Potato Virus Y Infection Alters Small RNA Metabolism and Immune Response in Tomato
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant and Virus Material
2.2. RNA Extraction
2.3. Primer Design and RT-qPCR Analysis for Detection of Viral RNA, mRNAs and miRNAs
2.4. Sequencing and sRNA Data Analysis
2.5. Identification of Differentially Expressed miRNAs
2.6. Target Gene Prediction and Functional Analysis
2.7. Degradome-Seq Target Validation
2.8. Validation of miRNA Targets with RLM 5´-RACE
2.9. Data Availability and Retrieval
3. Results
3.1. Tomato plants Exhibit Symptom Recovery at Later Stages of PVYC-to Infection
3.2. Viral siRNA Accumulation Levels Correlate with Viral RNA Titer
3.3. Known and Novel miRNAs and Their isomiR Variants Were Identified in Tomato Leaf Tissues
3.4. Regulation of Known and Novel Tomato mRNA upon PVYC-to Infection
3.5. RT-qPCR Expression Analysis of Known and Novel miRNAs and Their Target mRNAs
3.6. Target Genes Prediction and Functional Characterization of Differentially Expressed Tomato miRNAs
3.7. PVYC-to Infection Induces Secondary Phased siRNA Accumulation
3.8. miRNAs Mediate Cleavage of Transcripts Encoding NLRs and RLPs
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Mk-21 1 | PVY-21 1 | Mk-30 1 | PVY-30 1 | |||||
Redundant Reads Number | Nonredundant Reads Number | Redundant Reads Number | Nonredundant Reads Number | Redundant Reads Number | Nonredundant Reads Number | Redundant Reads Number | Nonredundant Reads Number | |
raw reads (library size) | 28.222.339 | 6.959.896 | 23.645.346 | 3.744.089 | 17.602.784 | 4.531.582 | 12.801.610 | 3.164.947 |
low-complexity sequences | 195 | 171 | 105 | 93 | 172 | 148 | 134 | 118 |
clean reads 18–26 nt | 22.463.045 | 6.376.853 | 21.507.027 | 3.392.422 | 13.973.890 | 4.137.134 | 10.945.324 | 2.845.983 |
Length Distribution of Clean Reads | Redundant Reads Number | % 2 | Redundant Reads Number | % 2 | Redundant Reads Number | % 2 | Redundant Reads Number | % 2 |
18 | 207.038 | 0.92 | 201.139 | 0.94 | 151.081 | 1.08 | 137.259 | 1.25 |
19 | 280.484 | 1.25 | 288.476 | 1.34 | 175.548 | 1.26 | 182.771 | 1.67 |
20 | 495.843 | 2.21 | 484.206 | 2.25 | 300.467 | 2.15 | 281.912 | 2.58 |
21 | 2.133.848 | 9.5 | 12.018.765 | 55.88 | 1.661.996 | 11.89 | 4.418.744 | 40.37 |
22 | 2.226.996 | 9.91 | 3.473.057 | 16.15 | 1.622.666 | 11.61 | 1.679.439 | 15.34 |
23 | 3.475.721 | 15.47 | 1.266.933 | 5.89 | 2.060.721 | 14.75 | 1.018.064 | 9.3 |
24 | 12.562.212 | 55.92 | 3.488.376 | 16.22 | 7.372.172 | 52.76 | 2.930.233 | 26.77 |
25 | 761.854 | 3.39 | 197.821 | 0.92 | 425.439 | 3.04 | 198.083 | 1.81 |
26 | 319.049 | 1.42 | 88.254 | 0.41 | 203.800 | 1.46 | 98.819 | 0.9 |
tomato SL3.0 genome mapped reads (100% identity) | 20.289.212 | 90.32 | 10.134.178 | 47.12 | 12.632.820 | 90.40 | 6.506.699 | 59.45 |
noncoding RNAs | 1787544 | 7.96 | 558036 | 2.59 | 1157563 | 8.28 | 554155 | 5.06 |
rRNAs | 1653355 | 7.36 | 519130 | 2.41 | 1034369 | 7.40 | 516761 | 4.72 |
tRNAs | 73891 | 0.33 | 25743 | 0.12 | 95440 | 0.68 | 22052 | 0.20 |
snoRNA | 48703 | 0.22 | 6736 | 0.03 | 20753 | 0.15 | 10516 | 0.10 |
snRNA | 6507 | 0.03 | 3602 | 0.02 | 4221 | 0.03 | 2674 | 0.02 |
lncRNA | 5088 | 0.02 | 2825 | 0.01 | 2780 | 0.02 | 2152 | 0.02 |
Tot. | 1787544 | 7.96 | 558036 | 2.59 | 1157563 | 8.28 | 554155 | 5.06 |
tomato phasiRNAs all | 53902 | 0.27 | 50163 | 0.49 | 20991 | 0.17 | 19981 | 0.31 |
tomato phasiRNAs PVY specific | 0 | 0 | 313 | 0.003 | 0 | 0 | 219 | 0.003 |
known mature miRNAs | 758288 | 3.38 | 883482 | 4.11 | 810148 | 5.80 | 455002 | 4.16 |
vsiRNAs (100% identity) | 928 | 0.004 | 10040027 | 46.68 | 590 | 0.004 | 3593569 | 32.83 |
sequence length of vsiRNAs | abs | % 3 | abs | % 3 | abs | % 3 | abs | % 3 |
18 | 2 | 0.22 | 17649 | 0.18 | 7299 | 0.20 | ||
19 | 5 | 0.54 | 24487 | 0.24 | 1 | 0.17 | 9812 | 0.27 |
20 | 9 | 0.97 | 66285 | 0.66 | 5 | 0.85 | 24028 | 0.67 |
21 | 726 | 78.23 | 8209564 | 81.77 | 469 | 79.49 | 2914094 | 81.09 |
22 | 179 | 19.29 | 1652385 | 16.46 | 109 | 18.47 | 616990 | 17.17 |
23 | 4 | 0.43 | 41968 | 0.42 | 4 | 0.68 | 13838 | 0.39 |
24 | 3 | 0.32 | 24511 | 0.24 | 2 | 0.34 | 6138 | 0.17 |
25 | 2405 | 0.02 | 984 | 0.03 | ||||
26 | 773 | 0.01 | 386 | 0.01 |
Normalized Read Counts (RPM) | Relative Accumulation (PVY vs. Mk) | ||||||||
---|---|---|---|---|---|---|---|---|---|
Cluster 1 | Length | miRNA ID | Mk-21 | PVY-21 | Mk-30 | PVY-30 | Log2fc 21 dpi | Log2fc 30 dpi | PVY-Associated 2 |
1 | 22 | sly-miR6023 | 257.9 | 161.2 | 118.1 | 177.5 | −0.7 | 0.6 | |
1 | 23 | sly-miR6023.30 | 4.3 | 154.3 | 1.3 | 185.2 | 5.2 | 7.2 | |
1 | 19 | sly-miR6023.3 | 0.2 | 46.1 | 0.1 | 31.0 | 7.9 | 9.0 | |
1 | 23 | sly-miR6023.14 | 0.0 | 4.1 | 0.1 | 4.5 | 6.8 | 5.2 | YES |
1 | 23 | sly-miR6023.16 | 0.1 | 2.9 | 0.1 | 5.6 | 4.3 | 6.5 | YES |
1 | 22 | sly-miR6023.7 | 0.0 | 2.6 | 0.0 | 2.5 | 7.1 | 6.4 | YES |
1 | 22 | sly-miR6023.13 | 0.1 | 1.7 | 0.0 | 1.1 | 4.5 | 5.2 | YES |
1 | 22 | sly-miR6023.21 | 0.0 | 1.7 | 0.0 | 2.5 | 6.5 | 6.4 | YES |
1 | 21 | sly-miR6023.29 | 0.7 | 0.5 | 0.4 | 0.6 | |||
2 | 22 | sly-miR6023.1 | 0.6 | 59.3 | 0.6 | 51.0 | 6.6 | 6.4 | |
2 | 21 | sly-miR6023.4 | 0.2 | 1.4 | 0.2 | 1.1 | 2.9 | 2.7 | |
2 | 24 | sly-miR6023.5 | 3.4 | 0.6 | 2.3 | 1.3 | −2.4 | −0.8 | |
3 | 22 | sly-miR6023.28 | 13.5 | 87.2 | 13.9 | 96.9 | 2.7 | 2.8 | |
3 | 21 | sly-miR6023.18 | 0.7 | 15.3 | 1.5 | 12.0 | 4.4 | 3.0 | |
3 | 21 | sly-miR6023.17 | 0.1 | 14.8 | 0.0 | 9.1 | 7.6 | 8.2 | YES |
3 | 22 | sly-miR6023.10 | 0.1 | 9.0 | 0.0 | 12.2 | 6.3 | 8.7 | YES |
3 | 21 | sly-miR6023.19 | 0.4 | 6.3 | 0.8 | 5.2 | 3.8 | 2.7 | |
3 | 21 | sly-miR6023.27 | 0.6 | 5.8 | 0.5 | 8.9 | 3.4 | 4.1 | |
3 | 21 | sly-miR6023.25 | 0.1 | 5.3 | 0.1 | 6.1 | 5.6 | 5.7 | YES |
3 | 21 | sly-miR6023.2 | 0.3 | 3.8 | 0.5 | 3.6 | 3.7 | 2.9 | |
3 | 22 | sly-miR6023.26 | 0.0 | 3.6 | 0.0 | 6.0 | 7.6 | 7.7 | YES |
3 | 23 | sly-miR6023.11 | 0.0 | 1.3 | 0.3 | 1.3 | 5.1 | 2.1 | YES |
4 | 21 | sly-miR6023.15 | 0.1 | 2.2 | 0.1 | 4.2 | 4.9 | 5.1 | YES |
4 | 21 | sly-miR6023.12 | 0.0 | 1.4 | 0.0 | 1.1 | 6.2 | 5.1 | YES |
5 | 19 | sly-miR6023.23 | 1.6 | 43.1 | 2.6 | 47.7 | 4.7 | 4.2 | |
5 | 20 | sly-miR6023.24 | 0.1 | 5.9 | 0.0 | 7.1 | 5.3 | 7.9 | YES |
5 | 20 | sly-miR6023.8 | 0.1 | 5.4 | 0.1 | 6.3 | 5.6 | 5.7 | YES |
5 | 18 | sly-miR6023.22 | 0.1 | 2.4 | 0.1 | 2.0 | 4.0 | 5.1 | YES |
5 | 21 | sly-miR6023.20 | 0.3 | 2.0 | 1.1 | 1.5 | 2.8 | 0.4 | |
5 | 22 | sly-miR6023.6 | 0.1 | 1.3 | 0.0 | 1.5 | 3.6 | 5.7 | YES |
5 | 21 | sly-miR6023.9 | 0.0 | 1.3 | 0.1 | 2.3 | 6.2 | 5.2 | YES |
Normalized Read Counts (RPM) | Relative Accumulation (PVY vs. Mk) | ||||||||
---|---|---|---|---|---|---|---|---|---|
Sequence | miRNA ID 1 | Identical miRNA ID in miRBase 2 | miRNA Family | Mk-21 | PVY-21 | Mk-30 | PVY-30 | Log2fc 21 dpi | Log2fc 30 dpi |
GGAGGCAGCGGTTCATCGATC | novel_sly-miR162a* | aly-miR162a-5p | miR162 | 6.2 | 130.8 | 3.2 | 96.9 | 4.4 | 4.9 |
AGATCATGTGGTTGCTTCACC | novel_sly-miR167d | NO | miR167 | 1.0 | 36.0 | 0.5 | 30.1 | 5.1 | 5.8 |
TTCCAAAGCTGCAGAAATGAGT | novel_sly-miR8033 | stu-miR8033-5p | miR8033 | 1.2 | 17.8 | 2.8 | 12.7 | 3.9 | 2.2 |
TTTTGTAGTAACTGTACCACA | novel-sly-miR1 | NO | novel | 0.1 | 2.9 | 0.0 | 2.3 | 4.3 | 6.2 |
GGGGCAACTTGAGATCACATG | novel-sly-miR11 | NO | novel | 295.5 | 19,873 | 184.5 | 4511.7 | 6.1 | 4.6 |
TGTGTTCTCAGGTTACCCCTG | novel-sly-miR11* | NO | novel | 254.1 | 9302.3 | 187.6 | 4058.3 | 5.2 | 4.4 |
AACTGTCGGGAGACATTAGCT | novel-sly-miR2 | NO | novel | 1.1 | 8.4 | 1.6 | 7.1 | 2.9 | 2.2 |
TACTATCTGATTTAAGATTAG | novel-sly-miR3 | NO | novel | 0.0 | 4.5 | 0.0 | 3.6 | 7.9 | 6.9 |
CCTGAACTATCACCATCTATG | novel-sly-miR5 | NO | novel | 0.1 | 7.8 | 0.0 | 5.3 | 6.1 | 7.5 |
TGGCAAGTAAGCGCTCCAACT | novel-sly-miR6 | NO | novel | 0.0 | 4.3 | 0.2 | 3.8 | 6.8 | 4.4 |
AGGGGAGATAGATGAAGTTAGG | novel-sly-miR7 | NO | novel | 0.2 | 7.3 | 0.2 | 7.1 | 5.0 | 5.3 |
TTATTTGGGGTAGATGAGCTC | novel-sly-miR8 | NO | novel | 1.0 | 14.3 | 0.4 | 9.5 | 3.9 | 4.7 |
AACAACTGATAGTTGAGGTGT | novel-sly-miR9 | NO | novel | 0.3 | 7.5 | 0.0 | 4.8 | 4.8 | 7.3 |
ATTTACCCCAAGTTCGTTGTC | sly-miR10537 | sly-miR10537 | miR10537 | 1.2 | 14.1 | 0.9 | 8.4 | 3.6 | 3.2 |
ATAATAACTATTAGTTGAATG | sly-miR10540 | sly-miR10540 | miR10540 | 0.0 | 1.5 | 0.0 | 1.2 | 6.3 | 5.3 |
CATGTGCCTGTTTTCCCCATC | sly-miR164a-3p | sly-miR164a-3p | miR164 | 0.9 | 30.8 | 1.0 | 14.9 | 5.1 | 4.0 |
GGGATGTTGTCTGGCTCGACA | sly-miR166c-5p | sly-miR166c-5p | miR166 | 155.7 | 1440.6 | 99.9 | 905.8 | 3.2 | 3.2 |
AGGTCATCTAGCAGCTTCAAT | sly-miR167b-3p | sly-miR167b-3p | miR167 | 0.2 | 9.5 | 0.1 | 7.0 | 5.4 | 6.9 |
CCTGCCTTGCATCAACTGAAT | sly-miR168a-3p | sly-miR168a-3p | miR168 | 67.3 | 441.4 | 32.9 | 201.2 | 2.7 | 2.6 |
CCCGCCTTGCATCAACTGAAT | sly-miR168b-3p | sly-miR168b-3p | miR168 | 220.1 | 1731.6 | 121.5 | 794.4 | 3.0 | 2.7 |
TTGAGCCGTGCCAATATCACG | sly-miR171b-3p | sly-miR171b-3p | miR171 | 3.4 | 28.0 | 1.4 | 22.0 | 3.0 | 4.0 |
TATTGGCCTGGTTCACTCAGA | sly-miR171f | sly-miR171f | miR171 | 36.6 | 371.0 | 25.1 | 226.4 | 3.3 | 3.2 |
ACGAGAGTCATCTGTGACAGG | sly-miR1919a|sly-miR1919c-3p|sly-miR1919b|novel_sly-miR1919d | sly-miR1919a | miR1919 | 9.0 | 93.2 | 9.0 | 52.8 | 3.4 | 2.6 |
AGGAAACTGTTTAGTCCAACC | sly-miR319d | sly-miR319d | miR319 | 0.0 | 1.2 | 0.0 | 1.6 | 5.0 | 5.8 |
CGCTATCCATCCTGAGTTTTA | sly-miR390a-3p | sly-miR390a-3p | miR390 | 0.5 | 3.9 | 0.2 | 6.1 | 3.0 | 4.7 |
AAGCTCAGGAGGGATAGCACC | sly-miR390a-5p | sly-miR390a-5p | miR390 | 75.9 | 524.5 | 70.9 | 378.0 | 2.8 | 2.4 |
CGCTATCCATCCTGAGTTTCA | sly-miR390b-3p | sly-miR390b-3p | miR390 | 0.1 | 1.6 | 0.0 | 1.1 | 4.4 | 5.2 |
ATCATGCGATCTCTTCGGAAT | sly-miR393 | sly-miR393 | miR393 | 10.0 | 120.2 | 6.7 | 76.1 | 3.6 | 3.5 |
AGGTGGGCATACTGTCAACA | sly-miR394-3p | sly-miR394-3p | miR394 | 1.3 | 16.4 | 1.6 | 28.5 | 3.7 | 4.2 |
GTTCAATAAAGCTGTGGGAAG | sly-miR396a-3p | sly-miR396a-3p | miR396 | 49.9 | 1727.2 | 45.5 | 1132.1 | 5.1 | 4.6 |
ATTGAGTGCAGCGTTGATGA | sly-miR397-5p | sly-miR397-5p | miR397 | 10.0 | 359.3 | 15.5 | 208.7 | 5.2 | 3.7 |
TATGTTCTCAGGTCGCCCCTG | sly-miR398a | sly-miR398a | miR398 | 834.8 | 13869 | 925.1 | 5806.1 | 4.1 | 2.6 |
CGTTTGTGCGTGAATCTAACA | sly-miR403-5p | sly-miR403-5p | miR403 | 0.7 | 11.9 | 0.2 | 9.7 | 4.1 | 5.3 |
ACGGGGACGAGCCAGAGCATG | sly-miR408 | sly-miR408 | miR408 | 1.6 | 14.5 | 1.0 | 40.2 | 3.2 | 5.4 |
TGTGGGTGGGGTGGAAAGATT | sly-miR482e-5p | sly-miR482e-5p | miR482 | 6.0 | 89.3 | 9.6 | 202.5 | 3.9 | 4.4 |
AGGTGTAGGTGTTCATGCAGA | sly-miR530 | sly-miR530 | miR530 | 0.4 | 48.7 | 0.4 | 18.7 | 6.8 | 5.7 |
ATGGGTAGCACAAGGATTAATG | sly-miR6027-5p | sly-miR6027-5p | miR6027 | 313.9 | 4006.6 | 281.9 | 3442.1 | 3.7 | 3.6 |
TGAAATCCATGAGCCTAAACT | sly-miR9470-5p | sly-miR9470-5p | miR9470 | 0.7 | 13.6 | 0.5 | 5.3 | 4.2 | 3.5 |
TTTCAGTAGACGTTGTGAATA | sly-miR9472-5p | sly-miR9472-5p | miR9472 | 0.2 | 4.3 | 0.2 | 2.9 | 4.5 | 4.0 |
TGTAGAAGTCATGAATAAAATG | sly-miR9474-5p | sly-miR9474-5p | miR9474 | 6.3 | 26.5 | 6.0 | 33.6 | 2.1 | 2.5 |
AAAAAGATGCAGGACTAGACC | sly-miR9476-3p | sly-miR9476-3p | miR9476 | 114.0 | 675.9 | 112.5 | 493.2 | 2.6 | 2.1 |
AACAACATACTTACTGAAATGCCA | novel_sly-miR8020 | NO | miR8020 | 6.6 | 1.3 | 5.5 | 2.7 | −2.3 | −1.0 |
GAATTTCATTGAGTATGTTGTTGT | novel_sly-miR8020* | NO | miR8020 | 1.6 | 0.3 | 0.6 | 0.1 | −2.6 | − |
AGTGGACAAGTAAAGGTGGATGGA | novel-sly-miR4 | NO | novel | 5.2 | 0.9 | 3.3 | 1.1 | −2.6 | −1.5 |
AACGAGTGAGACTTGCTCAGTTGG | sly-miR10529 | sly-miR10529 | miR10529 | 1.6 | 0.3 | 0.4 | 0.8 | −2.6 | − |
ACGTCCCTTCCCCATCGTTCAACA | sly-miR10530 | sly-miR10530 | miR10530 | 3.7 | 0.7 | 2.7 | 1.3 | −2.3 | −1.1 |
TTTTAGCAAGAGTTGTTTTACC | sly-miR6024 | sly-miR6024 | miR6024 | 365.2 | 56.1 | 158.0 | 90.1 | −2.7 | −0.8 |
AAGTGTGTCTCTGGAATTTCGGGC | sly-miR7981f | sly-miR7981f | miR7981 | 5.2 | 1.1 | 3.4 | 2.5 | −2.2 | −0.4 |
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Prigigallo, M.I.; Križnik, M.; De Paola, D.; Catalano, D.; Gruden, K.; Finetti-Sialer, M.M.; Cillo, F. Potato Virus Y Infection Alters Small RNA Metabolism and Immune Response in Tomato. Viruses 2019, 11, 1100. https://doi.org/10.3390/v11121100
Prigigallo MI, Križnik M, De Paola D, Catalano D, Gruden K, Finetti-Sialer MM, Cillo F. Potato Virus Y Infection Alters Small RNA Metabolism and Immune Response in Tomato. Viruses. 2019; 11(12):1100. https://doi.org/10.3390/v11121100
Chicago/Turabian StylePrigigallo, Maria I., Maja Križnik, Domenico De Paola, Domenico Catalano, Kristina Gruden, Mariella M. Finetti-Sialer, and Fabrizio Cillo. 2019. "Potato Virus Y Infection Alters Small RNA Metabolism and Immune Response in Tomato" Viruses 11, no. 12: 1100. https://doi.org/10.3390/v11121100
APA StylePrigigallo, M. I., Križnik, M., De Paola, D., Catalano, D., Gruden, K., Finetti-Sialer, M. M., & Cillo, F. (2019). Potato Virus Y Infection Alters Small RNA Metabolism and Immune Response in Tomato. Viruses, 11(12), 1100. https://doi.org/10.3390/v11121100