Background
Methods
Plant cultivation and virus inoculation
RNA extraction, small RNA sequencing and analysis of siRNAs
Prediction and annotation of target genes
Reverse transcription-quantitative PCR
Results
Profiling of SRBSDV-derived siRNAs in infected rice
Distribution of vsiRNAs in the SRBSDV genome
vsiRNA No. | Sequence (5’ to 3’) | Read count | Strand & position (nt) | GC% | Number of putative rice targets |
---|---|---|---|---|---|
vsiRS1_1 | UAAUCGAGAUACACUCUGCGCC | 336 | (−) 4304-4325 | 50.0 | 2 |
vsiRS1_2 | AAUCGAGAUACACUCUGCGCC | 167 | (−) 4304-4324 | 52.4 | 1 |
vsiRS1_3 | UUGCGCAUUGUACUGACCUUGG | 114 | (+) 1400-1421 | 50.0 | 2 |
vsiRS1_4 | UCGAGAUACACUCUGCGCCCA | 104 | (−) 4302-4322 | 57.1 | 1 |
vsiRS3_1 | UUAGACGCAGAAUUGAAGAAUC | 237 | (+) 176-197 | 36.4 | 4 |
vsiRS3_2 | UUCAUUAGGUACUUGAUCUGA | 112 | (−) 3466-3486 | 47.6 | 14 |
vsiRS4_1 | UACAGGUAGUGAACACAAGCC | 112 | (+) 2684-2704 | 33.3 | 7 |
vsiRS4_2 | UACGAGACAAUGCAAACUGUA | 106 | (−) 465-485 | 38.1 | 9 |
vsiRS5_1 | ACACUCGUGACUCAAUUCUGCC | 209 | (+) 176-197 | 50.0 | 5 |
vsiRS5_2 | CACUCGUGACUCAAUUCUGCC | 176 | (+) 177-197 | 52.4 | 5 |
vsiRS5_3 | CACGAACAAGCAGACAACACU | 162 | (+) 112-132 | 47.6 | 2 |
vsiRS5_4 | UAGCGUUGUGAUUGUGAUCAAA | 128 | (−) 2927-2948 | 36.4 | 7 |
vsiRS5_5 | UUCACGACUUUGAAGACACCCA | 125 | (+) 155-176 | 45.5 | 0 |
vsiRS5_6 | UUCACGACUUUGAAGACACCC | 116 | (+) 155-175 | 47.6 | 0 |
vsiRS6_1 | UUGAGAGAACAAAGUGAUCGUU | 122 | (+) 1846-1867 | 36.4 | 9 |
vsiRS6_2 | UGAGAGAACAAAGUGAUCGUU | 119 | (+) 1847-1867 | 38.1 | 8 |
vsiRS6_3 | UGAGAUCUCUGUCCGUUAAAGA | 119 | (−) 2540-2561 | 40.9 | 14 |
vsiRS6_4 | UGGGCCGACGUAGUUGAAAGAA | 111 | (+) 1579-1600 | 50.0 | 7 |
vsiRS7_1 | UUGAAUAACAUUCUGUAGGAA | 228 | (−) 1851-1871 | 28.6 | 13 |
vsiRS7_2 | UCCAAUCAAGAUUCAAGAGCU | 109 | (+) 344-364 | 38.1 | 7 |
vsiRS8_1 | UCACUAGAAUCUACAUCGACCU | 217 | (−) 1882-1903 | 40.9 | 32 |
vsiRS8_2 | UCGCAAUGGCACGAGUAGGACU | 160 | (−) 1648-1669 | 54.5 | 4 |
vsiRS8_3 | AUGGCACGAGUAGGACUUAAUA | 120 | (−) 1643-1664 | 40.9 | 2 |
vsiRS8_4 | AAUGGCACGAGUAGGACUUAAU | 111 | (−) 1644-1665 | 40.9 | 1 |
vsiRS9_1 | AGAGAAUGGCAGACCUAGAGCG | 159 | (+) 47-68 | 54.5 | 0 |
vsiRS9_2 | UACAGUACCUCCAUUGAACACU | 147 | (−) 1746-1767 | 40.9 | 13 |
vsiRS9_3 | ACAGAUCAAUUGGACUUGGCU | 143 | (+) 349-369 | 42.9 | 8 |
vsiRS10_1 | UUAUGGAUAAGAUCGGGCGCUA | 134 | (−) 44-65 | 45.5 | 3 |
Prediction of putative rice target genes of vsiRNAs
vsiRNA No.a)
| ID of predicted target gene | Expectation score b)
| Target Accessibility (UPE) b)
| Alignment | Inhibition manner | Putative function of rice target gene |
---|---|---|---|---|---|---|
vsiRS1_1 | LOC_Os08g29710.1 | 3.0 | 14.152 | miRNA 20 GCGUCUCACAUAGAGCUAAU 1 .::::: :::::::.:::: Target 1652 UGCAGAUUGUAUCUUGAUUU 1671 | Cleavage | galactosyltransferase family protein |
LOC_Os06g39650.1 | 3.0 | 19.7 | miRNA 20 GCGUCUCACAUAGAGCUAAU 1 .::::.::::: ::.::::. Target 1557 UGCAGGGUGUAGCUUGAUUG 1576 | Translation | pentatricopeptide | |
vsiRS2_1 | LOC_Os02g07310.1 | 2.5 | 20.512 | miRNA 21 UCACAAUAGUAUAAGAUACCU 1 .:: :::::::.::::.:::. Target 2336 GGUAUUAUCAUGUUCUGUGGG 2356 | Cleavage | AGO17 |
vsiRS3_1 | LOC_Os09g25330.1 | 3.0 | 7.741 | miRNA 22 CUAAGAAGUUAA-GACGCAGAUU 1 : :::::.:::: ::::::.::: Target 158 GUUUCUUUAAUUGCUGCGUUUAA 180 | Translation | Bric-a-Brac, Tramtrack, Broad Complex BTB domain with non-phototropic hypocotyl 3 NPH3 domain, BTBN19 |
vsiRS4_2 | LOC_Os01g63710.2 | 3.0 | 11.886 | miRNA 20 UGUCAAACGUAACAGAGCAU 1 :::::::::::::: :: :: Target 1668 ACAGUUUGCAUUGUAUCUUA 1687 | Cleavage | Putative DNA replication initiation protein, CDC6 |
LOC_Os03g51270.1 | 3.0 | 14.253 | miRNA 20 UGUCAAACGUAACAGAGCAU 1 :::: :: :::: ::::::: Target 1029 ACAGCUUACAUUUUCUCGUA 1048 | Cleavage | F-box domain containing protein, OsFBX108 | |
vsiRS5_1 | LOC_Os03g36790.2 | 3.0 | 21.375 | miRNA 20 GUCUUAACUCAGUGCUCACA 1 ::::::: :::..:::::: Target 204 CAGAAUUCAGUUGCGAGUGC 223 | Cleavage | tobamovirus multiplication protein |
vsiRS5_3 | LOC_Os07g35750.1 | 2.5 | 18.456 | miRNA 20 CACAACAGACGAACAAGCAC 1 ::::::: ::::::::.::: Target 3702 GUGUUGU-UGCUUGUUUGUG 3720 | Cleavage | DUF26 kinases homologous to DUF26 containing loci, TKL_IRAK_DUF26-ld.3 |
vsiRS6_1 | LOC_Os07g36280.1 | 2.5 | 16.42 | miRNA 22 UUGCUAGUGA-AACAAGAGAGUU 1 :: ::::::: :::::.:::::: Target 1053 AAAGAUCACUGUUGUUUUCUCAA 1075 | Cleavage | F-box domain containing protein, OsFBX241 |
LOC_Os05g50890.3 | 3.0 | 23.237 | miRNA 22 UUGCUAGUGAAACAAGAGAGUU 1 ..::.::.:::: :::::::.. Target 319 GGCGGUCGCUUUCUUCUCUCGG 340 | Translation | Probable indole-3-acetic acid-amido synthetase, OsGH3.5 | |
vsiRS7_1 | LOC_Os02g54500.1 | 2.5 | 14.148 | miRNA 20 AGGAUGUCUUACAAUAAGUU 1 :: ::::::::::::::: : Target 3812 UCAUACAGAAUGUUAUUCUA 3831 | Cleavage | WD40-like, putative, expressed |
LOC_Os08g12780.1 | 3 | 17.599 | miRNA 21 AAGGAUGUCUUACAAUAAGUU 1 :::::.:::.: :::::::: Target 1935 UUCCUGCAGGAAAUUAUUCAA 1955 | Translation | chloroplast envelope membrane protein | |
vsiRS8_1 | LOC_Os06g25560.1 | 2.5 | 13.472 | miRNA 20 CAGCUACAUCUAAGAUCACU 1 :.::::: ::::::::::: Target 454 CUUGAUGUUGAUUCUAGUGA 473 | Cleavage | retrotransposon protein |
LOC_Os04g11830.1 | 3.0 | 16.405 | miRNA 20 CAGCUACAUCUAAGAUCACU 1 :: :::::.:: :.:::::: Target 1708 GUGGAUGUGGAAUUUAGUGA 1727 | Translation | TCP family transcription factor | |
vsiRS8_2 | LOC_Os12g40279.2 | 2.5 | 17.285 | miRNA 20 AGGAUGAGCACGGUAACGCU 1 ::.::::: :::.::::::. Target 3774 UCUUACUCUUGCUAUUGCGG 3793 | Cleavage | protein kinase domain containing protein |
vsiRS9_3 | LOC_Os06g20050.1 | 3.0 | 18.094 | miRNA 20 CGGUUCAGGUUAACUAGACA 1 ::::::.:::::::: ::: Target 2505 UCCAAGUUCAAUUGAUAUGU 2524 | Cleavage | leucine-rich repeat receptor protein kinase EXS precursor |
vsiRS9_3 | LOC_Os05g07820.1 | 3.0 | 17.222 | miRNA 20 CGGUUCAGGUUAACUAGACA 1 :.:.:::::: ::::::::: Target 586 GUCGAGUCCA-UUGAUCUGU 604 | Translation | NBS type disease resistance protein |
vsiRS10_1 | LOC_Os08g40170.2 | 3.0 | 19.095 | miRNA 21 UCGCGGGCUAGAAUAGGUAUU 1 ::.:.:.::::::.:::::.. Target 723 AGUGUCUGAUCUUGUCCAUGG 743 | Cleavage | cyclin-dependent kinase B2-1 |