Potato virus Y (PVY) is one of the most important pathogens of pepper, potato, tobacco, tomato and other solanaceous and non-solanaceous hosts. Two PVY isolates, PVY-Cto and PVYSON41, induce very different disease phenotypes on tomato (Solanum lycopersicum), the former being responsible of leaf distortions and growth reduction and the latter inducing only very mild symptoms. With the aim of investigating the role of RNA silencing mechanisms in the different pathogenic behaviour of the two isolates, we performed a systematic analysis of expression profiles of Dicer-like (DCL), Argonaute (AGO) and RNA-dependent RNA polymerase (RDR) genes that form the core components of the RNA silencing-based plant immune system. The effects on expression profiles of some of these genes were significantly higher in tomato plants infected by the aggressive isolate PVY-Cto in comparison with PVY-SON41-infected and healthy plants. We further investigated on whether effects on expression of silencing genes could lead to a differential accumulation of small RNAs (sRNAs) in plants. Both endogenous microRNA (miRNA) and viral small interfering RNA (vsiRNA) populations were analysed by high-throughput sequencing. SRNAs from tomato plants, healthy or infected by PVY-Cto and PVY-SON41, were isolated at 21 and 30 days post-inoculation (dpi) and sequenced using the Illumina platform at the NGS Laboratory, IGA (Udine, Italy). Total reads from samples at 21 dpi ranged between 13 to 17 million, and at 30 dpi between 23 to 28 million. From these data, vsiRNA and miRNA populations were analysed separately. VsiRNA from PVY-Cto-infected samples were abundant and corresponded to 44% and 29% of total reads at 21 and 30 dpi, respectively. Surprisingly, a negligible number of vsiRNA were found in PVY-SON41-infected samples, representing less than 1% of total reads at both timepoints. As expected, virus infection affected also the composition of the miRNAome. The total number of sRNAs corresponding to known plant miRNAs was proportional to each library size and varied in a narrow range from 2.8 % ot total reads in mock-inoculated at 21 dpi to 4,8% in PVY-Cto samples at the same timepoint. A total of 156 known miRNA was identified in the six library following stringent criteria. To compare miRNA abundance in different libraries, the count of each miRNA was normalized to transcripts per million (TPM) and subjected to a Z-test. Change in miRNA read counts between virus- and mock-inoculated plants was scored as fold change and recorded. On this basis, PVY-Cto infection was associated to the differential accumulation (either up- or downregulated) of 71 and 39 miRNA at 21 and 30 dpi, respectively. The mild PVY-SON41 isolate affected the composition of miRNA population at significantly lower extent, and a differential accumulation was observed only for 25 (21 dpi) and 6 (30 dpi) miRNAs. Our results provide a comparative analysis, via deep sequencing, of changes in the small RNA profiles and in the genes of RNA silencing machinery induced by different isolates in an economically important crop plant. They uncover new mechanisms and complexity of virus-host interactions that may have important implications for further studies on infection, pathogenesis, and plant defense responses.
Two Potato Virus Y Isolates Have Different Effects on Expression of RNA Silencing Genes and Small RNA Profiles in Solanum lycopersicum
FinettiSialer MM;De Paola D;Cillo F
2014
Abstract
Potato virus Y (PVY) is one of the most important pathogens of pepper, potato, tobacco, tomato and other solanaceous and non-solanaceous hosts. Two PVY isolates, PVY-Cto and PVYSON41, induce very different disease phenotypes on tomato (Solanum lycopersicum), the former being responsible of leaf distortions and growth reduction and the latter inducing only very mild symptoms. With the aim of investigating the role of RNA silencing mechanisms in the different pathogenic behaviour of the two isolates, we performed a systematic analysis of expression profiles of Dicer-like (DCL), Argonaute (AGO) and RNA-dependent RNA polymerase (RDR) genes that form the core components of the RNA silencing-based plant immune system. The effects on expression profiles of some of these genes were significantly higher in tomato plants infected by the aggressive isolate PVY-Cto in comparison with PVY-SON41-infected and healthy plants. We further investigated on whether effects on expression of silencing genes could lead to a differential accumulation of small RNAs (sRNAs) in plants. Both endogenous microRNA (miRNA) and viral small interfering RNA (vsiRNA) populations were analysed by high-throughput sequencing. SRNAs from tomato plants, healthy or infected by PVY-Cto and PVY-SON41, were isolated at 21 and 30 days post-inoculation (dpi) and sequenced using the Illumina platform at the NGS Laboratory, IGA (Udine, Italy). Total reads from samples at 21 dpi ranged between 13 to 17 million, and at 30 dpi between 23 to 28 million. From these data, vsiRNA and miRNA populations were analysed separately. VsiRNA from PVY-Cto-infected samples were abundant and corresponded to 44% and 29% of total reads at 21 and 30 dpi, respectively. Surprisingly, a negligible number of vsiRNA were found in PVY-SON41-infected samples, representing less than 1% of total reads at both timepoints. As expected, virus infection affected also the composition of the miRNAome. The total number of sRNAs corresponding to known plant miRNAs was proportional to each library size and varied in a narrow range from 2.8 % ot total reads in mock-inoculated at 21 dpi to 4,8% in PVY-Cto samples at the same timepoint. A total of 156 known miRNA was identified in the six library following stringent criteria. To compare miRNA abundance in different libraries, the count of each miRNA was normalized to transcripts per million (TPM) and subjected to a Z-test. Change in miRNA read counts between virus- and mock-inoculated plants was scored as fold change and recorded. On this basis, PVY-Cto infection was associated to the differential accumulation (either up- or downregulated) of 71 and 39 miRNA at 21 and 30 dpi, respectively. The mild PVY-SON41 isolate affected the composition of miRNA population at significantly lower extent, and a differential accumulation was observed only for 25 (21 dpi) and 6 (30 dpi) miRNAs. Our results provide a comparative analysis, via deep sequencing, of changes in the small RNA profiles and in the genes of RNA silencing machinery induced by different isolates in an economically important crop plant. They uncover new mechanisms and complexity of virus-host interactions that may have important implications for further studies on infection, pathogenesis, and plant defense responses.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
