HAIRPIN RNA INTERFERENCE CONSTRUCTS AGAINST Tomato Leaf Curl Sudan Virus: TRANSFORMATION, AND EVALUATION IN Nicotiana benthamiana PLANTS

INTRODUCTION

In the world of vegetable crops, tomato (Solanum lycopersicum L.; family: Solanaceae) is one of the most widely consumed and economically significant crops. Growing in various places, tomatoes are regarded as one of the most commercially significant vegetable crops in Saudi Arabia, grown on approximately more than 12,000 hectares of land, yielding about 351,000 tons of fresh tomato fruits annually. Numerous phytopathogens greatly decrease crop production when they infect tomato plants (Agrios, 2005; Jones et al., 2016; FAO, 2020). Viruses belonging to the family Geminiviridae and genus Begomovirus seriously harm a variety of crops and lower their intended productivity (Malathi et al. 2017). Tomato Leaf Curl Sudan Virus (ToLCSDV) is a unique begomovirus that, like other leaf curl viruses, is spread by the whitefly B. tabaci, however it is occasionally mistaken for tomato yellow leaf curl virus (TYLCV). Begomoviruses have circular single-stranded DNA genomes that are either mono- (DNA-A) or bipartite (DNA-A and DNA-B) (Zhai et al., 2022). According to previous reports, ToLCSDV causes tomato leaf curl disease, which results in notable yield losses because of symptoms including, yellowing, reduced development, and leaf curling. The virus is a danger to tomato production in the area. Depending on the crop type and infection stage, the infection might result in yield losses of up to 100 % (Odeh et al., 2025; Mohammed et al., 2018; Akhtar et al., 2014; Idris et al., 2011, 2012, 2014; Khan et al., 2013a, b; Al-Saleh et al., 2014; Khan et al., 2008; Ajlan et al., 2007). Numerous reports have documented the link between begomovirus and leaf curl disease on a variety of crops. The world's economically significant agricultural crop production and food security are seriously threatened by begomoviruses and their associated ssDNA satellites due to their high diversity, recombination potential, limited knowledge of alternative hosts, and transmission by B. tabaci. As a result, further research and deeper understanding are required (Saeed et al., 2017).

Hence, a sequence-specific RNA regulation mechanism called RNA silencing (RNA silencing) has been identified in eukaryotic organisms (Agrawal et al., 2003). It is a crucial protective system for hosts that counters resistance against foreign nucleic acids, including transposons, transgenes, and viruses (Voinnet, 2001; Lippman et al., 2003). It is important in regulating gene expression (Shabalina and Koonin, 2008). Currently, it has been demonstrated that almost every class of virus can encode proteins that interfere with RNA silencing (Li and Ding, 2006). The presence of these suppressors is the strongest evidence supporting the contention that an adaptable defence against infections is RNA silencing. A potential solution for the management of diseases caused by begomovirus is RNA interference (RNAi) (Prins et al., 2008; Chowda-Reddy et al., 2008). RNAi was utilized to help tomatoes become resistant to TYLCV, Bean golden mosaic virus (BGMV) (Bonfim et al., 2007) and cotton leaf curl geminivirus complex (Mubin et al., 2011). This resistance lessens the severity of disease and virus titer, but it is not immunity. Additionally, the betasatellite might weaken resistance, which would raise the percentage of plants that eventually exhibit symptoms (Ammara et al., 2015).

Whereas Helper component protease (HC-Pro) stops Dicer from processing dsRNA and prevents duplex siRNA from unwinding (Chapman et al., 2004). These examples illustrate that numerous procedures have emerged in viruses for overcoming RNA silencing by interfering at distinct points in the pathway. A common eukaryotic gene regulatory mechanism called "RNA silencing" hinders the gene expression of a gene by inhibiting transcription and triggering the degradation of RNA specific to a certain region. A specific RNA degradation process called post-transcriptional gene silencing (PTGS) takes place in the cytoplasm and results in the accumulation of 21–24 nt-long siRNAs that correspond to the silenced gene (Fagard and Vaucheret, 2000). Sometimes, because of RNA silencing, plants previously infected with a milder virus strain offer cross-protection (resistance) against a more severe virus with positive sense RNA genomes. Most plant viruses are replicated by using dsRNA intermediates, which significantly induce RNA silencing (Ahlquist, 2002). It has been observed that both DNA and RNA viruses have virus-derived siRNAs (Xie et al., 2004; Akbergenov et al., 2006). When it comes to RNA viruses, secondary structures in the single-stranded viral RNA or replication intermediate can both produce dsRNA (Molnár et al., 2005). In the present study, the major objective was to develop RNAi-based resistance in the model plant, Nicotiana benthamiana to prove the efficiency of RNAi technology against ToLCSDV and pave the way for the development of broad-spectrum transgenic resistance against dominant begomovirus species infecting tomatoes and other crops of commercial significance.

MATERIALS AND METHODS

Sense and anti-sense CP gene construction: PCR amplification of the capsid protein gene was performed using the ToLCSDV clone from a previous study (Amer et al. 2025) as a template. A fragment of 686 bp was amplified using Sense (CPS-F/CPS-R) and Anti-Sense (CPAS-F/CPAS-R) primers orientations (Table 1).

Hairpin RNAi construct of the CP gene and transformation into competent E. coli: The restriction enzymes XhoI and NcoI were used to digest the PCR product's CP sense and the RNAi binary plasmid vector pFGC5941 to clone the amplified CP sense. Using the restriction enzymes BamHI and XbaI, clone the amplified CP antisense + RNAi binary plasmid vector (CPS-pFGC5941) that has been inserted with CP sense. Linear vector DNA, insert DNA, and T4 DNA ligase (New England Biolabs) were used in the ligation mixture. On a 1% agarose gel, the quality of the DNA fragments was assessed. Transformation of competent Escherichia coli was performed using an adapted calcium chloride procedure (Chang et al., 2017).

Hairpin RNAi construct of the CP gene and transformation into Agrobacterium cells: DNA isolation of the Hairpin RNAi construct of the CP gene was carried out according to the Gene JET Plasmid Miniprep Kit manufactured by Thermo Scientific^TM. Two pairs of primers were utilized for the sense (35S-Pro-F/chsA-intr-R) and antisense orientation (chsA-intr-F/OCS-term-R). Restriction (XhoI and NcoI for the sense fragment and BamHI and XbaI for the antisense fragment) analysis of three plasmid clones were used to validate and confirm the hairpin CP construct orientation (hp-CP-pFGC5941) (Table 1). After that, 5 μl was added to a 1% agarose gel to validate the recombinant plasmid DNA. Electroporation technique for Agrobacterium tumefaciens GV3101 was conducted according to Mersereau et al., 1990 and Kámán-Tóth et al., 2018.

The verified transformed Agrobacterium colony was selected and cultivated in LB broth that was enhanced with a suitable combination of antibiotics (Kanamycin and Rifampicin). The culture grew in a shaker incubator at 28°C for 24 hours to prepare the inoculum. PCR amplification using specific primers (CPSF/CPSR and CPAS-F/CPAS-R) was employed to confirm the transformation of hairpin RNAi construct into Agrobacterium.

Agro-infiltration of plants and symptom observation: A single Agrobacterium colony with a recombinant plasmid was selected, inoculated in a 50-ml LB culture with appropriate antibiotics, vigorously shaken, and kept at 28°C for 48 hours. After centrifuging the culture for 10 min at 4,000 rpm, the pellet was resuspended in a solution containing 100 μM acetosyringone and 10 mM MgCl2 until the optical density (OD600) achieved at 1.0. Before N. benthamiana inoculation, the culture was left for two to three hours. Next, a needleless disposable syringe was used to infiltrate the Agrobacterium suspension on the underside of a leaf disc. Ten plants of N. benthamiana were inoculated with three different clones in biological experiment to evaluate the resistance response compared to the control plants. For each experiment, five plants were inoculated with ToLCSDV (infectious clone) plus hpCP-pFGC5941 (hpRNAi construct) and five plants were inoculated with ToLCSDV (infectious clone) alone. Five plants were inoculated with pFGC5941 vector without insert and five plants were kept without any inoculation as a negative control. These experiments were carried out three times. All these plants were kept in environmentally controlled and completely isolated in an insect-proof cages in a greenhouse at 25-28 °C and symptoms were observed after 21-30 days. Three weeks after the inoculation, the severity of the symptoms was documented. According to Al-Shahwan et al., 1995, the following scale was used to assess the intensity of the symptoms: mild (+), moderate (++), severe (+++), very severe (++++), and symptomless (-). All the inoculated and control plants were being tested by PCR against ToLCSDV using specific primers (CPSF/CPSR). 

 

Table 1. List of primers used to clone CP sense and antisense orientation and on vector pFGC5941 sequence for clone confirmation.

Primer Name	Primer Sequence	Restriction site	Amplicon Size
CPS-F	5´-ggctcgagatgttgaagcgtccc-3´	XhoI	686 bp
CPS-R	5´-caccatgggagcgttctcagtatga-3´	NcoI
CPAS-F	5´-caggatccgagcgttctcagtatga-3´	BamHI	686 bp
CPAS-R	5´-gctctagaatgttgaagcgtcccg-3´	XbaI
35S-Pro-F	5´-ccactgacgtaagggatgacgcacaat-3´		341 bp
chsA-intr-R	5´-acaattcgtcggccaccccaacccaaa-3´
chsA-intr-F	5´-gcacctataaacacttacttacacttgccttggag-3´		429 bp
OCS-term-R	5´-ttgttattgtggcgctctatcatagatgtcgc-3´

RESULTS

PCR Amplification of CP Sense and anti-sense genes Hairpin RNAi construct: PCR amplification of capsid protein gene sense and anti-sense orientations was done using a ToLCSDV clone as a template. The CPS-F and CPS-R and CPAS-F and CPAS-R primers were used to amplify a 686 bp fragment of the CP gene. No PCR amplified product from uninfected sample was used as a negative control. (Figure 1).

Figure 1: PCR amplification of CP sense (A) and CP antisense (B) using a ToLCSDV clone as a template. Lane M shows the DNA Ladder (Hyper Ladder^TM 50 bp; Meridian Bio-Science Inc.), Lanes shows the bands of CP sense and CP antisense (approximately 686 bp), and Lane N shows the negative control.

For the construction of the hairpin RNAi construct of the CP region of ToLCSDV, first we amplified the CP gene fragment in sense orientation and antisense orientation using CPS-F and CPS-R and CPAS-F and CPAS-R pairs of primers. Then both strands (sense and antisense orientation) were ligated into pFGC5941 as a binary vector in MCS1 and MCS2, respectively, to make the hairpin RNAi construct (hpCP-pFGC5941). Restriction of pFGC5941 vector and CPS PCR product with XhoI and NcoI showing the specific bands for vector and coat protein sense. Lane 1 shows pFGC5941 vector (size is 11,406 bp) and lane 2 shows CPS (approx. 686 bp) (Figure 2).

Figure 2: XhoI and NcoI-restricted pFGC5941 vector and CPS PCR product. Lane M is showing DNA Ladder, Lane 1 is showing the pFGC5941 vector (size is 11,406 bp), and Lane 2 is showing CPS (approx. 686 bp).

For restriction of CP antisense in the CPS-pFGC5941941 vector, the amplified CP antisense fragment was restricted into the vectCPS-pFGC5941, which already had the CP gene using BamHI and XbaI. The results obtained showed bands at specific size for each product: pFGC5941 vector (size is 11,406 bp), CPAS (~ 686 bp) and CPS-pFGC5941 vector (~ size is 12.1Kb) (Figure 3).

Figure 3: Restriction of CPS-pFGC5941 vector and CPAS PCR product with BamHI and XbaI. Lane M is showing DNA Ladder (Thermo Scientific™ Gene Ruler 1 kb DNA Ladder), lane 1 is showing pFGC5941 vector (size is 11,406 bp), lane 2 showing CPAS (approx. 686 bp) and lane 3 is showing CPS-pFGC5941 vector (approx. size is 12.1Kb).

Clone confirmation of hairpin CP in the pFGC5941 vector using PCR and restriction enzyme analysis: Three clones for sense and antisense are shown in panels 1-3 (1Kb) after PCR validation of clones of CP sense and CP antisense using 35S-Pro-F/chsA-intr-R and chsA-intr-F/OCS-term, respectively. No PCR amplified product (lane N) from uninfected sample was used as a negative control (Figure 4).

Analysis of restriction of the hp-CP RNAi construct in the pFGC5941 vector. The samples were digested using XhoI and NcoI for the sense fragment and BamHI and XbaI for the antisense fragment. The restriction reactions were analyzed on a 1% agarose gel and the expected size of bands for the vector as well as the insert were obtained. Samples of sense and antisense segments are displayed in lanes 1–7, respectively. (Figure 5). Hyper Ladder^TM 50bp (Meridian Bioscience Inc.) was utilized to determine the product sizes. Finally, one clone was chosen for transformation into the A. tumefaciens GV3101 strain, and the clone was named hpCP-pFGC5941.

Figure 4: PCR confirmation of clones of CP sense and CP antisense using 35S-Pro-F and chsA-intr-R primers (for sense) and chsA-intr-F and OCS-term-R primers (for antisense). The panel M is showing DNA Ladder (Hyper Ladder™ 50bp; Meridian Bioscience Inc.) and panels 1-3 are showing three clones for sense and antisense respectively. Panel N is showing negative control.

Figure 5: Restriction analysis of hp-CP RNAi construct in pFGC5941 vector. For sense fragment the samples were digested with XhoI and NcoI and for antisense fragment the samples were digested with BamHI and XbaI. The panel M is showing DNA Ladder (Thermo Scientific™ Gene Ruler 1 kb DNA Ladder) and panels 1-7 are showing samples for sense and antisense fragments respectively.

Transient analysis of the efficiency of the hairpin RNAi construct to develop resistance against ToLCSDV: To assess the capacity of the hairpin RNAi construct to resist the infection of ToLCSDV, a transient assay was designed, and the construct was transformed by Agrobacterium into N. benthamiana. The transient assay was done by co-inoculation of the agro-inoculum of the hairpin RNAi construct as well as the agro-inoculum of ToLCSDV infectious clones into N. benthamiana. Agroinfiltration of N. benthamiana plants was done in three different experiments. 10 plants of N. benthamiana were inoculated in each experiment: 5 plants with hpCP-pFGC5941 (hpRNAi construct) + ToLCSDV (infectious clone) and 5 plants with ToLCSDV (infectious clone) only (Table 2). After 30 days of inoculation, 15/15 plants (100%) plants were found to be positive for ToLCSDV inoculated with infectious clone of ToLCSDV only. The plants developed typical disease symptoms, including leaf curling, vein swelling, and stunted growth (Figure 6). However, 4/15 (27%) plants developed mild symptoms that were inoculated with hpCP-pFGC5941 (hpRNAi construct) + ToLCSDV (infectious clone) while the 11/15 plants (73%) remained asymptomatic (Figure 7 A, B). The results obtained revealed that hpRNAi plants exhibited resistance compared to the controls hpRNAi targeting CP of ToLCSDV reduced infection from 100% to 27% in treated plants.

Figure 6: N. benthamiana plants inoculated with ToLCSDV-Infectious clone only showing leaf curling, vein swelling, and stunted growth symptoms. After 30 post inoculation, all plants (100%) inoculated with ToLCSDV (infectious clone) were found positive for ToLCSDV.

Figure 7: N. benthamiana plants co-inoculated with hpCP-pFGC5941 (hpRNAi construct and ToLCSDV-Infectious Clone showing asymptomatic and mild symptoms. After 30 post inoculation, 4/15 plants (27%) were found positive for ToLCSDV (A). However, 11/15 plants (73%) had remained asymptomatic (B).

Table 2. Transient assay of hairpin RNAi construct against ToLCSDV in N. benthamiana plants

Inoculum	Infected plants/Inoculated plants experiments				PCR detection of ToLCSDV		PCR results
	I	II	III
hpRNAiconstruct+ToLCSDV (Infectious Clone)	1/5	1/5	2/5			4/15		4/15
ToLCSDV (Infectious Clone only)	5/5	5/5	5/5			15/15		15/15
pFGC5941 vector (without insert)	0/5	0/5	0/5			0/15		0/15
Healthy plants	0/5	0/5	0/5			0/15		0/15

DISCUSSION

The primary pathogens that significantly reduce tomato crop quality and productivity are plant viruses. Begomovirus, Tobamovirus, Orthotospovirus, Crinivirus, and Potyvirus are the five genera that comprise the major viruses that infect tomatoes globally. Through hybrid breeding, tomato resistance genes against viruses, such as the Ty gene resistance against begomoviruses, the Sw gene resistance against orthotospoviruses, the Tm gene resistance against tobamoviruses, and the Pot 1 gene resistance against potyviruses, have been detected from indigenous germplasm and introduced into commercial cultivars. Tomato antiviral breeding is severely restricted by the fact that these resistance genes mostly display qualitative resistance driven by single genes, which cannot defend against virus mutations, recombination, mixed infection, or new viruses. Future research on tomato viral resistance breeding should concentrate on quickly, safely, and effectively producing broad-spectrum genetic materials resistant to several viruses based on the epidemic features of tomato viruses.

To develop resistant germplasm against tomato viruses, modern methods of tomato breeding are recommended. These methods include gene editing based on CRISPR/Cas, transgenic breeding based on RNA interference and hybrid breeding based on marker-assisted selection (MAS) (Shahriari et al., 2023). The results presented here have shown the potential of hpRNAi approach for generating resistance in model plants, N. benthamiana targeted against conserved part of CP gene the virus genome, revealed the promising resistance against the ToLCSDV; a begomovirus virus causing huge losses to tomato production in Arabian Peninsula (Ammara et al., 2015). Increasing tomato crop yield is crucial to minimizing production losses caused by various viral diseases. Conventional methods for developing resistance are either confined to selective breeding or are not sustainable over time, making it difficult to find a line that is resistant to various viral infections (Sajid and Elçi, 2024). The results presented involve the cloning of hairpin RNAi construct targeting CP gene of ToLCSDV, cloned in a binary vector pFGC5941 and evaluation of the efficiency of the construct against the virus in model plants N. benthamiana. pFGC5941 binary vector was used for cloning the hpRNAi construct to develop the RNAi resistance against Tomato spotted wilt orthotospovirus (TSWV) species (Sajid and Elçi, 2024). In a related investigation, the conserved sequences corresponding to the two ORFs begomovirus genome, AC1 (which codes for replication associated protein), AC2 (which codes for transcriptional activator protein), and the bC1 ORF of betasatellite genome (which determines pathogenicity) were found. These ORFs encode proteins that are crucial for viral pathogenicity, movement, replication, and suppression of gene silencing. These proteins' ability to multitask makes their transcript suitable targets for begomovirus resistance development. Off-target effects are frequently linked to RNAi-mediated gene silencing, where transgene- generated siRNA may silence the transcript of additional host genes that are not targeted, resulting in aberrant and harmful phenotypes (Fedorov et al., 2006; Jackson et al., 2003).

In our study we targeted of CP region of ToLCSDV, this is a novel approach to develop resistance against this virus. For other viruses’ similar approaches has also been used to develop RNA interference-based resistance for example Ammara et al., (2015) used similar method for Tomato yellow leaf curl virus-Oman (TYLCV-OM) and its related betasatellite (Ammara et al., 2015). A potential technique that has been proven to be successful against a few infections is RNA interference (RNAi). To achieve long-lasting resistance, various geminivirus genomic regions have been targeted via RNA interference. However, one limiting factor is the transgene's silence after virus infection. In this instance, we have developed the first amplicon-based RNA interference construct to target the βC1 gene of the betasatellite linked to cotton leaf curl virus.

Rep-based activation or looping out of the construct caused by virus infection not only produces short interfering (si) RNAs but also multiple copies of transgene, which leads to the accumulation of defective betasatellite molecules. Subsequent transcription gives rise to increased number of siRNAs that gives enhanced resistance. Cotton leaf curl Multan betasatellite (CLCuMB) and Cotton leaf curl Khokran virus (CLCuKV) were tested against transgenic N. benthamiana plants carrying RCβ (RNAi construct for betasatellite). Southern blot hybridization revealed a decreased titter of the virus and betasatellite (Akhtar et al., 2021). In another promising study, to express dsRNA homologous to the intergenic region (IR) of CLCuRV, an intron hairpin (ihp) RNAi construct was developed. Nine independent lines of transformed cotton were generated when transformation of cotton plant carried out using ihpRNAi construct with the help of Agrobacterium tumefaciens. PCR paired with Southern hybridization verified the existence of the possible IR stretch in the transformed cotton. The transgenic plants demonstrated a high level of resistance when infected with viruliferous whiteflies (Khatoon et al., 2016). Further studies and large experiment will be needed to develop hpRNAi-CP based resistance against ToLCSDV in elite tomato varieties to get economic benefits of RNAi-based broad-spectrum resistance.

Conclusion: The current study results are highly promising for developing resistance against begomoviruses like TLCSVD and may be useful in the transformation of tomato varieties to develop broad-spectrum transgenic resistance against begomoviruses. Further studies are recommended to develop RNAi constructs targeting multiple segments of the genome of begomoviruses, and stacking multiple RNAi constructs under a single promoter may be initiated to make durable and broad-spectrum resistance. Different inducible and tissue-specific promoters can be investigated to avoid off-targeting of RNAi constructs. In a study concerning TLCSDV infection, the implementation of hairpin RNA interference (hpRNAi) targeting the coat protein (CP) resulted in a significant reduction of infection. Specifically, in treated plants, the infection rate decreased from 100% in the untreated control group to 27% in the plants that received the hpRNAi treatment. More research is required to enhance the design of RNA interference (RNAi) constructs for more precise and efficient gene silencing. The development of combinatorial RNAi (coRNAi) tactics to overcome viral resistance, the creation of improved viral vectors for precise delivery and tissue specificity, and the optimization of RNAi trigger design including the use of longer small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs)-are all examples of this.

Acknowledgement: The authors extend their appreciation to the Ongoing Research Funding Program (ORF-2026-1755-1), King Saud University, Riyadh, Saudi Arabia for financial support.

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