Assessment of co-infection with BNYVV and BSCTV on resistance against Rhizomania disease in transgenic sugar beet plants

DOI: https://doi.org/10.21203/rs.3.rs-2059865/v1

Abstract

Sugar beet is an economically important crop and one of the major sources of sucrose. Beet necrotic yellow vein virus (BNYVV) and Beet severe curly top virus (BSCTV) are two widespread viruses in sugar beet that cause severe damage to its performance. Previously, we have successfully achieved resistance to BNVYY by introducing coat protein-based DNA sequence constructs inducing gene silencing into sugar beet. However, the RNA silencing-mediated resistance of plants to a specific virus can be potentially broken down by another one as a part of synergistic interactions. In this study, we assayed the efficiency of the induced resistance of transgenic events to BNYVV and BSCTV-Ir under single or mixed infections. All the plants inoculated with just BSCTV-Ir displayed curly-leaf symptoms. However, partial resistance was observed in S3 events based on mild symptoms and low PCR amplification of the BSCTV-Ir coat protein sequence. Similarly, partial resistance to BSCTV-Ir was detected in the same transgenic plants under co-infection conditions. Based on the presented data, resistance to BNYVV was stable in almost all the transgenic plants co-infected with BSCTV-Ir, except for one event (S3-229) that was broken down. Overall, it seems that the co-infection with BNYVV and BSCTV-Ir does not affect the resistance of transgenic plants to both viruses. These findings demonstrated that RNA silencing-based resistance to BNYVV introduced in transgenic sugar beets of this study is very efficient and is not easily weakened after co-infection with a heterologous virus.

Introduction

As a primary source of sugar production, sugar beet (Beta vulgaris L.) is one of the most important industrial crops in the world. Due to the increased global demand for sugar, the sustainability of sugar beet production is essential (Stevanato et al. 2019). Rhizomania, caused by Beet necrotic yellow vein virus (BNYVV), is one of the most devastating and widespread diseases of sugar beet that could diminish sugar beet yield by up to 80% (McGrann et al. 2009; Biancardi and Lewellen 2016). The virus is transmitted to sugar beet roots by Polymyxa betae Keskin, a plasmodiophorid vector, that can remain viable in the soil for over 15 years by forming a resting spore (Pferdmenges 2007; Biancardi and Lewellen 2016). While the use of chemicals is now phased out as part of the Montreal Protocol (McGrann et al. 2009), current resistant cultivars, such as those carrying Rz1 and/or Rz2 genes are reported to be prone to break in some regions (Pferdmenges and Varrelmann 2009; Kutluk Yilmaz et al. 2018). Thus, it is necessary to find a way to effectively deal with this disease as soon as possible.

The advent of genetic engineering has opened up new ways to control Rhizomania by introducing novel resistance genes resources (Pavli et al. 2011; Dhir et al. 2019). In recent years, several methods based on RNA silencing and predominantly pathogen-induced resistance have emerged to strengthen plant defenses against viral invasions (Palukaitis 2011; Duan et al. 2012; Uslu1 and Wassenegger 2020). Virus-induced gene silencing (VIGS) is an RNA silencing-based mechanism that innately activates the plant’s natural defense mechanism against viruses (Lu et al. 2003; Duan et al. 2012). In this approach, part of the viral genome is introduced into plant cells which generates double-stranded RNAs (dsRNA) intermediates producing short interfering RNA (siRNA) which trigger RNA silencing against virus infections (Lu et al. 2003; Duan et al. 2012).

To date, RNA silencing-mediated resistance has been effectively applied in various plants (Duan et al. 2012; Jin et al. 2020; Jiang et al. 2022). In particular, through the RNA silencing mechanism, the transgenic N. benthamiana expressing the coat protein (CP) read-through domain of BNYVV revealed very low levels of virus after inoculation (Andika et al. 2005). In another study, an inverted cDNA repeat derived from the BNYVV replicase gene was transferred into the sugar beet genome and showed considerable resistance to the virus (Lennefors et al. 2006). Transgenic hairy roots of sugar beet exhibited a remarkable resistance against Rhizomania through expressing BNYVV-derived dsRNA (Pavli et al. 2010). In our recent publications, we have shown RNA silencing-mediated resistance against Rhizomania in sugar beet through the transient and stable transformation of several constructs expressing BNYVV-derived RNA which confirmed the effectiveness of this mechanism in the greenhouse and field experiments (Zare et al. 2015; Safar et al. 2021).

However, a sugar beet field may comprise several kinds of pathogens, and the co-infection of plants by two or more viruses is quite possible (Susi et al. 2015; Moreno and López-Moya, 2020). Co-infection often leads to interactions between viruses which can affect disease development in plants both negatively (antagonistic) and positively (synergistic) (Syller 2014; Syller and Grupa 2016; Mascia and Gallitelli 2016). Syller and Grupa (2016) suggested that synergistic interactions within plants mostly occur between unrelated viruses. Numerous viral synergistic interactions have been reported that enhance infection, particularly through the suppression of RNA-silencing machinery (Li et al. 2017; Liang et al. 2017; Aulia et al. 2019). For instance, rice tungro disease is caused by synergistic interactions of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV). It was revealed that combined actions of RTBV ORF-IV and RTSV CP3 proteins play a key role in tungro symptom development by suppressing the RNA silencing of rice (Anand et al. 2022). Therefore, some concerns have been raised over the efficiency of RNA silencing-based resistance of transgenic plants under co-infection conditions.

Beet curly top virus (BCTV), a member of the Curtovirus genus, is another common and destructive virus in sugar beet fields around the world. Beet severe curly top virus (BSCTV, recently called BCTV-Svr) is a strain of BCTV named for the severity of curly symptoms in infected sugar beet. Iranian isolate of Beet severe curly top virus (BSCTV-Ir) is one of the main causal agents of the curly top disease in sugar beet farms of Iran. The C2/L2 protein of BCTV has been described as a suppressor of RNA silencing machinery (Yang et al. 2007). Besides, it was recently revealed that V2 of BCTV can also act as an inhibitor of RNA silencing (Luna et al. 2017).

Previously, we have reported effective resistance to Rhizomania in transgenic sugar beets through the introduction of CP21 BNYVV-based constructs. Considering that BNYVV and BSCTV co-infection of sugar beets growing in Iran and perhaps other parts of the world is very likely. The present study was conducted to explore the possible influence of BNYVV and BSCTV-Ir co-infection on the resistance of transgenic sugar beet plants against Rhizomania; whether synergistic interactions between these viruses exist or whether the silencing effect of CP21 BNYVV-derived mediates also be effective in inhibiting BSCTV-Ir propagation.

Materials And Methods

Plant material

Based on our previous studies (Zare et al. 2015), a number of transgenic events carrying intron-hairpin RNA (ihpRNA) construct containing the 5' UTR with or without coding sequence of CP of BNYVV, called IHP-P (S3) and IHP-U (S6), respectively, were selected. Three T1 progenies of S3-12 and one of the S3-13.2 events were chosen named 227, 228, 229, and 219, respectively. Also, two T1 progenies of S6-2 and S6-44 events named 221 and 231 were selected. A diploid monogram cultivar as a wild-type parental plant, named ‘9597’, and a cultivar called ‘Dorothea’ carrying the Rz1 gene, a Holly-based resistant plant, served as the negative and positive controls, respectively. The Sugar Beet Seed Institute of Iran kindly provided these non-transgenic cultivars.

Vegetative propagation of transgenic events 

Transgenic plants were propagated through tissue culture to obtain a sufficient number of genetically identical individuals. The culture medium was composed of MS salts (Murashige and Skoog 1962) supplemented with 0.1 mg/l IBA, 1 mg/l BA, and 0.1 mg/l GA3. The root-inducing medium was MS containing 3 mg/l NAA hormone. Clonally propagated plants were transferred into the soil composed of peat, perlite, and vermiculite at a 1:1:1 ratio and adapted under the yellow-white fluorescent bulbs with 16 hours of light photoperiod. The temperature was 25-30°C and the humidity was adjusted to 40-60%.   

Molecular analysis for transgenic plants

To select progenies carrying the transgene, a Southern blot analysis was performed. Genomic DNA was isolated from 50 mg of sugar beet leaves, according to Dellaporta et al. (1983). Genomic DNA (30 µg) was directly spotted on a positively charged nylon membrane (Roche Diagnostics, Germany) using a vacuum-assisted dot blotter tool (Gentaur BVBA, Belgium). Probes were synthesized PCR and DIG-labeled dNTP mixture using a DIG DNA labeling and detection kit (Roche Biochemical, Germany). The temperature for hybridization was 65°C and the concentration of salt for the last wash was 0.1 mM NaCl in sodium citrate buffer. Detection was done by NBT/PCIP as instructed and the darkness of dots was inspected visually. 

For genotyping of the progenies, DNA extraction of transgenic events was performed from the leaves using a GTP kit (Gene Transfer Pioneers, Iran). The presence of the transgene in each progeny was monitored by PCR amplification using gene-specific pairs of primers (Table 1).  The reaction mixture contained 1 µl (50 ng) genomic DNA template, 2 pmol of each primer, 10 µl 2X PCR master mix (Thermo Fisher Scientific, USA), 2 mM MgCl2, 200 µM of each dNTPs, and 5 U Taq DNA polymerase (Cinagen, Iran) in a volume of 30 µl. Amplification cycles were as follows: denaturation cycle at 95°C for 5 min, 40 cycles of 94°C, 60°C, and 72°C (1 min each) with a final extension step at 72°C for 10 min. The PCR products were separated on 1% agarose gels, stained with ethidium bromide, and visualized by UV light. 

Viral challenges and bioassays 

The propagated clones with 6-8 leaves were challenged with BNYVV or BSCTV-Ir viruses individually or both. Plants were transplanted into the mixture of BNYVV-infested and sterile soil at a 1:1 ratio. BSCTV-Ir infection was done through agro-infection of sugar beet plants with a full-length recombinant BSCTV-Ir construct (Ebadzad Sahraei et al. 2008) using Agrobacterium tumefaciens strain C58. To this end, the Agrobacterium was cultured in LB medium supplemented with Rifampicin and Kanamycin at 50 µg/ml and grown to OD600 1.0. The bacteria were pelleted and resuspended in MS medium supplemented with 2% sucrose, 10 mM MgCl2, and 150 µM acetosyringone at pH 5.8 and diluted to OD600 0.5. After 3 hours of incubation at room temperature, it was injected into the back of the leaves.

Table 1

General details of primers used in this study.

Name

Primer sequence (5'-3')

Tm (°C)

Target

Amplicon Size (bp)

C-2

AGCTAATTGCTATTGTCCGGGT

60

CP21- 

736

CS-1

CGCATATCTCATTAAAGCAGGACTCTA

60

ORF

 

C-1

TTCTCATTAGTACCAGCAGTTTT

60

CP21-

460

U+2

CTCGAGAATAGAATTTCACCGTCTG

60

ORF

 

PIF

CAAGGTAACATGATAGATCATGTCATTGTG

67

CP21-

333

TOCS

AAACCGGCGGTAAGGATCTG

67

UTR

 

BSCTV-Ir

AGAAAATATACAAGAAATC

41

V1/CP

761

BSCTV-I

TTAATAAAAATAACATCTAC

41

 

 

After 30 days of BSCTV-Ir infection, the presence of the virus was detected by PCR for an expected band of 761 bp using a pair of primers (Table 1). Total DNA was isolated from 50 mg of sugar beet leaves using the i-Genomic Plant DNA Extraction Mini Kit (Intron Biotechnology, South Korea). The PCR reaction mixture and program were carried out as above.

After 60 days of inoculation, BNYVV titers for each event were estimated using the enzyme-linked immunosorbent assay (ELISA) either by a DAS-ELISA kit (BIOREBA, Switzerland) based on the instructions provided by the manufacturer or according to Clark and Adams (1977) using an anti-CP21 antibody provided by Dr. Izadpanah (Shiraz University, Iran). Following overnight incubation of the reaction mixture, absorbance for each sample was measured at 405 nm. The cut-off value was mean+3SD for non-infected wild-type plants. If the absorbance was more than two times the cut-off value, the plant was considered susceptible, whereas if it was below the cut-off, the plant was resistant, and if between one and two cut-off values, the plant was designated as tolerant. To assure the infection process, P. betae spores were stained with lactophenol fussing acid and observed microscopically.

Statistical analyses

Analysis of variance (ANOVA) for bioassay data was performed in a factorial experiment with a completely randomized design and three replications. In the following, the means were compared using Duncan’s multiple range test (P<0.05). All the statistical analyses were conducted with the use of SPSS software (IBM, USA).

Bioinformatics data analysis

To examine the possible similarity between the CP of BNYVV (GenBank Accession No. AY277887) and BSCTV (GenBank Accession No. X97203), their nucleotide sequences were pairwise aligned with MegAlign software in Lasergene package (DNASTAR, USA).

Results

Following previous studies (Zare et al. 2015; Safar et al. 2021), six T1 progenies of transgenic events with induced silencing against BNYVV CP21 were selected. As summarized in Table 2, the presence of the transgene and the expected effects on the selected events were verified using dot blot, PCR, and ELISA methods.

The data collected for the infected and non-infected plants with BNYVV and BSCTV-Ir have been briefed in Table 3. After agro-infection with recombinant BSCTV-Ir DNA constructs, S3 events showed mild leaf curl symptoms while severe symptoms were observed in S6 events, Dorothea, and wild-type ‘9597’ cultivar (Fig. 1). The same patterns of symptoms were also observed in co-infected plants. Accordingly, lower levels of PCR products were detected in S3 plants using BSCTV-Ir primer pairs (Fig. 2, Table 3).  

Since partial resistance was observed in some transgenic events infected with BSCTV-Ir, the possible sequence identity between the coding sequence of BNYVV and BSCTV-Ir coat proteins was investigated using pairwise alignment. As shown in Fig. 3, substantial sequence identities were observed in some regions between the nucleotide sequence of BNYVV and BSCTV-Ir.

Table 2

 Summarized data of genotyping by dot blots and PCR and viral propagation inhibition by ELISA for the selected events.

Plant No.

Construct

Event

Dot Blot1

PCR

ELISA2

227

IHP-P

S3-12

++

+

0.14

228

IHP-P

S3-12

++

+

0.07

229

IHP-P

S3-12

+++

+

0.10

219

IHP-P

S3-13.2

ND3

+

0.32

221

IHP-U

S6-2

+

+

0.16

231

IHP-U

S6-44

+

+

0.01

95974

-

-

-

-

0.79

1. The plants that had a darker spot compared to the non-transgenic wild-type parent (9597) were shown by positive marks and the numbers of these marks indicate the rate of darkness.

2. Average ELISA value as an indicator for detectable viral propagation.

3. ND: not determined.

4. 9597 is the non-transgenic parental plant used as a control.

The root infection of clonally propagated plants challenged with BNYVV and BSCTV-Ir viruses, individually or together, was assessed after 60 days. The BNYVV infection was confirmed as P. betae spores were detected in the roots of all examined plants by microscopic observations (Table 3). Based on the ELISA data, fourteen S3 plants were challenged with only BNYVV, almost all of which remained resistant or tolerant for the duration of the experiment. Among those plants with BNYVV and BSCTV-Ir co-infection, 6 plants were resistant and 4 tolerant to BNYVV, while 2 plants showed susceptibility when they were infected by both BNYVV and BSCTV-Ir. For S6 construct, 13 plants were infected by BNYVV or co-infected by both BNYVV and BSCTV-Ir. Just one plant was susceptible to BNYVV when it was infected with BNYVV only. In all S6 plants, the co-infection of BNYVV and BSCTV-Ir did not affect the symptoms of the latter virus.

To reduce the positional effects of gene insertion and genotype variations, the mean of the 6 selected transgenic events (4 events of S3 and 2 from S6) was compared (Fig. 4). In all plants, except S3-229, no significant difference was observed between BNYVV single infection and its co-infection with BSCTV-Ir. For S3-229 case, the BNYVV accumulation was significantly higher in co-infection treatments than in single infections. The wild-type cultivar also showed higher BNYVV titers under co-infection conditions, though it was not significant.

Table 3

 The summarized data of infected and non-infected plants with BNYVV and/or BSCTV-IR viruses.

Event

Progeny

Treatments

Repeats

BSCTV-IR

Spore2

BNYVV

Symptom1

PCR

No. of Susceptible

No. of Resistance

No. of Tolerant

Positive

Negative

S3

227

None

3

-

0

3

-

-

-

-

 

BNYVV only

4

-

-

-

+

0

3

1

 

BSCTV-Ir only

3

+

2

1

-

-

-

-

 

BNYVV & BSCTV-Ir

3

+

3

0

+

0

2

1

S3

228

None

3

-

0

3

-

-

-

-

 

BNYVV only

4

-

-

-

+

0

3

1

 

BSCTV-Ir only

3

+

3

0

-

-

-

-

 

BNYVV & BSCTV-Ir

3

+

1

2

+

0

2

1

S3

229

None

3

-

0

3

-

-

-

-

 

BNYVV only

3

-

-

-

+

0

2

1

 

BSCTV-Ir only

3

+

3

0

-

-

-

-

 

BNYVV & BSCTV-Ir

3

+

2

1

+

2

0

1

S3

219

None

3

-

0

3

-

-

-

-

 

BNYVV only

3

-

-

-

+

1

2

0

 

BSCTV-Ir only

3

+

3

0

-

-

-

-

 

BNYVV & BSCTV-Ir

3

+

0

3

+

0

2

1

S6

221

None

3

-

0

3

-

-

-

-

 

BNYVV only

3

-

-

-

+

0

2

1

 

BSCTV-Ir only

3

++

3

0

-

-

-

-

 

BNYVV & BSCTV-Ir

4

++

3

0

+

0

4

0

S6

231

None

3

-

0

3

-

-

-

-

 

BNYVV only

3

-

-

-

+

1

2

0

 

BSCTV-Ir only

3

++

3

0

-

-

-

-

 

BNYVV & BSCTV-Ir

3

++

3

0

+

0

3

-

Wild type

(9597)

 

None

3

-

0

3

-

-

-

-

 

BNYVV only

5

-

-

-

+

4

0

1

 

BSCTV-Ir only

3

+++

3

0

-

-

-

-

 

BNYVV & BSCTV-Ir

5

+++

3

0

+

3

0

2

Dorothea

 

None

3

-

0

3

-

-

-

-

 

BNYVV only

6

-

-

-

+

0

6

0

 

BSCTV-Ir only

3

+++

3

0

-

-

-

-

 

BNYVV & BSCTV-Ir

7

+++

3

0

+

1

4

2

1. The symptoms of the BSCTV-Ir virus. The number of + symbols indicates the severity of symptoms.

2. Microscopic slides were obtained from the infected sugar beet roots to confirm BNYVV inoculation. The plus sign means the existence of P. betae spores.

 

Discussion

In order to explore the possible effects of viral co-infection on the efficiency of RNA Silencing-mediated resistance, transgenic events were exposed to BNYVV and BSCTV-Ir individually and together. Almost all transgenic events were resistant to single infections of BNYVV. Consistent with our previous studies (Zare et al. 2015; Safar et al. 2021), the reduced spread of BNYVV in transgenic events indicates the effectiveness of the CP21-based inserts in inducing resistance against Rhizomania. Similarly, other researchers have already shown that the introduction of BNYVV-based constructs can be an effective way to control Rhizomania (Mannerlöf et al. 1996; Lennefors et al. 2006; Lennefors et al. 2008). Considerable resistance against Rhizomania disease was achieved through the transformation of the BNYVV replicase gene-derived dsRNA into sugar beet plants (Pavli et al. 2010). Transgenic Nicotiana benthamiana plants encoding CP readthrough protein exhibited high resistance to BNYVV (Andika et al. 2005).

On the other hand, the transgenic plants carrying S6 constructs revealed severe curly top symptoms, when subjected to BSCTV-Ir. However, S3 events with IHP-P construct moderately resisted BSCTV-Ir compared to control and S6 event carrying IHP-U. Such resistance to a particular virus infection in transgenic plants while containing the insert derived from another virus is generally referred to as heterologous resistance (Dinant et al. 1993). So far, several cases of heterologous resistance in different transgenic plants have been reported (Dinant et al. 1993; Hassairi et al. 1998; Peng et al. 2014; Ali et al. 2019). Medina-Hernández et al. (2013) evaluated the efficiency of Tomato Chino La Paz virus (ToChLPV)-derived construct for resistance against Pepper Golden Mosaic virus (PepGMV) in Nicotiana benthamiana plants. It was shown that the severity of PepGMV symptoms was reduced to 45% in transgenic plants with this structure which is in line with our results. As shown (Fig. 3), there are noticeable homologies between BNYVV and BSCTV-Ir coat proteins in some regions of their coding sequences. Therefore, the slight resistance to BSCTV-Ir observed in S3-based events may be induced by some BNYVV CP-derived siRNAs.

Likewise, all control and transgenic plants exhibited severe curly top symptoms when co-infected with BNYVV and BSCTV-Ir except for S3 events which slightly resisted it. On the other hand, almost all transgenic events showed stable resistance to BNYVV compared to wild-type plants under co-infection conditions. Yet, the high titer of BNYVV in 3 out of 28 S3 progenies (Table 3) needs further investigations. It might be due to either interaction of a suppressor protein encoded by the BSCTV-Ir virus, rearrangement of the transgene, or co-infection with another soil-borne virus. Regardless of this exception, overall, co-infection with BNYVV and BCTV-Ir does not appear to affect the RNA-silencing-based resistance of transgenic sugar beets. Similar to our findings, it was revealed that co-infection with heterologous viruses does not always suppress the resistance of transgenic plants (Vassilakos, 2012). For instance, the co-infection Plum pox virus (PPV) with either Apple chlorotic leaf spot virus (ACLSV) or Prune dwarf virus (PDV) did not suppress RNA silencing-based resistance in transgenic plum (Prunus domestica L.) with the PPV coat protein gene (Singh et al. 2019). The RNA silencing-based resistance against the BNYVV was not affected by co-infection with either Beet Soil-Borne virus, Beet Virus Q, Beet Mild Yellowing virus, or Beet Yellows virus (BYV) in transgenic sugar beets (Lennefors et al. 2008).

In summary, the results indicate that BNYVV CP21-based constructs are highly efficient in inducing resistance to Rhizomania and can be used as a powerful means in breeding programs for the control of this disease in sugar beet. Moreover, the siRNAs generated from them can be fairly effective in inducing heterologous resistance to other sugar beet viruses like BSCTV-Ir. It was also demonstrated that the co-infection of BSCTV-Ir with BNYVV does not affect the efficiency of inducing silencing by the constructs producing RNA with hair-pin structures. Overall, the induced RNA silencing-based resistance was stable in transgenic plants under both single and multiple infection conditions and, therefore, it can be a suitable alternative to the conventional breeding cultivars for BNYVV resistance.

Declarations

Acknowledgments

This research was supported and funded by the National Institute of Genetic Engineering and Biotechnology (NIGEB) of Iran (Grant Nos. 167 and 102M) and Green Transgene Technology Development Company (Tehran, Iran).  

Conflict of interest

The authors have no relevant financial or non-financial interests to disclose. 

Ethics approval

Not applicable. 

Consent to participate

Not applicable. 

Consent for publication

Not applicable.

References

  1. Ali I, Khurshid M, Iqbal Z, Shafiq M, Amin I, Mansoor S, Briddon R (2019) The antisense 5'end of the V2 gene confers enhanced resistance against the monopartite begomovirus cotton leaf curl Kokhran virus-Burewala strain. Acta Virologica 63(1):26-35. https://doi.org/10.4149/av_2019_101
  2. Anand A, Pinninti M, Tripathi A, Mangrauthia SK, Sanan-Mishra N (2022) Coordinated action of RTBV and RTSV proteins suppress host RNA silencing machinery. Microorganisms 10(2):197. https://doi.org/10.3390/microorganisms10020197
  3. Andika IB, Kondo H, Tamada T (2005) Evidence that RNA silencing-mediated resistance to Beet necrotic yellow vein virus is less effective in roots than in leaves. Molecular Plant-Microbe Interactions 18(3):194-204. https://doi.org/10.1094/MPMI-18-0194
  4. Aulia A, Andika IB, Kondo H, Hillman BI, Suzuki N (2019) A symptomless hypovirus, CHV4, facilitates stable infection of the chestnut blight fungus by a coinfecting reovirus likely through suppression of antiviral RNA silencing. Virology 533:99-107. https://doi.org/10.1016/j.virol.2019.05.004
  5. Biancardi E, Lewellen RT (2016) Introduction. In: Biancardi E, Tamada T (eds) Rhizomania. Springer, Cham, pp 3-28. https://doi.org/10.1007/978-3-319-30678-0_1
  6. Clark MF, Adams A (1977) Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. Journal of General Virology 34(3):475-83. https://doi.org/10.1099/0022-1317-34-3-475
  7. Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Molecular Biology Reporter 1(4):19-21. https://doi.org/10.1007/BF02712670
  8. Dhir S, Srivastava A, Yoshikawa N, Khurana S (2019) Plant viruses as virus induced gene silencing (VIGS) vectors. In: Khurana SMP, Gaur RK (eds) Plant Biotechnology: Progress in Genomic Era. Springer, Singapore, pp 517-26. https://doi.org/10.1007/978-981-13-8499-8_22
  9. Dinant S, Blaise F, Kusiak C, Astier-Manifacier S, Albouy J (1993) Heterologous resistance to potato virus Y in transgenic tobacco plants expressing the coat protein gene of lettuce mosaic potyvirus. Phytopathology 83(8):818-24. https://doi.org/10.1094/Phyto-83-818
  10. Duan C-G, Wang C-H, Guo H-S (2012) Application of RNA silencing to plant disease resistance. Silence 3:5. https://doi.org/10.1186/1758-907X-3-5
  11. Ebadzad Sahraei G, Behjatnia SAA, Izadpanah K (2008) Infectivity of the cloned genome of Iranian isolate of Beet severe curly top virus in experimental hosts. Iranian Journal of Plant Pathology 44(2):176-83
  12. Hassairi A, Masmoudi K, Albouy J, Robaglia C, Jullien M, Ellouz R (1998) Transformation of two potato cultivars ‘Spunta’and ‘Claustar’(Solanum tuberosum) with lettuce mosaic virus coat protein gene and heterologous immunity to potato virus Y. Plant Science 136(1):31-42. https://doi.org/10.1016/S0168-9452(98)00078-8
  13. Jiang L, Du Z, Zhang G, Wang T, Jin G (2022) Advances in RNA-Silencing-Related Resistance against Viruses in Potato. Genes 13(5):731. https://doi.org/10.3390/genes13050731
  14. Jin F, Song J, Xue J, Sun H, Zhang Y, Wang S, Wang Y (2020) Successful generation of anti-ToCV and TYLCV transgenic tomato plants by RNAi. Biologia Plantarum 64:490-6. https://doi.org/10.32615/bp.2020.069
  15. Kutluk Yilmaz ND, Uzunbacak H, Arli-Sokmen M, Kaya R (2018) Distribution of resistance-breaking isolates of beet necrotic yellow vein virus differing in virulence in sugar beet fields in Turkey. Acta Agriculturae Scandinavica, Section B — Soil & Plant Science 68(6):546-54. https://doi.org/10.1080/09064710.2018.1441432
  16. Lennefors B-L, Savenkov EI, Bensefelt J, Wremerth-Weich E, van Roggen P, Tuvesson S, Valkonen J, Gielen J (2006) dsRNA-mediated resistance to Beet Necrotic Yellow Vein Virus infections in sugar beet (Beta vulgaris L. ssp. vulgaris). Molecular Breeding 18(4):313-25. https://doi.org/10.1007/s11032-006-9030-5
  17. Lennefors B-L, van Roggen PM, Yndgaard F, Savenkov EI, Valkonen J (2008) Efficient dsRNA-mediated transgenic resistance to Beet necrotic yellow vein virus in sugar beets is not affected by other soilborne and aphid-transmitted viruses. Transgenic Research 17(2):219-28. https://doi.org/10.1007/s11248-007-9092-0
  18. Li S, Zhang T, Zhu Y, Zhou G (2017) Co-infection of two reoviruses increases both viruses accumulation in rice by up-regulating of viroplasm components and movement proteins bilaterally and RNA silencing suppressor unilaterally. Virology Journal 14(1):1-8. https://doi.org/10.1186/s12985-017-0819-0
  19. Liang Q-l, Wei L-x, Xu B-l, Calderón-Urrea A, Xiang D (2017) Study of viruses co-infecting white clover (Trifolium repens) in China. Journal of Integrative Agriculture 16(9):1990-8. https://doi.org/10.1016/S2095-3119(16)61606-4
  20. Lu R, Martin-Hernandez AM, Peart JR, Malcuit I, Baulcombe DC (2003) Virus-induced gene silencing in plants. Methods 30(4):296-303. https://doi.org/10.1016/s1046-2023(03)00037-9
  21. Luna AP, Rodríguez-Negrete EA, Morilla G, Wang L, Lozano-Durán R, Castillo AG, Bejarano ER (2017) V2 from a curtovirus is a suppressor of post-transcriptional gene silencing. Journal of General Virology 98(10):2607-14. https://doi.org/10.1099/jgv.0.000933
  22. Mannerlöf M, Lennerfors B-L, Tenning P (1996) Reduced titer of BNYVV in transgenic sugar beets expressing the BNYVV coat protein. Euphytica 90(3):293-9. https://doi.org/10.1007/BF00027479
  23. Mascia T, Gallitelli D (2016) Synergies and antagonisms in virus interactions. Plant Science 252:176-92. https://doi.org/10.1016/j.plantsci.2016.07.015
  24. McGrann GR, Grimmer MK, Mutasa-Göttgens ES, Stevens M (2009) Progress towards the understanding and control of sugar beet rhizomania disease. Molecular Plant Pathology 10(1):129-41. https://doi.org/10.1111/j.1364-3703.2008.00514.x
  25. Medina-Hernández D, Rivera-Bustamante RF, Tenllado F, Holguín-Peña RJ (2013) Effects and effectiveness of two RNAi constructs for resistance to Pepper golden mosaic virus in Nicotiana benthamiana plants. Viruses 5(12):2931-45. https://doi.org/10.3390/v5122931
  26. Moreno AB, López-Moya JJ (2020) When viruses play team sports: mixed infections in plants. Phytopathology 110(1):29-48. https://doi.org/10.1094/PHYTO-07-19-0250-FI
  27. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15(3):473-97. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
  28. Palukaitis P (2011) The road to RNA silencing is paved with plant-virus interactions. The Plant Pathology Journal 27(3):197-206. https://doi.org/10.5423/PPJ.2011.27.3.197
  29. Pavli OI, Panopoulos NJ, Goldbach R, Skaracis GN (2010) BNYVV-derived dsRNA confers resistance to rhizomania disease of sugar beet as evidenced by a novel transgenic hairy root approach. Transgenic Research 19(5):915-22. https://doi.org/10.1007/s11248-010-9364-y
  30. Pavli OI, Stevanato P, Biancardi E, Skaracis GN (2011) Achievements and prospects in breeding for rhizomania resistance in sugar beet. Field Crops Research 122(3):165-72. https://doi.org/10.1016/j.fcr.2011.03.019
  31. Peng J-C, Chen T-C, Raja JA, Yang C-F, Chien W-C, Lin C-H, Liu F-L, Wu H-W, Yeh S-D (2014) Broad-spectrum transgenic resistance against distinct tospovirus species at the genus level. PloS One 9(5):e96073. https://doi.org/10.1371/journal.pone.0096073
  32. Pferdmenges F (2008) Occurrence, spread and pathogenicity of different Beet necrotic yellow vein virus (BNYVV) isolates. Cuvillier Verlag, Göttingen
  33. Pferdmenges F, Varrelmann M (2009) Breaking of Beet necrotic yellow vein virus resistance in sugar beet is independent of virus and vector inoculum densities. European Journal of Plant Pathology 124(2):231-45. https://doi.org/10.1007/s10658-008-9408-9
  34. Safar S, Bazrafshan M, Khoshnami M, Behrooz AA, Hedayati F, Maleki M, Mahmoudi SB, Malboobi MA (2021) Field evaluation for rhizomania resistance of transgenic sugar beet events based on gene silencing. Canadian Journal of Plant Pathology 43(1):179-88. https://doi.org/10.1080/07060661.2020.1783575
  35. Singh K, Neubauerová T, Kundu JK (2019) Quantitative analysis of the interaction of heterologous viruses with Plum pox virus in C5 HoneySweet transgenic plums. Journal of Integrative Agriculture 18(10):2302-10. https://doi.org/10.1016/S2095-3119(18)62136-7
  36. Stevanato P, Chiodi C, Broccanello C, Concheri G, Biancardi E, Pavli O, Skaracis G (2019) Sustainability of the sugar beet crop. Sugar Tech 21(5):703-16. https://doi.org/10.1007/s12355-019-00734-9
  37. Susi H, Barrès B, Vale PF, Laine A-L (2015) Co-infection alters population dynamics of infectious disease. Nature Communications 6:5975. https://doi.org/10.1038/ncomms6975
  38. Syller J (2014) Biological and molecular events associated with simultaneous transmission of plant viruses by invertebrate and fungal vectors. Molecular Plant Pathology 15(4):417-26. https://doi.org/10.1111/mpp.12101
  39. Syller J, Grupa A (2016) Antagonistic within‐host interactions between plant viruses: molecular basis and impact on viral and host fitness. Molecular Plant Pathology 17(5):769-82. https://doi.org/10.1111/mpp.12322
  40. Uslu VV, Wassenegger M (2020) Critical view on RNA silencing-mediated virus resistance using exogenously applied RNA. Current Opinion in Virology 42:18-24. https://doi.org/10.1016/j.coviro.2020.03.004
  41. Vassilakos N (2012) Stability of transgenic resistance against plant viruses. In: Çiftçi YO (ed) Transgenic Plants: Advances and Limitations. IntechOpen, London, pp 219-36. https://doi.org/10.5772/33133
  42. Yang X, Baliji S, Buchmann RC, Wang H, Lindbo JA, Sunter G, Bisaro DM (2007) Functional modulation of the geminivirus AL2 transcription factor and silencing suppressor by self-interaction. Journal of Virology 81(21):11972-81. https://doi.org/10.1128/JVI.00617-07
  43. Zare B, Niazi A, Sattari R, Aghelpasand H, Zamani K, Sabet M, Moshiri F, Darabie S, Daneshvar M, Norouzi P, Kazemi-Tabar SK, Khoshnami M, Malboobi MA (2015) Resistance against rhizomania disease via RNA silencing in sugar beet. Plant Pathology 64(1):35-42. https://doi.org/10.1111/ppa.12239