Abstract
RNA silencing is a conserved mechanism in plants that targets viruses. Viral small RNAs (vsiRNAs) can be generated from viral double-stranded RNA replicative intermediates within the infected host, or from host RNA-dependent RNA polymerases activity on viral templates. The abundance and profile of vsiRNAs in viral infections have been reported previously. However, the involvement of vsiRNAs during infection of the Geminiviridae family member cotton leaf curl virus (CLCuD), which causes significant economic losses in cotton growing regions, remains largely uncharacterized. Cotton leaf curl Multan virus (CLCuMuV) associated with a betasatellite called Cotton leaf curl Multan betasatellite (CLCuMuB) is a major constraint to cotton production in South Asia and is now established in Southern China. In this study, we obtained the profiles of vsiRNAs from CLCuMV and CLCuMB in infected upland cotton (Gossypium hirsutum) plants by deep sequencing. Our data showed that vsiRNA that were derived almost equally from sense and antisense CLCuD DNA strands accumulated preferentially as 21- and 22-nucleotide (nt) small RNA population and had a cytosine bias at the 5′-terminus. Polarity distribution revealed that vsiRNAs were almost continuously present along the CLCuD genome and hotspots of sense and antisense strands were mainly distributed in the Rep proteins region of CLCuMuV and in the C1 protein of CLCuMuB. In addition, hundreds of host transcripts targeted by vsiRNAs were predicted, many of which encode transcription factors associated with biotic and abiotic stresses. Quantitative real-time polymerase chain reaction analysis of selected potential vsiRNA targets showed that some targets were significantly down-regulated in CLCuD-infected cotton plants. We also verified the potential function of vsiRNA targets that may be involved in CLCuD infection by virus-induced gene silencing (VIGS) and 5′-rapid amplification of cDNA end (5′-RACE). Here, we provide the first report on vsiRNAs responses to CLCuD infection in cotton.
Highlights
Cotton leaf curl disease (CLCuD) is a serious disease of cotton across Africa and South Asia (Rajagopalan et al, 2012)
To obtain vsiRNAs profiles produced during CLCuD infection, sRNAs derived from cotton plants that were inoculated with CLCuD at 0 dpi (CK) and 20 dpi for two biological replicates were analyzed using deep sequencing on the Illumina Hiseq2000 platform
In CLCuD-infected cotton plants, 21- and 22-nt vsiRNAs were most, representing 40 and 36% of total vsiRNAs, respectively (Figure 2C; Supplementary Table S2), which suggested that the cotton DCL4 and DCL2 proteins could be predominant dicer ribonucleases with roles in vsiRNA biogenesis
Summary
Cotton leaf curl disease (CLCuD) is a serious disease of cotton across Africa and South Asia (Rajagopalan et al, 2012). CLCuD is caused by a pathogen complex of monopartite begomoviruses (single-stranded DNA viruses of the family Geminiviridae that are transmitted by the whitefly Bemisia tabaci) and a specific DNA betasatellite (DNA-β) molecule, cotton leaf curl Multan betasatellite (CLCuMB; Briddon et al, 2001; Mansoor et al, 2003). Betasatellites encode for a dominant symptom determinant (known as βC1) which is a suppressor of post-transcriptional gene silencing (PTGS). It may be involved in movement of the virus with in plant tissues and in increasing viral DNA levels in planta (Qazi et al, 2007; Amin et al, 2011)
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