Host recovery and reduced virus level in the upper leaves after Potato virus Y infection occur in tobacco and tomato but not in potato plants

Abstract

In this study, the recovery phenomenon following infection with Potato virus Y (PVY) was investigated in tobacco (Nicotiana tobaccum), tomato (Solanum lycopersicum) and potato (Solanum tuberosum) plants. In tobacco plants, infection of severe strains of PVY (PVYN or PVYN:O) induced conspicuous vein clearing and leaf deformation in the first three leaves above the inoculated leaves, but much milder symptoms in the upper leaves. The recovery phenotype was not obvious in tobacco plants infected with PVY strain that induce mild symptoms (PVYO). However, regardless of the virus strains, reduction in PVY RNA levels was similarly observed in the upper leaves of these plants. Removal of the first three leaves above the inoculated leaves interfered with the occurrence of recovery, suggesting that the signal(s) mediating the recovery is likely generated in these leaves. In PVYN or PVYN:O but not in PVYO-infected tobacco plants, the expression of PR-1a transcripts were correlated with the accumulation level of PVY RNA. Reduced level of PVY RNA in the upper leaves was also observed in infected tomato plants, whereas such phenomenon was not observed in potato plants. PVY-derived small RNAs were detected in both tobacco and potato plants and their accumulation levels were correlated with PVY RNA levels. Our results demonstrate that the recovery phenotype following PVY infection is host-specific and not necessarily associated with the expression of PR-1a and generation of PVY small RNAs.

Figure 1

Recovery phenotype in tobacco plant after infection with PVY^N:O-Mb58. (A) Symptoms in Potato virus Y (PVY)-infected plant (right) at 14 days post-inoculation (dpi). Left, mock-inoculated plant; (B,C) RT-PCR-Southern blot analysis of PVY level and COX1 in +1 to +6 leaves as well as stem cortex at the bottom (B) and top (T); (D) Real-time RT-PCR analysis of the relative PVY level (RQ) after normalization with the COX1 level in leaves and stem segments.

Figure 2

Effects of leaf age on the recovery phenotype in latterly emerging leaves of tobacco plants infected with PVY^N:O-Mb58. (A) Symptoms in +1 to +6 leaves at the leaf age of 8, 12 and 15 days post-emergence (dpe); (B) Real-time RT-PCR analysis of the relative PVY level (RQ) after normalization with the COX1 level in the leaves.

Figure 3

Symptoms and virus level in leaves of tobacco plants infected with different strains/isolates of PVY. (A) Symptoms in +2, +4 and +6 leaves of plants infected with PVY^O (Y^O, isolate RB), PVY^N:O (Y^N:O, isolates MB146, Mb55 and Mb58) and PVY^N (Y^N, isolate N-Jg) at the leaf age of 12 days post-emergence; (B) Relative PVY level (RQ) in leaves. The virus level was obtained using real-time RT-PCR analysis of PVY upon normalization with COX1 level in the leaves.

Figure 4

Effects of removal of earlier leaves on symptoms and virus level in latter leaves of tobacco plants infected with PVY^N:O-Mb58. (A) Symptoms in plants with (R) and without (NR) the removal of the +1 to +3 leaves; (B) Leaves at +4 to +7 node position at the leaf age of 12 days post-emergence (dpe) from plants with and without the removal of the leaves at +1 to +3 node position; (C) Relative PVY level (RQ) in leaves. The virus level was obtained using real-time RT-PCR analysis of PVY upon normalization with the COX1 level in the leaves.

Figure 4

Effects of removal of earlier leaves on symptoms and virus level in latter leaves of tobacco plants infected with PVY^N:O-Mb58. (A) Symptoms in plants with (R) and without (NR) the removal of the +1 to +3 leaves; (B) Leaves at +4 to +7 node position at the leaf age of 12 days post-emergence (dpe) from plants with and without the removal of the leaves at +1 to +3 node position; (C) Relative PVY level (RQ) in leaves. The virus level was obtained using real-time RT-PCR analysis of PVY upon normalization with the COX1 level in the leaves.

Figure 5

Northern blot analysis of the expression of ACO1, Hsp90 and PR-1a in leaves emerged after inoculation in plants infected with different strains/isolates of PVY. (A) Gene expression in +2, +4 and +6 leaves of plants infected with PVY^O (Y^O, isolate RB), PVY^N:O (Y^N:O, isolates MB146, Mb55 and Mb58) and PVY^N (Y^N, isolate N-Jg) at the leaf age of 12 days post-emergence (dpe); (B) Gene expression in +4 to +7 leaves in plants with (+) and without (−) the removal of the +1 to +3 leaves. Northern blots were performed using total RNA extracted from the pooled leaves of 4 plants. ACO1, ACC oxidase 1; Hsp90, heat-shock protein 90; PR-1a, pathogenesis-related protein 1a; H, leaves from healthy plants. The experiments were repeated two times, and similar results were obtained.

Figure 6

Virus level in leaves emerged after inoculation in potato and tomato plants infected with PVY^N:O-Mb58. (A) Leaves of potato (cv. “Ranger Russet”) at 30 days post-inoculation (dpi) and at the leaf age of 30 days post-emergence (dpe); (B) Leaves of tomato (cv. “Sheyenne”) at 30 dpi and at the leaf age of 30 dpe. The relative virus level (RQ) was obtained by real-time RT-PCR analysis of PVY RNA upon normalization with COX1 mRNA in leaves.

Figure 7

Northern blot detection of PVY-derived small RNA (siRNA) in leaves of tobacco and potato plants infected with PVY^N:O-Mb58. (A) Detection of PVY-siRNA in leaves (12 dpe) of PVY (Y)- and mock (M)-inoculated tobacco plants using fragments covering various segments of the complete PVY genome; (B) Detection of PVY-siRNA in leaves of PVY-inoculated potato plants using probes of PVY fragments 1–3; (C) Detection of PVY-siRNA in different leaves of PVY-inoculated tobacco plants using probes of PVY fragment 1; (D) Detection of PVY-siRNA in leaves (30 dpe) of PVY- and mock-inoculated potato plants using fragments covering various segments of the complete PVY genome; (E) Detection of PVY-siRNA in various leaves of PVY-inoculated potato plants using probes of PVY fragments 1–3. The PVY fragments 1–9 cover the nucleotide segment of PVY^N:O genome 5'end–1200, 1100–2400, 2300–3500, 3400–4600, 4500–5700, 5600–6800, 6700–7900, 7800–9000, 8900–3'end, respectively. The far left lane in low-molecular-weight (LMW) gels is 10 bp DNA step ladder (Promega. Bottom to top: 10 bp to 100 bp in exactly 10 bp increments). M, mock; Y, PVY; RR, potato cv. “Ranger Russet”; CW, potato cv. “Cal White”.