Use of RNAi technology to develop a PRSV-resistant transgenic papaya

Papaya ringspot virus (PRSV) seriously limits papaya (Carica papaya L.) production in tropical and subtropical areas throughout the world. Coat protein (CP)- transgenic papaya lines resistant to PRSV isolates in the sequence-homology-dependent manner have been developed in the U.S.A. and Taiwan. A previous investigation revealed that genetic divergence among Hainan isolates of PRSV has allowed the virus to overcome the CP-mediated transgenic resistance. In this study, we designed a comprehensive RNAi strategy targeting the conserved domain of the PRSV CP gene to develop a broader-spectrum transgenic resistance to the Hainan PRSV isolates. We used an optimized particle-bombardment transformation system to produce RNAi-CP-transgenic papaya lines. Southern blot analysis and Droplet Digital PCR revealed that line 474 contained a single transgene insert. Challenging this line with different viruses (PRSV I, II and III subgroup) under greenhouse conditions validated the transgenic resistance of line 474 to the Hainan isolates. Northern blot analysis detected the siRNAs products in virus-free transgenic papaya tissue culture seedlings. The siRNAs also accumulated in PRSV infected transgenic papaya lines. Our results indicated that this transgenic papaya line has a useful application against PRSV in the major growing area of Hainan, China.

have previously reported that PRSV isolates from Hainan province, China, are highly variable 16 . The high levels of genetic divergence in PRSV isolates from Hainan is likely to be the cause of the failure of transgenic papaya lines that targets specific viral CP genes 16 .
The RNA-mediated resistance in papaya is based on post-transcriptional gene silencing (PTGS), a host defense response to foreign RNA 11 . PTGS-mediated transgenic resistance depends on the sequence homology between a transgene and the corresponding viral genome 17 . Transgenic papaya has been proven to have effective resistance to PRSV isolates from Hawaii, Taiwan, and other. In this work, we use the RNAi strategy to construct a transgene that targets the conserved region of the PRSV CP genes and confers broad-spectrum resistance to the diverse Hainan PRSV isolates.

Results
Vector construction and plant transformation. The genetic variability among PRSV isolates in Hainan was investigated in a previous study 16 . Sequence analysis of the 544-bp region of the Hainan PRSV isolates shared 97 to 100% identity (Supplementary Figure 1) were used to design RNAi hairpin structures. The constructed CP hairpin structure was inserted into pCAMBIA2300-35S-OCS to construct the plant expression RNAi vector (Fig. 1). The hairpin structure was validated by digestions with Sal I and Pst I (Supplementary Figure 2).

Identification of transgenic lines by PCR. All plants regenerated from embryogenic callus under anti-
biotic selection were screened for transgenic events by PCR (Fig. 2). Primers amplifying a 760-bp fragment containing the 35 S promoter elements and complete CP hairpin region were used to verify presence of the target transgene (Table 1 and Fig. 2). Regenerated lines 242, 422, 474, and 537 were confirmed to be transgenic by PCR, while line 1280 was not transgenic.

Southern blot analysis to confirm single insert in transgenic line. Digestion of line 474 genomic
DNA with either Hind III or BamH I produced a Southern blot that showed a single band in each digest, indicating that there is only one insertion site in transgenic line 474 (Fig. 3).
Using digital PCR to determine copy number in the transgenic lines. Analysis by ddPCR showed that the transgenic insertion in papaya line 474 contained 0.83 copy (about 1) of the CP gene. SunUp is homozygous for the CP gene at a single insertion site (CP/CP) 18 and therefore has 2 copies of the transgene (Supplementary Figure 4). hiTAIL-PCR used to locate the insertion site. The genomic location of the insert in transgenic line 474 was determined by hiTAIL-PCR (Fig. 4A). The result showed the insertion site was anchored on papaya chromosome VII, within supercontig_61 at base pair 717,141 (Carica papaya ASGPB v4.0, https://phytozome.jgi.doe. gov). The insertion did not lie within any predicted open reading frames (ORFs) (Fig. 4B).
Transformation-site-specific PCR to confirm the insertion. To further verify the authenticity of the insert, two primers, 474-61 F/474-61 R, were designed to amplify a 192-bp product containing and centered approximately on the insertion site (Fig. 4C). The site-specific primers produced one band of the expected size (192 bp), when amplifying genomic DNA of a non-transgenic control line, but no band was produced when genomic DNA of line 474 was used as template, due to the large size of the inserted sequence. Furthermore, nested  PCR containing both a site-specific primer and CP reverse primers was designed to confirm the insertion site. PCR products from line 474 contain a 1.2-kb band (CP inserts + 35 S promoter + plasmid right border + plant genome right wing). Additionally, we also confirmed that line 474 was hemizygous (CP/-), since the nested primers produced both the 1.2-kb site-specific band and the 192-bp band of the unaltered genome (Fig. 4C).
Bioassays for resistance to Hainan PRSV isolates in transgenic line 474. Transgenic line 474 and a non-transgenic line 1280 were challenged with severe PRSV isolates of Hainan subgroups I, II and III, as well as a mixture of all three. The accumulation of viruses in inoculated plants was monitored every 4 days from 0 to 28 days after inoculation (DAI) (Fig. 5). Typical PRSV symptoms were observed in line 1280 at 12 DAI, and severe symptoms were observed at 24 DAI. In contrast, line 474 had no visual symptoms (Fig. 5A). Reverse transcription PCR (RT-PCR) using primers specific for the HC-Pro gene of PRSV was employed to semi-quantitatively measure PRSV replication in inoculated papaya plants. PRSV accumulation increased dramatically in line 1280 by 12 DAI, but there was no evidence of PRSV in line 474 over the course of the experiment (Fig. 5).
The PRSV resistance of line 474 was also measured quantitatively by qPCR (Fig. 6). Virus accumulation within plants inoculated with individual isolates F61d, F10d, and F21d, representing PRSV subgroups I, II and III, respectively, as well as with a mixture of all three isolates, was investigated 0, 12, and 24 days after inoculation. Line 474 suppressed replication of all three PRSV isolates, whether applied individually or as a mixture,, while the non-transgenic line 1280 accumulated the PRSV isolates dramatically. Virulence of the three PRSV isolates, in terms of virus accumulation within the host, was not significantly different within either line 474 or line 1280.

Northern blotting to detect accumulation of siRNA in transgenic papaya. Evidence for PRSV
resistance based on transgene silencing was strengthened by detection via Northern blotting of siRNAs in transgenic papaya seedlings (Fig. 7). Two siRNAs (<50 bp) were found in uninoculated transgenic papaya leaves. Our results also showed there were no corresponding siRNA products in non-trangenic line 1280 or traditional papaya cultivars 'Zhongbai' and 'Suizhonghong 48' .

Discussion
Breakdown of transgenic PRSV resistance, which depends on sequence homology between the transgene and attacking virus strain, is a major concern facing papaya cultivation, since genetically distinct strains of PRSV have been identified throughout the world 18 . In Hawaii, due to geographical isolation and the relative homology of PRSV strain existing there, the transgenic resistance to PRSV introduced in papaya cultivars Rainbow and SunUp in 1998 is still effective against local strains 8 . However, in areas like Hainan, China, where more diverse PRSV strains exist, a narrowly based transgenic resistance is expected to drive the emergence of more virulent virus strains and eventually succumb to the adapting virus population 16 . Consequently, papaya cultivars with resistance to a broad spectrum of PRSV strains are urgently needed by the papaya industry in Hainan.
Other researchers have suggested introducing the HC-Pro gene into transgenic lines to overcome the potential emergence of more virulent virus strains selected by transgenic crops currently using a CP target gene 11 . Our approach to achieving broad transgenic resistance to PRSV involved selecting a conserved region of the CP gene  Table 1. Primers used in this work. * siRNA probes were designed and synthesized to hybridize to the CP gene (sense strand) sequence in 50-bp steps. For example: CP544-1 is from 1 bp to 50 bp, CP544-2 is from 51 bp to 100 bp, … CP544-10 is from 451 bp to 500 bp and its reverse complimentary sequence is CP544-10-10; CP544-11 is from 501 bp to 544 bp and its reverse complimentary sequence is CP544-11-11. The latter two probes (CP544-10/CP544-10-10 and CP544-11/CP544-11-11) have efficient hybridization signal.
as the RNAi target sequence. Analysis of 53 PRSV isolates from Hainan revealed a 544-bp region of the CP gene that shared 97 to 100% identity among all isolates. Particle bombardment has been commonly used as a gene transformation method [19][20][21] . Some researchers report that particle bombardment tends to produced multiple or damaged insertions 22 . However, the flexibility of the particle gun method allows control of the frequency of insertions and ability to produce either low-or high-copy events by altering the quantity of cassette DNA used in the bombardment 22 .
The use of PTGS to produce PRSV-resistant papaya is a proven approach to management of PRSV. We used an RNAi strategy to target a conserved region of the PRSV CP gene during virus replication to develop resistance to a broad spectrum of PRSV isolates from Hainan. Our results showed that transgenic line 474 produced two siR-NAs with molecular weights of less than 50 bp. In short term (4-week) greenhouse experiments, transgenic line 474 showed resistance to PRSV strains that caused disease symptoms in non-transgenic controls, but evaluation of resistance durability must await longer term field trials using inoculated and uninoculated plants grown from seedling generations (Supplementary Figure 8). Embryogenic callus tissue from seeds of the papaya cultivar 'Sunrise' was cultured on the induction medium 23 . Papaya transformation and regeneration were carried out by particle bombardment as described previously 24 .

Methods
PRSV isolates and inoculation bioassay. The PRSV isolates from Hainan province are divided into subgroup I, II and III 16 . Isolates F61d, F10d, and F21d from each subgroup, respectively, were maintained −80 °C. Virus inoculum was prepared by inoculating local papaya plants 'Suizhonghong 48' prior to each bioassay. Papaya leaf samples with the well-developed symptoms of PRSV were homogenized with phosphate buffer solution (PBS) 1:10 (w/v), followed by centrifugation at 6000 × g, 4 °C. The supernatant was used as inocula within 48 hours or stored at −80 °C.
Transgenic line and non-transgenic papaya plants with four to six leaves were used to evaluate plant resistance. The third or fourth leaf from the top was gently rubbed with quartz sand using a blade tip. A 10-μl aliquot of inoculum was applied to each rubbed spot. Inoculum stayed on the leaf surface 5 min prior to washing with distilled water. PBS buffer was used as a control inoculum. All inoculated plants were kept in a growth chamber with 16 h light at 28 °C and 8 h dark at 25 °C. The rate of infection from two independent tests, each with 12 plants per line,

Vector construction and plant transformation. The genetic variability among PRSV isolates in Hainan
was investigated in a previous study 16 . In this study, in order to use hairpin construct to generate a higher efficiency of RNA silencing, we selected the conserved region of the CP genes of the different Hainan isolates as the RNAi target sequence (Supplementary Figure 1). Sequence analysis of the 544-bp region of the Hainan PRSV isolates shared 97 to 100% identity.
The conserved fragment of PRSV CP gene was used to design RNAi hairpin structures. The constructed CP hairpin structure was inserted into pCAMBIA2300-35S-OCS to construct the plant expression RNAi vector (Fig. 1).
The total RNA from papaya leaves infected with isolate F61d (PRSV I) was extracted with RNeasy Plant Mini Kit (QIAGEN). The first strand of cDNA was synthesized by using the ImProm-II Reverse Transcription System (Promega). The targeted CP fragment with restriction sites (Table 1) was amplified by high-fidelity DNA polymerase (NEB). Two "directional" CP fragments (CP sense strand was digested with Xho I and Bgl II, and CP reversed strand was digested with Sal I and BamH I) were ligated to pUCCRNAi plasmid 25 to form the hairpin structure with a 201-bp intron. The hairpin structure was digested with Sal I and Pst I and inserted into pCAM-BIA2300-35S-OCS to form the plant expression vector ( Fig. 1 and Supplementary Figure 2) 26 .
Plasmid DNA was applied to 1.6 μm gold particles (Bio-Rad, Hercules, Calif.) that were inserted into embryogenic callus using a PDS 1000 Helium (Bio-Rad) particle bombardment system as previously described 24 . Each target plate, containing approximately 1 g fresh weight of embryogenic callus, was bombarded three times. The bombarded callus was placed on half-strength MS medium, pH 5.8, containing 10 mg/L 2, 4-dichlorophenoxyacetic acid, 3% sucrose, and 2.5% Phytagel for 10 days to recover, and then treated with 100 mg/L Kanamycin.

PCR validation the transgenic lines. PCR was used to detect transformation events in plants regenerated
from the callus lines. DNA was extracted from 100 mg of papaya leaves using the E.Z.N.A. High Performance (HP) Plant DNA Kit (Omega) according to the manufacturer's instructions. We used target-gene-specific primers, CP1/CP2, and construct-specific primers, 35S-F/CP2, to detect inserted sequences, and endogenous papain gene primers, papainF/papainR, as a check for proper function of PCR assays (Table 1). PCR was performed on a thermocycler (Biometra) as follows: one cycle of initial denaturation at 94 °C for 3 min; 35 cycles of denaturation The QX100 TM Droplet Digital PCR system (ddPCR, Bio-Rad, USA) was used with target-gene primers CP1/ CP2 referenced to the endogenous papain gene to estimate the target-gene copy number in transgenic lines 474 and SunUp, following the manufacturer's instructions. The ddPCR amplification cycling program began with an initial denaturation at 95 °C for 5 min, followed by 45 cycles of denaturation at 95 °C for 20 s, annealing and elongation at 58 °C for 30 s; and the final cycle of incubation at 98 °C for 10 min. QuantaSoft (V1.3.2.0) software was used to analyze the experimental data and quantify the PCR products. Three biological replicates per transgenic genotype x primer-set treatment, and three technical replicates per biological sample, were assayed.
Southern blot analysis to confirm transgenic events. Papaya DNA was extracted with the E.Z.N.A.
High Performance Plant DNA Maxi Kit (Omega) and used for Southern blotting. CP-gene probes were prepared by amplification with specific primers CP1 and CP2 (Table 1) and labeled with Digoxigenin-11-dUTP using DIG-High-Prime Kit (Roche). About 60 μg of genomic DNA was digested with Hind III and BamH I, separately. Electrophoresis of the genomic DNA was performed in 1.0% agarose gel. The DNA was transferred to nylon Northern blot analysis to investigate siRNA accumulation. Tissue cultured transgenic and non-transgenic papaya plants, virus free or 24 days after inoculation with PRSV, provided leaf RNA extracts via RNeasy Plant Mini Kit (QIAGEN) for investigation of siRNA accumulation. The integrity of RNA was checked with formaldehyde denaturing agarose gel electrophoresis 27 . The conserved 544-bp sequence of the PRSV CP gene was fragmented into 11 probes averaging 50 bp in length, of which probe CP544-10 (from 451 bp to 500 bp) and probe CP544-11 (from 501 bp to 544 bp) were later confirmed to produce efficient hybridization signals ( Table 1). The Northern blot probes were labeled with Digoxigenin-11-dUTP using DIG-High-Prime Kit. Both 15% formaldehyde denaturing agarose gels (lab made) and 15% Mini-PROTEAN TBE-Urea Gels (Bio-Rad, USA) were used to separate the small RNAs by loading approximately 60-80 μg of RNA per sample, and transferring the separated bands to nylon membranes (GE Healthcare) using the capillary siphon blot method 27 . Hybridization  was performed overnight at 42 °C in a hybridization incubator (Model 2000, Robbins Scientific). Immunological detection was the same as for the Southern blot, except that the containers, reagents and buffers were pre-treated with 0.1% DEPC (v:v) water to remove contaminating RNAase.

High-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR). The candidate transgenic lines
were screened for insertion sites using the hiTAIL-PCR method 28 . The primers used are listed in Table 1. PCR products were cut from the agarose gel and purified with the E.Z.N.A. Gel Extraction Kit (Omega). The purified amplicons were cloned in a pMD18 vector (TaKaRa) following the manufacturer's protocol, and then transformed into Escherichia coli DH5α competent cells. Three positive colonies from each transformation were sequenced with an ABI 3130xl Genetic Analyzer (Hitachi), and a result of identical sequences from all three colonies was considered confirmation of the actual sequence.
RT-PCR and qPCR to detect PRSV-resistant lines. PRSV accumulation in leaves of transgenic and non-transgenic papaya plants was investigated after inoculation with individual PRSV isolates (F61d, F10d, or F21d), as well as a mixture of all three isolates. Each papaya genotype x PRSV isolate treatment had 5 biological replicates. The tissue cultured papaya plants were cultivated in the greenhouse and observed every four days for symptom development.
PRSV accumulation in papaya leaves after inoculation with the mixture of PRSV isolates was monitored in a time series of 0, 4, 8, 12, 16, 20, 24 and 28 days after inoculation (DAIs). For this assay, we used the PRSV HC-Pro gene as an indicator of the virus accumulation. Plant RNA was extracted with an RNeasy Plant Mini Kit (QIAGEN). First-strand cDNA was synthesized using the ImProm-II Reverse Transcription System (Promega) by starting with total RNA and oligo (dT)15. HC-Pro gene-specific primers were used for second-strand amplification and were able to amplify PRSV I, II and III ( Table 1). The polymerase chain reaction (PCR) mixture contained 1 µl of cDNA, 0.5 µl of each primer (10 µM), 2 µl of 10 × PCR buffer, 1 µl of dNTP, 0.25 U of Taq DNA polymerase (Takara), and nuclease-free water to a final volume of 20 µl. The amplification consisted of 35 cycles at 94 °C (30 s), 50 °C (1 min), and 72 °C (1 min); the initial cycle incorporated a melting step of 94 °C for 3 min and the final cycle a synthesis step of 72 °C for 10 min. PCR products were electrophoresed on 1.5% agarose gel, and visualized after ethidium bromide staining.
Quantitative real-time PCR investigated the PRSV accumulation in papaya leaves at 0, 12 and 24 days after the inoculation with individual isolates (F61d, F10d, and F21d) and PRSV mix. A Mx3005 P Thermocycler (Strata Gene) was used in the amplification. The accumulation of PRSV was detected by SYBR Premix Ex Taq (TaKaRa) with signal correction using ROX reference dye.