| Maize rough dwarf disease(MRDD) is a worldwide viral disease of maize, particularly in the Yellow and Huai River summer maize-growing regions of China. MRDD has been proposed to be caused by rice black-streaked dwarf virus(RBSDV) in China, which is transmitted in a persistent manner by the small brown planthopper(SBPH, Laodelphax striatellus). Analysis of genetic variation and pathopoiesis pathway of RBSDV in maize will be the key way for germplasm improvement and breeding for MRDD resistance. In this study, we collected the plants with typical sympotoms of MRDD or rice black-streaked dwarf disease(RBSDD) from nine different locations in China. Genetic variation and evolution of RBSDV were characterized by analyzing the genome sequences of RBSDV, and furthermore, the pathopoiesis pathway of RBSDV was investigated by analyzing the differentially expressed genes and mi RNAs followed RBSDV-infected maize. The main research results were as follows:1. The plants with typical symptoms of MRDD or RBSDD were collected from nine locations where the diseases prevailed in 2012 to 2014, and genetic vaiation of S1-S10 segments of RBSDV was investigated.(1) There was 9bp deletion mutantion in S8, while no deletions or insertions were found in other segments. The ratios of mutations between two bases were the highest from 93.73% to 96.11%. Transition ratios ranged from 76.87% to 85.92% were more than transversion from 8.69% to 17.43%, and the transitions between pyrimidines were higher 47.72%-59.60%. S9 with the highest nucleotide diversity(π = 0.0626) were siginicantly higher(P < 0.01) than S4 with the lowest level of diversity(π = 0.0225). S9(π = 0.0755) and S7(π = 0.0391) of isolates from Jinan, Shandong province with the highest diversity were significantly higher than S9(π = 0.0391) and S7(π = 0.0090) from Nanjing and Yancheng, Jiangsu province with the lowest level of nucleotide diversity. The difference of nucleotide diversity of RBSDV derived from maize and rice host was not significant(P > 0.05). Nucleotide diversity(π) of S7 sequences with a value of 0.0307, of isolates collected in 2013, was significantly higher than that in 2014 with a value of 0.0244(P = 0.0226).(2) Three recombinants in S9 and one recombinant in S7 were detected among different hosts or geographical origins. The phylogenic analysis based on S9 sequences of 46 RBSDV isolates from this study and 23 from Gen Bank indicates that the sequence is genetically related to RBSDV. Phylogenetic analyses showed that the RBSDV isolates could be classified into two groups according to S9 and S7 sequences, and further classified into two subgroups in each group of S7, respectively, regardless of host or geographical origin.(3) At amino acid level, P5-2 showed the highest variation, with an average of one mutation site per ten amino acids, but P2 showed the lowest variation, with an average of one mutation site per 45 amino acids. The variation of different ORFs was not the same even in the same segment.(4) The conservative sequences among S1-S10 segments of RBSDV genome were predicted. Three vectors of RNAi were designed and constructed in different target positions for each segments of RBSDV.2. The selection response of RBSDV was investigated based on 49 S9 sequences and 111 S7 sequences. The S7 containing two ORFs named as S7-1 and S7-2 appears to be the A+U rich, and overall codon usage is biased toward A-(A3s, S7-1: 32.64%, S7-2: 29.95%) and U-ending codons(U3s, S7-1: 44.18%, S7-2: 46.06%). Average Nc values of 45.63 in S7-1 and 39.96 in S7-2 showed low degrees of codon usage bias, and mutational bias was likely to be major force driving RBSDV-S7 codon usage bias, regardless of year, host or geographical origin. Twelve optimal codons were detected in S7-1 and S7-2. Furthermore S7-1 and S7-2 were found under negative and purifying selection, with Ka/Ks ratios from 0.0179 to 0.0589. ORFs in the same segment were under different levels of selection. The selection pressure on S9-2 was higher than that on S9-1, and S7-1 was higher than that on S7-2(P < 0.01). Ka/Ks ratios of S9 and S7 were no significantly different between hosts or between geographic locations.The variation of RBSDV populations were in a state of expansion(P < 0.01) in maize, and frequent gene flow were happened among hosts, years or geographic locations.3. Morphology changes of tissue infected by RBSDV in maize were investigated using the thin slice of electron microscope. The RBSDV particles were found mianly in cytoplasm and chloroplast, and the morphology in phloem, cell wall and chloroplast were all found altered after infecting by RBSDV. It was found that pathogenesis-related, cell wall-related, gibberellin-related, phloem-related, chloroplas-related and ubiquitin-related genes were detected in maize infected by RBSDV using RNA-sequcened method. Thirty-one known mi RNAs from seventeen mi RNA familes were detected in reponse to RBSDV, and the expression levels of these mi RNA were more than two folds higher. Among them, seventeen mi RNAs were significantly up-related expressed and fourteen mi RNAs were siginificantly down-related expressed. The target genes were identified using degradome sequencing, and target genes were mainly in nucleus and ribosome. The functions of these target genes were found to be regulation of transcription, DNA-temptated and DNA binding. The target genes were also related to metal ion transmembrane transporter activity, hydrolase activity, acting on acid anhydrides or in phosphorus, stress-response and photosynthesis. The target genes were also enriched in the photosynthesis metabolism pathway. The different expression of transcriptome and mi RNA-target gene showed that genes GRMZM2G069316 and GRMZM2G031169 play important roles in the response to RBSDV infection in maize. |
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