RNA silencing-related genes contribute to tolerance of infection with potato virus X and Y in a susceptible tomato plant

Abstract

Background In plants, the RNA silencing system functions as an antiviral defense mechanism following its induction with virus-derived double-stranded RNAs. This occurs through the action of RNA silencing components, including Dicer-like (DCL) nucleases, Argonaute (AGO) proteins, and RNA-dependent RNA polymerases (RDR). Plants encode multiple AGOs, DCLs, and RDRs. The functions of these components have been mainly examined in Arabidopsis thaliana and Nicotiana benthamiana. In this study, we investigated the roles of DCL2, DCL4, AGO2, AGO3 and RDR6 in tomato responses to viral infection. For this purpose, we used transgenic tomato plants (Solanum lycopersicum cv. Moneymaker), in which the expression of these genes were suppressed by double-stranded RNA-mediated RNA silencing. Methods We previously created multiple DCL (i.e., DCL2 and DCL4) (hpDCL2.4) and RDR6 (hpRDR6) knockdown transgenic tomato plants and here additionally did multiple AGO (i.e., AGO2 and AGO3) knockdown plants (hpAGO2.3), in which double-stranded RNAs cognate to these genes were expressed to induce RNA silencing to them. Potato virus X (PVX) and Y (PVY) were inoculated onto these transgenic tomato plants, and the reactions of these plants to the viruses were investigated. In addition to observation of symptoms, viral coat protein and genomic RNA were detected by western and northern blotting and reverse transcription-polymerase chain reaction (RT-PCR). Host mRNA levels were investigated by quantitative RT-PCR. Results Following inoculation with PVX, hpDCL2.4 plants developed a more severe systemic mosaic with leaf curling compared with the other inoculated plants. Systemic necrosis was also observed in hpAGO2.3 plants. Despite the difference in the severity of symptoms, the accumulation of PVX coat protein (CP) and genomic RNA in the uninoculated upper leaves was not obviously different among hpDCL2.4, hpRDR6, and hpAGO2.3 plants and the empty vector-transformed plants. Moneymaker tomato plants were asymptomatic after infection with PVY. However, hpDCL2.4 plants inoculated with PVY developed symptoms, including leaf curling. Consistently, PVY CP was detected in the uninoculated symptomatic upper leaves of hpDCL2.4 plants through western blotting. Of note, PVY CP was rarely detected in other asymptomatic transgenic or wild-type plants. However, PVY was detected in the uninoculated upper leaves of all the inoculated plants using reverse transcription-polymerase chain reactions. These findings indicated that PVY systemically infected asymptomatic Moneymaker tomato plants at a low level (i.e., no detection of CP via western blotting). Conclusion Our results indicate that the tomato cultivar Moneymaker is susceptible to PVX and shows mild mosaic symptoms, whereas it is tolerant and asymptomatic to systemic PVY infection with a low virus titer. In contrast, in hpDCL2.4 plants, PVX-induced symptoms became more severe and PVY infection caused symptoms. These results indicate that DCL2, DCL4, or both contribute to tolerance to infection with PVX and PVY. PVY CP and genomic RNA accumulated to a greater extent in DCL2.4-knockdown plants. Hence, the contribution of these DCLs to tolerance to infection with PVY is at least partly attributed to their roles in anti-viral RNA silencing, which controls the multiplication of PVY in tomato plants. The necrotic symptoms observed in the PVX-infected hpAGO2.3 plants suggest that AGO2, AGO3 or both are also distinctly involved in tolerance to infection with PVX.

Tolerance Even to Lethal Strain of Potato Spindle Tuber Viroid Found in Wild Tomato Species Can Be Introduced by Crossing

“…Regarding the low accumulation of PSTVd in wild tomato relatives, it is possible that PSTVd replication and intercellular/systemic transfer were suppressed or that the accumulated PSTVd was degraded by the anti-viroid defense mechanism, including RNA silencing. In previous reports, suppression of DCL2 and DCL4 in tolerant ‘Moneymaker’ tomatoes increased the accumulation and sensitivity during PSTVd, PVX, or PVY infection [ 46 , 47 ]. It has also been reported that the combined activity of DCL2 and DCL3 in N. benthamiana is important for suppressing the disease symptom expression of PSTVd [ 48 ].…”

Artificially Edited Alleles of the Eukaryotic Translation Initiation Factor 4E1 Gene Differentially Reduce Susceptibility to Cucumber Mosaic Virus and Potato Virus Y in Tomato

“…Cucumber mosaic virus strain yellow (CMV-Y) and PVY strains N and O (PVY N and PVY O ) ( Kwon et al, 2020 ) were initially propagated on Nicotiana benthamiana , and infected upper leaves were used as inoculum for mechanical inoculation. Tomato seedlings at the second or third true leaf stage were mechanically inoculated with crude inoculum, which was viral-infected leaf tissue ground in 0.1 M phosphate buffer (pH 7.0).…”

“…Following the time-course observations of symptom expression, viral coat protein (CP) accumulation in non-inoculated upper leaves at 25 or 35 days post inoculation (dpi) was investigated with a double antibody sandwich-enzyme linked immunosorbent assay (ELISA) using anti-CP polyclonal antibody (Japan Plant Protection Association). Reverse transcription PCR (RT-PCR) analysis to detect CMV-Y genomic RNA was performed with a pair of primers, 5'-gtacagagttcagggttgagcg-3' and 5'-agcaatactgccaactcagctcc-3', as described in our previous study ( Kwon et al, 2020 ).…”

The Carboxyl Terminal Regions of P0 Protein Are Required for Systemic Infections of Poleroviruses

“…1 The proportion of BrYV P0∆235-249 detected in the inoculated leaves at 2 dpi. 2 The proportion of BrYV P0∆235-249 detected in the upper leaves at 14 dpi. 3 The proportion of successful systemic infections by BrYV P0∆235-249 .…”

“…RNA silencing is a natural host antiviral defense pathway at the nucleic acid level [1,2]. It occurs through the processing of double-stranded RNAs into complementary short (21-24 nucleotides) interfering RNAs (siRNAs) by the Dicer-like enzyme RNAse III [3,4].…”