Expression and Role of Biosynthetic, Transporter, Receptor, and Responsive Genes for Auxin Signaling during Clubroot Disease Development
Abstract
:1. Introduction
2. Results
2.1. Properties of Auxin-Related Proteins
2.2. Variation in Expression between Leaves and Roots
2.3. Expression Profiles of Auxin-Responsive GH3 Family and BrDCK1 Genes
2.4. Expression Profiles of BrIAA and BrRACK Genes
2.5. Expression Profiles of BrSAUR and BrNIT Signaling Genes
2.6. Expression Profiles of Other Auxin-Related Genes
3. Discussion
3.1. Expression Level Difference in Leaf vs. Root Tissue
3.2. Expression Level Difference and Role of Auxin Signaling and Biosynthesis (BrGH3, BrIAA, BrNIT, and BrSAUR) Genes
3.3. Expression Level Difference and Role of Auxin Transporter (BrPIN, BrPILS, and BrLAX) Genes
3.4. Expression Level Difference and Role of Auxin Receptor (BrDCK and BrRACK) Genes
3.5. Expression Level Difference and Role of BrARP1 Gene
4. Materials and Methods
4.1. Preparation of Plant Materials
4.2. Gall Collection, Spore Preparation, and Inoculation of Plant Materials
4.3. Sampling of Inoculated Roots and Leaves for Expression Analysis
4.4. Extraction of RNA
4.5. In Silico Analysis of Auxin-Related Genes
4.6. Quantitative RT-PCR for Expression Analysis
4.7. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Gene Name | BRAD ID | Chromosomal Location | Start Codon | Stop Codon | St. | Gene Identity | (aa) | Pi | MW (kDa) | Subcellular Localization |
---|---|---|---|---|---|---|---|---|---|---|
BrDCK1 | Bra008038 | A02 | 11763540 | 11765539 | − | Receptor kinase | 447 | 9.06 | 50.44 | Cell membrane |
BrDCK1.1 | Bra016050 | A07 | 22960115 | 22961810 | − | Protein kinase | 451 | 9.24 | 50.77 | Cell membrane |
BrDCK1.2 | Bra003875 | A07 | 18779483 | 18780917 | + | 396 | 8.74 | 44.61 | Cell membrane | |
BrLAX1 | Bra008325 | A02 | 13732347 | 13734803 | − | Auxin efflux carrier/ Transporter | 472 | 8.95 | 53.53 | Cell membrane |
BrLAX1.1 | Bra003674 | A07 | 17668825 | 17671143 | + | 471 | 8.86 | 53.45 | Cell membrane | |
BrPIN1 | Bra015983 | A07 | 23234264 | 23237329 | + | 618 | 9.14 | 66.82 | Cell membrane | |
BrPIN1.1 | Bra008105 | A02 | 12154076 | 12156932 | + | 616 | 9.10 | 66.61 | Cell membrane | |
BrPILS1 | Bra014265 | A08 | 2101045 | 2103020 | − | 431 | 9.26 | 46.66 | Lysosome | |
BrPILS1.1 | Bra030441 | A05 | 11329601 | 11331470 | + | 423 | 8.86 | 46.08 | Cell membrane | |
BrRACK1 | Bra032261 | A05 | 12304734 | 12306049 | + | Receptor C kinase | 327 | 6.31 | 35.70 | Nucleus |
BrRACK1.1 | Bra018745 | A06 | 2234607 | 2235718 | + | 298 | 6.06 | 32.47 | Nucleus | |
BrRACK1.2 | Bra022282 | A05 | 19371512 | 19372720 | + | 325 | 7.59 | 35.46 | Nucleus | |
BrRACK1.3 | Bra021294 | A01 | 21899789 | 21900926 | + | 326 | 8.33 | 35.58 | Nucleus | |
BrGH3.1 | Bra039186 | A09 | 33153634 | 33155891 | + | Auxin-responsive GH3 (auxin signaling) | 596 | 6.28 | 67.77 | Cytoplasm |
BrGH3.2 | Bra008833 | A10 | 12836737 | 12839205 | − | 594 | 7.81 | 67.08 | Cytoplasm | |
BrGH3.3 | Bra041046 | Scaffold000414 | 2738 | 4832 | − | 587 | 5.98 | 66.03 | Cytoplasm | |
BrGH3.31 | Bra023405 | A02 | 2218215 | 2221058 | + | 595 | 5.80 | 67.02 | Cytoplasm | |
BrGH3.4 | Bra034205 | A09 | 1271960 | 1274617 | − | 610 | 6.28 | 68.54 | Cytoplasm | |
BrGH3.41 | Bra013086 | A03 | 20567114 | 20569305 | − | 557 | 5.43 | 62.48 | Cytoplasm | |
BrGH3.5 | Bra019060 | A03 | 26541024 | 26543324 | + | 612 | 5.98 | 69.39 | Cytoplasm | |
BrGH3.51 | Bra026365 | A01 | 9560154 | 9562651 | + | 612 | 6.05 | 69.36 | Cytoplasm | |
BrGH3.52 | Bra022725 | A02 | 6752640 | 6754845 | + | 612 | 5.87 | 69.09 | Cytoplasm | |
BrGH3.53 | Bra029023 | A03 | 5931112 | 5933213 | + | 608 | 5.74 | 68.63 | Cytoplasm | |
BrIAA1 | Bra039855 | A08 | 6303677 | 6305559 | − | Auxin signaling | 233 | 8.68 | 25.85 | Nucleus |
BrIAA2 | Bra033886 | A05 | 15764615 | 15766543 | − | 242 | 8.78 | 26.43 | Nucleus | |
BrIAA2.1 | Bra001900 | A03 | 19263301 | 19265148 | + | 237 | 8.54 | 26.16 | Nucleus | |
BrIAA3 | Bra001634 | A03 | 17624668 | 17625966 | − | Phytochrome -associated | 272 | 7.68 | 30.43 | Nucleus |
BrIAA3.1 | Bra021184 | A01 | 22676134 | 22677514 | + | 269 | 9.20 | 30.28 | Nucleus | |
BrIAA4 | Bra009867 | A06 | 18399781 | 18400911 | + | Auxin signaling | 176 | 8.80 | 20.33 | Nucleus |
BrIAA4.1 | Bra036557 | A09 | 2815416 | 2816543 | − | 175 | 9.24 | 20.00 | Cytoplasm | |
BrIAA5 | Bra032521 | A09 | 38191234 | 38191967 | + | Transcriptional regulator | 180 | 5.11 | 20.36 | Cytoplasm |
BrIAA5.1 | Bra015297 | A10 | 2137104 | 2137826 | + | 186 | 5.31 | 21.10 | Cytoplasm | |
BrSAUR1 | Bra011560 | A01 | 1680948 | 1681271 | + | Auxin-responsive protein (auxin signaling) | 107 | 6.82 | 12.11 | Mitochondrion |
BrSAUR1.1 | Bra034651 | A08 | 11488514 | 11488828 | + | 104 | 8.60 | 12.03 | Mitochondrion | |
BrSAUR1.2 | Bra017676 | A03 | 29740606 | 29740929 | − | 107 | 6.82 | 12.14 | Mitochondrion | |
BrSAUR1.3 | Bra013061 | A03 | 20715121 | 20715447 | − | 108 | 7.73 | 12.36 | Mitochondrion | |
BrSAUR2 | Bra039345 | Scaffold000164 | 28744 | 29145 | − | 133 | 9.03 | 15.34 | Mitochondrion | |
BrSAUR3 | Bra039264 | A04 | 18962112 | 18962471 | + | 119 | 7.81 | 13.67 | Cell membrane | |
BrSAUR3.1 | Bra004515 | A05 | 560573 | 560941 | − | 122 | 8.79 | 14.06 | Cell membrane | |
BrSAUR4 | Bra014476 | A04 | 882244 | 882756 | − | 170 | 10.96 | 19.75 | Mitochondrion | |
BrNIT1 | Bra028932 | A03 | 5467727 | 5469454 | − | Auxin biosynthesis | 343 | 5.54 | 37.73 | Cytoplasm |
BrNIT1.1 | Bra021681 | A04 | 13934901 | 13936735 | + | 350 | 5.48 | 38.48 | Cytoplasm | |
BrNIT2 | Bra020184 | A02 | 4417472 | 4419242 | + | 357 | 5.30 | 38.94 | Cytoplasm | |
BrARG10-like | Bra026348 | A01 | 9659581 | 9660796 | − | Auxin responsive | 250 | 5.52 | 27.55 | Cytoplasm |
BrARG10-like1 | Bra010404 | A08 | 13579603 | 13580693 | + | 250 | 6.07 | 27.49 | Cytoplasm | |
BrARP1 | Bra013191 | A03 | 19982046 | 19983742 | + | Auxin repressed protein | 403 | 9.70 | 43.86 | Cell membrane |
Sources of Variation | df | Genes | MS | F Statistic | p Value | Genes | MS | F Statistic | p Value | Genes | MS | F Statistic | p Value |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Time-point | 4 | BrGH3.1 | 2.68 | 83.2 | < 0.01 | BrIAA4 | 1.01 | 63.4 | < 0.01 | BrDCK1 | 132.7 | 76.3 | < 0.01 |
Treatment | 1 | 0.94 | 29.2 | < 0.01 | 1.28 | 80.2 | < 0.01 | 22.1 | 12.7 | < 0.01 | |||
Tm × Tr | 4 | 1.34 | 41.8 | < 0.01 | 1.35 | 84.5 | < 0.01 | 28.5 | 16.4 | < 0.01 | |||
LvR (Tm Tr) | 10 | 1.21 | 37.7 | < 0.01 | 2.14 | 134.3 | < 0.01 | 265 | 152.9 | < 0.01 | |||
Error | 40 | 0.032 | 0.016 | 1.74 | |||||||||
Time-point | 4 | BrGH3.2 | 1519 | 19.8 | < 0.01 | BrIAA5 | 15.5 | 71.7 | < 0.01 | BrRACK1 | 11.67 | 69.7 | < 0.01 |
Treatment | 1 | 2169 | 28.2 | < 0.01 | 1.14 | 5.28 | 0.027 | 0.116 | 0.70 | 0.409 | |||
Tm × Tr | 4 | 1184 | 15.4 | < 0.01 | 16.7 | 77.3 | < 0.01 | 1.11 | 6.66 | < 0.01 | |||
LvR (Tm Tr) | 10 | 1408 | 18.3 | < 0.01 | 14.2 | 65.9 | < 0.01 | 8.06 | 48.2 | < 0.01 | |||
Error | 40 | 76.8 | 0.22 | 0.167 | |||||||||
Time-point | 4 | BrGH3.3 | 4,699,114 | 2767 | < 0.01 | BrSAUR1 | 0.268 | 406.6 | < 0.01 | BrLAX1 | 14.2 | 128.2 | < 0.01 |
Treatment | 1 | 1,822,177 | 1073 | < 0.01 | 0.345 | 522.1 | < 0.01 | 3.52 | 31.7 | < 0.01 | |||
Tm × Tr | 4 | 4,623,051 | 2723 | < 0.01 | 0.533 | 805.8 | < 0.01 | 0.80 | 7.24 | < 0.01 | |||
LvR (Tm Tr) | 10 | 6,320,827 | 3723 | < 0.01 | 0.505 | 765.3 | < 0.01 | 9.12 | 82.2 | < 0.01 | |||
Error | 40 | 1698 | 0.0007 | 0.11 | |||||||||
Time-point | 4 | BrGH3.4 | 1978.9 | 582.8 | < 0.01 | BrSAUR2 | 3.56 | 46.24 | < 0.01 | BrARP1 | 0.726 | 158 | < 0.01 |
Treatment | 1 | 54.83 | 17.0 | < 0.01 | 3.63 | 47.22 | < 0.01 | 0.422 | 91.8 | < 0.01 | |||
Tm × Tr | 4 | 161.8 | 50.2 | < 0.01 | 2.93 | 38.03 | < 0.01 | 0.243 | 52.9 | < 0.01 | |||
LvR (Tm Tr) | 10 | 1369.7 | 424.8 | < 0.01 | 5.61 | 72.92 | < 0.01 | 0.368 | 80.1 | < 0.01 | |||
Error | 40 | 3.22 | 0.077 | 0.004 | |||||||||
Time-point | 4 | BrGH3.5 | 396.0 | 72.82 | < 0.01 | BrSAUR3 | 4.84 | 355.6 | < 0.01 | BrARG | 52.95 | 741.5 | < 0.01 |
Treatment | 1 | 71.8 | 13.21 | < 0.01 | 3.37 | 247.5 | < 0.01 | 2.62 | 36.6 | < 0.01 | |||
Tm × Tr | 4 | 215.8 | 39.69 | < 0.01 | 2.06 | 151.0 | < 0.01 | 26.39 | 369.6 | < 0.01 | |||
LvR (Tm Tr) | 10 | 357.9 | 65.82 | < 0.01 | 2.96 | 218.0 | < 0.01 | 65.54 | 917.8 | < 0.01 | |||
Error | 40 | 5.44 | 0.013 | 0.071 | |||||||||
Time-point | 4 | BrIAA1 | 200.9 | 192.5 | < 0.01 | BrSAUR4 | 0.518 | 121.1 | < 0.01 | BrPIN1 | 260.1 | 13.2 | < 0.01 |
Treatment | 1 | 76.18 | 73.0 | < 0.01 | 0.057 | 13.44 | < 0.01 | 298.8 | 15.1 | < 0.01 | |||
Tm × Tr | 4 | 213.8 | 204.8 | < 0.01 | 0.121 | 28.35 | < 0.01 | 168.5 | 8.54 | < 0.01 | |||
LvR (Tm Tr) | 10 | 266.2 | 255.1 | < 0.01 | 0.857 | 200.5 | < 0.01 | 206.0 | 10.4 | < 0.01 | |||
Error | 40 | 1.04 | 0.004 | 19.7 | |||||||||
Time-point | 4 | BrIAA2 | 0.017 | 12.15 | < 0.01 | BrNIT1 | 14844 | 165.8 | < 0.01 | BrPILS1 | 0.969 | 233.9 | < 0.01 |
Treatment | 1 | 0.013 | 9.57 | < 0.01 | 9378 | 104.8 | < 0.01 | 0.789 | 190.5 | < 0.01 | |||
Tm × Tr | 4 | 0.173 | 120.9 | < 0.01 | 6064 | 67.7 | < 0.01 | 0.695 | 167.6 | < 0.01 | |||
LvR (Tm Tr) | 10 | 0.129 | 90.6 | < 0.01 | 15511 | 173.3 | < 0.01 | 0.584 | 140.9 | < 0.01 | |||
Error | 40 | 0.0014 | 89.5 | 0.004 | |||||||||
Time-point | 4 | BrIAA3 | 47.3 | 632.4 | < 0.01 | BrNIT2 | 1.55 | 22.95 | < 0.01 | ||||
Treatment | 1 | 8.28 | 110.7 | < 0.01 | 0.85 | 12.67 | < 0.01 | ||||||
Tm × Tr | 4 | 8.16 | 109.2 | < 0.01 | 1.05 | 15.52 | < 0.01 | ||||||
LvR (Tm Tr) | 10 | 40.1 | 535.5 | < 0.01 | 2.78 | 41.08 | < 0.01 | ||||||
Error | 40 | 0.075 | 0.067 |
Sample ID | Time Point | Treatment | Sample Type |
---|---|---|---|
1 | Day 1 | Mock | Leaf |
2 | Day 1 | Mock | Root |
3 | Day 1 | Seosan-inoculated | Leaf |
4 | Day 1 | Seosan-inoculated | Root |
5 | Day 3 | Mock | Leaf |
6 | Day 3 | Mock | Root |
7 | Day 3 | Seosan-inoculated | Leaf |
8 | Day 3 | Seosan-inoculated | Root |
9 | Day 14 | Mock | Leaf |
10 | Day 14 | Mock | Root |
11 | Day 14 | Seosan-inoculated | Leaf |
12 | Day 14 | Seosan-inoculated | Root |
13 | Day 28 | Mock | Leaf |
14 | Day 28 | Mock | Root |
15 | Day 28 | Seosan-inoculated | Leaf |
16 | Day 28 | Seosan-inoculated | Root |
17 | Day 35 | Mock | Leaf |
18 | Day 35 | Mock | Root |
19 | Day 35 | Seosan-inoculated | Leaf |
20 | Day 35 | Seosan-inoculated | Root |
Arabidopsis Homolog (Accession Number) | Gene Name | BRAD ID | cDNA Size (bp) | Primer Forward (F) and Reverse(R) | Product Size (bp) |
---|---|---|---|---|---|
At2g23170 | BrGH3.1 | Bra039186 | 1791 | AAGGACTTGAAAGCTCTCAG | 156 |
CTTAAATGTTTTCCGGTCGG | |||||
At1g72540 | BrDCK1 | Bra008038 | 1344 | ATCCAGACAAACCTCTGTTC | 177 |
AATCCTCCTTCACCGAGATA | |||||
At5g13360 | BrGH3.2 | Bra008833 | 1758 | AGTGAGACAGCCTCTAGAAT | 177 |
TCGTTGTATAAGCCCACAAA | |||||
At4g34760 | BrSAUR1 | Bra011560 | 324 | TTCCACTCGATGTACCAAAG | 197 |
TGAGGTTAGGGTCTGAAAGA | |||||
At1g77690 | BrLAX1 | Bra008325 | 1419 | CTGTGGAGATAATGCATGCCA | 161 |
GAGTAGAGAGAGTGCATTGG | |||||
At1g73590 | BrPIN1 | Bra015983 | 1857 | GGAAACAGAAAAGCAGCATT | 187 |
GAGTAGAGAGAGTGCATTGG | |||||
At1g48630 | BrRACK1 | Bra032261 | 984 | ACATGATCGTCACTTCTTCC | 243 |
AGAGAACGTCTTTCGTGTGT | |||||
At3g12830 | BrSAUR2 | Bra039345 | 402 | GAATCTTTCCTCCGGTCTTC | 187 |
TTTGCTCGTAACCATACTCC | |||||
At3g44300 | BrNIT1 | Bra028932 | 1032 | GAATCAGAGGGTCTCATCAC | 151 |
TCGAAACGAATGTAACTGGT | |||||
At5g22300 | BrNIT2 | Bra020184 | 1074 | GCAAGTATCTTGCTTCTGCT | 157 |
TGACCATGTGAGTCAAAGAA | |||||
At5g01990 | BrPILS1 | Bra014265 | 1296 | GAAGCAAATCATCCAACCTG | 167 |
GTGCCAGTAATATACAGGGG | |||||
At1g59500 | BrGH3.3 | Bra041046 | 1764 | TAAGCTTCCTCCAGAACAAC | 157 |
GAGCAAACCTCTACCAAGAA | |||||
At4g14550 | BrIAA1 | Bra039855 | 702 | CTCTACCAAAGAAGGATCCG | 163 |
TTCGCTACACTTCTGATGAG | |||||
At4g03400 | BrGH3.4 | Bra034205 | 1833 | ATGTTGAAGAGATGGACGAC | 208 |
CTATGGCAAGTAAACGGAAC | |||||
At3g23050 | BrIAA2 | Bra033886 | 729 | AGCTGCATTGGTTAAGGTAT | 184 |
CAGATCCATTAGCTTGCTCT | |||||
At4g27260 | BrGH3.5 | Bra019060 | 1839 | AAGAACGTGATGCCTGTTAT | 183 |
TGATCTCCGGTCTAGTTCTT | |||||
At4g27450 | BrARG10-like | Bra026348 | 753 | CTCTGACAACACTTTCTCCA | 175 |
GGTCAGACCATATTGCTTG | |||||
At3g16500 | BrIAA3 | Bra001634 | 819 | GAGGTCAAGTCAACAAGAGT | 171 |
CTCTGAAGAGTTGGTCTACG | |||||
At2g04850 | BrARP1 | Bra013191 | 1212 | GAATGTGTTCCAAGGGTTTG | 189 |
CTGCTGCATCTATCATCAGT | |||||
At2g46690 | BrSAUR3 | Bra039264 | 360 | GCTCTGAAAGGCTTCCATTTAC | 222 |
AACGTCGCATGGAATTGTGATG | |||||
At5g25890 | BrIAA4 | Bra009867 | 531 | TTCGTCGGATCTAAACAGAC | 167 |
CCTTGCAGTTCTTTCTAGGT | |||||
At1g04240 | BrIAA5 | Bra032521 | 543 | ATCCCCTCCTAGAAAGACTC | 152 |
TTTTCCTCAAGTATGGTGCA | |||||
At3g60690 | BrSAUR4 | Bra014476 | 513 | ATACCAATTGGGAAGGAACC | 172 |
CAAACTCTTCCTCTGCTTCT | |||||
XM_009147610.2 | ACTIN1 | 1371 | F: CAACCAATCGTCTGTGACAA | 105 | |
R: ATGTCTTGGCCTACCAACAA | |||||
FJ969844.1 | ACTIN2 | 491 | F: AATGGTACCGGAATGGTCAA | 119 | |
R: TCCTTCTGGTTCATCCCAAC |
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Robin, A.H.K.; Saha, G.; Laila, R.; Park, J.-I.; Kim, H.-T.; Nou, I.-S. Expression and Role of Biosynthetic, Transporter, Receptor, and Responsive Genes for Auxin Signaling during Clubroot Disease Development. Int. J. Mol. Sci. 2020, 21, 5554. https://doi.org/10.3390/ijms21155554
Robin AHK, Saha G, Laila R, Park J-I, Kim H-T, Nou I-S. Expression and Role of Biosynthetic, Transporter, Receptor, and Responsive Genes for Auxin Signaling during Clubroot Disease Development. International Journal of Molecular Sciences. 2020; 21(15):5554. https://doi.org/10.3390/ijms21155554
Chicago/Turabian StyleRobin, Arif Hasan Khan, Gopal Saha, Rawnak Laila, Jong-In Park, Hoy-Taek Kim, and Ill-Sup Nou. 2020. "Expression and Role of Biosynthetic, Transporter, Receptor, and Responsive Genes for Auxin Signaling during Clubroot Disease Development" International Journal of Molecular Sciences 21, no. 15: 5554. https://doi.org/10.3390/ijms21155554