Quantitative analysis of the interaction of heterologous viruses with Plum pox virus in C5 HoneySweet transgenic plums

Khushwant Singh, Tereza Neubauerová, Jiban Kumar Kundu

Plant Virus and Vector Interactions Group, Division of Crop Protection and Plant Health, Crop Research Institute, Prague 16106,Czech Republic

Abstract Stone fruits are an important crop in most parts of the world and are heavily challenged by several viruses including Plum pox virus (PPV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), and Apple chlorotic leaf spot virus (ACLSV).We validated the PPV resistance in C5 plum plants (commercially known as HoneySweet) grown in the Czech Republic for more than 16 years in a field trial experiment under natural environmental conditions. We quantified single (PPV-Rec) and mixed viruses (PPV-Rec+ACLSV, PPV-Rec+PDV and PPV-Rec+ACLSV+PDV) in C5 transgenic plums inoculated for the period 2016 to 2018. The accumulation of PPV-Rec was high (～5.43E+05 copies) compared with that of ACLSV (～8.70E+04 copies) in the inoculated graft of C5 transgenic plants. Leaves close to the inoculum sources showed a differential level of virus titre in single and mixed infections (～10 to ～5×102 copies). C5 plants with permanent virus pressure showed 103- to 105-fold fewer copies of viruses than those of the inoculated graft. We observed high accumulation of conserved miRNAs such as miR167, miR69 and miR396 in C5 plants co-infected with PPV, ACLSV and PDV that are associated with its resistance against viruses. Overall, i) C5 transgenic plums showed high resistance to PPV infection, and a low level (～32 copies) of PPV only accumulated in some grafted plants, ii) high accumulation of PPV was found in inoculated grafts in single PPV infection and mixed infections, iii) heterologous virus infection sustained by ACLSV or PDV did not suppress PPV resistance, and iv) high and low conserved microRNAs accumulated in C5 plants.

Keywords: PPV, PDV, ACLSV, C5, HoneySweet, resistance

1. Introduction

Prunus spp. are affected by many viruses, which most frequently occur in the genera Ilarvirus, Potyvirus, and Trichovirus. The following viruses are the most important for stone fruit trees: Plum pox virus (PPV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV),Apple chlorotic leaf spot virus (ACLSV), Apple mosaic virus(ApMV), Apricot latent virus (ApLV), Plum bark necrosis stem pitting associated virus (PBNSPaV), Peach latent mosaic viroid (PLMVd), and Hop stunt viroid (HSVd)(Bouani et al. 2004; Gümüs et al. 2007). Among the viruses, PPV causes the most devastating impact on stone fruits (Sihelská et al. 2017). PPV disease management strategies are based on the use of virus-free plant material,recurring inspection of orchards, elimination of diseased trees and insecticide treatment to control aphid populations(Šubr and Glasa 2013; García et al. 2014). However, the development of PPV-resistant transgenic plants remains the best approach to control PPV (Marandel et al. 2009;Rubio et al. 2010). Thus, implementing an RNA-silencing approach, a transgenic plum clone C5 of Prunus domestica L. was successfully engineered with the PPV coat protein gene (PPV-CP) (Scorza et al. 1994). The resistance in C5 is conferred by RNA silencing through regulation of short interfering RNAs (siRNAs, duplex) (Hily et al. 2005; Kundu et al. 2008). The resistance of C5 (commercial name is cv.HoneySweet) to PPV has been evaluated in the greenhouse conditions (Ravelonandro et al. 1998; Scorza et al. 1998,2001) and in four field tests in Europe, i.e., in Poland, Spain(Malinowski et al. 2006), Romania (Zagrai et al. 2008) and the Czech Republic (Polák et al. 2017). The efficacy and stability of PPV resistance in C5 plants have been tested with three viruses PDV, PNRSV and ACLSV, including different combinations (Polák et al. 2005; Ravelonandro et al.2007; Zagrai et al. 2008). These studies all showed that the challenging heterologous viruses (e.g., PDV, PNRSV and ACLSV) in a mixed infection with PPV affect either the symptom development or the stability of resistance in C5 plants (Zagrai et al. 2008; Polák et al. 2008, 2017). In the present study, we focused on the quantitative analysis of heterologous virus titre in C5 trees inoculated with the different combinations of PPV with PDV and ACLSV to determine the level of resistance. In addition to virus titre,the expression profile of microRNAs (miRNAs) in C5 trees was analyzed to determine the regulation of the low and high conserved Prunus miRNAs in regards to heterologous virus interaction.

2. Materials and methods

2.1. Experimental design and collection of samples for quantification

A field trial with the plum clone C5 (cv. HoneySweet approved in the USA in 2011) was established in the Czech Republic. Budwood of the plum P. domestica clone C5 was prepared in the USDA-ARS Appalachian Fruit Research Station, Kearneysville, WV, USA, and sent to the Crop Research Institute (CRI), Prague-Ruzyně, Czech Republic.The C5 buds were grafted onto virus-free rootstocks of cv.St. Julien in a greenhouse in March 2002. Fifty-five trees of C5 were obtained. C5 transgenic trees were inoculated in August 2002 by budding with the single virus PPV-Rec strain (CRI), dual viruses PPV+ACLSV and PPV+PDV and the triple combination PPV+PDV+ACLSV. Grafted infection buds produced new shoots, permanently growing in transgenic trees, creating permanent infection pressure.Eleven plum trees were used for each virus combination,and 11 non-inoculated trees of C5 were used as the controls(Polák et al. 2017). The collected sample leaves were then in three groups: group 1, C5 plants inoculated with water;group 2, C5 plants with nonpermanent viral pressure (graft and nongraft) from 2003 to 2011; and group 3, plum trees with permanent virus pressure (graft) from 2003 to 2018.The PDV graft was removed completely from all trees in 2011.

2.2. RNA isolation and cDNA synthesis

Total RNA was extracted from leaves using a Spectrum™Plant Total RNA Kit (Sigma Aldrich, St. Louis, Missouri,USA) according to the manufacturer’s instructions with minor modifications and as previously described in Singh and Kundu (2017). cDNA was synthesized using a Reverse Transcription System (Promega, Madison, Wisconsin, USA).A reaction mixture composed of 1 μg of total RNA and 0.5 μg of random primers was incubated at 70°C for 5 min and chilled on ice for 2 min. Then, the following components were added in the given order: 5× reaction buffer, 10 mmol L-1dNTP mix, 20 U μL-1RNasin®RNase inhibitor and 200 U μL-1of M-MLV reverse transcriptase enzyme. The mixture was incubated at 37°C for 1 h, and then the reaction was stopped by heating to 70°C for 10 min and subsequent chilling on ice (Singh and Kundu 2017).

2.3. Quantification of PPV, PDV and ACLSV

Real-time PCR was performed using a LightCycler®480(Roche, Basilej, Switzerland) with SYBR Green I. The thermal cycling protocol was as follows: initial denaturation for 10 min at 95°C followed by 40 cycles of 10 s at 95°C,10 s at 60°C and 10 s at 72°C. The fluorescence signal was measured at the end of each extension step at 72°C.After the amplification, a melting curve analysis with a temperature gradient of 0.1°C s-1from 70 to 95°C was performed to confirm that only the specific products were amplified. Finally, the samples were cooled down to 40°C for 30 s. The ‘Fit Points Method’ in the software determined the threshold cycle (CT). The PCR Mastermix comprised the primers (0.6 μL of primer pair mix of 10 μmol L-1primer pair stock), 6 μL of LightCycler®480 SYBR Green I Master(Roche, Basilej, Switzerland), and sterile nuclease-free water to a final volume of 12 μL. Finally, 2 μL of cDNA was added to this mixture. The primers used in study is listed in Table 1. Primers were evaluated to calibration curves composed of 10 dilution standards. Data obtained from the standards were used to plot a standard curve of crossing point (Cp) vs. log concentration. Care was required in designing, preparing, and calibrating standards, because the absolute quantity of an unknown sample is based on the performance and consistency of the standards. Standards and samples were measured in triplicates.

2.4. Quantitative polymerase chain reaction (qPCR)standard preparation and data analyses

The virus titre in the samples was calculated according to a 10-fold standard dilution curve constructed using PCR-amplified from cDNA using high-fidelity Pfu DNA Taq polymerase (Thermo Scientific, Waltham, MA, USA). The primer pairs transgene PPV-CP (PPV-F3 and PPV-RR) and inoculum PPV-Rec (mM3 and mD5) were used for PCR(Table 1). The reaction conditions were as follow: initial denaturation at 94°C for 2 min; followed by 40 amplification cycles of 94°C for 15 s, 55°C for 30 s and 72°C for 1 min; and a final extension at 72°C for 10 min. The PCR products were separated on a 2% agarose gel and stained with SYBR Safe DNA Gel Stain (Invitrogen, Carlsbad, CA,USA). Subsequently, the amplicon was eluted from the gel using a GenElute PCR Clean-Up Kit (Sigma Aldrich,St. Louis, Missouri, USA) and then cloned into a pGEMT-T Easy vector (Promega, Madison, USA) as described by Singh and Kundu (2017). Plasmid DNA was isolated with a GeneJET Plasmid Miniprep Kit (Thermo Scientific, USA).Two independent clones per amplicon were commercially sequenced (Macrogen, Amsterdam, the Netherlands) using reverse and forward primers of each virus of several isolates.Sequences were analyzed using the software Clustal W2 version 2.0 (Larkin et al. 2007) (http://www.clustal.org/clustal2/), Sequencher 4.8 (Gene Codes Corporation, MI,USA) and BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi).RT-qPCR calculations were performed using LightCycler®480 Real-Time PCR System Software. The following mathematical formula was applied to count virus titre:dsDNA (pmol)=dsDNA (μg)×106pg μg-1/660 pmol pg-1/Nb.Avogadro’s constant was used 6.023×1023molecules mol-1(Chalupniková et al. 2017). A graphical representation of the data was constructed using the program Microsoft Excel.

2.5. Expression analysis of miRNAs

Total RNA (including miRNA) was isolated from leaves using TRI Reagent (Thermo Scientific, USA). cDNA was generated from each miRNA with specific primers following the manufacturer’s protocol using a TaqMan®MicroRNA Reverse Transcription Kit (Thermo Scientific,USA). The reaction mixture was composed of 10 mmol L-1dNTP mix (100 mmol L-1), 50 U μL-1MultiScribe™ reverse transcriptase enzyme, 10× reaction buffer, 20 U μL-1RNase inhibitor, 1-10 ng of purified total RNA and 5× RT primer.The reaction mixture was incubated on ice for 5 min. The mixture was then incubated in thermal cycler at 16°C for 30 min, 42°C for 30 min, and then the reaction was stopped by heating to 85°C for 10 min and subsequent chilling on ice.The real-time PCR mixture comprised 20× TaqMan®Small RNA Assay (Small RNA-Specific TaqMan®MGB Probe),3 μL of cDNA, TaqMan®Universal PCR Master Mix II (2×),and no uracil-N glycosylase (UNG) in a final volume of 20 μL.RT-qPCR was performed (in triplicate) employing the 7300™Real-Time PCR System and software (Applied Biosystems,Foster City, California, USA). The reaction conditions were 95°C (10 min), and 40 cycles of 15 s at 95°C and 1 min at 60°C. Relative quantification of miRNA was calculated using the comparative CT(ΔΔCT) value method (Schmittgen and Livak 2008) using Prunus miR172 as the internal control(Zhu et al. 2012) as previously described (Tripathi et al.2015). For the statistical analyses, ANOVA (one-way) was conducted using a general linear model implemented in R Software (R Core Team 2014) (Singh et al. 2014). Pairwisecomparisons were performed using Tukey’s method at the 95% significance level (P-value＜0.05) (Tukey 1949).

Table 1 List of primers used in this study

3. Results

3.1. Detection of viruses in C5 plants

Plum pox virus isolates belonging to two strains, PPV-Rec(inoculum) and PPV-D (transgene), were detected in the 48 total samples of leaves from 2016 to 2018. We used PCR (for detection of PPV) and qPCR for the absolute quantification of the virus (Fig. 1). PPV-D (PPV-CP) in the C5 plants was used to normalize the abundance of virus(PPV-Rec) in the samples. No PPV-Rec was detected in the control C5 transgenic plants. Very low copies (～1) of the virus were found in C5 transgenic plants, viz., trees 2, 3 and 4, with nonpermanent virus pressure (PPV graft removed in 2011) from 2016-2018. Trees 1 and 5 accumulated～30 copies of PPV in 2016 (data not shown). However,the accumulation of virus decreased in 2018 to fewer than 3 copies. The C5 transgenic trees 6, 7, 8 and 9 with permanent virus pressure (PPV graft remained) showed few copies (＜4) of the virus from 2016 to 2018 (Fig. 1).

C5 transgenic plants inoculated with PPV showed a high level of virus in the graft part, i.e., part 0. Tree 6 (graft 6/0) showed increased accumulation of virus titre (2.8E+03 copies) during 2016. Tree 7 (graft 7/0) accumulated a higher level of virus (3.5E+03 copies) during 2017 than that in 2016 and 2018 (Fig. 1). Tree 8 (graft 8/0) showed a low level of virus (2.0E+03 copies) during all years. The abundance of virus in tree 9 (graft 9/0) was 2.7E+03 copies in 2016.PPV graft parts (graft 6/0, graft 7/0, graft 8/0, and graft 9/0)for three of four trees (except tree 7; graft 7/0) showed a decrease in abundance of virus titre over the period (Fig. 1).

3.2. Quantification of virus titre in C5 plants

In addition to PPV-Rec and PPV-D, two other stone fruits viruses, namely, ACLSV and PDV, were detected in C5 transgenic plants and in graft inoculated with dual infections PPV+ACLSV and PPV+PDV and triple infection(PPV+ACLSV+PDV).

In the first combination, for C5 transgenic plants graftinoculated with PPV (PPV-Rec) and ACLSV (PPV+ACLSV),63 total samples of leaves were tested. No PPV-Rec or ACLSV was detected in the control C5 transgenic plants.In the C5 transgenic plants with the PPV graft removed in 2011, trees 1 and 5 showed ～50-60 copies of PPV-Rec in 2017, which was reduced to 0 (tree 1) and ～32 copies (tree 5) in 2018 (data not shown). Few copies (～5) of PPV-Rec were quantified in trees 1, 3 and 4 (graft removed in 2011).In C5 transgenic plants with the ACLSV graft removed in 2011, ～70 copies of ACLSV were detected in 2017 (tree T1).In 2018, only 2 copies of ACLSV were accumulated in trees 1-5. The abundance of PPV-Rec in the graft of tree 7 (graft T7/0 P) was the highest (6.0E+04 copies) followed by that of tree 11 (graft T11/0 P; 2.02E+04 copies), tree T9 (graft T9/0 P; 1.33E+04 copies), tree 6 (graft T6/0 P; 5.01E+03 copies), and tree T8 (graft T8/0 P; 9.83E+02 copies). Very low numbers of copies (～6) of PPV-Rec were detected in C5 transgenic plants with permanent virus pressure (trees 6, 7,8, 9 and 10) (data not shown). No PPV-Rec was detected in ACLSV-infected grafts (Fig. 2-B). The amount of ACLSV detected in the ACLSV grafts was high; tree T9 showed the highest accumulation of virus (graft T9/0 A; 7.86E+03 copies), followed by tree 8 (graft T8/0 A; 2.29E+03 copies)and tree 11 (graft T11/0 A; 1.16E+03 copies) (Fig. 2-A). We found ＜10 copies of ACLSV in C5 transgenic plants with continuous disease pressure until 2018.

Fig. 1 Detection and absolute quantification using real-time polymerase chain reaction (RT-qPCR) of Plum pox virus (PPV-Rec)in C5 transgenic plums grown in the Czech Republic from 2016 to 2018. Virus titre (copy number) was detected in a healthy noninoculated C5 transgenic plants (control, C1-C3) and C5 transgenic plums graft-inoculated with Plum pox virus (T1-T9). High Plum pox virus copy numbers were observed in the graft part (part 0) compared with those in C5 plants.

In the second combination, for C5 transgenic plants graft-inoculated with PPV (PPV-Rec) and PDV (PPV+PDV),54 total samples of leaves were tested. No PPV-Rec or PDV was detected in control C5 transgenic plants. In C5 transgenic plants with the PPV graft removed in 2011, ～10 copies (tree T2) of PPV-Rec were detected in 2016, which were reduced to 0 copies in 2018 (data not shown). No PDV was detected in PDV graft-removed transgenic C5 plants. The highest abundance of PPV-Rec was in the graft of tree 7 (graft T7/0; 11.2E+04 copies), followed by tree 6(graft T6/0; 9.88E+04 copies), tree T9 (graft T9/0; 9.23E+03 copies), tree 10 (graft T10/0; 9.14E+03 copies) and tree T8(graft T8/0; 8.80E+02 copies). PPV-Rec was not detected in the presence of both viruses with permanent pressure in trees 6, 7, 8, 9 and 10 (Fig. 2-B). No PDV accumulated in C5 plants with the PPV graft (PDV graft was removed in 2011) (Fig. 2-D).

Fig. 2 Detection and absolute quantification using real-time polymerase chain reaction (RT-qPCR) of Plum pox virus (PPV-Rec),Apple chlorotic leaf spot virus (ACLSV) and Prune dwarf virus in C5 transgenic plants. A, accumulation of PPV in graft inoculated with ACLSV (PPV+ACLSV). B, ACLSV accumulation in PPV+ACLSV-infected C5 transgenic plants. C and D, PPV and Prune dwarf virus (PDV) accumulation in dual-infected (PPV+PDV) C5 transgenic plants. E, F and G, abundance of PPV, ACLSV and PDV in triple virus (PPV+ACLSV+PDV)-infected C5 transgenic plants. C1-C3, control; T1-T11, C5 transgenic plants; T6/0 P-T11/0 P,PPV-infected C5 transgenic plants; T8/0 A, T9/0 A and T11/0 A, ACLSV-infected C5 transgenic plants.

In the triple combination, for C5 transgenic plants graft-inoculated with PPV (PPV-Rec), ACLSV and PDV(PPV+ACLSV+PDV), 63 total samples of leaves were tested. PPV-Rec, ACLSV and PDV were not detected in control C5 transgenic plants. In C5 transgenic plants with the PPV graft removed in 2011, tree 5 showed ～5×102copies of PPV-Rec in 2016, which were reduced to ～150 copies in 2018. Tree 3 showed ～210 copies in 2016 (data not shown), and in 2018, ～20 copies were detected. In C5 transgenic plants with the ACLSV graft removed in 2011,～160 copies of ACLSV were detected in 2017 (tree T5). In 2018, the number of copies (＜2) of ACLSV accumulated in trees 1 to 5 was not significant. PDV was not detected in any of the trees (trees 1 to 5). The highest abundance of PPV-Rec was in the PPV graft of tree 7 (graft T7/0 P;5.43E+05) compared with that in trees 9 (graft T9/0 P;3.53E+05), 11 (graft T11/0 P; 1.40E+05) and 8 (graft T8/0 P;1.38E+05) (Fig. 2-E). However, ＜3×102copies of PPV-Rec were detected in trees 6, 7, 8, 9 and 10 (data not shown).PPV-Rec was not detected in ACLSV-infected grafts, and ACLSV was not detected in PPV-infected grafts. High ACLSV accumulation was detected in trees 8 (graft T8/0 A;8.70E+04), 9 (graft T9/0 A; 6.32E+05) and 11 (graft T11/0 A;3.32E+05) (Fig. 2-F). PDV did not accumulate in PPV+ACLSV+PDV-infected C5 transgenics, either in the graft part or in C5 transgenic plants (Fig. 2-G).

3.3. Expression profile of microRNAs

Differential expression profile of conserved microRNAs from Prunus (Zhu et al. 2012) was observed in C5 transgenic plants. Relative expression of highly conserved miRNAs such as miR167, miR169 and miR396 and less conserved miRNAs, miR828 and miR858, were observed using TaqMan®Small RNA Assays (Material and methods). The fold change (FC, log2Fold change) in expression observed under these conditions has been shown as bar graphs(Fig. 3). miR167 showed upregulation in C5 transgenic plants (control) (PPV: 1.9 FC), C5 transgenic plants infected with only PPV showed 2.42 FC, in co-infected combination:PPV and ACLS (PPV+ACLSV; 4.55 FC) and PPV and PDV(PPV+PDV; 3.24 FC), and in triple infection in combination of PPV+ACLS+PDV (3.91 FC) (Fig. 3-A). Expression of miR169 showed upregulation in C5 transgenic plants: control(0.71), PPV (0.91 FC), PPV+ACLS (2.13 FC), followed by upregulation in PPV+PDV (1.22 FC) combination and PPV+ACLSV+PDV (0.89 FC) (Fig. 3-B). Expression profile of miR396 showed high upregulation in dual combination PPV+ACLS (3.13 FC) followed by in co-infection of triple virus, PPV+ACLSV+PDV (1.9 FC) and PPV+PDV (1.8 FC)and similar concentration in PPV infected plants and control (Fig. 3-C). Among less-conserved miRNAs, miR828 and miR858 showed downregulation in C5 transgenic plants (control) as well as when infected with only PPV,PPV+ACLSV, PPV+PDV and PPV+ACLS+PDV (Fig. 3-D and E). In C5 transgenic plums, conserved miRNA showed high upregulation in dual infection PPV+ACLSV (～2.4-fold higher than the control C5 plants), followed by PPV+PDV(～1.7 fold higher than the control plants). C5 plants with triple viral infection (PPV+ACLSV+PDV) showed ～1.55-fold higher expression than the C5 plants. However, lower level of expression was observed in case of only PPV-infected C5 transgenic plants.

Fig. 3 Expression profile of microRNAs (miR) in C5 transgenic plums. The expressions of highly conserved miRNAs, miR167(A), miR169 (B) and miR396 (C), and less conserved microRNAs, miR828 (D) and miR858 (E) were analyzed. Prunus miR172 was used as the endogenous control to normalize the expression data. Healthy non-inoculated C5 transgenic plants were used as the controls. C5 transgenic plums with dual inoculation: Plum pox virus (PPV)+Apple chlorotic leaf spot virus (ACLSV) (P+A)and PPV+Prune dwarf virus (PDV) (P+D); C5 transgenic plums with triple inoculation: PPV+ACLSV+PDV (P+A+D). Letters (a,b, c, d and e) indicate statistically significant differences (P-value＜0.05) according to Tukey’s HSD (honest significant difference)test. Error bars represent the SD based on at least five biological replicates.

4. Discussion

Quantitative analysis of virus uptake in C5 transgenic plants performed by RT-qPCR demonstrated resistance not only to a single virus (PPV) but also to the combinatorial infection of two additional viruses (ACLSV and PDV) in different combinations (PPV+ACLSV, PPV+PDV and PPV+ACLSV+PDV). Our results provided absolute quantification of PPV, ACLSV and PDV (copy number)in addition to the presence or absence of virus close to the inoculum. However, the virus copy number varied in the individual cases. In C5 transgenic plums grafted with the single virus (only PPV), a very low copy number of the virus was accumulated (＜32 copies), whereas the copy number increased to ～60 in the dual combination(PPV+ACLSV) and by ～8.3-fold (～5×102copies) in the triple combination (PPV+ACLSV+PDV). Similarly, we observed a low copy number (～70) of ACLSV in the dual combination PPV+ACLSV, whereas the number increased 2-fold(～1.6×102copies) in PPV+ACLSV+PDV. Thus, the virus titre analysis demonstrated the stability and status of resistance of these C5 plants challenged with PPV and heterologous viruses (ACLSV and PDV), which were previously observed through long-term symptoms evaluation and virus detection by ELISA and RT-PCR (Polák et al. 2005, 2017). The level of resistance status was similar in graft-inoculated C5 transgenic plums from a field experiment in Romania in which the plants were challenged by multiple viruses such as ACLSV, PNRSV and PDV in different combinations with PPV(Zagrai et al. 2008). However, systemic infection of PPV in C5 plants under a permanent PPV infection pressure was shown previously, which occurred through graft-inoculation and with mild symptoms sometimes appearing close to the inoculum sources (Kundu et al. 2008). The significant increase in virus titre (PPV-Rec: ～5.43E+05 copies and ACLSV: ～8.70E+04 copies) in the inoculum source grafts showed that the virus remained active and was exerting pressure with time. The accumulation of PPV-Rec found to be higher due to the fact that a highly susceptible graft material such as cv. Emma Leppermann was used as PPV inoculum source. The differential viral pressure in the infected grafts could correspond to the long-term exposure of plants and environmental conditions in the field. Only a few C5 transgenic plants with nonpermanent pressure(graft removed in 2011) showed very few copies of viruses,whereas others were completely virus-free. The significantly very low copy number (103to 105) of virus compared with the inoculated graft showed that these viruses (PPV, ACLSV and PDV) did not affect significantly the efficacy and stability of posttranscriptional gene silencing (PTGS)-based resistance in C5 transgenic plants. Viral synergism occurs in nature and is caused by the interaction of two unrelated viruses in the same host, resulting in increased symptoms and an accumulation of one or both viruses (Pruss et al. 1997;Syller 2012). The mixed infection of viruses may lead to both synergetic and antagonistic response as well in stone fruit trees (Maliogka et al. 2018). In our study, such an effect was not recorded in C5 plums with either ACLSV or PDV co-infected with PPV. Hence, the stability of resistance in C5 plants was confirmed even with permanent PPV and other heterologous viruses tested in our field experiment and elsewhere (Hily et al. 2004; Malinowski et al. 2006;Scorza et al. 2013).

miRNAs are among the most important gene regulators that control plant growth and development, maintenance of genome integrity, signal transduction, innate immunity,hormone signaling pathways, and response to abiotic and biotic stress in plants (Sunkar et al. 2007; Sun 2012). Plant miRNAs have antiviral activity and support the siRNA-based antiviral defense (Simon-Mateo and Garcia 2006; Pérez-Quintero et al. 2010). Both virus and host utilize miRNAs as efficient weapons to fight against one another (Peng et al. 2018). Tomato miRNAs exhibit altered expression in response to leaf curl disease caused by Tomato leaf curl New Delhi virus (ToLCNDV), suggesting their role in basal defense activity and leaf morphogenesis (Naqvi et al. 2010).In ToLCNDV-agroinfected leaves and flowers, most of the miRNAs including miR159, miR160, miR162, miR166a and b, miR167, miR171, miR172, miR395, and miR397 are up-regulated compared with expression in healthy plants.Co-infection of Nicotiana benthamiana by PVX and PPY or PPV produced the most severe symptoms and altered the accumulation of miRNAs (miR156, miR171, miR398,and miR168) and/or target transcription to a greater extent as compared to single infections (Pacheco et al. 2012).Our data showed significantly higher accumulation of conserved miRNAs such as miR167, miR69 and miR396 in C5 transgenic plants in co-infected C5 transgenic plants(PPV+ACLSV, PPV+PDV and PPV+ACLSV+PDV) as compared to single infected PPV plants. However, it would be intriguing to identify other miRNAs that are differentially regulated during virus infection in the C5 transgenic plants using high-throughput sequencing methods (Ma et al. 2015).The conserved and abundantly expressed plant miRNAs are associated with the resistance to virus infection in plant (Ai et al. 2011; Ramesh et al. 2014). It has been suggested that the miRNA-guided processing contribute similar manner of plant defense like siRNA-guided silencing by interfering with virus infection (Simon-Mateo and Garcia 2006). The miRNAs profile of C5 further confirm the RNA silencing based resistance in C5 plant to PPV (Hily et al. 2005; Kundu et al. 2008) as well as multipole heterologous virus infection such as ACLSV and PDV.

5. Conclusion

Quantitative analysis of virus uptake in C5 transgenic plants demonstrated complete resistance not only to a single virus (PPV) but also to the combinatorial infections of two additional viruses ACLSV and PDV, even with the continuous infection pressure that occurred in graft-inoculated plants in the field. The significantly very low copy number (103to 105)of virus compared with that of the inoculated graft showed that these viruses did not affect the efficacy and stability of PTGS-based resistance in C5 transgenic plants either to PPV or to the heterologous viruses ACLSV and PDV. The stability of resistance in C5 plants was further confirmed by the miRNAs expression profile, suggesting the upregulated miRNAs may be associated with siRNA regulation in this transgenic plum. Hence, the present study demonstrated the long-term efficacy of RNAi-mediated virus resistance in C5 transgenic plants to these viruses.

Acknowledgements

Projects from the Ministry of Agriculture of the Czech Republic (NAZV QJ1610186 and MZE-RO0418) supported this work.