily. The Walker-A “P-loop” motif is proposed to coordinate ATP hydrolysis with DNA translocation. The Walker A-like motif (lavender color), with the consensus sequences RGRGSSET, interacts with the β- and γ-phosphates of the bound ATP, whereas the conserved Asp of Walker B (consensus sequence hhhhDG), located next to a β-strand, binds to a metal ion and helps in ATP hydrolysis. The Walker-B (blue) Asp coordinates with the Mg2+ cation, while another conserved catalytic Gly residue primes a water molecule for the nucleophilic attack on the γ-phosphate of the bound ATP. Our sequence and structure analysis found that all the critical motifs are either present at the tip of the strand or part of the loop, indicating relatedness of CPs to ASCE P-loop ATPases (Fig. 1). Furthermore, we observed that the arginine fingers I and II (red), which are located 9–10 residues downstream of sensor I (green) motif and 5–6 residues downstream of Walker A′ (lavender), and the sensor motif II (green) present about five residues further downstream of the Arginine finger II motif are strictly conserved across various strains of PLRV analyzed in this study (Fig. 1). Sensor motif I (green) is located 48 residues downstream of Walker B and flanking of arginine finger I, II and Walker A′ with sensor motifs I and II represents the uniqueness of this novel P-loop-containing ATPase. Our structure analysis revealed that the active site of PLRV CP comprises all the motifs necessary for ATP interaction except Arginine finger II, which is situated far from the rest of the motifs in their folded structure. Arginine finger is a classical hallmark of ATPases and is conserved across many ATPases. It completes the active site from a distinct location, forming contacts with the γ-phosphate of the nucleotide (Guo et al. 2019).

Fig. 1.

Fig. 1

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A Multiple sequence alignment of the ATPase domain of CP from different variants pf PLRV. The alignment was generated using Clustal W and manually corrected for domain superimpositions. The number(s) in brackets represent the number of amino acids. (Accession Numbers: PLRV China: QEE13716.1, PLRV Shimla: AFJ11896.1, PLRV Beijing: QEE13717.1, PLRV Japan: BBN91709.1, PLRV Yunnan: ACO92389.1, PLRV France: AAL77948.1, PLRV Saudi Arabia: AGR87640.1, PLRV Iran: ACK99523.1, PLRV Mongolia: AHA43773.1, PLRV Pakistan: ASB17109.1, PLRV Punjab: ASB17109.1, PLRV Canada: AYA73305.1, PLRV Netherlands: CAA54535.1, PLRV Czech Republic: ABY49848.1, PLRV Kunming: ACO92404.1, PLRV Harbin: AKN09918.1, PLRV UK: CAA54537.1, PLRV India: AFJ11862.1, PLRV Germany: QBO24571.1, PLRV Himachal Pradesh: AAN31764.1, PLRV Islamabad: ASB17111.1, PLRV Jordan: ACF33055.1, PLRV Quedlinberg: AFI93516.1, PLRV Ireland: QJF11910.1, PLRV Korea: AAG14887.1, PLRV South Africa: AAB80766.1, PLRV Korea: AAD00221.1, PLRV Argentina: ARS33719.1, PLRV Egypt: ATV90882.1, PLRV Assam: QDA76478.1, PLRV Patna: MW027216.1, PLRV Peru: APC60287.1)

Binding mode and interaction of CP with ATP molecule

Blind docking was performed to explore the putative binding mode of PLRV CP with the ATP molecule. Capsid protein was enclosed in a grid box with 126 × 126 × 126 grid points and the grid spacing was fixed to 0.375 Å. We maintained CP in rigid confirmation and ATP as a flexible molecule. A total of 5 independent dockings were performed with 100 runs each. For each run, ATP was positioned at a random location around the CP. Total 500 output conformations were generated by applying the Lamarckian Genetic Algorithm (LGA) with the default parameters. These output conformations were further clustered using an all-atom RMSD with a cut-off 4 Å. The clusters were further analyzed thoroughly based on binding, van der Waals, and intermolecular energies. The analysis of docking reveals that the least energy conformation of ATP binds near the sensor I region of the CP (Fig. 2). Furthermore, we performed local docking to get the least energy conformation targeting the sites observed in the blind docking. For local docking, a grid box of 80 × 80 × 80 grid points with grid spacing 0.375 Å was built around the ATP-binding pocket of CP as observed during the blind docking. We run 100 output conformation to get the least energy docked conformation of ATP. The least energy docked conformation of ATP was further used for the analysis of hydrogen bonding, van der Waals, π–π type, π-alkyl type of interactions using Discovery Studio visualizer (BIOVIA 2016), and PyMOL (DeLano 2002), respectively (Fig. 3). Interestingly, superimposed structure of docked PLRV CP-ATP complex revealed the presence of active site at the interface of each monomer (shown in red dotted circle in Fig. 2C).

Fig. 2.

Fig. 2

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Molecular docking of PLRV CP with the ATP molecule. A The atomic model of PLRV CP (6SCO.pdb) with all the ATP binding and catalysis motifs (Walker A and A′ in lavender; Walker B in blue; sensor I and II in green and arginine finger I and II in red color). Walker B (blue) is not visible in this structure and it is present behind the atomic model. The enlarge view of binding pocket for ATP is represented in inset. B Superimposed structure of docked PLRV CP-ATP complex representing the presence of active site at the interface of each monomer. C PyMoL view of solvent accessible surface area of CP with ATP-binding pocket at the interface of each monomer (shown in red dotted circle)

Fig. 3.

Fig. 3

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Two-dimensional interaction of CP residues with the ATP molecule. CP and ATP complex is being stabilized by the bonding interaction with residues such as Lys159 and Gln162. While the residues Lys159, Tyr161, and Asn149 make carbon–hydrogen interactions, whereas the residues Glu134 and Val148 make π-anion and π-alkyl types of interactions, respectively

Construction of the pART27 binary vector

Specific primers were designed for the amplification of the gene sequence of CP in sense and antisense orientations. The generation of siRNA construct is illustrated in Fig. 4A. The total RNA extracted from infected potato leaves was subjected to RT-PCR to generate cDNA, and the desired sequences were amplified from the cDNA using oligo dT primers. This PCR yielded two 627 bp bands: one of sense orientation and another antisense (Fig. 4B; lanes 1 and 2). These fragments were cloned into the pHANNIBAL vector (5.8 kb) after digestion with the appropriate set of enzymes (Fig. 4C; lane 1). The cloning of the CP gene in pHANNIBAL in the sense orientation (pHANNIBAL-sense CP) and antisense orientation (pHANNIBAL-antisense CP) was carefully analyzed using different sets of restriction enzymes (Fig. 4D; lanes 1–7). Furthermore, the orientation of genes was confirmed through sequencing (IDT). Another round of sequencing was performed for the cloning vector carrying both sense and antisense CP (pHANNIBAL-CP). The resulting siRNA constructs were further digested by Not I and introduced into the digested linear (~ 11.6 kb) binary vector pART27 (Fig. 4E; lane 1) to generate the pART27-CP-siRNA construct. The accuracy of siRNA constructs in pHANNIBAL (Fig. 4D) and pART27 (Fig. 4F) binary vector was further confirmed through restriction digestion analysis and sequencing.

Fig. 4.

Fig. 4

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A Schematic representation of the generation of pART27-CP siRNA constructs used for transient expression by agroinfiltration. CP genes were first cloned in pHANNIBAL plasmid in sense and antisense orientations. Subsequently, the complete gene cassette was transferred into the binary pART27 plasmid. B RT-PCR was performed to amplify sense and antisense PLRV CP sequences from the leaves of infected potato plants. Amplified cDNA fragments were analyzed by electrophoresis on 0.8% agarose gel. M: 100 bp DNA ladder; lanes 1 and 2: PCR products of sense and antisense CP (627 bp). (C) The pHANNIBAL plasmids were purified from E. coli DH5α cultures and analyzed by electrophoresis on 0.8% agarose gel. M: 1 kb DNA ladder; lanes 1: pHANNIBAL plasmid (5.8 kb). (D) Confirmation of siRNA constructs in pHANNIBAL and pART27 binary vector through restriction analysis. M: 1 kb DNA ladder; lane 1: undigested pHANNIBAL; lane 2: antisense CP ligated with pHANNIBAL (pHANNIBAL-antisense CP construct); lane 3: both antisense and sense CP ligated with pHANNIBAL (pHANNIBAL-CP-siRNA construct); lane 4: Hind III and Xba I digestion of pHANNIBAL-antisense CP construct shows the release of a 627 bp fragment; lane 5: Xho I and Xba I digestion of pHANNIBAL-CP siRNA construct shows the release of a ~ 1500 bp fragment; and lane 6: Not I digestion of pHANNIBAL-CP siRNA construct shows the release of two fragments of ~ 4 kb (including sense CP, intron, and antisense CP sequence) and 3.5 kb. E The pART27 binary plasmid was purified from E. coli DH5α culture and analyzed by electrophoresis on 0.8% agarose gel. M: 1 kb DNA ladder; lane 1: pART27 plasmid (11.6 kb). F The whole siRNA cassette (~ 4 kb) was transferred from pHANNIBAL to pART27, and this was further confirmed by Not I restriction analysis of recombinant pART27

Transient expression of CP siRNA

To determine whether the knockdown of CP encoding a novel ATPase domain could interfere the genome packaging and assembly of PLRV in potato, the pART27-CP-siRNA constructs were agroinfiltrated into S. tuberosum cv. Khufri Ashoka. Control plants (PLRV-infected) (Fig. 5A; lane 1) and those agroinfiltrated with the empty vector (Fig. 5A; lane 2) showed rolling symptoms of PLRV infection in the upper leaves, whereas plants that were agroinfiltrated with the CP siRNA constructs (pART27-CP) did not show any symptoms of viral infection (Fig. 5A; lane 5). Rolling symptoms were also observed in the upper leaves of plants agroinfiltrated with pART27-sense CP and pART27-antisense CP construct (Fig. 5A; lane 3 and 4, respectively). These findings indicated that siRNA constructs suppressed viral genome packaging and assembly in the plants.

Fig. 5.

Fig. 5

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A Symptoms observed in the tertiary leaves of the PLRV-infected potato plants. (1) PLRV-infected control without agroinfiltration; (2) agroinfiltrated with the empty vector pART27; (3) agroinfiltrated with the plasmid containing only antisense sequence (pART27-antisense CP); (4) agroinfiltrated with the plasmid containing only sense sequence (pART27-sense CP); (5) agroinfiltrated with the pART27-CP siRNA construct; B Detection of PLRV RNA by RT-PCR. Amplified cDNA fragments (627 bp) were analyzed by electrophoresis on 0.8% agarose gel. M: 100 bp marker; lane 1: PLRV was observed in control plant; lane 2: PLRV from tertiary leaves of the plant containing the empty vector pART27; lane 3: PLRV from tertiary leaves of the plant containing the antisense construct (pART27-antisense CP); lane 4: PLRV from tertiary leaves of the plant containing the sense construct (pART27-sense CP); lane 5: no PLRV in tertiary leaves of the plant containing CP siRNA (pART27-CP). C Actin PCR was performed as an internal control. D Confirmation of expression of siRNA using Northern blotting analysis. Higher expression of siRNA in the leaves at 10 dpi (lane 1) was observed as compared to 5 dpi (lane 2), whereas empty vector (mock) agro-infected leaves did not show any siRNA (lane 3)

RT-PCR analysis of the CP gene

RT-PCR for analysis of PLRV capsid protein was carried out by following our previously developed protocol (Kumari et al. 2020). Total RNA isolated from the upper tertiary leaves of agroinfiltrated plants and control plants were subjected to RT-PCR using PLRV-CP-specific primers. The 627 bp amplified DNA fragment corresponding to the CP gene of PLRV was detected in the tertiary leaves of plants agroinfiltrated with the empty vector and control leaves; however, it was not detected in the tertiary leaves of plants agroinfiltrated with the CP siRNA constructs. Thus, RT-PCR confirmed that PLRV multiplication was suppressed by these siRNA constructs (Fig. 5B). RT-PCR for the actin gene was also performed simultaneously as an internal control (Fig. 5C).

Expression analysis of siRNA in agroinfiltrated plants

Analysis of siRNA construct in agroinfiltrated potato plants was carried out with modifications in previously developed protocol (Kumari et al. 2020). The expression of siRNA was analyzed by Northern blotting using an equal amount of total RNA from the leaf samples of pART27-CP construct and empty vector harboring plants, respectively, to ascertain whether RNA silencing was initiated in agro-infected potato leaves by pART27-CP construct. The agro-infected regions of the leaf were used to isolate total RNA, which was subsequently analyzed for the accumulation of siRNAs at 5 and 10 dpi. The siRNAs were detected in the agro-infected region at 5 and 10 dpi (Fig. 5D). The RNA band intensity suggested higher expression of siRNA in the leaves at 10 dpi (Fig. 5D; lane 1) than that at 5 dpi (Fig. 5D; lane 2), whereas empty vector (mock) agro-infected leaves did not show any presence of siRNA (Fig. 5D; lane 3).

Detection of PLRV using DAS-ELISA in host plants

The presence of PLRV in host plants was detected by DAS-ELISA using an anti-PLRV CP antibody. The absorbance values of all the samples were normalized to that of the positive control. The mean absorbance values for all the samples are presented in Fig. 6A. We noted that the relative absorbance values of the samples from plants containing the CP siRNA construct were similar those of the negative controls (healthy plants). Overall, PLRV CP was undetectable in plants agroinfiltrated with siRNA, whereas PLRV was present in high concentration in the plants agroinfiltrated with the empty vector, with only the sense construct, or only the antisense construct.

Fig. 6.

Fig. 6

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A ELISA to determine the titer of PLRV. Absorbance values of all the samples were normalized to that of the positive control (PLRV-infected plant). A healthy uninfected plants were considered as negative control. The vertical bar represents mean ± S.E. of three replicates. B Hypothetical model for the genome packaging and assembly in PLRV. CP recognizes the end of the genome (pac site) and nucleates over the genome. CP further oligomerizes and encapsidates the unit length genome, and leads to the formation of mature virion particle. Knockdown of the ATPase domain of CP resulted in either inhibition of genome packaging or generation of non-functional/immature virus particle

Discussion

Mechanism of genome packaging is reasonably well understood in viruses belonging to the type II and III packaging systems (Al-Zahrani et al. 2009; Chelikani et al. 2014a, b; Feiss and Catalano 2005). However, the detailed mechanism of genome packaging in plant viruses with type I packaging system remains unclear. Whether plant viruses should be placed in a group of energy-dependent packaging system or energy-independent one was debatable until we proposed a subgroup of the type I system. In this subgroup, which we named as type IA, majority of plant viruses such as PLRV, potato virus X, and other geminiviruses were finally classified (Chelikani et al. 2014a; Ranjan et al. 2021). The plant viruses that possess ATPase domain in their CP were placed under this subgroup, and for these viruses, direct role of CP in genome packaging was hypothesized (Ranjan et al. 2021). Our thorough sequence analysis (Fig. 1) of CP revealed the presence of a novel P-loop-containing ATPase motif. The Walker-A motif is a phosphate-binding loop (P-loop) found in possibly the most ancient and abundant protein class, the so-called P-loop ATPases (Ortiz et al. 2019; Romero et al. 2018). Researchers have proposed that the Walker-A “P-loop” motif coordinates ATP binding and hydrolysis with DNA translocation during active genome packaging in plant viruses and that it is a part of the ATPase motor (Ortiz et al. 2014; Rakitina et al. 2005). During the assembly of viruses, a powerful ATP-driven motor translocates and packages DNA into a capsid coat. The crucial Arginine finger II motif is distantly placed in the atomic model from the two Walker A, B, sensor, and arginine finger I motifs, which form a cleft or active site for the binding of ATP. Once ATP occupies its position in the active site, the arginine finger comes into the vicinity of the γ-phosphate for hydrolysis. The unique arrangement of ATP-binding and hydrolysis motifs in the primary (Fig. 1) and tertiary structure of CP (Fig. 2A) indicates the structural similarity of CP with the members of the well-known classical P-loop ATPase superfamily (Iyer et al. 2004; Ortiz et al. 2014; Rakitina et al. 2005). The role of two Walker A-like motifs (i.e., Walker A and Walker A′) during ATP binding and catalysis needs to be experimentally investigated. Replacing the critical amino acid residues of these ATPase motifs through site-directed mutagenesis (SDM) is underway to further dissect the function of these motifs, especially in terms of active genome packaging and assembly.

Our molecular docking study reveals that the ATP molecule prefers to bind PLRV CP and the least energy conformation was observed at − 5.51 kcal/mol (Fig. 2A). Intriguingly, we found that PLRV CP has an ATP-binding pocket (Fig. 2A and B ) at the interface of each monomer, as shown in Fig. 2B and C. Moreover, the analysis of binding site residues presents within the 4 Å distance revealed that the ATP is surrounded by both hydrophobic and hydrophilic residues and stabilized by hydrogen-bonding interactions with the residues include Lys159 (2.47 Å) and Gln162 (2.13 Å) (Fig. 3 and Table 1). Additionally, Lys159 (2.75 Å), Tyr161 (2.05 Å), and Asn149 (2.53 Å and 2.42 Å) form carbon–hydrogen-bonding interactions with atoms O1A and O3G of the phosphate backbone of ATP. However, Lys159 forms electrostatic interactions with O2B (1.93 Å) and O3G (1.93 Å) of phosphate backbone atoms of ATP (Fig. 3 and Table 1). While, Tyr133, Leu135, Asp136, and Met165 form van der Waals interactions with ATP (Fig. 3). Moreover, residues such as Lys159, Thr160, Tyr161, Gln162, Arg164 form bonding interactions, while Leu135, Asp136, and Met165 form van der Waals interactions with the phosphate backbone atoms of ATP (Fig. 3), and could be responsible for hydrolysis of ATP to ADP and Pi. Thus, silencing of ATPase domain of PLRV CP could offer us an exciting strategy toward the development of virus-resistant crops.

Table 1.

Hydrogen-bonding interactions of PLRV CP with ATP molecule

Complex Binding energy
Kcal/mol		 Atoms involved in the bonding Distance (Å) Angle (°) Fig. ref
PLRV-ATP  − 5.51 LYS159:HZ1—ATP102:O1A 136.91 127.79 12
LYS159:HZ2—ATP102:O2G 131.22 127.64
LYS159:HZ1—ATP102:O2B 126.71 94.60
GLN162:HN—ATP102:O3G 143.95 154.31
LYS159:HE2—ATP102:O1A 91.63 159.48
TYR161:HA—ATP102:O3G 155.27 144.89
ATP102:H2ʹ—ASN149:OD1 153.34 100.02
ATP102:H8—ASN149:OD1 170.96 122.30
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In the present study, the efficiency of CP siRNA constructs to silence and inhibit PLRV assembly was assessed. The siRNA constructs were designed against CP of PLRV and agroinfiltrated into infected potato plants. The agroinfiltrated plants did not show PLRV infection (Fig. 5). Suppression of viral infection could be attributed to the reduced expression of CP due to its silencing by the siRNA constructs. The present study indicated that the transient expression of CP constructs resulted in specific and efficient inhibition of PLRV. Northern blotting and RT-PCR also confirmed that the PLRV CP mRNAs were not detected in the leaves of plants agroinfiltrated with CP siRNA, whereas these mRNAs were detected at high levels in tertiary leaves of control plants (naturally infected) and those agroinfiltrated with the empty vector (Fig. 5). These findings further confirm the earlier reports and suggest that CP ATPase has a direct role in the genome packaging, and suppression of genome packaging may lead to genome deficient and non-functional viral particles (Ranjan et al. 2021; Rakitina et al. 2005). Thus, the presence of three basic types of viral DNA packaging motors probably indicates independent innovations.

To the best of our knowledge, this is the first study that presents a novel P-loop containing ATPase fold of CP comprising several repeated motifs and demonstrates that the suppression of CP expression using siRNA constructs can lead to resistance against PLRV (Fig. 6B). The inhibition of the expression of CP by gene silencing is an efficient and promising method to introduce resistance to PLRV (Arif et al. 2009; Kumar et al. 2020a; Kumari et al. 2020; Ranjan et al. 2021). As PLRV causes severe crop yield losses in the potato growing regions worldwide (Munoz et al. 1975; Polder et al. 2019), our findings will be helpful for developing PLRV-resistant potato crops. This approach can also be applied to an extensive range of plant species to develop resistance against various viral diseases (Kumar et al. 2020b) (Fig. 6B). We are developing strategies for the development of PLRV-resistant potato varieties using the RNAi approach by targeting multiple genes.

Acknowledgements

We are grateful to CSIRO, Australia, for the pHANNIBAL vectors. This work was supported by grants from the Science and Engineering Research Board (SERB), Department of Science and Technology, Govt. of India, New Delhi (India) (File No. SRG/2019/002223) to TR. We would also like to thank Bihar Agricultural University (BAU), Sabour, for providing the basic infrastructure for conducting the research works. This article bears BAU Communication No. 981/210602.

Author contributions

The study was conceived by TR and RRK. Molecular experiments and in silico works were carried out by JK, AM, SK, and NK, and data were analyzed by TR, RRK, DKD, VK, and KR. Docking experiment and their analysis was performed by BVK. The manuscript was written through contributions from all authors. All authors have given approval to the final version of the manuscript.

Funding

This work was funded by Science and Engineering Research Board (SERB), Govt. of India, New Delhi (India) (File No. SRG/2019/002223).

Declarations

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

Not applicable.

Consent to participate

Not applicable.

Footnotes

Jitesh Kumar and Ravi Ranjan Kumar contributed equally to this work and share equal authorship.

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