Global food security requires innovative strategies for sustainable crop improvement. Gene editing offers a precise and rapid approach to plant modification, but its success depends on efficient delivery and robust expression systems. Geminivirus-derived replicons (GVRs) enhance transient expression by amplifying introduced DNA within plant cells. In this study, we evaluated three previously deconstructed geminiviral backbones -Bean yellow dwarf virus (BeYDV), Tomato leaf curl virus (ToLCV), and Wheat dwarf virus (WDV)- against a non-replicating T-DNA control for their ability to sustain GFP expression in tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum). Constructs were delivered via Agrobacterium tumefaciens, and in planta GVR circularization was verified, with accumulation levels dependent on the specific replicon and host species. GFP RNA and protein accumulation was assessed by RT-qPCR, fluorescence imaging, and ELISA; all GVRs prolonged GFP fluorescence relative to control. In tobacco, transcript levels increased significantly by 3 days post infiltration (dpi), reaching up to 221- fold by 6 dpi with BeYDV, while BeYDV and ToLCV produced approximately fivefold higher protein levels. In tomato, ToLCV and WDV showed the strongest enhancement, with transcript and protein levels increasing up to 6.3-fold and 2.4-fold, respectively. These results demonstrate that GVRs markedly enhance and extend transient gene expression in solanaceous hosts, with performance dependent on the replicon and plant species. ToLCV and BeYDV were most effective in tobacco, whereas ToLCV and WDV performed best in tomato. Overall, GVRs represent versatile tools for transient protein production and for improving the delivery and efficiency of genome-editing reagents in plants.

Crop protection against viruses is a challenging task and different approaches were developed that are environmentally friendly and safe for human and animal health. The non-transgenic dsRNA-mediated strategy exploiting the RNA interference (RNAi) has given very good results for the plant-virus pathosystems to which it has been applied. Previously, dsRNA for the helper-component (dsHC) of zucchini yellow mosaic virus (ZYMV) conferred a low 18% protection when applied in zucchini. Four dsRNAs derived from P1, P3, 6k2-VPg, and VPg cistrons of ZYMV were produced and topically applied on zucchini for a comparative analysis. All four dsRNAs protected the zucchini against ZYMV; the dsP1, dsP3, ds6k2, and dsVPg gave 62%, 21%, 25%, 25% protection, respectively, whereas dsHC gave only 16% protection. Therefore, topical application of dsP1, providing a high protection (62%) in zucchini against ZYMV, is a significant improvement in disease control. Comparison of the dsP1 with the amiZYMV_HC-196as (previously shown to convey 54% protection to ZYMV) indicated that both approaches/treatments provide high resistance in zucchini against ZYMV. Lastly, it is confirmed that dsHC, applied as root drenching, can be absorbed by roots, transported to leaves, and remained in leaves for more than six days depending on the cucurbit host.

Plant viruses utilize a subset of host plasmodesmata-associated proteins to establish infection in plants. In the present study, we aimed to understand the role of two plant genes, one encoding a putative plasmodesma located protein (PDLP) and a homolog of soybean gene regulated by cold 2 protein (SRC2) during Cucumber mosaic virus (CMV) infection. Virus-induced gene silencing (VIGS) was used to silence PDLP and SRC2 genes in Nicotiana benthamiana and in two related solanaceous plants, N. tabacum and Capsicum chinense Jacq. (Bhut Jolokia). Up to 50% downregulation in the expression of the PDLP gene using the TRV2-PDLP VIGS construct was observed in N. benthamiana and N. tabacum while, using the same gene construct, 30% downregulation of the target mRNA was observed in C. chinense. Similarly, using the TRV2-SRC2 VIGS construct, a 60% downregulation of the SRC2 mRNA was observed in N. benthamiana, N. tabacum, and a 40% downregulation in C. chinense as confirmed by qRT-PCR analysis. Downregulation of the PDLP gene in N. benthamiana resulted in delayed symptom appearance up to 7-12 days post inoculation with reduced CMV accumulation compared to the control plants expressing TRV2-eGFP. In contrast, SRC2-silenced plants showed enhanced susceptibility to CMV infection compared to the control plants. Our data suggest that the PDLP gene might facilitate infection of CMV, thus being a susceptibility factor, while the SRC2 gene could play a role in resistance to CMV infection in N. benthamiana.

Histone acetyltransferases (HATs) modify the amino-terminal tails of the core histone proteins via acetylation, regulating chromatin structure and transcription. GENERAL CONTROL NON-DEREPRESSIBLE 5 (GCN5) is a HAT that specifically acetylates H3K14 residues. GCN5 has been associated with cell division and differentiation, meristem function, root, stem, foliar, and floral development, and plant environmental response. The flowers of gcn5 plants display a reduced stamen length and exhibit male sterility relative to the wild-type plants. We show that these effects may arise from gibberellin (GA)-signaling defects. The signaling pathway of bioactive GAs depends on the proteolysis of their repressors, DELLA proteins. The repressor GA (RGA) DELLA protein represses plant growth, inflorescence, and flower and seed development. Our molecular data indicate that GCN5 is required for the activation and H3K14 acetylation of genes involved in the late stages of GA biosynthesis and catabolism. We studied the genetic interaction of the RGA and GCN5; the RGA can partially suppress GCN5 action during the whole plant life cycle. The reduced elongation of the stamen filament of gcn5–6 mutants is reversed in the rga–t2;gcn5–6 double mutants. RGAs suppress the GCN5 effect on the gene expression and histone acetylation of GA catabolism and GA signaling. Interestingly, the RGA and RGL2 do not suppress ADA2b function, suggesting that ADA2b acts downstream of GA signaling and is distinct from GCN5 activity. In conclusion, we propose that the action of GCN5 on stamen elongation is partially mediated by RGA and GA signaling.

Our objective was to identify differentially expressed watermelon genes in its interaction with ZYMV through a bioinformatics analysis of differentially expressed miRNAs. Small RNAseq data analysis of healthy and ZYMV-infected watermelon (21 dpi) identified 353 miRNAs from which 22 known and 331 new miRNAs. Important information about their precursors, their length, the loci of which they originated on watermelon genome are provided. The ZYMV genome could be a target for mir396a-3p, miR8706a, and miR1886i-5p from the miRbase, but none of them was identified in the watermelon miRNAome. Furthermore, watermelon miRNAome does not contain a miRNA targeting ZYMV genome with an expectation score ≤ 3.5. In healthy watermelon bioinformatically predicted targets of 32 miRNAs were 34 resistance genes, as CC-NBS-LRR, TIR-NBS-LRR, TIR-NBS. For nine differentially expressed miRNAs the respective target genes (10 in total) were bioinformatically predicted. For cla-new_miR307 (upregulated upon ZYMV infection) and cla-miR166h-3p (downregulated upon ZYMV infection) the targets were predicted to be ClaATRIP and ClaRBOHB, respectively, with ClaATRIP downregulated and ClaRBOHB upregulated upon ZYMV infection. ALSV-mediated VIGS of ClaATRIP rendered watermelon plants more resistant to ZYMV, whereas VIGS of ClaRBOHB resulted in higher levels of ZYMV titer in watermelon. These data suggest that ClaATRIP and ClaRBOHB are a susceptibility and resistant gene, respectively. Our results provide new insights in watermelon miRNAome and could propose new strategies for generating resistant watermelon to ZYMV.

Main conclusion Silencing of an ascorbate oxidase ( AO ) gene in N. benthamiana enhanced disease severity from cucumber mosaic virus (CMV), showing higher accumulation and expansion of the spreading area of CMV. Abstract A Nicotiana benthamiana ascorbate oxidase ( NbAO ) gene was found to be induced upon cucumber mosaic virus (CMV) infection. Virus-induced gene silencing (VIGS) was employed to elucidate the function of AO in N. benthamiana . The tobacco rattle virus (TRV)-mediated VIGS resulted in an efficient silencing of the NbAO gene, i.e., 97.5% and 78.8% in relative quantification as compared to the control groups (TRV::eGFP- and the mock-inoculated plants), respectively. In addition, AO enzymatic activity decreased in the TRV::NtAO-silenced plants as compared to control. TRV::NtAO-mediated NbAO silencing induced a greater reduction in plant height by 15.2% upon CMV infection. CMV titer at 3 dpi was increased in the systemic leaves of NbAO -silenced plants (a 35-fold change difference as compared to the TRV::eGFP-treated group). Interestingly, CMV and TRV titers vary in different parts of systemically infected N. benthamiana leaves. In TRV::eGFP-treated plants, CMV accumulated only at the top half of the leaf, whereas the bottom half of the leaf was “occupied” by TRV. In contrast, in the NbAO -silenced plants, CMV accumulated in both the top and the bottom half of the leaf, suggesting that the silencing of the NbAO gene resulted in the expansion of the spreading area of CMV. Our data suggest that the AO gene might function as a resistant factor against CMV infection in N. benthamiana .

Histone acetyltransferases (HAT) modify the amino-terminal tails of the core histone proteins via acetylation, regulating chromatin structure and transcription. The GENERAL CONTROL NON-DEREPRESSIBLE 5 (GCN5) is a HAT that specifically acetylates H3K14 residues. GCN5 has been associated with cell division and differentiation, meristem function, root, stem, foliar and floral development, and plant environmental response. The flowers of gcn5–6 plants display reduced length of stamen and exhibit male sterility relative to the wild-type plants. We show these effects may arise from gibberellin (GA) signaling defects. The signaling pathway of bioactive GAs depends on the proteolysis of their repressors, DELLA proteins. The DELLA protein, REPRESSOR OF GA (RGA), represses plant growth, inflorescence, flower and seed development. Our molecular data indicate that GCN5 is required for activation and H3K14 acetylation of genes involved in the late stages of GA biosynthesis and catabolism. We studied the genetic interaction of RGA and GCN5; RGA can partially suppress GCN5 action. The reduced elongation of the stamen filament of gcn5–6 mutants is reversed in the rga–t2;gcn5–6 double mutants. This mechanism involved suppressing the GCN5 effect on the expression and histone acetylation in GAI -locus by RGA. Interestingly, RGA and RGL2 do not suppress ADA2b function, suggesting that ADA2b acts downstream in GA signaling and is distinct from GCN5 activity. In conclusion, we propose that the action of GCN5 on stamen elongation is mediated partially by RGA and GA signaling.

AbstractSmall RNA sequencing of healthy and ZYMV-infected watermelon (21 dpi) was performed and bioinformatics analysis identified 353 miRNAs from which 22 known and 331 new miRNAs. Important information about their precursors, their length, the loci of which they originated on watermelon genome are provided. The ZYMV genome could be a target for mir396a-3p, miR8706a, and miR1886i-5p from the miRbase, but none of them was identified in the watermelon miRNAome. Furthermore, watermelon miRNAome does not contain a miRNA targeting ZYMV genome with an expectation score ≤ 3.5. There were 34 resistance genes (CC-NBS-LRR, TIR-NBS-LRR, TIR-NBS) predicted as targets of 32 miRNAs in healthy watermelon. For nine differentially expressed miRNAs the respective target genes (10 in total) were bioinformatically predicted. For cla-new_miR307 (upregulated upon ZYMV infection) and cla-miR166h-3p (downregulated upon ZYMV infection) the targets were predicted to beClaATRIPandClaRBOHB, respectively, withClaATRIPdownregulated andClaRBOHBupregulated upon ZYMV infection. ALSV-mediated VIGS ofClaATRIPrendered watermelon plants more resistant to ZYMV, whereas VIGS ofClaRBOHBresulted in higher levels of ZYMV titer in watermelon. These data suggest thatClaATRIPandClaRBOHBare a susceptibility and resistant gene, respectively. Our results provide new insights in watermelon miRNAome and could propose new strategies for generating resistant watermelon to ZYMV.HighlightsBioinformatics analysis in healthy and ZYMV-infected watermelon plants gave 353 miRNAs, 22 known and 331 new.The watermelon miRNAome identified in the present study does not appear to target the ZYMV genome (sense and reverse complement).The differential expression of ten genes of watermelon in relation to ZYMV infection was validated.The silencing of the two genes,ATRIPandRBOHB, through VIGS strongly suggested thatATRIPis a gene of susceptibility andRBOHBis a gene of resistance.

Zucchini yellow mosaic virus (ZYMV) infects cucurbits causing yellow mosaic in leaves, malformations in fruits, and degradation of the product quality. RNA interference (RNAi) is a cellular mechanism in eukaryotes and it is exploited to protect them against viruses. The artificial micro RNA (amiRNA) mediated approach was employed to develop resistance against ZYMV. Four amiRNAs, amiZYMV_HC-115s and amiZYMV_HC-1162s (sense), amiZYMV_HC-182as and amiZYMV_HC-196as (antisense), were computationally designed and introduced into the AtMIR390a backbone. At four days post agroinfiltration (dpa) of zucchini cotyledons the corresponding pre- and the mature amiRNAs were identified in local tissue. Upon ZYMV inoculation of zucchini, ZYMV titer was significantly lower where amiZYMV_HCs were applied in relation to control starting at two days post inoculation (dpi). Control zucchini plants exhibited symptoms at 5-8 dpi, whereas the amiZYMV_HC-treated zucchini had symptoms at 14 dpi; at 21 dpi treated zucchini exhibited a 16 %, 19 %, 32 %, and 42.5 % protection, respectively. For luffa, we observed a lower protection (0 %, 17 %, 22.5 %, and 31 % at 21 dpi). Nicotiana benthamiana DCL4 knock-down mutants were infected by ZYMV, whereas when the amiZYMV_HC-196as was agroinfiltrated ZYMV was not detected by RT-PCR. These results indicate that amiRNA-mediated resistance could be applied against ZYMV in zucchini.

Plant viruses cause nearly half of the emerging plant diseases worldwide, contributing to 10-15% of crop yield losses. Control of plant viral diseases is mainly accomplished by extensive chemical applications targeting the vectors (i.e., insects, nematodes, fungi) transmitting these viruses. However, these chemicals have a significant negative effect on human health and the environment. RNA interference is an endogenous, cellular, sequence-specific RNA degradation mechanism in eukaryotes induced by double-stranded RNA molecules that has been exploited as an antiviral strategy through transgenesis. Because genetically modified crop plants are not accepted for cultivation in several countries globally, there is an urgent demand for alternative strategies. This has boosted research on exogenous application of the RNA-based biopesticides that are shown to exhibit significant protective effect against viral infections. Such environment-friendly and efficacious antiviral agents for crop protection will contribute to global food security, without adverse effects on human health.

The phytophagy of the predator Nesidiocoris tenuis (Hemiptera: Miridae) can trigger defense responses in tomato plants against pests, such as two spotted spider mite Tetranychus urticae (Acari: Tetranychidae) and South American leaf miner Tuta absoluta (Lepidoptera: Gelechiidae). The expression of genes governing Jasmonic Acid (JA) biosynthesis pathway and fluctuations in the levels of underlying metabolites have been rarely studied in mirid-infested plants. In the present study, fifteen 3rd instar nymphs of N.tenuis were caged on each top and lower leaf of tomato plants for 4 d to induce plant defense; after this period the predators were removed. With regard to T. absoluta, oviposition preference; larval period; and pupal weight were significantly reduced in N. tenuis-punctured plants. T. urticae adults exhibited a significantly higher escape tendency and reduced survival on punctured plants. Metabolomics confirmed such observations revealing substantial differences between N. tenuis-punctured and unpunctured (control) plants. Metabolites directly associated with the activation of the JA defense pathway, such as the precursor α-linolenic acid, had increased concentrations. The expression of the defense-related genes PI-II, MYC2, VSP2, and HEL was increased in the top leaves and only VSP2 and MBP2 in the lower leaves; interestingly, in the middle (unpunctured) leaves VSP2, HEL, and MBP2 were also upregulated, indicating systemic signaling. Collectively, phytophagy of N. tenuis caused adverse effects on T. absoluta and T. urticae, whereas the multi-omics approach (phenomics, metabolomics, and genomics) offered valuable insights into the nature of the plant defense responses and provided useful evidence for future applications in integrated pest management, plausibly resulting in the reduction in the required pesticide volumes.

Exogenous application of double-stranded RNA (dsRNA) in the tobacco– Tobacco mosaic virus (TMV) pathosystem was shown previously to induce resistance against TMV providing an alternative approach to transgenesis. In the present study, we employed proteomics technology to elucidate the effect of TMV on tobacco as well as the effect of exogenous application of TMV p126 dsRNA molecules (dsRNAp126) at an early stage of the tobacco–TMV interaction. The proteome of tobacco leaf at 15 min post inoculation (mpi) in the presence or absence of dsRNAp126 molecules was studied. Thirty-six tobacco proteins were differentially accumulated in TMV-infected vs. healthy tobacco leaf tissue. The identified main differential TMV-responsive proteins were found to be involved in photosynthesis, energy metabolism, stress, and defense responses. Most of the virus-induced changes in the tobacco leaf proteome were not observed in the leaves treated with dsRNAp126 + TMV. The results indicated that the protein changes induced by TMV infection were counteracted by the exogenous application of dsRNAp126 molecules. Moreover, using small RNA sequencing, we showed that the exogenously applied dsRNAp126 was efficiently processed in tobacco as early as 15 min post application (mpa) to produce small interfering RNAs (siRNAs); the dicing pattern was not affected by the presence of TMV. The presence of dsRNAp126 reduced TMV p126 RNA abundance suggesting virus titer reduction via a sequence-specific mechanism, since a non-homologous dsRNA did not protect from TMV infection nor affect TMV accumulation.

RNAi-mediated insect pest management has recently shown promising results against the most serious pest of tomato, the tomato leafminer, Tuta absoluta. This study aimed to investigate whether dsRNA (dsTa-αCOP) designed to target the T. absoluta-αCOP gene could cause adverse effects to its biocontrol agent, the mirid predator, Nesidiocoris tenuis. Oral exposure of N. tenuis to dsRNA (dsNt-αCOP) designed to target N. tenuis-αCOP resulted in a 61%, 67% and 55% reduction in its transcript level in comparison to the sucrose, dsGFP and dsTa-αCOP treatments, respectively. In addition, significantly higher mortality of 57% was recorded in dsNt-αCOP-treated N. tenuis when compared to the sucrose (7%), dsGFP (10%) and dsTa-αCOP (10%) treatments. Moreover, the predation rate of ~33–39 Ephestia kuehniella eggs per N. tenuis adult dramatically reduced to almost half in the surviving dsNt-αCOP-treated N. tenuis. This worst-case exposure scenario confirmed for the first time that the RNAi machinery is functional in this species and that the risk of exposure through the oral route is possible. In contrast, dsTa-αCOP did not cause any sub-lethal effects to N. tenuis upon oral exposure. Oral exposure of T. absoluta to dsTa-αCOP resulted in 50% mortality. In the context of a biosafety risk assessment of RNAi-mediated insect management, investigating the effects on non-target organisms is essential in order to include this method as part of an integrated pest management strategy. Based on our laboratory assays, RNAi-mediated control is compatible with the biological control of T. absoluta by its natural enemy N. tenuis, adding the RNAi approach in the armoire of integrated pest management of T. absoluta.

Citrus yellow mosaic badnavirus (CMBV) causes mosaic disease in all economically important citrus cultivars of India, with losses reaching up to 70%. CMBV belongs to the genus Badnavirus, family Caulimoviridae, possessing a circular double-stranded (ds) DNA genome with six open reading frames (ORFs I to VI), whose functions are yet to be deciphered. The RNA-silencing suppressor (RSS) activity has not been assigned to any CMBV ORF as yet. In the present study, it was found that ORFI exhibited RSS activity among all the six CMBV ORFs tested. Studies were done by employing the well-established Agrobacterium-mediated transient assay based on the transgenic Nicotiana benthamiana 16c plant line expressing the green fluorescent protein (GFP). The RSS activity of ORFI was confirmed by the analysis of the GFP visual expression in the agroinfiltrated leaves, further supported by quantification of GFP expression by RT-PCR. Based on the GFP visual expression, the CMBV ORFI was a weak RSS when compared to the p19 protein of tomato bushy stunt virus. In contrast, the ORFII, ORFIV, ORFV, ORFVI, and CP gene did not exhibit any RSS activity. Hence, ORFI is the first ORF of CMBV to be identified with RNA-silencing suppression activity.

Tomato yellow leaf curl virus (TYLCV), a whitefly-transmitted single-stranded DNA (ssDNA) virus, causes the most important viral disease of tomato worldwide. TYLCV-mediated disease is mainly controlled via extensive insecticide sprays aiming at the whitefly vector. RNA-based vaccination was proven to be a non-transgenic approach leading to efficient plant virus control. In this work, double-stranded RNA (dsRNA) molecules deriving from sequences of the C4 and V2 genes of TYLCV-Mild were produced in vitro and topically applied onto tomato plants along with the virus (via agroinfiltration). DsC4 and dsV2 application reduced disease incidence to 23 and 46 %, respectively, while TYLCV positive control reached 64 %. Bioinformatics analysis of the virus-specific small interfering RNAs (vsiRNAs) from TYLCV-infected tomato revealed 'hot' and 'cold' spots in the TYLCV-Mild genome. Interestingly, the viral C-strand had twofold siRNA reads when compared to that of the V-strand. Overall, vsiRNAs of negative and positive polarity were almost equal (53.5 vs. 46.6 %); vsiRNAs of negative polarity prevailed at the V-strand. Stem-loop RT-PCR validated the presence of six vsiRNAs (hot or cold spots) in TYLCV-Mild-infected and dsRNA-treated tomato. The exogenously applied dsRNA was found to rapidly move systemically in tomato and was detected for 54 days post treatment (dpt). The applied dsRNA molecules were successfully processed by the Dicer-like proteins (DCLs) in tomato since small interfering RNAs (siRNAs) deriving from the dsRNA were detected for at least 54 dpt. This consists the first report of dsRNA-based vaccination applied against a monopartite geminivirus.

RNA interference (RNAi) is a sequence-specific, gene silencing mechanism, induced by double-stranded RNA (dsRNA). It is a defense mechanism that protects eukaryotic cells from invasive nucleic acids such as viruses and transposons. In this study, we used a non-transgenic strategy in order to activate the antiviral RNAi mechanism against Sesbania mosaic virus (SeMV) in its natural host Sesbania grandiflora. DsRNA molecules from SeMV coat protein (CP) and movement protein (MP) (sobemovirus MP is considered a silencing suppressor) genes were produced by a two-step PCR approach followed by in vitro transcription and exogenously applied on sesbania plants along with SeMV. DsRNA for CP and MP conferred 53% and 64% resistance against SeMV, respectively based on the disease incidence data. The effect of dsRNA molecules against SeMV infection on sesbania plants was confirmed by direct antigen coating ELISA (DAC-ELISA). The strategy employed demonstrated the applicability of the RNA-based vaccination method, for the first time, to control sobemoviruses in a simple, highly specific and environmentally safe way.

The Tomato leaf curl virus (ToLCV) is a bipartite (DNA) geminivirus whereas Cucumber mosaic virus (CMV) is a tripartite RNA virus, both infecting a wide range of host plants. RNAi that is triggered by double-stranded RNA (dsRNA) molecules, is a powerful means to control plant viruses in transgenic plants, as well as in a non-transgenic manner in a process designated as ‘RNA-based vaccination’. DsRNA molecules were made for AC1/AC4, AV1/AV2 (overlapping regions of ToLCV), AC1/AC4_AV1/AV2 (designated as fusion construct), CMV-2b and CMV-2b_ToLCV-AV1/AV2 (designated as hybrid construct, containing regions from two different viruses). In tomato (Solanum lycopersicum), dsRNAs for AC1/AC4, AV1/AV2, AC1/AC4_AV1/AV2 and CMV-2b_ToLCV-AV1/AV2 conferred 45%, 60%, 50% and 55% protection against ToLCV, respectively. Experiments with tobacco (Nicotiana tabacum) showed that the dsRNA construct CMV-2b_ToLCV-AV1/AV2 conferred 33.3% protection against CMV, while CMV-2b provided 40% protection. The present study reported that the dsRNA exhibits systemic transport in tomato. This is the first case where RNA-based vaccination is functional for a bipartite geminivirus and where a single dsRNA molecule could protect against two tomato-infecting viruses, namely a DNA (ToLCV) and an RNA (CMV) plant virus.

Sri Lankan cassava mosaic virus (SLCMV) is the principal causal agent of cassava mosaic disease in the Indian subcontinent. To gain resistance against the virus, the coat protein (CP) gene, namely the AV1 of SLCMV-Adivaram isolate, was cloned in either sense or antisense orientation under the Cauliflower mosaic virus 35S promoter, and transgenic Nicotiana benthamiana plants were obtained through Agrobacterium-mediated transformation. A total of eight T1 transgenic lines, four harboring the CP-sense construct and four harboring the CP-antisense construct were challenged with agro-infectious clones of SLCMV DNA-A and DNA-B. Based on symptom exhibition at 20 days post inoculation, 3 out of the 4 CP-sense transgenic lines and all 4 CP-antisense transgenic lines showed a high level of resistance against SLCMV. In addition, a delay in symptom initiation was observed in all the transgenic lines inoculated with a high viral load at agro-dilution 1: 625 from an absorbance (A(600)) of 1. However, the resistance was more prominent at a lower viral load of 1: 1000 agro-dilution. The viral titer was lower in the SLCMV-challenged transgenic lines compared to the non-transgenic N. benthamiana plants as confirmed by quantitative PCR and dot blot analysis. Furthermore, small RNA Northern blot analysis revealed lowered amounts of virus-specific small interfering RNAs in the resistant transgenic lines as compared to the non-transgenic plants upon SLCMV infection, which correlates to lower virus titers due to resistance against the virus.

Papaya ringspot virus (PRSV) infections in papaya result in heavy yield losses, severely affecting the papaya industry worldwide, and hence warranting for effective control measures. In the past, transgenic papaya cultivars were developed that overexpressed parts of the PRSV genome and exhibited high levels of virus resistance. In the present study, a non-transgenic approach was employed, in which in vitro produced dsRNA molecules derived from a PRSV isolate from South India (PRSV-Tirupati) was tested for dsRNA-mediated protection against two isolates of PRSV through topical application of the dsRNA on papaya. The results showed that the dsRNA molecules from both the coat protein (CP) and helper component-proteinase (HC-Pro) genes of the PRSV-Tirupati isolate conferred 100 % resistance against PRSV-Tirupati infection. Further, the same dsRNA molecules were highly effective against the PRSV-Delhi isolate on the papaya cv. Pusa Nanha, conferring a resistance of 94 % and 81 %, respectively. Systemic papaya leaves of the dsRNA-treated plants were virus-free at 14 days post-inoculation, confirming the robustness of this non-transgenic virus control strategy. In contrast, the control TMV dsRNA did not protect against the PRSV infection. This study on the topical application of dsRNA opened up a new avenue for the control of papaya ringspot disease worldwide.

Due to the rapidly growing global population, food production and security is the major challenge of agriculture. Plant viruses are obligate parasites that in some instances could cause up to 100% losses in a crop (e.g. maize streak disease). Although difficult to accurately determine the global economic impact that plant viruses have on agriculture, it is estimated that US$60 billion loss in crop yields worldwide each year is due to plant viral diseases. RNA silencing (RNA interference, RNAi) is a conserved endogenous pathway of all higher eukaryotes, which controls gene expression. RNAi is induced by double-stranded RNA (dsRNA) and allows the cell to recognize aberrant genetic material in a highly sequence-specific manner ultimately leading to its degradation, thus protecting the cell from subcellular pathogens, such as viruses and transposons. DsRNA-mediated resistance has been exploited in transgenic plants to convey resistance to viruses and against insects, vectors of plant viruses, via host induced gene silencing (HIGS). A non-transgenic approach employing RNAi has been used where enzymatically synthesized specific dsRNA molecules, when applied directly onto plant tissue, induce resistance to the cognate virus; as a result dsRNA molecules could be efficacious antiviral agents for crop protection. Next generation sequencing and bioinformatics analyses have provided a plethora of information and useful tools for the design and study of dsRNA application. In this chapter, the different methods for dsRNA production, both in vitro and in vivo, the means of direct application of the dsRNA molecules onto plants and several examples of non-transgenic dsRNA-mediated resistance are presented.