Tobacco RNA-dependent RNA polymerase 1 affects the expression of defence-related genes in Nicotiana benthamiana upon Tomato leaf curl Gujarat virus infection

RNA-dependent RNA polymerase 1 of Nicotiana tabacum modulates ToLCGV pathogenesis by influencing a number of defence-related genes in N. benthamiana plants. Key means of plants protecting themselves from the invading viruses is through RNA silencing. RNA-dependent RNA polymerase-1 (RDR1) is one of the crucial proteins of the RNA silencing pathway, which is induced after infection by viruses. RDR1 functions in the generation of small interfering RNAs (siRNAs) against the viral genome, thus it is antiviral in nature. Here, we used the transgenic Nicotiana benthamiana plant expressing N. tabacum NtRDR1 and observed reduced susceptibility towards Tomato leaf curl Gujarat virus (ToLCGV) infection compared to the wild-type N. benthamiana plants. To understand the reason for such reduced susceptibility, we prepared high-definition small RNA (sRNA) cDNA libraries from ToLCGV-infected wild-type N. benthamiana and NtRDR1 expressing N. benthamiana lines and carried out next-generation sequencing (NGS). We found that upon ToLCGV infection the majority of siRNAs generated from the host genome were of the 24 nucleotide (nt) class, while viral siRNAs (vsiRNAs) were of the 21-22-nt class, indicating that transcriptional gene silencing (TGS) is the major pathway for silencing of host genes while viral genes are silenced, predominantly, by post transcriptional gene silencing (PTGS) pathways. We estimated the changes in the expression of various defence-related genes, such as Constitutively Photomorphogenic-9 (COP9) signalosome (CSN) complex subunit-7, Pentatricopeptide repeat containing protein (PPRP), Laccase-3, Glutathione peroxidase-1 (GPX-1), Universal stress protein (USP) A-like protein, Heat shock transcription factor B4 (HSTF-B4), Auxin response factor-18 (ARF18), WRKY-6 and Short chain dehydrogenase reductase-3a. The differential expression of these genes might be linked with the enhanced tolerance of NtRDR1 N. benthamiana transgenic plants to ToLCGV. Our study suggests that reduced expression of subunit-7 of CSN complex and WRKY6, and increased expression of USPA-like protein might be linked with the reduced susceptibility of NtRDR1-transgenic N. benthamiana plants to ToLCGV.

Keywords: Defence-related genes; Geminivirus; High-definition adapters; Host siRNA; Next-generation sequencing; Nicotiana tabacum; NtRDR1; Plant virus; RNA-dependent RNA polymerase; Small RNA; Tomato leaf curl gujarat virus.