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. 2017 Oct 3;7(1):12636.
doi: 10.1038/s41598-017-13049-0.

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

Ruizong Jia  1   2 , Hui Zhao  1 , Jing Huang  1   3 , Hua Kong  1 , Yuliang Zhang  1 , Jingyuan Guo  1 , Qixing Huang  1 , Yunling Guo  1 , Qing Wei  1   4 , Jiao Zuo  1 , Yun J Zhu  5   6 , Ming Peng  7 , Anping Guo  8
Affiliations

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

Ruizong Jia et al. Sci Rep. .

Erratum in

Abstract

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.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Components of the constructed RNAi vector pCAMBIA2300-35S-OCS. 35 S promoter indicated Cauliflower mosaic virus (CaMV) promoter. NOS terminator = nopaline synthase gene terminator, OCS terminator = octopine synthase terminator. NPTII = neomycin phosphotransferase gene. Sense and Anti-sense = conserved 544 bp fragment of CP gene and inverted repeat sequence.
Figure 2
Figure 2
PCR validation of the transgenic event in difference lines. CP1/CP2 primers amplified the 544-bp conserved region of the CP gene in sense orientation. 35S-F/CP2 primers were vector-specific, amplifying the 35 S promoter and conserved sense region of the CP gene. Papain F/ Papain R primers amplified the papaya reference gene. M = DNA molecular weight, P = plasmid DNA, SU = ‘SunUp’ DNA, SR = ‘SunRise’ DNA before transformation. The other numbers are candidate transgene lines. Arrows on the right panel indicated the expected PCR products. The full-length gels are presented in Supplementary Figure 3.
Figure 3
Figure 3
Southern blot analysis confirmed a single insertion site in transgenic line 474. Plasmid and line 474 were digested with BamH I and Hind III separately, and the 544-bp CP gene conserved region was produced by PCR as a reference control. (A) Agarose gel image before transfer to nylon membrane. (B) Southern blot. The probe of CP fragment and DNA marker were labeled with Digoxigenin-11-dUTP individually, and mixed together before hybridization.
Figure 4
Figure 4
High-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR) verified the insertion site. (A) Two rounds of hiTAIL-PCR for line 474. M = molecular marker; lane 1 = first round PCR; lane 2 = second round PCR; arrow indicates two products that were subsequently sequenced. (B) Structural diagram of the specific insertion site located in chromosome VII, supercontig 61 at 717141 bp. Primer 474-61 F/R were designed to amplify 192 bp in non-transgenic line. (C) Nested PCR with primers 474-61 F/61 R bracketing the insertion site and primers CP1/CP2 used to verify the insertion of the CP gene. Line 474 samples amplified as a PCR template produced two bands; one band (192 bp) was the same as the negative control for the insertion event, while the other band was about 1.2 kb.
Figure 5
Figure 5
Bioassays of transgenic line 474 to determine resistance response to inoculation with a mixture of PRSV isolates F61d, F10d and F21d. (A) Plant inoculation bioassay. Typical PRSV symptoms were observed in non-transgenic line 1280 at 24 DAIs, while transgenic line 474 had no visual symptoms. IL indicates the inoculated leaf, DL indicates the detected leaf. (B) Reverse-transcription PCR of the HC-Pro gene (484 bp) was used to detect PRSV. Papain was used as internal reference gene (198 bp). PRSV accumulation in the transgenic and non-transgenic papaya was monitored 0, 4, 8, 12, 16, 20, 24, and 28 days after inoculation. P = plasmid as positive control. H2O = nuclease-free water as negative control. M = DNA molecular marker.
Figure 6
Figure 6
Quantitative real-time PCR was used to investigate the PRSV accumulation in transgenic and non-transgenic papaya leaves. Individual isolates F61d, F10d, and F21d represented PRSV Groups I, and II and III, respectively. PRSV MIX indicated the mixture of same amount of PRSV I, and II and III. The PRSV accumulation was measured at 0, 12 and 24 days after the inoculation.
Figure 7
Figure 7
Northern blot analysis to investigate the siRNA accumulation on virus free papaya leaves (A) and virus challenged papaya leaves (B). The full-length gels are presented in Supplementary Figure 5 and Supplementary Figure 6. (A) Young tissue culture seedlings regenerated from embryogenic callus considered as virus free materials. Lane 1, 3 and 4 are non-transgenic line 1280. Lane 2, 5 and 6 are transgenic line 474. Lane 7 and 8 are traditional cultivars ‘ZhongBai’ and ‘Suizhonghong 48’ germinated from seeds as references. (B) PRSV challenged (24 days) papaya leaves were also estimated the siRNA accumulation. Lane 1 and 2 are transgenic lane 474 challenged with PRSV I isolate F61d. Lane 3 and 4 are transgenic lane 474 challenged with PRSV II isolate F10d. Lane 5 and 6 are transgenic lane 474 challenged with PRSV III isolate F21d. Lane 7 and 8 are pooled RNA mixture of non-transgenic line 1280 challenged with PRSV I, II and III respectively. Arrow indicated the probe self-hybridization dot (50 bp).
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