Background

Maize (Zea mays L.) is an essential crop for human food, animal feed, and industrial material, which is also a classic model plant for genetics research (Gore et al. 2009). Sugarcane mosaic virus (SCMV) is a positive-sense single-stranded RNA (+ ssRNA) virus in the genus Potyvirus of the family Potyviridae. SCMV can infect maize, sorghum (Sorghum vulgare), sugarcane (Saccharum sinensis), and many other Gramineae crops, causing significant losses in various field crops worldwide (Shi et al. 2005). SCMV is the primary causal agent of maize dwarf mosaic disease in China, which initially causes chlorotic symptoms at the base of the leaf and then extends to the whole leaf until a stripe mosaic pattern appears on maize (Jiang and Zhou 2002). Breeding and planting resistant maize varieties are the most economical and effective methods to control SCMV infection. Therefore, exploring the interaction between SCMV and maize plants is important for develo** effective virus-control strategies and cultivating disease-resistant varieties.

RNA silencing is a natural antiviral mechanism in plants, which is triggered by double-stranded RNAs (dsRNAs) with different sources and lengths (Pumplin and Voinnet 2013). Plant Dicer-like (DCL) and Argonaute (AGO) proteins play crucial antiviral roles in the RNA silencing pathway (Bouché et al. 2006; Schuck et al. 2013). The dsRNAs are cleaved by DCL proteins into virus-derived small interfering RNAs (vsiRNAs) of 21 to 24 nucleotides (nt) (Ding and Voinnet 2007). Previous studies have shown that plants infected with positive-strand RNA viruses mainly generate 21-nt vsiRNAs processed by DCL4, while 22-nt vsiRNAs produced by DCL2 are accumulated when the activity of DCL4 is reduced or inhibited (Bouché et al. 2006; Ding 2010). DCL1 and DCL3 can produce 21- and 24-nt vsiRNAs in dcl2/dcl3/dcl4 and dcl2/dcl4 mutant plants, respectively (Bouché et al. 2006; Qu et al. 2008). The vsiRNAs are loaded into RNA-induced silencing complexes (RISCs), which contain Argonaute (AGO) proteins, guiding the degradation of viral RNAs and host target mRNAs in a sequence-specific manner (Baumberger and Baulcombe 2005). In Arabidopsis, AGOs 1, 2, 4, 5, 7, and 10 can bind vsiRNAs upon different virus infections (Carbonell and Carrington 2015). AGO1 is most important in plant antiviral defense (Harvey et al. 2011). Moreover, AGO2 can protect against suppressor-defective tomato bushy stunt virus (TBSV) in Nicotiana benthamiana (Scholthof et al. 2011). AGO4 also shows antiviral function in CMV-infected N. benthamiana (Ye et al. 2009). AGO1 and AGO18 are the main antiviral AGOs against rice stripe virus (RSV) and rice dwarf virus (RDV) in rice (Carbonell and Carrington 2015). In plants, the effect of RNA silencing can be amplified by cellular RNA-dependent RNA polymerases (RDRs), which synthesize dsRNAs and produce secondary vsiRNAs (Wang et al. 2010).

Previous studies have shown that vsiRNAs are mainly responsible for RNA silencing mediated antiviral immunity (Zhu et al. 2011). Recently, more and more studies suggest that vsiRNAs play potential regulatory roles in the expression of host genes, which determines the manifestation of viral symptoms in host plants (Wang et al. 2022). The first report of this phenomenon was the vsiRNAs derived from CMV Y-satellite that specifically regulate the expression of chlorophyll-related gene (ChlI) and modulate the typical yellowing symptoms in N. benthamiana (Smith et al. 2011; Shimura et al. 2011). It has also been reported that Chinese wheat mosaic virus (CWMV) RNA1-derived vsiRNA-20 can cleave the mRNA of TaVP to maintain a weak alkaline environment in the cytoplasm to enhance CWMV infection in wheat (Yang et al. 2020; Huang et al. 2022).

Recently, competing endogenous RNAs (ceRNAs) have been widely accepted as a new mode of gene regulation, of which circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs) can act as ceRNAs to regulate miRNA or vsiRNA activity (Song et al. 2021). Based on this mechanism, many studies have analyzed the regulatory network of ceRNA-miRNA/siRNA-target gene in plants (Salmena et al. 2011). For example, maize lncRNA PILNCR1 inhibits miR399-guided cleavage of PHOSPHATE2 (PHO2) to regulate plant tolerance to low phosphate (Du et al. 2018). Tomato lncRNA08489-miR482e module was reported to enhance host resistance to Phytophthora infestans through the reactive oxygen species (ROS)-scavenging system (Liu et al. 2022). In addition, LINC-AP2 contributes to the formation of shorter stamen in the flowers of A. thaliana plants infected with turnip crinkle virus (TCV) by anti-cis downregulating the expression of AP2 gene (Gao et al. 2016). Tomato lncRNA SlLNR1 interacts with vsiRNAs derived from a non-coding intergenic region (IR) of tomato yellow leaf curl virus (TYLCV) and suppresses disease development during viral infection (Yang et al. 2019). However, there is still a lack of comprehensive research on the interactions among circRNA, lncRNA, vsiRNA, and mRNA in virus-infected maize plants.

Our previous studies have reported the vsiRNA expression profiles in SCMV-infected maize inbred lines Zong 31 and B73 plants (** and transcriptome assembly

The raw data of whole-transcriptome RNA sequencing were processed using SOAPnuke v1.5.2 (Chen et al. 2018), and sequence quality was verified with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). For small RNA sequencing data, clean reads from each sample were screened in a range of length (18–36 nt). Next, these small RNA sequences were mapped to SCMV genome (Accession number: AY042184), and only those sequences that were identical or complementary to the viral genome sequences within two mismatches were identified as vsiRNAs. For RNA sequencing data, clean reads were mapped to B73 reference genome (ftp://ftp.ensemblgenomes.org/pub/release-45/plants/fasta/zea_mays/dna/, B73_RefGen_v4) using HISAT2 software (http://ccb.jhu.edu/software/hisat2/index.shtml). StringTie v1.3.0 was used to assemble the mapped reads of each sample (Pertea et al. 2015).

Degradome library construction and sequencing analysis

Total RNA from both treatment and control groups was pooled together to prepare the degradome library. The process of library construction was as follows: (a) mRNA fragments with poly (A) sequences were specifically captured with poly (T) magnetic beads; (b) 5′ RNA adapters were ligated to RNAs containing 5′ monophosphates; (c) The ligated products were purified and reverse-transcribed into cDNAs using biotinylated random primers; (d) The cDNAs were amplified by PCR to construct the degradome libraries; (e) Single-end (36 bp) sequencing was then performed on an Illumina Hiseq 2500 (LC Bio, Hangzhou, China). The original data of degradome sequencing (BioProject: GSE234274) have been uploaded to NCBI database.

The raw reads were processed using ACGT101-DEG (LC Sciences, Houston, Texas, USA) and potential siRNA editing sites were identified using the small RNA sequencing data by CleaveLand4 software (Addo-Quaye et al. 2009). Thereafter, based on the characteristics and abundance of maize RNA sequencing data, T-plots were established for high efficiency analysis of potential siRNA targets.

GO and KEGG pathway analysis

The predicted target genes of vsiRNAs were aligned based on BLAST (http://blast.ncbi.nlm.nih.gov/). GO analysis was performed to construct annotations of vsiRNA targets using AgriGO v2.0 (Tian et al. 2017). KEGG pathway analysis was implemented to understand the function among targets of vsiRNA by KOBAS 2.0 (**e et al. 2011). The threshold of significant GO terms and KEGG pathways was set to p < 0.05.

Target gene prediction and visualization of ceRNA regulatory network

To understand the potential molecular functions of the candidate vsiRNAs, according to the ceRNA theory, the interaction of ncRNA-vsiRNA and vsiRNA-mRNA pairs were predicted simultaneously by psRNATarget (Dai and Zhao 2011). The ceRNAs networks regulatory network was visualized using Cytoscape v3.7.2 software (Shannon et al. 2003) to display the potential relationships between circRNAs, lncRNAs, vsiRNAs, and mRNAs.

qRT-PCR analysis

Total RNA was extracted from samples by TRIzol Reagent (Vazyme, Nan**g, China) according to the manufacturer’s instructions. About 2 μg of total RNA was reverse-transcribed into cDNA with PrimeScript RT Reagent (TaKaRa, Dalian, China). The qRT-PCR reactions were performed on StepOne plus real time PCR platform (Applied Biosystems, Foster City, USA) using SYBR Green PCR Master Mix (Vazyme, Nan**g, China) as instructed. ZmUBI (XM_008647047) gene was used as an internal control, and relative gene expression levels in different samples were calculated by the 2−ΔΔCT method (Schefe et al. 2006). All primers were listed in Additional file 1: Table S19.