Molecular characterization of virus-derived small RNAs in Nicotiana benthamiana plants infected with tobacco curly shoot virus and its β satellite

Highlights

• TbCSB may influence the accumulation of small RNAs, while it encodes a suppressor of RNA silencing.

• TbCSB did not change the nucleotide preference of vsiRNAs, which derived from TbCSV.

• TbCSB may affect the function of DCL2 and DCL4 to recognize and cleave viral genome.

• TbCSV induces the expression of NbRDRs, but TbCSB does not influence the expression of NbRDRs.

Abstract

Tobacco curly shoot virus (TbCSV) is a monopartite DNA virus of the genus Begomovirus, which causes leaf curl symptoms in tobacco and tomato. The β satellite of TbCSV (TbCSB induces more severe symptoms and enhanced virus accumulation when co-infects the host plants with TbCSV. Small interfering RNAs derived from virus(vsiRNAs) induce disease symptoms and promote virus invasion by target and guide the degradation of host transcripts The vsiRNAs derived from TbCSV and TbCSV + TbCSB remained to be explored to elucidate the molecular mechanism of symptoms development in plants. In the present work, two libraries of small RNA from TbCSV-infected and TbCSV + TbCSB-infected N. benthamiana plants were constructed and the vsiRNAs in both samples shared the same characteristics. The size of the vsiRNAs ranged from 18 to 30 nucleotides (nt), with most of them being 21 or 22 nt, which accounted for 29.11% and 23.22% in TbCSV plants and 29.39% and 21.82% in TbCSV + TbCSV plants, respectively. The vsiRNAs with A/U bias at the first site were abundant in both the TbCSV-treated and TbCSV + TbCSB-treated plants. It is discovered that the vsiRNAs continuously, but heterogeneously, distributed through bothe the TbCSV and TbCSB sequences. And the distribution profiles were similar in both the treatments such as mainly in the overlapping region of the AC2/AC3 coding sequences. The host transcripts targeted by vsiRNAs were predicted, and the targeted genes were found to be involved in varied biological processes. It is indicated that the presence of TbCSB does not significantly affect the production of vsiRNAs from TbCSV in plants, the distribution hotsopt of TbCSV vsiRNAs could be useful in designing effective targets for TbCSV resistance exploiting RNA interference.

Introduction

Plant microRNAs (miRNAs), play vital roles in controlling development and productivity. For examples, miR156 and miR172 act as major factors in the plant growth and propagation (Teotia and Tang, 2015; Xie et al., 2006; Sha et al., 2014), and miR164 modulates leaf development by regulating growth, which determines leaf shape (Byrne, 2012). Plant miRNAs also play roles in plant defense against pathogens by regulating the expression of resistance genes (Shivaprasad et al., 2012; Yin et al., 2015) and genes associated with viral symptoms (Wang et al., 2018). For example, miR6019-mediated innate immunity during plant growth is regulated by the expression level of NLR (Deng et al., 2018), and osa-miR171b contributes to stunting and yellowing symptoms in rice stripe virus (RSV)-infected rice plants (Tong et al., 2017).

Pants viruses infection usually cause production of small interfering RNAs derived from the viruses genomes in host plants. These virus-derived small interfering RNAs (vsiRNAs), which range in length from 21- to 24- nucleotides (nt), are generated by Dicer-like (DCL) proteins that recognize and cleave the double-stranded viral RNA (dsRNA) and base-paired single-stranded RNA (ssRNA) (Hamilton and Baulcombe, 1999; Molnar et al., 2005). In plants, DCL4 and DCL2 are most important DCLs for the production of 21- and 22-nt vsiRNAs, respectively (Bouche et al., 2006; Donaire et al., 2008; Garcia-Ruiz et al., 2010). The antiviral immunity is mainly conferred by DCL4-dependent 21-nt vsiRNAs with DCL2 as a surrogate (Bouche et al., 2006; Brodersen et al., 2008). Some vsiRNAs are loaded into the Argonaute (AGO)-containing complexes to inactivate both viral genomes and in some cases the host mRNAs (Miozzi et al., 2013; Shimura et al., 2011; Jaubert et al., 2011; Zhu et al., 2011). Additionally, the vsiRNAs can be amplified by RNA-dependent RNA polymerases (RDRs), which help to enhance their functions. This process leads to production of secondary vsiRNAs that further reinforce the activity of the RNA silencing. Host-encoded RDRs including RDR1, RDR2 and RDR6 are responsible for this amplification process (Csorba et al., 2015; Wang et al., 2010). These vsiRNAs are recruited into the RNA-induced silencing complex by the AGO proteins, and this process is conducted by their 5′-terminal nucleotides (Brodersen et al., 2008; Fang and Qi, 2016; Mi et al., 2008).

It is well known that vsiRNAs target and degrade viral RNAs in plants, which benefits the host as a protection mechanism against viral infection. Recently, reports have shown that some vsiRNAs guide the degradation of host transcripts at the post-transcriptional level by base-pairing mechanisms to induce disease symptoms and promote virus invasion (Shi et al., 2016; Shimura et al., 2011; Smith et al., 2011). The vsiRNA from the Y satellite of cucumber mosaic virus (CMV) cleaves Ch1I mRNA in N. benthamiana and causes yellowing symptoms (Shimura et al., 2011; Smith et al., 2011). The vsiRNA from RSV RNA4 targets host eukaryotic translation initiation factor 4 A (eIF4 A) which leads to leaf curling and wilting (Shi et al., 2016). Furthermore, vsiRNAs from CMV target abundant host genes that participate in metabolic processes, cellular processes, and single-organism processes, as shown by analysis using Gene Ontology (GO) annotation, and genes that are related to metabolic activities, as shown by analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) (Qiu et al., 2017). Southern rice black-streaked dwarf virus (SRBSDV)-derived vsiRNAs, most of which are 21- or 22- nt in length, target a series of rice genes related to host defense, pathogenesis, and symptomology (Xu and Zhou, 2017). Moreover, many plant factors can be targeted by vsiRNAs, such as resistance proteins, kinase proteins, transcription factors, F-box proteins, and pathogen-related proteins (Miozzi et al., 2013).

Tobacco curly shoot virus (TbCSV), a member of the genus Begomovirus, consists of DNA (TbCSV) and DNA-β (tobacco curly shoot β satellite, TbCSB) and is transmitted exclusively by whitefly (Bemisia tabaci) as vector in tobacco, tomato, and weeds. Leaf curl disease caused by co-infection of TbCSV and TbCSB (TbCSV + TbCSB) complex in tobacco and tomato have been resulted in great economic losses. TbCSV is a circular single-stranded DNA (ssDNA) helper virus of approximately 2.7 kilonucleotides (knt) that encodes the AV1, AV2, AC1, AC2, AC3, and AC4 genes. TbCSB is ssDNA of about 1.3 knt and encodes the βC1 gene. Most begomoviruses require a β satellite to induce typical disease symptoms (Mansoor et al., 2003; Tao and Zhou, 2008). However, the TbCSV + TbCSB disease complex differs from other begomovirus/β satellite disease complexes in its pathogenicity and its role in regulating disease symptoms. TbCSV alone is able to induce severe symptoms, such as upward leaf curling, in tomato and tobacco plants. However, TbCSB was found to intensify the symptoms induced by TbCSV in Nicotiana spp., while the symptoms changed to downward leaf curling (Ding et al., 2009; Li et al., 2005). Although the functions of the viral genes are well known, few studies have focused on vsiRNA profiles from geminiviruses, especially TbCSV/TbCSB pathotypes. With the development of deep sequencing technology, an increasing number of vsiRNAs have been identified and the functions of their target genes have been characterized (Moyo et al., 2017; Qiu et al., 2017; Xu and Zhou, 2017). In this study, we used small RNA deep sequencing to comparatively analyze the vsiRNA profiles of Nicotiana benthamiana plants exhibiting the typical symptoms upon inoculation infection with TbCSV or TbCSV + TbCSB.