Distinguishing resistances of transgenic sugarcane generated from RNA interference and pathogen-derived resistance approaches to combating sugarcane mosaic virus

Sugarcane mosaic virus (SCMV) is a causative agent that reduces growth and productivity in sugarcane. Pathogen‐derived resistance (PDR) and RNA interference (RNAi) are the most common approaches to generating resis‐ tance against plant viruses. Two types of transgenic sugarcane have been obtained by PDR and RNAi methods using a gene‐encoding coat protein (CP) of SCMV (SCMVCp). This research aimed to distinguish resistance of the two transgenic sugarcanes in combating SCMV through artificial viral inoculation. The experiment was conducted using transgenic sugar‐ cane lines validated by PCR analysis. Insertion of gene‐encoding CP in the transgenic lines was confirmed by amplification of 702 bp of DNA fragment of SCMVCp. After viral inoculation, mosaic symptoms appeared earlier, at 21 days post inoculation (dpi) in PDR transgenic lines, but was at 26 dpi in RNAi transgenic lines. Symptom observation showed that 77.8% and 50% of the inoculated plants developed mosaic symptoms in PDR and RNAi transgenic lines, respectively. RT‐PCR analysis revealed that the nuclear inclusion protein b (Nib) gene of SCMV was amplified in the symptomatic leaves in plants classified as susceptible lines. Immunoblot analysis confirmed presence of viral CP with a molecular size of 37 kDa in the susceptible lines. Collectively, these results indicated that the RNAi approach targeting the gene for CP effectively produces more resistance against the SCMV infection in transgenic sugarcane compared to the PDR approach.


Introduction
Sugarcane (Saccharum officinarum L.) is an important agricultural commodity for sugar production in Indone sia. The productivity of sugarcane was affected by several factors, such as topography, climate, soil fertility, insects, fungal, bacterial, and viral infections (Srivastava 2012). Sugarcane mosaic virus (SCMV) is one of the most de structive viruses for sugarcane in Indonesia with disease incidence up to 78% (Addy et al. 2017) and reduce yield up to 45% . The SCMV infection inhibits the development of stem diameter and length of intern ode from the early growth to the harvest period. The im pact of viral infection could be attributed to disturbance of gene expression associated with the photosynthesis pro cess (Chen et al. 2017a) and chlorosis on sugarcane leaves showing mosaic patterns. In addition, upon infection in host plants, the virus is distributed from cell to cell using plasmodesmata, then the virus moves from source to sink tissue through the vascular system to establish systemic infection (Anurag 2013).
SCMV belongs to the genus of Potyvirus, the family of Potyviridae, which has a positivesense singlestranded RNA (+ssRNA) genome type. The genome has an open reading frame (ORF) encoding 10 proteins, such as P1, HCpro, P3, 6K1, CI, 6K2, NIaVPg, NIapro, NIb, and CP (Gao et al. 2011). In plants, Potyvirus is transmitted by vectorlike aphids during the feeding process via stylets. The virus transmission occurs through two different strate gies, in the capsid strategy, coat protein (CP) directly in teracts with binding sites (receptors) in the aphid stylet. While, in the helper strategy, nonstructural protein HC Pro (Helper component proteinase) facilitates the binding by creating a reversible molecular bridge between CP and aphid receptors (Gadhave et al. 2020). Thus, the CP is the widely studied infection of the virus at a molecular level in plants.
CP plays a role in the systemic spread of viruses in plant tissues and regulates the assembly process of intact virus particles (BesongNdika et al. 2015). CP was known to provide resistance to protect from Tobacco mosaic virus (TMV) infection in transgenic tobacco through the CP mediated resistance (CPMR) technique. This technique induces resistance when the Cp gene was expressed in plant cells and forms an aggregate of CP (Sharma et al. 2020). Transgenic tobacco that expressing the CP has been shown to inhibit TMV virus infection by interfering with the accumulation of movement protein (MP) responsible for carrying viral particles from cell to cell and block ing the virus particle assembly (Bendahmane et al. 2002). The expressing CP could prevent TMV virions from un dergoing cotranslational disassembly, which is an early event of infection. The protective mechanism of trans genic CP is immediately recoated disassembling virus par ticles to prevent their infection (Lu et al. 1998). Resistance against virus using CP was also successfully performed in peanuts (Arachis hypogea L.) (Mehta et al. 2013), in egg plant (Solanum melongea L.) (Pratap et al. 2011), and sug arcane (Saccharum officinarum L.) (Apriasti et al. 2018). The defense against viruses through the expression of nu cleotide sequences derived from viruses to produce the vi ral protein in plant cell is known as pathogenderived resis tance (PDR) Dougherty 1992; Sharma et al. 2020).
RNA interference (RNAi) or RNA silencing is also known as a defense mechanism that protects plants from pathogen infections and downregulate the viral gene ex pression in a specific manner (Muhammad et al. 2019). Transgenic RNAi has been developed as a molecular tool for enhancing disease resistance in plants. The resistance mechanism of RNAi has effectively synthesized small interfering RNA (siRNA) to downregulate toxic fungal genes in Aspergillus and Fusarium fungi based on the for mation of hairpin RNA (hpRNA) to control mycotoxigenic fungi (Majumdar et al. 2017). The dsRNA or hpRNA is processed into small interfering RNA (siRNAs) of 21 28 nucleotide in lenght by the activity of RNase IIIlike enzymes called Dicer. The siRNAs bind to argonaute (AGO) a ribonuclease Hlike protein, then incorporate into a RNAinduced silencing complex (RISC) (Widyan ingrum et al. 2021). Small RNA complexes could rec ognize RNA targets through complementary base pairs, while the AGO protein functions as an effector to mod ulate target activity (Campo et al. 2016). Therefore, the RNAi resistance mechanism has a high efficiency that sup presses viral infection through dsRNA expression in the transgenic plant by targeting the Cp gene in Plum pox virus (Montes et al. 2014). Recently, the RNAi mechanism has been proven workable to combat virus infection in trans genic sugarcane (Widyaningrum et al. 2021).
The transgenic sugarcane resistance to SCMV has been successfully produced by the PDR (Apriasti et al. 2018) and RNAi (Widyaningrum et al. 2021) approach targeting the coat protein (Cp) gene of SCMV. In this re search, we compared the two types of transgenic sugarcane lines and determined the level of effectiveness in prevent ing SCMV infection through the viral challenge. The re sults showed that RNAi more effective to combat the virus infection compared to the PDR technique. This is the first report for the comparative study of the response SCMV infection of two different transgenic sugarcane generated by the PDR and RNAi approach.

Transgenic sugarcane plant materials
To distinguish the resistances of transgenic sugarcane lines generated by PDR and RNAi methods, the transgenic lines were grown in the greenhouse. The lateral buds were iso lated from previous T2 transgenic lines generated from both PDR (Apriasti et al. 2018) and RNAi (Widyaningrum et al. 2021), then germinated and cultured in the pot con taining soil media. Totally three (A10, A11, A13) and four (C16, C18, U1, U8) transgenic lines generated from PDR and RNAi with three replicates were cultivated in a green house for three months, respectively. The plants were sub jected to artificial SCMV inoculation at the indicated time and observed the development of mosaic symptoms, fol lowed by molecular analysis.

Viral inoculation and mosaic symptom observation
To determine the resistances against SCMV, six weeks cultured the transgenic lines were challenged with a vi ral inoculum prepared from the symptomatic sugarcane leaves infected by the virus. The virus inoculum was pre pared as plant sap (plant fluid) according to the method previously described (Apriasti et al. 2018). Two grams of the sugarcane leaves were ground using a sterile mortar and pestle on ice, and added with 2% of polyvinylpyrroli done (PVP). The crushed leaves were transferred into a falcon tube containing 20 ml of 0.1 M phosphate buffer pH 7.0 and the solution was immediately used as inocu lum. Before virus inoculation, the transgenic plants were incubated in dark conditions for a night. The virus inoc ulant was mechanically inoculated on leaves by carefully scratching the 2nd and 3rd of the youngest leaves using 600 mesh of carborundum, followed by rubbing the leaves with sap and stand for 5 min. The plant was rinsed using sterile distilled water to remove the remaining carborun dum and sap liquid and then was continuously cultivated in the greenhouse.
The mosaic symptom was daily observed on the sug arcane leaves after the viral inoculation over 45 d post inoculation (dpi). The mosaic pattern on leaves was deter mined and grouped according to the Cobb scale (Kiss and Veres 2017). The number of symptomatic plants, includ ing the incubation period, was recorded as previously de scribed (Apriasti et al. 2018). Plant with or without symp toms was used for further molecular analysis.

DNA extraction and PCR analysis
Genomic DNA of sugarcane was extracted from leaf tis sue using the SDS protocol according to the method pre viously described (Widyaningrum et al. 2021). One g of the leaf tissue was ground using liquid nitrogen in a mortar and pestle, and the frozen leaf powder was added with 1 mL of extraction buffer containing 100 mM TrisHCl (pH 8), 50 mM EDTA, 500 mM NaCl, 1% SDS, and 5 mM 2 mercaptoethanol and then incubated at 65°C for 10 min. The mixture was centrifugated at 12,000× g at 4°C for 10 min and the supernatant was added with 0.8 vol. of iso propanol. After incubation at 20°C for an hour, the DNA was collected by centrifugation and rinsed with 70% cold ethanol. The remaining ethanol was evaporated and DNA was dissolved in 20 µl of buffer containing 10 mM Tris HCl (pH 7.5), 1 mM EDTA (TE buffer). The DNA con centration was determined by NanoVue spectrophotome ter (GE Healthcare, UK) at 260 nm and stored at 20°C for analysis.
PCR analysis was conducted to confirm the insertion of the Cp gene in the transgenic lines. The PCR analysis was conducted using a master mix kit (GoTaq Green Mas ter Mix, Promega, USA), 1 µg DNA genome, and a primer pair of SCMVCp (F1R1) according to the method previ ously described (Apriasti et al. 2018). The PCR ampli fication cycle was carried out using a T100 thermocycler (BioRad, USA) with the condition of predenaturation at 95°C for 5 min, followed by the 35 cycles of denaturation at 95°C for 30 s, at 58°C annealing for 30 s, extension at 72°C for 1 min, and the final extension at 72°C for 5 min. The PCR product was separated on 1% agarose gel electrophoresis and visualized using the GelDoc (Major Science, USA).

Viral detection by RT-PCR and immunoblot analysis
To detect viral infection RTPCR analysis was conducted for amplification DNA fragment of Nib gene after the viral infection in sugarcane. Total RNA was extracted from 0.5 g of the sugarcane leaves using the RNAprep Pure Kit for plant (Tiangen, China). The isolated total RNA was then dissolved into sterile ddH 2 O and measured the RNA con centration using a NanoVue spectrophotometer at a wave length of 260 nm (GE Healthcare, USA). One µg of total RNA was converted into cDNA using reverse transcriptase and oligo˗dT primer according to the manufacture proto col (Bio˗Rad, USA). The cDNA was used as a template for PCR amplification of the Nib DNA fragment using a primer pair of F4˗R4. To ensure the total isolated RNA has the same concentration, the Actin gene as an internal con trol was determined by employing a pair of F3˗R3 primers (Table 1).
To confirm the viral infection, immunoblot analysis was conducted using a polyclonal antibody against CP ). The sugarcane leaves (2 g) were ground using liquid nitrogen, and the protein was extracted using a buffer containing 50 mM of Tris˗HCl (pH 7.5), 5 mM of EDTA, 1 mM of phenylmethylsulfonyl fluoride, 10 mM of 2˗mercaptoethanol, and 2% polyvinylpolypyrroli done (PVP). The mixture was centrifuged at a speed of 14,000× g, 4°C for 10 min. Insoluble protein was solu bilized from the pellet using a buffer containing 50 mM Tris˗HCl (pH 8.5), 1 mM EDTA, 2% SDS, and 30% su crose and separated using centrifugation at 12,000× g for 10 min. The insoluble protein was then separated us ing SDSPAGE (sodium dodecyl sulfatepolyacrylamide gel electrophoresis) at 12% acrylamide and transferred to immobilonP transfer membrane (Millipore) using a semi˗dry trans˗blotter (Bio˗Rad, USA). The membrane was washed three times with TrisBuffer Saline (TBS), followed by blocking using 0.5% skim milk. The mem branes were incubated with the polyclonal antibody di luted with 3000× TBS buffer containing 0.5% skim milk in a shaker overnight at room temperature. The membrane was rinsed three times using TBS buffer and incubated with a secondary antibody of goat antirabbit IgG alkaline phosphatase (AP)˗conjugate (Bio˗Rad) with 3000× dilu tion for an hour at room temperature. The targeted CP protein was visualized with a solution mixture of BCIP (5˗Bromo˗4˗chloro˗3˗Indolyl˗phosphate) and NBT (nitro blue tetrazolium) (BioRad, USA).

Validation of transgenic sugarcane by PCR analysis
To validate the transgene insertion, the DNA genome was isolated from sugarcane leaves and used for PCR analy sis using F1˗R1 primers pair to amplify inserted Cp gene. The PCR analysis showed amplification of the 725 bp rep resenting the partially inserted Cp DNA fragment in both PDR and RNAi transgenic lines (Figure 1a, Figure 1b). The corresponding Cp DNA fragment was also amplified with the same molecular size in the control plasmid. The results validated the presence of the Cp gene in the trans genic lines used for the experiment.

Artificial Inoculationand symptom development
To evaluate the viral resistance, the transgenic lines gener ated from PDR and RNAi methods were challenged with SCMV inoculation. As expected, symptom development was early observed at 21 dpi and then clearly appeared at 45 dpi. Morphological symptom observation showed that among nine PDR transgenic lines, seven or 77.8% were symptomatic, but only six among 12 lines or 50% were developed the mosaic symptom in RNAi transgenic lines. Furthermore, disease assessment after artificial in oculation was also determined by observation of incuba tion periods and mosaic symptom patterns. In terms of the incubation period (time between host infection and ex pression of disease symptoms), the first mosaic symptom was appeared at 26 dpi in RNAi transgenic lines but was earlier at 21 dpi in PDR transgenic lines (Table 2). Ac cording to the Cobb scale, the mosaic distribution pattern in leaves of PDR transgenic lines (Figure 2a) were grouped into 90˗100%, 20˗30%, and less than 5% which predicted as highly susceptible, moderately susceptible, and resis tant, respectively (Table 2). While in RNAi transgenic lines showed less mosaic distribution pattern (Figure 2b) with 11˗20%, 5˗10%, and less than 5% and suggested as moderately susceptible, moderately resistant, and resistant plants respectively. Collectively, these results indicated that compared to RNAi transgenic lines, the PDR trans genic lines were more severe developed the symptom due to SCMV infection.

Viral detection using molecular analysis
The presence of SCMV after the viral inoculation was de tected using RTPCR and immunoblot analysis.  (Figure 3a), where the symptoms distribu tion pattern were found less than 5% (Table 2). Actin DNA fragment that was used as a reference gene for normaliz ing the RNA content with molecular size at 568 bp was found in all examined transgenic lines (Figure 3b). These results indicated that symptomatic leaves less than 5%, the SCMV were not found and classified as resistant plants.
To confirm the presence of viral infection in trans genic sugarcane, immunoblot analysis was conducted with a specific polyclonal antibody against CP of SCMV (Dar sono et al. 2018). Immunoblot analysis revealed that the corresponding CP band with a molecular size of 37 kDa was found in the susceptible lines of transgenic PDR (A10.1, A10.2, A11.1, A11.2, A11.3, A13.1, A13.2) and transgenic RNAi (C18.1, C18.2, C18.3, U1.1, C16.2, C16.3). In parallel with the RTPCR analysis, among 9 PDR transgenic lines, 7 lines (77.8%) showed the CP band (Figure 3a), and among 12 RNAi transgenic lines, only 6 lines (50%) appeared the corresponding protein band (Figure 3b). These molecular analyses indicated that the SCMV was detected in susceptible transgenic lines in con sistent with the appearance of the symptom. Compared to RNAi transgenic lines, the PDR lines were more fre quently infected by SCMV both in terms of symptom dis tribution and the appearance in molecular analysis.

Discussion
There are some techniques to produce resistant plants against viruses through transgene techniques. The most common approaches to generate the resistant transgenic plant against the virus are PDR such as overexpressing coat proteinmediated resistance (CPMR) and RNAi ap proach by generating siRNA (Majumdar et al. 2017). We have successfully developed transgenic sugarcane resis tance to SCMV using PDR (Apriasti et al. 2018) as well as RNAi methods (Widyaningrum et al. 2021). In this study, these transgenic sugarcane lines were examined and com pared to their resistances against SCMV.
Plants provide different respond for defense in the virus infection, when the interaction is compatible makes plant susceptible and cause disease. However, if the inter action is incompatible, the plant prevents virus infection and the emergence of diseases so which causes resistance (Soosaar et al. 2005). The mosaic symptoms on leaves of transgenic lines appeared at different incubation peri ods that might depend on the plant's response to the virus infection. The incubation period of the virus in the PDR transgenic lines was showed at 21 dpi but was at 26 dpi in RNAi transgenic lines ( Figure 2). These results indicated that RNAi transgenic lines have delayed the appearance of the symptom and more effectively prevent virus infection.
The mosaic symptoms that appear in the leaves of sugarcane caused by SCMV infection have been proven by RTPCR and immunoblot analysis to detect the pres  ence of the virus (Figure 3, Figure 4). These results im plied that the virus successfully replicates and disrupts the plant defense system in the symptomatic plants. The im munoblot and RTPCR are commonly used to detect the presence of a virus in the infected plant, such as detec tion of Zucchini yellow mosaic virus (ZYMV) in cucur bitaceous plants (Chen et al. 2017b), detection of SCMV in sugarcane (Addy et al. 2017), detection Citrus yellow mosaic virus (CYMV) in citrus (Kumar et al. 2018), and detection of Banana bunchy top virus (BBTV) in banana (Thomson and Dietzgen 1995). Based on molecular anal ysis showed that the RNAi approach targeting the gene for CP effectively produces more resistance against the SCMV infection in sugarcane compared to the PDR ap proach.
Upon artificial viral inoculation, the PDR and RNAi transgenic sugarcane showed mosaic symptoms incidence reach 77% and 50%, respectively. Moreover, the mosaic pattern and molecular analysis indicated that the RNAi transgenic lines are more resistant than PDR transgenic lines ( Figure 3, Figure 4). The ability of PDR and RNAi methods to abolish pathogen infection has been reported by some studies (Lindbo and Falk 2017). One of the pos sible mechanisms of the PDR transgenic plant in control ling plant viruses is delaying the symptoms. For example, the tomato transgenic plant delays symptom appearances and could recover phenotype from symptoms expression (Sengoda et al. 2012). Moreover, the PDR mechanism in a plant to block viral replication depends on the level of accumulation of CP expression (Sengoda et al. 2012; Mishra et al. 2014. The higher expression results in the higher antiviral capability to block viral replication. So that, CP˗mediated resistance probably not be totally ef fective against virus infection. On the other hand, the RNAi transgenic plant probably reduces disease severity and virus titer. This suggestion is based on RNAi methods that develop transgenic tomato resistance against tomato TYLCV˗OM. This resistance was due to the genetic virus replication is inhibited, resulting in lower virus concentra tion and disease development symptoms (Ammara et al. 2015). The siRNA generated by dsRNA could confer re sistance to trigger gene silencing that has a direct rela tionship with viral replication. In a highlevel expression of siRNA, the virus replication is avoided (Kumari et al. 2018). This is the new strategy to genetically engineer virus resistance to mitigate several concerns of environ mental risk associated with PDR resistance.
In our study, the transgenic PDR showed highly sus ceptible symptoms in some lines. Meanwhile, transgenic RNAi lines showed moderately susceptible symptoms. The resistance mechanism mediated by RNAi can protect against inoculum very high because its target is specific. Proteinmediated resistance generally has a lower level of resistance, but its spectrum is wide. Furthermore, gene si lencing generated by hairpin RNA (hpRNA) was reported as a stable gene silencing method in plants (Helliwell and Waterhouse 2005), comparing with proteinmediated re sistance that the genetic stability is still unclear. There fore, the RNAi mechanism generated siRNA was worked and indicate that this strategy could controlled virus infec tion at a satisfactory level.

Conclusions
The results in this study revealed that the RNAi strategy could control viral infection at a satisfactory level than the PDR strategy. Symptoms still appeared in both strategies, but the highly susceptible symptoms were showed in PDR transgenic plants. On the contrary, RNAi could decrease viral infection based on the distribution mosaic pattern and incubation period. The symptom observation has been val idated by molecular analysis to confirm the presence of SCMV after viral inoculation.