Development of an attenuated potato virus Y mutant carrying multiple mutations in helper-component protease for cross-protection

Abstract

Tobacco (Nicotiana tabacum) is one of the major cash crops in China. Potato virus Y (PVY), a representative member of the genus Potyvirus, greatly reduces the quality and yield of tobacco leaves by inducing veinal necrosis. Mild strain-mediated cross-protection is an attractive method of controlling diseases caused by PVY. Currently, there is a lack of effective and stable attenuated PVY mutants. Potyviral helper component-protease (HC-Pro) is a likely target for the development of mild strains. Our previous studies showed that the residues lysine at positions 124 and 182 (K¹²⁴ and K¹⁸²) in HC-Pro were involved in PVY virulence, and the conserved KITC motif in HC-Pro was involved in aphid transmission. In this study, to improve the stability of PVY mild strains, K at position 50 (K⁵⁰) in KITC motif, K¹²⁴, and K¹⁸² were separately substituted with glutamic acid (E), leucine (L), and arginine (R), resulting in a triple-mutant PVY-HC_ELR. The mutant PVY-HC_ELR had attenuated virulence and did not induce leaf veinal necrosis symptoms in tobacco plants and could not be transmitted by Myzus persicae. Furthermore, PVY-HC_ELR mutant was genetically stable after six serial passages, and only caused mild mosaic symptoms in tobacco plants even at 90 days post inoculation. The tobacco plants cross-protected by PVY-HC_ELR mutant showed high resistance to the wild-type PVY. This study showed that PVY-HC_ELR mutant was a promising mild mutant for cross-protection to control PVY.

Keywords: Cross-protection; Helper component-protease; Potato virus Y; Site-directed mutagenesis; Stability.

Fig. 1

Symptoms and accumulation of attenuated variant of potato virus Y (PVY) in tobacco plants. (A) Schematic representation of PVY genome, showing all the mutations in helper component protease (HC-Pro). The conserved KITC and FRNK motifs in PVY HC-Pro were marked with black boxes. The red letters were the substituted residues in PVY HC-Pro. (B) Alignment of partial amino acid sequences of five potyviral HC-Pros using the BioEdit program version 7.2.5. The amino acid residues lysine at position 50 (K⁵⁰) in the conserved KITC motif, K at position 124 (K¹²⁴), and K at position 182 (K¹⁸²) in the conserved FRNK motif of HC-Pro were marked with red-dotted boxes. The analysis was performed with HC-Pro sequences of PVY isolate Guizhou (GenBank accession: MN381731), tobacco etch virus (TEV, GenBank accession: DQ986288), turnip mosaic virus (TuMV, GenBank accession: AB093596), papaya ringspot virus (PRSV, GenBank accession: MF085000), and sugarcane mosaic virus (SCMV, GenBank accession: AY042184). (C) Symptoms of tobacco (Nicotiana tabacum cv. Xanthi) plants infected with the wild-type PVY and its mutant. Mock, tobacco plants inoculated with the empty vector pCB301-Rz. PVY, tobacco plants infected with the wild-type PVY. The residues of K⁵⁰, K¹²⁴, and K¹⁸² were separately substituted with glutamic acid (E), leucine (L), and arginine (R) in HC-Pro of PVY-HC_ELR mutant. Leaf veinal necrosis of tobacco plants under daylight and staining with trypan blue. (D) Semi-quantitative reverse-transcriptase polymerase chain reaction (SqRT-PCR) analysis of the RNA accumulation levels of PVY CP in tobacco upper leaves infected with PVY and PVY-HC_ELR mutant at 15 days post agroinfiltration (dpai). 18S ribosomal RNA (18S rRNA) was used as an internal control. (E) Western blotting analysis of the wild-type and mutant PVY CP accumulation levels in tobacco upper leaves at 15 dpai. The sample loadings were shown with the ponceau S staining (PSS). Band intensities were measured using the ImageJ software. Numbers indicated PVY CP accumulation levels normalized to PSS staining. The experiments were repeated three times.

Fig. 2

Aphid transmissibility of attenuated variant of PVY in tobacco plants. (A) Symptoms under daylight and GFP fluorescent under UV light of tobacco plants inoculated with PVY-GFP or its mutant at 15 dpai. (B) Western blotting analysis of CP accumulation levels of PVY-GFP and its mutant in tobacco leaves at 15 dpai. The sample loadings were shown with PSS. Band intensities were measured using the ImageJ software. Numbers indicated PVY CP accumulation levels normalized to PSS staining. (C and D) Symptoms under daylight and GFP fluorescent under UV light of tobacco plants inoculated with PVY-GFP or its mutant by Myzus persicae at 5 d and 10 d. The numbers of symptomatic/inoculated tobacco plants were marked in brackets. (E) SqRT-PCR analysis of CP RNA accumulation in tobacco plants inoculated with PVY-GFP or its mutant by Myzus persicae at 10 d. Elongation factor 1-alpha (EF1α) was used as an internal control.

Fig. 3

The stability of PVY-HC_ELR mutant in tobacco plants. (A and B) Symptoms and the sequencing results of HC-Pro RT-PCR products from tobacco plants infected with PVY-HC_ELR mutant after three and six serial passages. The sites of point mutations in HC-Pro were underlined and the corresponding amino acid residues were indicated. The images represented at least 12 tobacco plants. (C) Symptoms of tobacco plants infected with the wild-type PVY and PVY-HC_ELR mutant at 90 dpai. The images represented at least 12 tobacco plants. (D) SqRT-PCR analysis of the wild-type and mutant PVY CP RNA accumulation levels in tobacco upper leaves at 90 dpai. 18S rRNA was used as an internal control. (E) Western blotting analysis of the wild-type and mutant PVY CP accumulation levels in tobacco leaves at 90 dpai. The sample loadings were shown with PSS. Band intensities were measured using the ImageJ software. Numbers indicated PVY CP accumulation levels normalized to PSS staining. The experiments were repeated three times.

Fig. 4

Attenuated mutant PVY-HC_ELR could protect tobacco plants against wild-type PVY infection. (A) Symptoms of tobacco plants challenged with PVY-GFP at 15 days post-challenge inoculation with intervals of 5 or 10 dpi. Mock, tobacco plants were inoculated with phosphate-buffered saline. Non-protected, tobacco plants inoculated with the empty vector pCB301-Rz. (B) SqRT-PCR analysis of GFP RNA accumulation of PVY-GFP in tobacco upper leaves at 15 days post-challenge inoculation. EF1α was used as an internal control. (C) Western blotting analysis of the GFP accumulation levels of PVY-GFP in tobacco top leaves at 15 days post-challenge inoculation. Band intensities were measured using the ImageJ software. Numbers indicated PVY CP accumulation levels normalized to PSS staining.