3 Biotech (2020) 10:278 https://doi.org/10.1007/s13205-020-02276-4

ORIGINAL ARTICLE

Efcient silencing gene construct for resistance to multiple common bean (Phaseolus vulgaris L.) viruses

Abdolbaset Azizi1 · Jeanmarie Verchot2 · Ahmad Moieni1 · Masoud Shams‑bakhsh1

Received: 14 January 2019 / Accepted: 25 May 2020 © King Abdulaziz City for Science and Technology 2020

Abstract One promising strategy to engineer plants that are resistant to plant pathogens involves transforming plants with RNA silencing constructs for resistance to multiple pathogens. Garden bean is signifcantly damaged by bean common mosaic virus (BCMV), bean common mosaic necrosis virus (BCMNV) and cucumber mosaic virus (CMV). In this study, we prepared constructs producing sense, antisense and hairpin RNA (hpRNA) structures to target single as well as multiple viruses. Silencing efciency of these constructions was analyzed using Agrobacterium (GV3101) transient expression in Nicothinia bethamiana and Phaseolus vulgaris plants. The results showed signifcantly reduced disease symptoms and virus accumulation in N. bethamiana plants. Generally, the efciency of the prepared constructs was hairpin, antisense and sense, respectively, and also, there was a signifcant diference between mono-gene and multiple-gene constructs for reducng virus accumulation and the multiple-gene constructs showed higher efectiveness. Experiments in this study showed that using Agrobacterium harboring binary constructs containing a Caenorhabditis elegans gene, Ced-9, or a plant gene, AtBag-4, anti-apoptosis gene as a mix suspension with an Agrobacterium containing pFGC-BNC.h, a plasmid containing multiple gene fragments consisting of BCMV-CP, BCMNV-HC-Pro and CMV-2b, improved the efciency of pFGC-BNC.h trans- formation. We showed reduced virus accumulation in these transgenic bean plans.

Keywords Viruses · RNA silencing · Breeding · Transgenic plant

Introduction manner, and by seeds (ICTV Description). CMV is the member of the genus Cucumovirus in the family Bromov- Bean common mosaic virus (BCMV), bean common mosaic iridae, has a broad host range of more than 1300 plant spe- necrosis virus (BCMNV) and cucumber mosaic virus cies, and is transmitted by aphids and mechanically. CMV is (CMV) are major concerns for common bean (Phaseolus transmitted through seeds in certain hosts such as common vulgaris L.) producers worldwide and cause considerable bean (García-Arenal and Palukatis 2008; Yang et al. 1997; damage in common bean plantings (El-Bramaw and El- Davis and Hampton 1986). Beshehy 2011; Faria et al. 1994). BCMV and BCMNV are This study uses RNAi to knock down the expression the related members of the genus Potyvirus in the family of viral suppressors of RNA silencing and to compromise Potyviridae, usually transmitted by aphids in nonpersistent viral response to host defenses (Diaz-Pendon et al. 2007; Goto et al. 2007; Kim et al. 2016). We target the CMV 2b, BCMNV-HC-Pro and BCMV-CP; CMV-2b protein has been Electronic supplementary material The online version of this reported to suppress plant RNA silencing machinery and can article (https​://doi.org/10.1007/s1320​5-020-02276​-4) contains interfere with the salicylic acid pathway which is involved supplementary material, which is available to authorized users. in systemic acquired resistance (Zhang et al. 2006; Ziebell * Masoud Shams‑bakhsh et al. 2011). The potyvirus HC-Pro is another silencing [email protected] suppressor protein that interacts with several viral and host proteins and plays an important role in virus transmission 1 Faculty of Agriculture, Tarbiat Modares University, Tehran, by aphids, viral polyprotein maturation and suppression of Iran RNA silencing (Valli et al. 2018). HCPro interferes with 2 Dallas Research, Texas A&M AgriLife Research, Dallas, the methylation of the viral-derived short interfering RNA Texas, USA

Vol.:(0123456789)1 3 278 Page 2 of 10 3 Biotech (2020) 10:278

(vsiRNA) (Ivanov et al. 2016), or by inhibition of HEN1 BCMNV was obtained by collecting common bean plants (Jamous et al. 2011). HC-Pro may also disturb the calcium- with typical mosaic symptoms from Tehran bean felds binding capacity of virus (Shen et al. 2010). With this expla- which were diagnosed by polymerase chain reaction (PCR) nation, HC-Pro has been suggested for plant resistance to and enzyme-linked immunosorbent assay (ELISA). potyviruses using RNAi. The potyviral coat protein (CP) has a diferent role including protection of viral genome, viral Bioinformatic analyses and designing of constructs movement, vector transmission (Atreya et al. 1995; Rojas et al. 1997), viral amplifcation (Mahajanet al. 1996) and Genome sequences of the viruses (BCMV, BCMNV and potyviral accumulation (Andrejeva et al. 1999). Potyviral CMV) were obtained from GenBank and multiple sequence CP termini afect long-distance movement (Lopez-Moya and alignments were performed using the GeneDoc software to Pirone 1998), and inhibit viral RNA translation in a dose- fnd the most conservative nucleic acid segment as an RNAi dependent system (Besong-Ndika et al. 2015). This gene has target sequence: (a) a 144-nt fragment of the CMV 2b gene been used efectively for the control of potyviruses (Kim (2442-2586-nt isolate of S72187); (b) a 163-bp fragment of et al. 2016, 2013). the BCMNV-HC-pro (2330-2493-nt isolate of AY138897); Incorporating genetic resistance from wild species into and (c) a 136-bp fragment of the BCMV CP gene (9570- breeding programs is the widely used approach for the con- 9706 nt of AY112735) RNAi scan was used (https​://bioin​ trol of plant viruses, but this process can take several years fo2.noble.org/RNAiS​ can/RNAiS​ can.htm​ ) to identify poten- to produce a commercial variety (Strausbaugh et al. 2003). tial of-target sequences within the host genome. Fragments Another approach is the use of genetic engineering and were analyzed for secondary structure using RNA structure pathogen-derived resistance (PDR) targets the plant post- software (Reuter and Mathews 2010), sequences were ana- transcriptional gene silencing (PTGS) machinery (Lenne- lyzed for secondary structure, because of the fact that the fors et al. 2006). Many reports show transgene-derived small sequences with low secondary structure are readily available RNAs efectively confer virus resistance (Frizzi and Huang for siRNA production (Duan et al. 2012). The simulations of 2010; Wang et al. 2000) and can be adapted for control- fragments cloning in a sense and antisense orientations were ling several plant viruses (Hu et al. 2011; Sun et al. 2010; run using Vector NTI (v11). Guo et al. 2015; Tan et al. 2012). Robost resistance against seven bean potyviruses was reported in soybean expressing Construction of the binary vectors for gene silencing an intron hairpin NIb RNA (Yang et al. 2017). It was shown that fve intron-spliced hairpin RNA (ihpRNA) constructs The consensus of selected sequences was synthesized by from seven genes of CMV induce resistance to CMV (Miao Jenray Co. (China) and fused as tandem sequences with et al. 2016). The purpose of the present investigation was to adding BamHI and XhoI endonuclease restriction sites at improved the efciency of transformation to common bean the 5′ and EcoRI and HindIII restriction sites at the 3′ end. and to generate efcient gene construct for resistance against The sequence of the fragment from 5′ to 3′ was BCMV-CP, single and multiple bean viruses including CMV, BCMV BCMNV-HC-Pro, and CMV 2b, respectively. Two extra and BCMNV using sense, antisense and hairpin RNA. nucleotides (GG) were placed at each end as an anchor (Fig. 1). Synthesized DNAs and pKannibal plasmids were digested with XhoI and EcoRI enzymes and ligated together Materials and methods for cloning of sense orientation. Synthesized antisense DANS and pKannibal plasmids were digested with BamHI Plant sources and virus isolates and HindIII restriction enzymes and ligated for cloning of antisense orientation. Table 1 contains a list of constructs. Nicotiana benthamiana and common bean (Phaseolus vul- These pKannibal constructs were then partially digested garis L.) plants were used for the assessment of the con- using XhoI and BamHI restriction enzymes for cloning struct efciency. Bean plants [Pinto (Lot: MPS127), Garden into the pFGC5941 binary vector (Fig. 1). The recombinant bean roma II (Lot: PU14010001), Mehali khomain, Goli, pFGC5941 plasmids were confrmed using endonuclease Shahzand)] were mechanically inoculated with virus prepa- restriction digestion and gene sequencing by Macrogene rations diluted in phosphate bufer [0.1 M K­ 2HPO4, 0.15% Company (South Korea). The gene constructs were prepared 2-β-mercaptoethanol, pH 8.5] (Yarwood 1952). Three bio- in E. coli DH5α and plasmid transformation was carried out logical replicates were carried out per construct and experi- using heat-shock method (Green and Sambrook 2012). Plas- ments were repeated twice. Plants were kept in the growth mid extractions were made using an alkaline lysis method chamber (20 ± 2 °C, 16 h day, 8 h night). In this research, (Green and Sambrook 2012). Using T4 ligase (Fermentas, CMV Fars (EF620777.1) and BCMV RU1 (AY863025) Lithuania), the ligation reactions were carried out in 10 µl isolates were used as the source of viruses. The isolated (0.3 ng plasmid, 0.5 ng DNA fragment, 1 µl ligase bufer,

1 3 3 Biotech (2020) 10:278 Page 3 of 10 278

Fig. 1 Schematic of cloning steps and vector maps. Frag- ments were cloned into pKan- nibal as sense and antisense, between pdk intron and then the hairpin structure cloned into pFGC5941

Table 1 List of primers used in Primers Sequences Annealing tempera- this research ture (°C)/fragment (bp)

BCMV-CP-F 5′-GC GGA​TCC​ CTC​GAG​ AAA​AAT​CTAG-3′ 54/138 BCMV-CP-R 5′-GC AAG​CTT​ GAA​TTC​ TTA​CCA​TCA​AGT​CC-3′ BCMNV-HC-pro-F 5′-GC GGA​TCC​ CTCGA​′ 54/165 BCMNV-HC-pro-R 5′-GC AAG​CTT​ GAA​TTC​ CCT​CCA​ACC​CTA​-3′ CMV-2b-F 5′-GC GGA​TCC​ CTC​GAG​ TGA​CAA​ACG​TCG​AAC-3′ 52/145 CMV-2b-R 5′-GC AAG​CTT​ GAA​TTC​ GGT​AGG​AAGC-3′ Nt actin-F 5′-GAG CGG GAA ATT GTT AGG G-3′ 56/100 Nt actin-R 5′-GGA GCT AAT GCA GTA ATT TC-3′ BCMV-6k2-F 5′-TAC AAA GCA AGG GAG CGA TA-3′ 56/164 BCMV-6k2-R 5′-ATA CGC TTC TTA CCT TGT GTG-3′

BamHI GGA​TCC​, XhoI CTC​GAG​, HindIII AAG​CTT​, EcoRI GAA​TTC​

10U enzyme, 0.5 µl PEG), and the reaction was kept at 4 °C RNA isolation and RT‑PCR for 16 h. pBAG404 contains Atbag4 gene and pPIN 253 con- tains a Caenorhabditis elegans gene, ced-9 gene, which were RNA was extracted using TRI Reagent­ ® (Sigma-Aldrich) obtained from Dr. Martin B. Dickman from Texas A&M and the second top leaf by grinding 0.2 g of tissue in liq- University. Binary plasmids were transferred to Agrobacte- uid nitrogen. Then cDNA synthesis was carried out using rium tumefaciens GV3101 by electroporation (2.0 kV, 25 μF High-Capacity cDNA Reverse Transcription kit (Applied and 200 Ω) as described by Bos et al. (2006). Biosystems, USA) in a 20 µl reaction volume containing 5 µl of RNA (50 ng/µl), 2 µl 10X bufer, 0.8 µl of the 25 mM dNTPs, 2 µl of random hexamer (20 mM), 1 µl of Multi- Agroinfltration and transient expression scribe RT and 9.2 µl of sterile distilled water. The cDNA synthesis was carried out at 25 °C for 10 min, 37 °C for 2 h Two leaves (third and fourth) of four N. benthamiana plants and 80 °C for 10 min. PCR was done using GoTaq Flexi and frst two leaves of each bean plant were injected using DNA Polymerase (Promega, USA) in 25 µl (5µ PCR bufer Agrobacterium ® ® ­OD600 = 0.6 GV3101 cultures containing ­GoTaq Green, 0.5 µl dNTPs, 1.2 µl ­MgCl2, 0.4 of ­GoTaq gene constructs and 100 µM acetosyringone. Finally, six DNA Polymerase, 1 µl of 10 mM reverse and forward prim- days after agroinfltration (Azizi et al. 2015; Hellens et al. ers, 5 µl of cDNA and 9 µl of sterile distilled water). The 2005), because of reports for systemic long‐distance trans- PCR amplifcation cycles were done at 94 °C for 3 min, port of small RNAs (Uddin and Kim 2013; Zhang et al. 94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min and the 2019), and no damage to the leaves expressing hairpin RNA, third step for fnal extension was at 72 °C for 5 min. one of the next leaves from each plant was mechanically inoculated with the virus as described by Zhao et al. (2006). Semi‑quantitative PCR Control plants were agroinfltrated by Agrobacterium sus- pension cells containing pFGC5941 without the viral gene Semi-quantitative PCR was carried out using BCMV 6k2-f fragment. and BCMNV 6k2-r primers for BCMV, and Nt actin-f and

1 3 278 Page 4 of 10 3 Biotech (2020) 10:278

Nt actin-r primers (Table 1) as internal control. PCR was a 30 carried out in a 25 µl reaction containing 2 µl of cDNA and a it was done in a diferent cycle from 20 to 45 cycles using b 25 b the above materials and programme. 20 c Evaluation of virus resistance and data processing ELISA Ab 15 FW DW d Evaluation of resistance and bioassay was performed 10 e using virus accumulation, and plant dry and fresh weights. Absorbance of each leaf sample was measured by plate 5 a bb reader in indirect ELISA, using a rabbit anti-CMV (1:350), b,c a e b,c d b,c c c b 0 BCMV and BCMNV (1:1000) polyclonal antisera, produced HpFGC.kpFGC.BNC.spFGC.BNC.apFGC.BNC.hpFGC.C.h by Plant Virology Research Center (PVRC), Shiraz, Iran. b 30 For this research, goat anti-rabbit-IgG–alkaline phosphatase a conjugate (Promega, USA) was used as the secondary anti- 25 body (1:7500). Three replicates were applied for each sam- 20 b ple and morphological bioassay was performed by meas- c uring the fresh and dry weights 3 weeks post-inoculation. ELISA Ab 15 Statistical analyses of data were carried out using SAS (SAS FW d DW Institute 2002) and the multiple mean comparisons were per- 10 e formed using LSD. e 5 a a b b e b e b d b b c 0 Results HpFGC.kpFGC.BNC.spFGC.BNC.apFGC.BNC.hpFGC.B.h c FGC 30 A 144-bp fragment of CMV-2b was cloned into p 5941 a a in a hairpin orientation and pdk intron between sense and 25 b antisense and the recombinant vector was named pFGC-C- h; for BCMV, a new recombinant vector containing 136-bp 20 BCMV-CP as hairpin and pdk intron was named pFGC-B-h; ELISA Ab and a 163-bp HC-Pro fragment of BCMNV was cloned as 15 c d FW hairpin into pFGC5941, and it was named pFGC-N-h. The DW multiple gene fragment containing BCMV-CP, BCMNV- 10 e HC-Pro and CMV-2b was named BNC, and it was cloned 5 into pFGC5941 in sense (pFGC-BNC.s), antisense (pFGC- a a c b BNC.a FGC-BNC.h b a e b d b b b ) and hairpin orientations (p ). All binary 0 constructions were transformed into Agrobacterium GV3101 HpFGC.kpFGC.BNC.spFGC.BNC.apFGC.BNC.hpFGC.N.h and they were used for bioassay using transient expression into N. benthamiana and common bean. The results are Fig. 2 Efect of transiently expressed diferent constructs on virus shown in Figs. 2, 3 and 4, supplementary Figs. (1–9) and titre, fresh and dry weights of Nicotiana benthamiana plants, 3 weeks post-inoculation by diferent viruses. Results for CMV, BCMV- Tables 1–6. Six days after Agrobacterium suspension injec- a b and c BCMNV-inoculated plants. Graphs with the same bar letters tion under N. benthamiana and common bean leaves, plants show no signifcant diference at P < 0.01 were inoculated with virus. Each virus bioassay was carried out separately and 15 days post-inoculation using viral con- struct, the efects on the disease symptoms, dry and fresh dry and fresh weights, compared to plant control that weights and virus accumulation were recorded. transiently expressed empty vector (pFGC-k) (Fig. 2a and Figs S1–3). Plants growth showed no diferences between Evaluation of transiently expressed plants pFGC-BNC-h and pFGC-C-h in dry and fresh weights. for resistance to CMV The ELISA results showed that hairpin construct for mul- tiple viruses (pFGC-BNC-h) can reduce virus accumula- Results for CMV are shown in Fig. 2a, Figs (S1–3) and tion more than hairpin construct for single virus (pFGC- Tables (S1–2). The results showed pFGC-BNC-h and C-h) (Fig. 2a). Bioassay results for fresh, dry weights pFGC-C-h reduced CMV impact on plants growth, vigor, and ELISA results showed the efect of the construct of

1 3 3 Biotech (2020) 10:278 Page 5 of 10 278

Fig. 3 Semi-quantitative RT-PCR for the analysis of the efect of are plants inoculated by virus and agroinfltrated using pFGC-BNC- pFGC-BNC-h on viral amplifcation using BCMV-6k2 (forward and h. PCR was carried out in diferent PCR cycles for quantifcation of reverse) primers and actin gene primers (forward and reverse) as the viral accumulation. (1 and 8) 20 cycles, (2 and 9) 25 cycles, (3 and internal control in Nicotiana benthamiana (a) and common bean 10) 30 cycles, (4 and 11) 35 cycles, (5 and 12) 40 cycles, (6–13) 45 (b), 1 week post-inoculation by BCMV. 1–6: plants inoculated by cycles, and (7) 1 Kb Plus DNA Ladder (Thermo Fisher Scientifc) virus and agroinfltrated using pFGC-k (negative control) and 8–13

Fig. 4 Measurement of the efect of pFGC-BNC-h and program cell death suppressor constructs (pBAG404 and pPIN 253) on BCMV accumulation in diferent common bean plants in transiently agroinfltrated bean plants 6 days post-inoculation by virus. PCR was done using BCMV-6k2 primers. Plants agroinfltrated using (a) pFGC- K, (b) pFGC-BNC-h, (c) pFGC- BNC.h & pBAG404, (d) pFGC- BNC.h & pPIN 253. (1) 1 Kb Plus DNA Ladder (Thermo Fisher Scientifc), (2) 20 PCR cycles, (3) 23 PCR cycles, (4) 25 PCR cycles, (5) 28 PCR cycles, (6) 32 PCR cycles

multiple viruses on level of resistance to CMV in hairpin, Evaluation of transiently expressed plants sense and antisense, respectively (Fig. 2a, Figs S3–4 and for resistance to BCMNV Table S2). The results for the efect of constructs on BCMNV was presented in Fig. 2c, Figs S7–9 and Tables S5–6. Same as Evaluation of transiently expressed plants the efectiveness of constructs for resistance to BCMV, the for resistance to BCMV constructs for BCMNV can signifcantly cause plant resist- ance, improve plant vigor and reduce virus accumulation. Bioassay analysis for BCMV was performed (Fig. 2b, Figs The ELISA absorption, fresh and dry weights showed that S4–6 and Tables S3–4) and the results showed signifcant plants that expressed pFGC-BNC.h can reduce BCMNV diferences between constructs’ efect on BCMV impact accumulation and viral efects on inoculated plants simi- on viral accumulation, dry and fresh weights. The efect of lar to healthy plants (Fig. 2c). Consequently, the level of pFGC-BNC-h was better than hairpin construct for single construct efects on plant health was pFGC-BNC.h, pFGC- virus (pFGC-B.h), and hairpin construct for multiple viruses N.h, pFGC-BNC.a, pFGC-BNC.s. can cause more resistance. For BCMV same as CMV, the efectiveness of constructs was pFGC-BNC-h, pFGC-B.h, pFGC-B-a and pFGC-B-s.

1 3 278 Page 6 of 10 3 Biotech (2020) 10:278

Efect of pFGC‑BNC.h infltration in common bean The results of this study showed that all constructs plant on viral accumulation and symptoms (pFGC-BNC-h, pFGC-N.h, pFGC-B.h, pFGC-C.h) can afect viral accumulation and improve plant growth. Based Although, infiltrations of common bean plants (Mehali on this research for three viruses, hairpin constructs were khomain, Goli, Pinto, Garden bean and Shahzand) with more efective than antisense and sense, and also antisense an Agrobacterium suspension with the OD600 = 0.6 caused constructs were more efcient than sense (Fig. 2 and Tables necrosis six days after Agrobacterium infltration, but not in S1–6). As shown in Fig. 2, plants that were transiently trans- FGC-BNC.h OD­ 600 = 0.2. The Agrobacterium-infltrated common bean formed by p and mechanically inoculated with plants were inoculated by viruses and results showed no sig- BCMNV had no signifcant diference in growth compared nifcant efect of pFGC-BNC-h on viral accumulation and with healthy plants, and ELISA results were also negative. symptoms. For understanding any efect of pFGC-BNC-h These results further confrmed the outcome of Duan et al. on the viral accumulation, semi-quantitative RT-PCR for (2012) who mentioned that the resistance incurred by sense BCMV accumulation before and after agroinfltration by and antisense in comparison with a hairpin was unstable and construct and 6 days after infltration using BCMV infec- siRNA association produced by sense and antisense orienta- tious clone was conducted. Semiquantitative RT-PCR tion was less than hairpin. Regarding the important role of was performed using BCMV 6k2 primers (BCMV-6k2-F CMV-2b in gene silencing suppressor and also inhibition and BCMV-6k2-R) and actin gene as the internal control of Argonaut 1 and SA defense pathway (Diaz-Pendon et al. (Table 1). The results showed that the virus was detected 2007), a fragment of this gene was used in the present study after 25 cycles in control N. benthamiana plants infltrated which showed highly efcient 2b silencing construct. As it by empty vector but it was detected after 30 cycles in N. was defned that CMV-2b can be attached to long dsRNA as benthamiana plants infltrated by pFGC-BNC-h. In common well, but it can lose attachment to miRNA, compared to p19 bean, no diferences between control and infltrated plants and for this reason, 2b has a less destructive efect on plant via pFGC-BNC-h and virus were detected in 20 cycles of genome (Goto et al. 2007) and that makes it a suitable can- PCR. However, results showed that although pFGC-BNC-h didate for preparing silencing resistance constructs. Further- can reduce viral accumulation in N. benthamiana, it can- more, it was reported that CMV-2b inhibits argonate1 cleav- not afect common bean plants compared to control plants age activity to counter plant defense system (Zhang et al. (Fig. 3). To clarify the reason, pBAG404 containing bag4 2006). The conserved HC-pro fragment for resistance toward gene and pPIN 253 containing ced-9 gene were used con- BCMNV was chosen in this research and it was considered comitantly with pFGC-BNC-h as mix suspension for infltra- that this fragment has a high similarity with BCMV HC-Pro tion of common bean leaves and RNA extraction was car- against BCMV. Previous research for transgenic plants has ried out 6 days post-inoculation by BCMV. Consequently, shown that HC-Pro can disrupt miRNA production and its results showed that these two genes can improve the efects action pathway (Soitamo et al. 2011), and also it was shown of pFGC-BNC-h on viral accumulation, and BCMV was that HC-Pro can interact with 26S proteasome and disrupt detected after 32 cycles in mix infltrated plants by pPIN 253 the endonuclease action of the 26S proteasome (Jin et al. and pFGC-BNC-h and also after 28 cycles for mix infltrated 2007; Ballut et al. 2005). In addition, it has been confrmed plants by pFGC-BNC-h and pBAG404, while the virus was that HC-Pro of potyviruses is a good candidate for resist- detected after 23 cycles in control plants (Fig. 4). However, ance against this group of viruses (Goswami et al. 2017; Ai pPIN 253 can improve the efect of pFGC-BNC-h on virus et al. 2011). Our results showed that all constructs can be accumulation better than pBAG404. efective against BCMNV, but hairpin constructs (pFGC- BNC-h and pFGC-N.h) were more efcient than sense and antisense (Fig. 2c and Tables S5, S6). In this research, a Discussion construct for conserve BCMV-CP sequence showed a reduc- tion of BCMV symptoms and viral accumulation. Potyviral Transient expression of silencing construct in diferent plants CP has a diferent role, including protection, movement, and is a fast way for testing the efciency of constructs and it was transmission by aphids and replication of the virus (Urcuqui- used in diferent researches. Testing of two artifcial micro- Inchima et al. 2001; King et al. 2012). Moreover, in some RNAs targeting grapevine fanleaf virus was transiently done previous study, it was shown that CP can efectively control in Grapevine (Jelly et al. 2012) and as in a previous study potyviruses using RNA silencing (Akbar et al. 2017; Fang it was shown the best time between Agro-infltration and and Grumet 1993; Joyce et al. 1998). the substrate infltration is up to 6 days (Hellens et al. 2005; Results of expression tests for pFGC-BNC-h, pFGC-B.h Azizi et al. 2015), we constructed and tested the silencing and pFGC-N.h have shown that the construct for multiple constructs transiently and with 6-day interval between Agro- viruses (pFGC-BNC-h) was more effective than single infltration and viral inoculation. virus constructs (pFGC-N.h for BCMNV and pFGC-B.h for

1 3 3 Biotech (2020) 10:278 Page 7 of 10 278

BCMV). This may be because of the sequence similarity of been reported by other researchers. These results suggest selected fragments between BCMV and BCMNV that can that many common bean plants resist against transforma- be efective against another virus, As shown in a previous tion by Agrobacterium because of apoptosis that can hap- research, exogenous application of dsRNAs targeting NIb pen in transformed cells. Consequently, in previous research protein of BCMNV or the coding region of BCMV-CP can with the same results, it has been reported that using some protect Nicotiana benthamiana and cowpea (Vigna unguic- genes like ced-9 and Bag4 can help in the survival of trans- ulata) plants against mechanical inoculation with BCMV formed plant cells after transformation using Agrobacterium (Worrall et al. 2019). They showed BCMV-CP-BioClay (Khanna et al. 2007). The CED-9, a Bcl-2 analogue from spraying of N. benthamiana leaves 5 days prior to inocu- Caenorhabditis elegans, was reported to improve plant sur- lation by viruliferous aphids can induce protection against vival under abiotic and biotic stresses and suppress plant cell BCMV. death (Xu et al. 2004), and BAG is another anti-apoptotic Because of the vector/Agrobacterium combination protein from Bcl-2 family (Elmore 2007). In this research, (Tan et al. 2012), the fragment length of the gene (Chen transient co-expression of pBAG404 and pFGC-BNC.h et al. 2004) and a sequence region that can afect resist- or pPIN 253 and pFGC-BNC.h in common bean leaves ance level (Jiang et al. 2011), the results of this research can showed a signifcant efect on pFGC-BNC.h efectiveness help in the development of transgenic resistant plants that on the viral accumulation (Fig. 4). The semi-quantitative are more efective and more durable. It was reported that PCR results showed that BCMV was detected after 32 and the target fragment length can afect the level of resistance 28 cycles for pPIN 253 construct containing Ced-9 and and percentage of transgenic plants that are resistant (Chen pBAG404 containing bag4, respectively (Fig. 4). These et al. 2004) and also mentioned that fragments containing results have shown that Ced-9 can reduce viral accumulation 100–800 bp can induce efective resistance (Pang et al. 1997; more efectively in common bean plants, compared to plants Sharma et al. 2015). The size of virus fragments used in this that expressed pFGC-BNC.h alone. Our results suggested research was in the range of 136–163 nt, and they efectively that common bean transformation using constructs contain- controlled viruses. As RNAs with a less secondary struc- ing Ced-9 can improve protocols for bean transformation and ture are more available for degradation by siRNA and the recovery of transformed new plantlets from tissue culture. RISC complex (de Alba et al. 2002; Ameres et al. 2007), In conclusion, the results of this research suggest sup- we tried to select fragments with less secondary structure. pression of PCD before Agrobacterium transformation for It was mentioned that constructs containing intron between the optimization of bean transformation, particularly using sense and antisense are more efcient than constructs with- Ced-9 gene, and also an efcient construct was constructed out intron (Smith et al. 2000; Lin and Ying 2018). for the control of three important bean viruses in transgenic In this research, pFGC5941 was used which contains pdk seed projects. This research suggests further research on intron. Though all constructs can efectively reduce the viral transgenic bean plants for the production of resistant com- accumulation and improve plant growth in N. benthamiana, mon bean to viruses and feld evaluation of resistance in especially pFGC-BNC.h construct, this construct did not transgenic plants. afect viral accumulation in common bean (Fig. 3). Semi- quantitative PCR showed that the virus can detect in P. vul- Acknowledgements The authors sincerely thank Dr. Martin B. Dick- garis N. man from Texas A&M University for providing pBAG404 and pPIN 6 days post-inoculation after 20 PCR cycles, but in 253 used in this study. benthamiana plants that transiently expressed pFGC-BNC.h, virus was detected after 30 cycles (Fig. 3). According to Compliance with ethical standards semi-quantitative PCR, in common bean (Fig. 3), results proved that pFGC-BNC.h cannot be expressed. The results Conflict of interest The authors declare no conficts of interest regard- were the same in diferent common bean cultivars including ing the publication of this article. Pinto, Garden bean, Mehali khomain, Goli and Shahzand using GV3101 and LBA4404 Agrobacterium strains. It has been reported that common bean plants were resistant to References the transformation using Agrobacterium (Hanatuszko-Konka et al. 2014), and bean transformation using biolistic meth- Ai T, Zhang L, Gao Z, Zhu CX, Guo X (2011) Highly efcient virus ods happened at a very low rate (Russell et al. 1993; Rech resistance mediated by artifcial microRNAs that target the sup- et al. 2008). Plant cell death for some plants like grapevine pressor of PVX and PVY in plants. Plant Biol 13:304–316. https​ (Perl et al. 1996), sorghum (Gao et al. 2005), wheat (Parrott ://doi.org/10.1111/j.1438-8677.2010.00374​.x et al. 2002), banana (Khanna et al. 2007) and some other Akbar S, Tahir M, Wang MB, Liu Q (2017) Expression analysis of hairpin RNA carrying Sugarcane mosaic virus (SCMV) derived plants (Van der Hoorn et al. 2000; Wroblewski et al. 2005; sequences and transgenic resistance development in a model rice Hasen 2000) after transformation using Agrobacterium has plant. Biomed Res Int. https​://doi.org/10.1155/2017/16461​40

1 3 278 Page 8 of 10 3 Biotech (2020) 10:278

Ameres SL, Martinez J, Schroeder R (2007) Molecular basis for target type II isolates from the Dominican Republic and Guatemala: RNA recognition and cleavage by human RISC. Cell 130:101– nucleotide sequences, infectious pseudorecombinants, and phy- 112. https​://doi.org/10.1016/j.cell.2007.04.037 logenetic relationships. Phytopathology 84:321–329. https://doi.​ Andrejeva J, Puurand U, Merits A, Rabenstein F, Jarvekulg L, org/10.1094/Phyto​-84-321 Valkonen JPT (1999) Potyvirus helper component-proteinase Frizzi A, Huang S (2010) Tapping RNA silencing pathways for plant and coat protein (CP) have coordinated functions in virus–host biotechnology. Plant Biotechnol J 8:655–677. https​://doi.org/1 interactions and the same CP motif affects virus transmis- 0.1111/j.1467-7652.2010.00505​.x sion and accumulation. J Gen Virol 80:1133–1139. https​://doi. Gao Z, Xie X, Ling Y, Muthukrishnan S, Liang GH (2005) Agrobac- org/10.1099/0022-1317-80-5-1133 terium tumefaciens-mediated sorghum transformation using a Atreya PL, Lopez-Moya JJ, Atreya CD, Pirone TP (1995) Mutational mannose selection system. Plant Biotechnol J 3:591–599. https​ analysis of the coat protein N-terminal amino acids involved in ://doi.org/10.1111/j.1467-7652.2005.00150​.x potyvirus transmission by aphids. J Gen Virol 76:265–270. https​ García-Arenal F, Palukaitis P (2008) Cucumber mosaic virus. In: ://doi.org/10.1099/0022-1317-76-2-265 García-Arenal F, Palukaitis P, Mahy BWJ, Van Regenmortel Azizi A, Shams-bakhsh M, Moeeni A (2015) Design and construction MHV (eds) Encyclopedia of virology, 3rd edn. Academic Press, of hairpin structure using sequence of CMV-2b gene for induc- Oxford, pp 614–619 tion of resistance to cucumber mosaic virus. Iran J Plant Pathol Goswami S, Kumar RR, Chinnusamy V, Praveen S (2017) In planta 51:115–128 In Persian silencing of NSs and Hc-Pro through RNAi constructs: to Ballut L, Drucker M, Pugniere M, Cambon F, Blanc S, Roquet F, develop durable resistance. Indian J Plant Physiol 22:577–586. Candresse T, Schmid HP, Nicolas P, Gall OL, Badaoui S (2005) https​://doi.org/10.1007/s4050​2-017-0344-6 HcPro, a multifunctional protein encoded by a plant RNA virus, Goto K, Kobori T, Kosaka Y, Natsuaki T, Masuta C (2007) Char- targets the 20S proteasome and afects its enzymic activities. J acterization of silencing suppressor 2b of Cucumber mosaic Gen Virol 86:2595–2603. https​://doi.org/10.1099/vir.0.81107​-0 virus based on examination of its small RNA-binding abilities. Besong-Ndika J, Ivanov KI, Hafrèn A, Michon T, Mäkinen K (2015) Plant Cell Physiol 48:1050–1060. https​://doi.org/10.1093/pcp/ Cotranslational coat protein-mediated inhibition of potyviral pcm07​4 RNA translation. J Virol 89:4237–4248. https​://doi.org/10.1128/ Green MR, Sambrook J (2012) Molecular cloning: a laboratory man- JVI.02915​-14 ual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor Bos JI, Kanneganti TD, Young C, Cakir C, Huitema E, Win J, Arm- Guo J, Gao S, Lin Q, Wang H, Que Y, Xu L (2015) Transgenic sug- strong MR, Birch PR, Kamoun S (2006) The C-terminal half of arcane resistant to sorghum mosaic virus based on coat protein Phytophthora infestans RXLR efector AVR3a is sufcient to trig- gene silencing by RNA interference. Biomed Res Int. https​://doi. ger R3a-mediated hypersensitivity and suppress INF1-induced org/10.1155/2015/86190​7 cell death in Nicotiana benthamiana. Plant J 48:165–176. https://​ Hansen G (2000) Evidence for Agrobacterium-induced apoptosis in doi.org/10.1111/j.1365-313X.2006.02866​.x maize cells. Mol Plant Microbe Interact 13:649–657. https​://doi. Chen Y, Lohuis D, Goldbach R, Prins M (2004) High frequency induc- org/10.1094/MPMI.2000.13.6.649 tion of RNA-mediated resistance against cucumber mosaic virus Hellens RP, Allan AC, Friel EN, Bolitho K, Grafton K, Templeton MD, using inverted repeat constructs. Mol Breed 14:215–226. https​:// Karunairetnam S, Gleave AP, Laing WA (2005) Transient expres- doi.org/10.1023/B:MOLB.00000​47769​.82881​.f5 sion vectors for functional genomics, quantifcation of promoter Davis RF, Hampton RO (1986) Cucumber mosaic virus isolates seed activity and RNA silencing in plants. Plant Methods 1:13. https​ borne in Phaseolus vulgaris: serology, host–pathogen relation- ://doi.org/10.1186/1746-4811-1-13 ships, and seed transmission. Phytopathology 76:999–1004. Hnatuszko-Konka K, Kowalczyk T, Gerszberg A, Wiktorek-Smagur A, https​://www.apsne​t.org/publi​catio​ns/phyto​patho​logy/backi​ssues​ Kononowicz AK (2014) Phaseolus vulgaris—recalcitrant poten- /Docum​ents/1986A​rticl​es/Phyto​76n10​_999.PDF tial. Biotechnol Adv 32:1205–1215. https://doi.org/10.1016/j.biote​ ​ de Alba AM, Flores R, Hernández C (2002) Two chloroplastic viroids chadv​.2014.06.001 induce the accumulation of small RNAs associated with post- Hu Q, Niu Y, Zhang K, Liu Y, Zhou X (2011) Virus-derived transgenes transcriptional gene silencing. J Virol 76:13094–13096. https​:// expressing hairpin RNA give immunity to tobacco mosaic virus doi.org/10.1128/JVI.76.24.13094​-13096​.2002 and cucumber mosaic virus. Virol J 8:41(1–11). https​://doi. Diaz-Pendon JA, Li F, Li WX, Ding SW (2007) Suppression of antivi- org/10.1186/1743-422X-8-41 ral silencing by cucumber mosaic virus 2b protein in Arabidop- Ivanov KI, Eskelin K, Basic M, De S, Lohmus A, Varjosalo M, Maki- sis is associated with drastically reduced accumulation of three nen K (2016) Molecular insights into the function of the viral classes of viral small interfering RNAs. Plant Cell 19:2053–2063. RNA silencing suppressor HC-Pro. Plant J 85:30–45. https​://doi. https​://doi.org/10.1105/tpc.106.04744​9 org/10.1111/tpj.13088​ Duan CG, Wang CH, Guo HS (2012) Application of RNA silenc- Jamous RM, Boonrod K, Fuellgrabe MW, Ali-Shtayeh MS, Krczal G, ing to plant disease resistance. Silence 3:5. https​://doi. Wassenegger M (2011) The helper component-proteinase of the org/10.1186/1758-907X-3-5 zucchini yellow mosaic virus inhibits the Hua Enhancer 1 meth- El-Bramaw MAEH, El-Beshehy EKF (2011) The resistance of bean yltransferase activity in vitro. J Gen Virol 92:2222–2226. https​:// yellow mosaic virus (BYMV) in faba bean (Vicia faba L.) with doi.org/10.1099/vir.0.03153​4-0 diallel analysis. J Biol Life Sci 2:1–15. https​://doi.org/10.5296/ Jelly NS, Schellenbaum P, Walter B, Maillot P (2012) Transient expres- jbls.v2i1.588 sion of artifcial microRNAs targeting Grapevine fanleaf virus Elmore S (2007) Apoptosis: a review of programmed cell death. Toxi- and evidence for RNA silencing in grapevine somatic embryos. col Pathol 35:495–516. https://doi.org/10.1080/01926​ 23070​ 13203​ ​ Transgenic Res 21:1319–1327. https​://doi.org/10.1007/s1124​ 37 8-012-9611-5 Fang G, Grumet R (1993) Genetic engineering of potyvirus resistance Jiang F, Song Y, Han Q, Zhu C, Wen F (2011) The choice of target using constructs derived from the Zucchini yellow mosaic virus site is crucial in artifcial miRNA-mediated virus resistance in coat protein gene. Mol Plant Microbe Interact 6:358–367. https://​ transgenic Nicotiana tabacum. Physiol Mol Plant Path 76:2–8. doi.org/10.1094/MPMI-6-358 https​://doi.org/10.1016/j.pmpp.2011.07.002 Faria JCd, Gilbertson RL, Hanson SF, Morales FJ, Ahlquist P, Lon- Jin Y, Ma D, Dong J, Jin J, Li D, Deng C, Wang T (2007) HC-Pro iello AO, Maxwell DP (1994) Bean golden mosaic geminivirus protein of potato virus Y can interact with three Arabidopsis

1 3 3 Biotech (2020) 10:278 Page 9 of 10 278

20S proteasome subunits in planta. J Virol 81:12881–12888. Russell D, Wallace K, Bathe J, Martinell B, McCabe D (1993) Stable https​://doi.org/10.1128/JVI.00913​-07 transformation of Phaseolus vulgaris via electric-discharge medi- Joyce PA, McQualter RB, Handley JA, Dale JL, Harding RM, Smith ated particle acceleration. Plant Cell Rep 12(3):165–169. https​:// GR (1998) Transgenic sugarcane resistant to sugarcane mosaic doi.org/10.1007/BF002​39099​ virus. In: Hogarth DM, ed. proceedings of the Australian soci- Sharma VK, Kushwaha N, Basu S, Singh AK, Chakraborty S (2015) ety of sugar cane technology, vol 20, pp 204–210 Identifcation of siRNA generating hot spots in multiple viral sup- Khanna HK, Paul JY, Harding RM, Dickman MB, Dale JL (2007) pressors to generate broad-spectrum antiviral resistance in plants. Inhibition of Agrobacterium-induced cell death by anti-apop- Physiol Mol Biol Plants 21:9–18. https​://doi.org/10.1007/s1229​ totic gene expression leads to very high transformation ef- 8-014-0264-0 ciency of banana. Mol Plant Microbe Interact 20:1048–1054. Shen W, Yan P, Gao L, Pan X, Wu J, Zhou P (2010) Helper component https​://doi.org/10.1094/MPMI-20-9-1048 proteinase (HC-Pro) protein of papaya ring spot virus interacts Kim HJ, Kim M, Pak JH, Im HH, Lee DH, Kim KH, Lee JH, Kim with papaya calreticulin. Mol Plant Pathol 11:335–346. https​:// DH, Choi HK, Jung HW, Chung YS (2016) RNAi-mediated soy- doi.org/10.1111/j.1364-3703.2009.00606​.x bean mosaic virus (SMV) resistance of a Korean soybean culti- Smith NA, Singh SP, Wang MB, Stoutjesdijk PA, Green AG, Water- var. Plant Biotechnol Rep 10:257–267. https://doi.org/10.1007/​ house PM (2000) Total silencing by intron-spliced hairpin RNAs. s1181​6-016-0402-y Nature 407:319–320. https​://doi.org/10.1038/35030​305 Kim HJ, Kim MJ, Pak JH, Jung HW, Cjoi HK, Lee YH, Back IY, Soitamo AJ, Jada B, Lehto K (2011) HC-Pro silencing suppres- Ko JM, Jeong SC, Pack IS, Ryu KH, Chung YS (2013) Charac- sor signifcantly alters the gene expression profle in tobacco terization of SMV resistance of soybean produced by genetic leaves and flowers. BMC Plant Biol 11:68. https​://doi. transformation of SMV-CP gene in RNAi. Plant Biotechnol Rep org/10.1186/1471-2229-11-68 7:425–433. https​://doi.org/10.1007/s1181​6-013-0279-y Strausbaugh C, Myers J, Forster R, McClean P (2003) A quantitative King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ (2012) Virus method to screen common bean plants for resistance to Bean com- taxonomy. In: Ninth Report of the International Committee on mon mosaic necrosis virus. Phytopathology 93:1430–1436. https​ Taxonomy of Viruses. Elsevier Academic Press, Amsterdam ://doi.org/10.1094/PHYTO​.2003.93.11.1430 Lennefors BL, Savenkov EI, Bensefelt J, Wremerth-Weich E, van Sun ZN, Song YZ, Yin GH, Zhu CX, Wen FJ (2010) HpRNAs derived Roggen P, Tuvesson S, Valkonen JP, Gielen J (2006) dsRNA- from diferent regions of the NIb gene have diferent abilities to mediated resistance to beet necrotic yellow vein virus infec- protect tobacco from infection with Potato virus Y. J Phytopathol tions in sugar beet (Beta vulgaris L. ssp. vulgaris). Mol Breed 158:566–568. https://doi.org/10.1111/j.1439-0434.2009.01650​ .x​ 18:313–325. https​://doi.org/10.1007/s1103​2-006-9030-5 Tan X, Zhang D, Wintgens C, Willingmann P, Adam G, Heinze C Lin SL, Ying SY (2018) Gene silencing in vitro and in vivo using (2012) A comparative testing of cucumber mosaic virus (CMV)- intronic MicroRNAs. MicroRNA Protocols. https​://doi. based constructs to generate virus resistant plants. Am J Plant Sci org/10.1007/978-1-4939-7601-0_9 3:461–472. https​://doi.org/10.4236/ajps.2012.34055​ Lopez-Moya JJ, Pirone TP (1998) Charge changes near the N ter- Uddin MN, Kim JY (2013) Intercellular and systemic spread of minus of the coat protein of two potyviruses afect virus move- RNA and RNAi in plants. WIREs RNA 4:279–293. https​://doi. ment. J Gen Virol 79:161–165. https​://hdl.handl​e.net/10261​ org/10.1002/wrna.1160 /10173​9 Urcuqui-Inchima S, Haenni AL, Bernardi F (2001) Potyvirus pro- Mahajan S, Dolja VV, Carrington JC (1996) Roles of the sequence teins: a wealth of functions. Virus Res 74:157–175. https​://doi. encoding Tobacco etch virus capsid protein in genome amplif- org/10.1016/S0168​-1702(01)00220​-9 cation: requirements for the translation process and a cis-active Valli AA, Gallo A, Rodamilans B, López-Moya JJ, García JA (2018) element. J Virol 70:4370–4379 The HC Pro from the Potyviridae family: an enviable multitasking Miao B, Wen-Ting C, Bing-Yan X, Guo-Shun Y (2016) A novel strat- Helper Component that every virus would like to have. Mol Plant egy to enhance resistance to cucumber mosaic virus in tomato by Pathol 19:744–763. https​://doi.org/10.1111/mpp.12553​ grafting to transgenic rootstocks. J Integr Agric 15(9):2040–2048. Van der Hoorn RAL, Laurent F, Roth R, De Wit PJGM (2000) Agro- https​://doi.org/10.1016/S2095​-3119(16)61330​-8 infltration is a versatile tool that facilitates comparative analy- Pang SZ, Jan FJ, Gonsalves D (1997) Nontarget DNA sequences reduce ses of Avr9/Cf-9-induced and Avr4/Cf-4-induced necrosis. Mol the transgene length necessary for RNA-mediated tospovirus Plant Microbe Interact 13:439–446. https​://doi.org/10.1094/ resistance in transgenic plants. Proc Natl Acad Sci USA 94:8261– MPMI.2000.13.4.439 8266. https​://doi.org/10.1073/pnas.94.15.8261 Wang MB, Abbott DC, Waterhouse PM (2000) A single copy of a Parrott DL, Anderson AJ, Carman JG (2002) Agrobacterium induces virus-derived transgene encoding hairpin RNA gives immunity plant cell death in wheat (Triticum aestivum L.). Physiol Mol Plant to Barley yellow dwarf virus. Mol Plant Pathol 1:347–356. https​ Path 60:59–69. https​://doi.org/10.1006/pmpp.2002.0378 ://doi.org/10.1046/j.1364-3703.2000.00038​.x Perl A, Lotan O, Abu-Abeid M, Holland D (1996) Establishment of an Worrall EA, Bravo-Cazar A, Nilon AT, Fletcher SJ, Robinson KE, Agrobacterium-mediated genetic transformation system for grape Carr JP, Mitter N (2019) Exogenous application of RNAi-induc- (Vitis vinifera L.): the role of antioxidants during grape–Agro- ing double-stranded RNA inhibits aphid-mediated transmission bacterium interaction. Nat Biotechnol 14:624–628. https​://doi. of a plant virus. Front Plant Sci 10:265. https​://doi.org/10.3389/ org/10.1038/nbt05​96-624 fpls.2019.00265​ Rech EL, Vianna GR, Aragao FJ (2008) High-efciency transforma- Wroblewski T, Tomczak A, Michelmore R (2005) Optimization of tion by biolistics of soybean, common bean and cotton transgenic Agrobacterium-mediated transient assays of gene expression in plants. Nat Protoc 3:410–418. https​://doi.org/10.1038/nprot​ lettuce, tomato and Arabidopsis. Plant Biotechnol J 3:259–273. .2008.9 https​://doi.org/10.1111/j.1467-7652.2005.00123​.x Reuter JS, Mathews DH (2010) RNA structure: software for RNA sec- Xu P, Rogers SJ, Roossinck MJ (2004) Expression of antiapoptotic ondary structure prediction and analysis. BMC Bioinform 11:129. genes Bcl-xLand CED-9 in tomato enhance tolerance to viral https​://doi.org/10.1186/1471-2105-11-129 induced necrosis and abiotic stress. Proc Natl Acad Sci USA Rojas MR, Zerbini FM, Allison RF, Gilbertson RL, Lucas WJ (1997) 101:15805–15810. https​://doi.org/10.1073/pnas.04070​94101​ Capsid protein and helper component proteinase function as poty- Yang X, Niu L, Zhang W, He H, Yang J, Xing G, Guo D, Du Q, Qian virus cell-to-cell movement proteins. Virology 237:283–295 X, Yao Y, Li Q, Dong Y (2017) Robust RNAi-mediated resistance

1 3 278 Page 10 of 10 3 Biotech (2020) 10:278

to infection of seven potyvirids in soybean expressing an intron inhibits Arabidopsis Argonaute1 cleavage activity to counter hairpin NIb RNA. Transgenic Res 26:665–676. https​://doi. plant defense. Genes Dev 20:3255–3268. https://doi.org/10.1101/​ org/10.1007/s1124​8-017-0041-2 gad.14955​06 Yang Y, Kim KS, Anderson EJ (1997) Seed transmission of cucumber Zhao MM, An DR, Zhao J, Huang GH, He ZH, Chen JY (2006) mosaic virus in spinach. Phytopathology 87:924–931. https://doi.​ Transiently expressed short hairpin RNA targeting 126 kDa org/10.1094/PHYTO​.1997.87.9.924 protein of tobacco mosaic virus interferes with virus infection. Yarwood CE (1952) The phosphate efect in plant virus inoculations. Acta Biochim Biophys Sin 38:22–28. https​://doi.org/10.111 Phytopathology 42:137–143 1/j.1745-7270.2006.00124​.x Zhang X, Laia T, Zhang P, Zhang X, Yuan C, Jin Z, Li H, Yu Z, Qin Ziebell H, Murphy AM, Groen SC, Tungadi T, Westwood JH, Lewsey C, Tör M, Ma P, Cheng Q, Hong Y (2019) Mini review: revisiting MG, Moulin M, Kleczkowski A, Smith AG, Stevens M (2011) mobile RNA silencing in plants. Plant Sci 278:113–117. https​:// Cucumber mosaic virus and its 2b RNA silencing suppressor doi.org/10.1016/j.plant​sci.2018.10.025 modify plant-aphid interactions in tobacco. Sci Rep 1:187. https​ Zhang X, Yuan YR, Pei Y, Lin SS, Tuschl T, Patel DJ, Chua ://doi.org/10.1038/srep0​0187 NH (2006) Cucumber mosaic virus-encoded 2b suppressor

1 3