A plasmid vector harboring the Pac1 gene was presented by Prof. Li (Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China). According to the known sequence of Pac1 gene from NCBI, a pair of cloning primers, Pac1 1F and 1R, were designed and synthesized Primer sequence shows in Table 1. The forward primer was inserted with BamH I restriction enzyme cutting site and the reverse primer was inserted with Sac I restriction enzyme cutting site. The Pac1 gene was then isolated using the presented vector as the template. PCR production was purified with the Gel Extraction Kit (AP-GX-P-50G, Axygen, China), and cloning was performed by using the TA cloning vector (T-Vector pMD™19, Takara, China). Then, the positive cloning vector (pMD19T-Pac1) was certified by sequencing.

Prokaryotic expression and purification of PAC1 protein were carried out by Detai Biologics Co., Ltd. (Nanjing, China). The main protocol was as follows. The Pac1 gene was synthesized artificially, and an N-His tag was added. Nde I and Hind III restriction enzyme cutting sites were added at the 5′ and 3′ sites, respectively. Then, by using Nde I and Hind III restriction enzymes, the Pac1 gene with the N-His tag was ligated to the prokaryotic expression vector (pET30a, Biodee, China). After verification, the positive prokaryotic expression vector pET30a-Pac1 was transformed into receptor bacteria BL21 (DE3) (EC1001S, WEIDI 2^nd lab, China). The positive BL21 bacterial cell was collected after 16 h of induced cultivation (ITPG.6 mmol/L). BL21 bacterial cell harboring empty pET30a vector was cultured and collected in the same way and set as a negative control. The bacterial cell was re-suspended in the reaction buffer (20 mmol/L Tris-HCl (pH 7.6), 100 mmol/L KCl, 10 mmol/L MgCl2, and 0.1 mmol/L DTT) and destroyed by ultrasonic wave. After centrifugation, the supernatant was derived, and the crude protein was analyzed by the SDS-PAGE gel segregation method. Then, the PAC1 protein was purified by affinity chromatography (Ni-IDA resin) method. The concentration of the purified protein was tested by the Bradford Protein Assay Kit (P0006, Beyotime Biotechnology, China). Western blot was conducted to test the activity of artificially synthesized PAC1 protein by using the Anti-His antibody.

Double-stranded RNA was artificially synthesized in vitro by using an RNA synthesize kit (MEGAscript^® RNAi Kit, Invitrogen, USA). PCR primers, SCSMV 1F and 1R, were synthesized to amplify part of the genomic fragment of SCSMV Primer sequence shows in Table 1. T7 promoter (23 bp) was added to the 5′ site of all PCR amplification primers. Each 25 μl of the PCR reactant contained 12.5 μl of PCR Master Mix buffer (2× Taq PCR Master Mix, ComWin Biotech, China), 1 μl of cDNA template synthesized from each kind of virus by reverse transcription reaction, 1 μl of forwarding primer (10 μmol/L), 1 μl reverse primer (10 μmol/L), and 9.5 μl of double-distilled (dd) H₂O. PCR amplification was conducted as follows. Initial polymerase activation was performed at 105°C for 5 min; 5 cycles at 95°C for 30 s, 66°C for 30 s, 72°C for 1 min; 25 cycles at 95°C for the 30 s, 70°C for 30 s, and 72°C for 1 min; and final extension at 72°C for 10 min. PCR production was purified by the Gel Extraction Kit (AP-GX-P-50G, Axygen, China). The purified PCR product and proteinase K (200 μg/mL) were mixed with the enzyme reaction buffer (10 mmol/L Tris-HCl at pH 8.0, 20 mmol/L EDTA at pH 8.0, 5% SDS, and 0.4 mmol/L NaCl), held at 50°C, and reacted for 30 min. Then, the phenol-chloroform (25:24) was added and maintained for 10 min. After centrifugation (13,000 r/min, 10 min), the supernatant was removed into a new tube (DNAse/RNAse free). Two-fold cold absolute ethyl alcohol was added and held at −20°C in the refrigerator for 1 h. After centrifugation (13,000 r/min, 10 min), the DNA sediment was washed with 70% DNAse/RNAse-free alcohol twice. After air drying, DEPC water was used to dilute the DNA sediment. By then, the DNA sediment was a double-stranded DNA that was connected with the T7 promoter.

Double-stranded DNA promoted by the T7 promoter was used as the template for the double-stranded RNA synthesis in vitro. According to the protocol that the RNA synthesis kit (MEGAscript^® RNAi Kit, Invitrogen, USA) provided, the double-stranded RNA synthesis reactant was found to contain 1–2 μg double-stranded DNA template, 2 μl 10 × T7 Reaction Buffer, 2 μl ATP Solution, 2 μl CTP Solution, 2 μ GTP Solution, 2 μ UTP Solution, and 2 μl T7 Enzyme mix. DNAse/RNAse-free water was added to achieve a total volume of 20 μl. The mixture was mixed gently and kept for 4 h at 37°C. Then, reactants were placed in a PCR device, and the temperature conditions were set as follows: 95°C for 3 min, 75°C for 3 min, 50°C for 3 min, and finally, room temperature for 5 min. Then, 5 μl of 10× Digestion Buffer, 2 μl of DNase I (2 U/μl), 2 μl of RNase (2 U/μl), and 21 μl of Nuclease-free water were added. The mixture was maintained at 37°C for 1 h to degrade all double-stranded DNA templates and any residue that the single-stranded RNA may have formulated during the synthesis process of the double-stranded RNA. After 1 h of degradation, 50 μl of 10× Binding Buffer, 250 μl of 100% Ethanol, and 150 μl of nuclease-free water were added. The 500-μl mixture was moved into the filter element provided by the RNA synthesis kit. After centrifugation (13,000 r/min, 10 min), the filtrate was removed, and 500 μl of 2× wash solution was added to the filter element. Centrifugation (13,000 r/min, 10 min) was performed, and the wash solution was removed. Pre-warmed (90°C) DEPC water (100 μl) was added to the filter element to elute the double-stranded RNA. Centrifugation was performed (13,000 r/min, 10 min), and the elution was transferred into a new nuclease-free tube. The elution (0.5 μl) was verified by 1% agarose gel electrophoresis. The concentration of the double-stranded RNA was tested by spectrophotometry.

Purified PAC1 protein and double-stranded RNA were mixed with enzyme reaction buffer (10 mmol/L of Tris-HCl at pH 8.0, 20 mmol/L of EDTA at pH 8.0, 5% SDS, and 0.4 mmol/L of NaCl) to achieve a total volume of 1,500 μl. The mixture was left to stand at 37°C for the degradation reaction. After 10 and 30 min, half of the reactant (750 μl) was removed, and 750 μl of phenol-chloroform was added; a ratio of 25:24 was obtained. Centrifugation was performed (12,000 r/min, 10 min), and the supernatant was transferred to a new nuclease-free tube. A second extraction was performed to eliminate PAC1 protein and to block the degradation reaction. NaAc (3 mol/L, pH 5.2) at 35 μl and 2-folds of cold 100% ethanol were added to the supernatant, and the mixture was left to stand at −20°C in the refrigerator for 1 h. Centrifugation was performed (13,000 r/min, 10 min), and the supernatant was removed. The sediment was washed twice by using 70% ethanol. The sediment was dissolved in 20-μl nuclease-free water. The degradation level of double-stranded RNA was checked by 1% agarose gel electrophoresis. The double-stranded RNA that was not mixed with purified PAC1 protein but subjected to the same process was set as the negative control.

By using the BamH I and Sac I sites, the Pac1 gene was isolated from the TA cloning vector (pMD19T-Pac1), ligated to the plant binary vector (pCAMBIA-3300-Ubi-GFP), and replaced the GFP gene on the vector. The binary vector was constructed in our previous research (Wang et al., 2017a,b). It comprised a selectable marker gene of the bar gene. The binary vector pCAMBIA3300 contained the npt II gene for bacterial selection. After recombination, the Pac1 gene was promoted by Ubi 1 promoter. After PCR and enzyme validation, the recombination binary vector (pCAMBIA-3300-Ubi-Pac1) was mobilized into Agrobacterium EHA105 via freeze-thawing with liquid nitrogen.

Transgenic plants were obtained by the bar/Basta transformation selection system, which was established in our previous research (Wang et al., 2017a,b). ROC22, which is the main variety cultivated in China, was chosen as the receptor for molecular genetic improvement. After two times of sub-culturing, light yellow and compact calli were selected and fragmented before the transformation. After streaking on the solid YEP medium in the Petri dish, a single colony of Agrobacterium EHA105 that harbored the target vector was cultured again in fresh liquid YEP medium at 28°C overnight. Bacterial cells were collected via centrifugation (5,000 r/min, 10 min) and then resuspended in a starter culture of liquid initiating medium and diluted to an optical density of ~0.6 at 600 nm. The starter culture was vortexed at 100 r/min for 2 h at 28°C before infection. The finely prepared calli were first air-dried and then transferred to an Erlenmeyer flask. An appropriate amount of initiated Agrobacterium suspension was added into the Erlenmeyer flask and mixed gently with the calli. After 30 min of infection, the Agrobacterium suspension was then pipetted out. The calli were transferred to a Petri dish, blotted dry with filter paper to remove the excess Agrobacterium suspension, and air-dried for approximately 30 min on the clean bench. The infected calli were transferred to a new empty Petri dish and co-cultivated in the dark at 22°C for 3 days. After co-cultivation, all calli were transferred for resting cultivation for ~7 days. Subsequently, all calli were transferred for selection cultivation for 30 days in the dark at 28°C. After about 30 days of selection by glufosinate, resistant calli were transferred for regeneration cultivation (14 days), and, finally, for elongation cultivation (28 days). Resistant shoots were then sampled for molecular analysis, and positive shoots were transferred to the soil. More details on the transformation procedure and the medium recipe have been presented in our published article (Wang et al., 2017a,b).

Total genomic DNA extracted from resistant shoots was first used for traditional PCR analysis. Primers Bar 1F and 1R, Pac1 2F and 2R, and VirG 1F and 1R were designed and synthesized for PCR detection of Bar gene, Pac1 gene, and VirG, respectively Primer sequence shows in Table 1. Genomic DNA from wild-type plants was used as the negative control, and binary vector (pCAMBIA-3300-Ubi-Pac1) was used as the positive control.

Total RNA of resistant shoots was extracted by an RNA extraction kit (E.Z.N.A^TM Plant RNA Kit, Omega, USA). The first strand's cDNA synthesis was conducted using a first strand cDNA synthesis kit (RevertAid First Strand cDNA Synthesis Kit, Thermo Fisher, USA). In the nuclease-free tube, 6 μl of the total RNA mix with 1 μl of Oligo (dT)₁₈ Primer and 5 μl nuclease-free water were mixed. The mixture was subjected to a temperature of 65°C for 5 min and then rapidly transferred to the ice for 2–3 min. The 4 μl of 5× Reaction Buffer, 1 μl of RiboLock RNase Inhibitor (20 U/μl), 2 μl of 10 mmol/L dNTP Mix, and 1 μl of RevertAid M-MuLV RT (200 U/μl) were added to the tube and mixed gently. The mixture was maintained at 42°C for 1 h and then at 70°C to end the reverse transcript reaction. Then, 2 μl of the reactant was used as the cDNA template for RT-PCR detection. Negative and positive control settings were the same as those used in traditional PCR detection.

Each traditional PCR and RT-PCR positive shoot were transplanted into a plastic pot for virus inoculation. SCSMV is a stronger pathogen compared with the other pathogens that induce sugarcane mosaic disease. So, single SCSMV strain infected sugarcane leaves were collected as the original pathogen for the transgenic shoot virus resistance testing. The inoculation protocol was described by Li et al. (2008). The SCSMV infected leaves were cut into small pieces and ground fully into muddy status with the help of silica sand. Phosphate buffer at 3:1 (v/g) (pH 7.2) was added to the ground leaves, and then, the mixture was filtrated through a cotton gauze. The filtered juice was used for artificial inoculation. Inoculation was conducted thrice in a gap of 1 week to increase the efficiency of successful inoculation. Wild-type plants were inoculated by the same pathogen and served as the negative control. Eight months later, leaf samples were collected from each transgenic shoot for total RNA extraction. RT-PCR detection of SCSMV was conducted to analyze the positive inoculation. Then, three axillary buds were cut from the shoots showing positive infection and propagated in plastic pots. Three months later, the symptoms and viral loads of the propagated plants were observed and interpreted. Symptoms on the leaves were recorded by a digital camera. RT-qPCR was conducted to compare the viral loads among different transgenic shoots. Ten months later (late growing period), the heights of all axillary buds of propagated shoots were determined.

Pac1 gene PCR production was purified by the Gel Extraction Kit and then successfully integrated into the TA cloning vector. The sequencing result shows that the target fragment ligated in the TA cloning vector was 1,092 bp in size, and the sequence showed 100% identity with the sequence that Prof. Li submitted in the GenBank (Sequence ID: AY695822.1). Thus, we successfully isolated the Pac1 gene from the presented plasmid vector from that of Prof. Li's. Otherwise, the sequencing result showed that BamH I and Sac I restriction enzyme-cutting sites had been added at the 5′ and 3′ sites, respectively, which could be utilized for plant expression vector construction.

By utilizing Nde I and Hind III restriction enzymes, the Pac1 gene with an N-His tag was ligated to the prokaryotic expression vector (pET30a, Biodee, China). Positive plasmid pET30a-Pac1 was successfully identified by restriction enzyme digestion (Figure 1A) and then transformed into receptor bacteria BL21 (DE3). After 16 h of inducing cultivation, crude protein was extracted from the positive BL21 bacterial cell and the negative control bacterial cell. After segregation by SDS-PAGE and in comparison with negative control, the BL21 bacterial cell harboring the pET30a-Pac1 vector expressed a specific protein that is similar to the target size 43 kDa (Figure 1B). Thus, we successfully expressed PAC1 protein by prokaryotic expression. Then, the PAC1 protein was purified by affinity chromatography (Ni-IDA resin) method (Figure 1C). The concentration of the purified protein was 0.151 mg/ml, which was tested by Bradford Protein Assay Kit (P0006, Beyotime Biotechnology, China). Western blot shows prokaryotic expressed PAC1 protein interacted with Anti-His Antibody strongly demonstrated the fine activity of purified PAC1 protein.

After the artificial synthesis, 985 bp of double-stranded RNA was obtained by using the genome of SCSMV as the original synthesis template. Mixing of 7.5 μg purified PAC1 protein with 3 μg of double-stranded RNA was conducted in the nuclease-free tube. After 10 and 30 min of degradation, 10 μl of the degraded double-stranded RNA was checked by 1% agarose gel electrophoresis, respectively. Results in Figure 2 showed that double-stranded RNA was degraded clearly by PAC1 protein in 10 min. However, the control double-stranded RNA maintained stability throughout the same treatment process. So, it means that the purified PAC1 protein could degrade double-stranded RNA efficiently.

After successful construction, the recombination Pac1 gene was promoted by the Ubi promoter and ended by the tNOS terminator (Figure 3). The selectable marker bar gene was promoted by CaMV 35S promoter and ended with 35S poly A. The positive recombination binary vector (pCAMBIA-3300-Ubi-Pac1) was identified by restriction enzyme analysis (Figure 4A). Then, the recombination binary vector was mobilized into Agrobacterium EHA105. The single colonies of the transformed bacterium were successfully analyzed by PCR assay individually (Figure 4B). Thus, the Pac1 gene plant expression vector was successfully constructed and transformed to the Agrobacterium EHA105 strain.

Figure 5A shows post-subculturing of light yellow, and compact calli were obtained and selected for transformation. Then, transformed calli were screened by selection medium supplemented with 2 mg/L of Basta. Figure 5B shows that after 30 days of selection, resistant calli sprouted and showed significant resistance on the screening medium. Figure 5C shows that after 14 days of regeneration, the resistant calli emerged quickly on the regeneration medium supplemented with 2 mg/L of Basta. Figure 5D shows that after more than 28 days of rooting cultivation, the resistant shoots were dark green and strong enough for transplanting. In the end, fifteen resistant shoots were obtained and successfully transplanted in the greenhouse (Figure 5E). All resistant shoots were sampled and evaluated for bar gene and Pac1 gene integration by PCR and RT-PCR assay.

Molecular assay results in Table 1 show that the 15 resistant shoots were positive for the bar gene. PCR detection showed that 14 resistant shoots were positive for the Pac1 gene. All the resistant shoots were PCR negative for the VirG gene, thereby suggesting that the resistant sugarcane plants were free from Agrobacterium. RT-PCR assay results showed that 13 resistant shoots were both PCR and RT-PCR positive for bar and Pac1 genes. Then, 13 resistant shoots that were positive for bar and Pac1 genes according to the PCR and RT-PCR results were chosen for further research.

After inoculation by strong pathogen SCSMV and cultivation in the greenhouse for 8 months, samples were collected from each transgenic shoot for the RT-PCR assay. The result in Table 1 shows that all the transgenic and wild-type shoots were successfully infected by the same pathogen SCSMV. Also, classic leaf mosaic symptoms were visible on all axillary buds of propagation shoots. However, upon closer observation, the mosaic symptoms on all transgenic shoots' leaves were mild compared with the serious mosaic symptoms on the wild-type shoots (Figure 6). All the transgenic shoots significantly attained greater heights compared with the wild-type shoots (Figures 6, 7). Transgenic shoots T5 and T7 were significantly shorter than normal uninfected shoots (without viral infection). However, other transgenic shoots T1, T2, T3, T4, T5, T6, T9, T12, T13, T14, and T15 were not significantly different from normal shoots. By comparison, wild-type shoots infected by the pathogen SCSMV were significantly shorter than normal shoots. RT-qPCR test results showed that all transgenic shoots had lower viral loads than wild-type shoots (Figure 8).