An efficient viral vector for functional genomic studies of Prunus fruit trees and its induced resistance to Plum pox virus via silencing of a host factor gene

Abstract

RNA silencing is a powerful technology for molecular characterization of gene functions in plants. A commonly used approach to the induction of RNA silencing is through genetic transformation. A potent alternative is to use a modified viral vector for virus-induced gene silencing (VIGS) to degrade RNA molecules sharing similar nucleotide sequence. Unfortunately, genomic studies in many allogamous woody perennials such as peach are severely hindered because they have a long juvenile period and are recalcitrant to genetic transformation. Here, we report the development of a viral vector derived from Prunus necrotic ringspot virus (PNRSV), a widespread fruit tree virus that is endemic in all Prunus fruit production countries and regions in the world. We show that the modified PNRSV vector, harbouring the sense-orientated target gene sequence of 100-200 bp in length in genomic RNA3, could efficiently trigger the silencing of a transgene or an endogenous gene in the model plant Nicotiana benthamiana. We further demonstrate that the PNRSV-based vector could be manipulated to silence endogenous genes in peach such as eukaryotic translation initiation factor 4E isoform (eIF(iso)4E), a host factor of many potyviruses including Plum pox virus (PPV). Moreover, the eIF(iso)4E-knocked down peach plants were resistant to PPV. This work opens a potential avenue for the control of virus diseases in perennial trees via viral vector-mediated silencing of host factors, and the PNRSV vector may serve as a powerful molecular tool for functional genomic studies of Prunus fruit trees.

Keywords: Prunus necrotic ringspot virus; eukaryotic translation initiation factor 4E; functional genomics; sharka; viral vector; virus-induced gene silencing.

Figure 1

Pathogenicity of the PNRSV‐derived vector and the PPV infectious clone pVPM in peach cv. ‘Loring’. (a) Schematic representation of the PNRSV vector derived from the PNRSV isolate Pch12. Both expression cassettes of genomic RNA1 and RNA2 are integrated into the same pCass4Rz backbone. Monocistronic RNA1 and 2‐encode replicase proteins P1 and P2 respectively. Domains for methyltransferase (MET), helicase (HEL) and RNA‐dependent RNA polymerase (RdRp) are indicated. Dicistronic RNA3 encodes 5′‐proximal movement protein (MP) and 3′‐proximal coat protein (CP), separated by a short intergenic region sequence. The Xba I restriction sequence ‘TCTAGA’ (TAG, stop codon of CP gene), located at the junction of CP and 3′ UTR in genomic RNA3, was employed to integrate foreign inserts for construction PNRSV‐based VIGS vectors. The self‐cleavage of ribozyme (Rz) is indicated by a bent arrow; T, 35S terminator. (b) Schematic representation of the infectious cDNA clone of the PPV isolate VPM. The expression cassette of full‐length PPV genomic RNA is integrated into a mini‐binary vector pCB301 backbone. 11 mature proteins encoded by PPV are shown. (c) Disease response induced by the PNRSV vector in ‘Loring’. At 16 days post bombardment (dpb), ‘Loring’ plants infected by PNRSV showed mild distortion and chlorosis symptoms on upper leaves. No visually detectable phenotypic differences were observed among PNRSV‐infected and mock plants at 105 dpb. (d) PPV induced severe vein yellowing, mosaic and distortion symptoms in ‘Loring’ plants at 18 dpb. The images on the right side were magnified from the portion of the corresponding leaf on the left side.

Figure 2

Disease responses triggered by the PNRSV vector in herbaceous hosts Cucumis sativus and Nicotiana benthamiana. (a) PNRSV‐infected cucumber plants showed extremely dwarfed and necrotic symptoms at 27 days post agroinfiltration (dpai). (b) No visible symptoms were observed in PNRSV‐infected N. benthamiana plants at 45 dpai.

Figure 3

Silencing of PDS or GFP mediated by the PNRSV‐based vectors, in wild‐type or gfp‐transgenic Nicotiana benthamiana plants. (a) PDS‐silenced N. benthamiana plants inoculated with different PNRSV‐based constructs harbouring foreign inserts of varied sizes in the sense or antisense orientation. At 20 days post agroinfiltration (dpai), N. benthamiana plants either agroinfiltrated with PNRSV‐NbPDS100(+) or PNRSV‐NbPDS200(+) showed the typical photo‐bleaching phenotype in the upper leaves, resulting from the reduction in the expression level of PDS. Symbols ‘+’ and ‘−’ represent the presence and absence of PNRSV by DAS‐ELISA analysis respectively. (b) The PNRSV‐based construct triggers systemic silencing of GFP in gfp‐transgenic N. benthamiana (16c). The green/red fluorescence in ‘16c’ plants inoculated with wtPNRSV or PNRSV‐GFP100(+) was excited under UV illumination. At 18 dpai, red fluorescence (chlorophyll autofluorescence), representing loss of GFP, was observed in upper leaves of the ‘16c’ plants inoculated with PNRSV‐GFP100(+). Subsequently, the GFP gene was silenced in the entire plants at 26 dpai. No GFP‐silenced phenotype was observed in ‘16c’ plants either inoculated with wtPNRSV or buffer‐treated (Mock) at all time points. (c) The relative expression level of PDS in N. benthamiana plants agroinfiltrated with PNRSV‐NbPDS100(+), PNRSV‐NbPDS200(+) or wtPNRSV control was determined by qRT‐PCR. Actin transcript levels were determined as an internal control. The leaf tissues showing photo‐bleaching were sampled for qRT‐PCR analysis at 20 dpai. Error bars denote standard errors of three independent biological replicates. Statistically significant differences, determined by an unpaired two‐tailed Student's t test, are indicated by brackets. *0.01 < P < 0.05. (d) Conventional RT‐PCR analysis of genetic stability of the foreign insert in the recombinant virus using primers RNA3‐CP‐F/RNA3‐3′UTR‐R. The predicted sizes of the RT‐PCR products derived from N. benthamiana plants infected with wtPNRSV, PNRSV‐NbPDS100(+) and PNRSV‐NbPDS200(+) are 269‐, 403‐ and 475‐bp respectively. However, the fragment amplified from plants inoculated with PNRSV‐NbPDS200(+) was approximately 400 bp but not 475 bp in length, indicating that the 200‐bp PDS fragment in recombinant virus was partially lost via a yet unidentified mechanism. (e) Northern blot analysis of the mRNA expression level of GFP in ‘16c’ plants infected with wtPNRSV, PNRSV‐GFP100(+) or buffer‐treated (mock) at 26 dpai. Ribosomal RNA (28S), stained with ethidium bromide, was used as a loading control. (f) Conventional RT‐PCR analysis of genetic stability of the foreign insert in the recombinant virus using primers RNA3‐CP‐F/RNA3‐3′UTR‐R. Consistent with the predicted sizes, RT‐PCR products obtained from ‘16c’ plants infected with wtPNRSV and PNRSV‐GFP100(+), were 269 and 375 bp in length respectively.

Figure 4

Down‐regulation of PDS in peach by a modified PNRSV‐based vector. (a, b) At 60 days post bombardment (dpb), ‘Loring’ plants infected with PNRSV‐pchPDS100(+) displayed typical photo‐bleaching phenotype in upper leaves (a) and stems (b), indicating the down‐regulation of the PDS gene in peach. The images on the right side (a) were magnified from the portion of the corresponding leaf on the left side. (c) The relative expression level of PDS in ‘Loring’ plants infected with wtPNRSV or PNRSV‐pchPDS100(+). qRT‐PCR was carried out with the Actin transcript levels as an internal control. The leaf tissues showing photo‐bleaching were sampled for qRT‐PCR analysis at 60 dpb. Error bars denote standard errors of three independent biological replicates. Statistically significant differences, determined by an unpaired two‐tailed Student's t test, are indicated by brackets. ***P < 0.001. (d) Conventional RT‐PCR analysis of genetic stability of the foreign insert in recombinant viruses using the primer set RNA3‐CP‐F/RNA3‐3′UTR‐R. PCR products of the predicted sizes 269 and 375 bp were amplified from ‘Loring’ plants infected with wtPNRSV and PNRSV‐pchPDS100(+) respectively.

Figure 5

The PNRSV‐based vector has the ability to knock down the expression of the eIF(iso)4E gene in peach. (a) The phenotype of PNRSV‐mediated knockdown of eIF(iso)4E in peach. At 22 days post bombardment (dpb), mild chlorosis and distortion symptoms were observed in plants either infected with wtPNRSV or PNRSV‐eIFiso4E120(+). Except these typical symptoms induced by PNRSV, no other physiological abnormality was observed among these plants. (b, c) The relative expression level of eIF(iso)4E or eIF4E in ‘Loring’ plants infected with wtPNRSV or PNRSV‐eIFiso4E120(+) at 16 (b) and 22 dpb (c). qRT‐PCR was performed, and the Actin transcript levels were used as an internal control. Error bars denote standard errors of three independent biological replicates. Statistically significant differences, determined by an unpaired two‐tailed Student's t test, are indicated by brackets. NS, no significant differences; *P < 0.05; **P < 0.01. (d, e) Conventional RT‐PCR analysis of genetic stability of the foreign insert in recombinant viruses using the primer set RNA3‐CP‐F/RNA3‐3′UTR‐R at 16 (d) and 90 dpb (e). PCR products of the predicted sizes 269 and 395 bp were amplified from ‘Loring’ plants infected with wtPNRSV and PNRSV‐eIFiso4E120(+), respectively.

Figure 6

Knockdown of eIF(iso)4E expression confers PPV resistance in peach. (a, b) Disease response induced by PPV in eIF(iso)4E‐silenced and wtPNRSV‐infected ‘Loring’ plants. At 22 days post bombardment with wtPNRSV or PNRSV‐eIFiso4E120(+), the infected ‘Loring’ plants were biolistically challenged with PPV. Also, mock plants were bombarded with PPV and used as a positive control. 16 days later, severe symptoms such as chlorotic spots and venation chlorosis were observed in all mock/PPV and wtPNRSV/PPV plants. In contrast, PNRSV‐eIFiso4E120(+)/PPV plants did not show any PPV symptom (a). Until 25 days later, no obvious symptoms were observed in PNRSV‐eIFiso4E120(+)/PPV plants (b). (c) Detection of PPV by semi‐quantitative DAS‐ELISA at 16 days post‐inoculation with PPV.