Identification Of Plant Viruses From Maize By Deep Sequencing And Analysis Of Small RNA In Virus Infected Maize

Maize is one of the most economically important crops in the world, while the continuous occurring of maize virus diseases causes serious damage to maize production. Identification of new maize viruses and determining their relationships with hosts are the primary tasks for preventing infectious and epidemic diseases caused by maize new viruses. Deep sequencing technology has broken out the limitations of traditional virus detection methods, which can simultaneously detect the known and unknown, high titer and low titer of all of the RNA viruses, DNA viruses and viroids in plant samples. This new sequencing technology has greatly revolutionized the methods for the detection of plant viruses, and has been considered as one of the most important methods for virus identification.In this study, field samples in Yunnan and Guizhou provinces were collected and used to detect viruses by small RNA deep sequencing. A total of seven maize viruses were identified through the contigs assembly, which were three unknown viruses and were named tentatively:Maize yellow mosaic virus, Maize-associated umbravirus and Maize-associated totivirus1; one DNA virus reported for the first time in China, Maize streak Reunion virus-YN isolate; three known RNA viruses, Maize chlorotic mottle virus, Sugarcane mosaic virus, and Southern rice black-streaked dwarf virus. The complete genomic sequences of the three unknown RNA viruses and MSRV-YN were obtained by RT-PCR/PCR using virus derived siRNAs and contigs for primer design.The MYMV genome is5,642nucleotides (nt) in length and has six open reading frames (ORFs) typical for the member of the genus Polerovirus, Luteoviridae. Phylogenetic analysis of whole genome sequences and amino acid sequences of the6ORFs between MYMV and the luteovirids showed that the MYMV grouped perfectly with members of Polerovirus, and gathered together with Maize yellow dwarf virus-RMV into a small cluster, which is a member of Polerovirus. The MYMV shared the highest nucleotide sequence homology to MYDV-RMV, with the similarity of73%, which indicated that the MYMV is a new member in genus Polerovirus. Furthermore, the full-length infectious cDNA clone of MYMV was successfully constructed. Both RT-PCR and Northern blot results showed that the infectious clone could systemiclly infect Nicotiana benthamiana plant. We also supplied evidence that the PO protein encoded by MYMV was a strong RNA silencing suppressor, and could cause local and systemic silencing of transgenic16c Nicotiana benthamiana plant. Subsequently, we collected maize leaves from the Yunnan and Guizhou provinces, and cloned the whole MYMV genome sequences of12isolates. The variation and evolution of complete genome nucleotide sequence of MYMV were analyzed. Our results showed that the genome nucleotide variation level was less than4%in the overall and an obvious fluctuation of the diversity genetic genome was observed, with a low variation in the conserved regions like CP and MP.The MAUV genome is3,040nt in length, encoding an unknown function protein by ORF1and a80kDa RdRp fusion protein. RdRp Phylogenetic tree analysis showed that MAUV grouped together with members of Umbravirus, Tombusviridae, and formed a small branch with Citrus yellow vein-associated virus, which is a member of Umbravirus. Both the nucleotide sequence similarity and amino acid similarity of RdRp between MAUV and Citrus yellow vein-associated virus are58.9%, suggesting that MAUV is a potential new member of Umbravirus.The MATV1genome is3,956nt in length, encoding an unknown function protein of ORF1and a92kDa RdRp fusion protein. Phylogenetic analysis of RdRp showed that MATV1grouped together with members of Totivirus, Totiviridae, and formed a small branch with Black raspberry virus F, which is a member of Totivirus. The nucleotide sequence similarity and amino acid similarity of RdRp between MAUV and Citrus yellow vein-associated virus are53.9%and46.0%, respectively, suggesting that MATV1is a potential new member of Totivirus.MSRV-YN is a circular single-stranded DNA virus with a length of2,880nt long, encoding a movement protein (MP/V1) and a coat protein (CP/V2) in the viral strand, a replication associated protein (Rep) and a RepA protein in the complementary strand, typical for the member of the genus Mastrevirus. Next, we analyzed the rolling circle amplification (RCA) product digested by BamHGI and obtained a single band of about3kilobase (kb). The phylogenetic tree analysis of the complete genome sequences showed that MSRV-YN grouped together with members of Mastrevirus. Geminiviridae, and formed a small branch with Maize streak Reunion virus. The nucleotide sequence similarity between MSRV-YN and Maize streak Reunion virus is96.3%.Small RNA is very important in regulating plant development and plant-environment interaction. Making an understanding of maize microRNA expression in MCMV-infected leaves at different time points would definitely facilitate the study of maize-MCMV interactions. In this study, we firstly constructed the small RNA libraries of maize leaves infected by MCMV in15dpi (MA-M) and30dpi (MA-Ml) by small RNA deep sequencing. After sequencing, a total of321known miRNAs were identified using bioinformatics tools, consisting of26known miRNA families, and379novel miRNAs. Subsequently, the target genes of the known and novel miRNAs and their corresponding functions were predicted, which provided the foundation for further research of the functions of these miRNAs. At the same time, the differentially expressed known miRNAs in this two time periods were analyzed. Our results showed that there were16differentially expressed known miRNAs with significant difference. Among them,7miRNAs showed down-regulated, and9showed up-regulated, all belonging to the10miRNA families. Next, quantitative real-time PCR (qRT-PCR) was used to analyze the expression level of miRNAs to verify the deep sequencing results. In addition, analysis of MCMV derived siRNAs in virus infected maize leaves was carried out, and we found that large amounts of virus-derived siRNAs could be produced in sample MA-M and MA-Ml, mainly derived from the viral strand. These virus-derived siRNAs might have some relationships with the host antiviral activity. We also found that along with the decline of virus titer in late infection, the amount of virus derived siRNAs also reduced, with the hotspot distribution also showing a certain degree of inconsistency. In15dpi, there were two hotspots in genomic loci nt1100and nt2899respectively. While in30dpi, these two hotspots turned into cold spots, with almost no virus-derived siRNAs.The synergism infection of plant viruses usually resulted in increased accumulation of one or two viruses than single infection and can cause more severe symptoms. In order to study the mechanism of Corn lethal necrosis disease caused by MCMV and SCMV mix infection, we constructed four maize leaves small RNA libraries, of which were MCMV infected only (MA-M), SCMV infected only (MA-S), MCMV and SCMV co-infected (MA-MS) and no virus infected (MA-H). respectively. Finally,321known miRNAs were identified by bioinformatics analysis. Next, the common and specific differentially expressed known-miRNAs among MA-M_MA-H, MA-S_MA-H and MA-MS_MA-H were analyzed, generating28common differentially expressed known-miRNAs and with the unique differentially expressed being8,6,28, respectively. GO enrichment analysis of these known miRNAs was also carried out. Meanwhile, qRT-PCR was used to analyze the expression level of miRNAs to verify the results from the deep sequencing assay. Furthermore,493novel miRNAs were predicted among the four sRNA libraries. A total of34common differentially expressed novel-miRNAs were analyzed among MA-M_MA-H, MA-S_MA-H and MA-MS_MA-H, with the unique differentially expressed being16,25,20, respectively. GO enrichment analysis of these miRNAs was also carried out, with significant GO enrichment in genes of targeting viral movement and virus-host interaction, which indicated that these novel-miRNAs might contribute a lot in virus infection and host antiviral process. Finally, we analyzed the distribution of viral siRNAs along viral genome, which provides basis for a better understanding of the pathogenic mechanism on synergism infection of plant virus and plant antiviral gene engineering.