Background
Methods
Virus sources and agroinoculation
DNA extraction and PCR detection
Materials for RNA sequencing
RNA extraction and cDNA synthesis
Library preparation for transcriptome sequencing
Quality control
Reads mapping to the reference genome
Quantification of gene expression level
Differential gene expression analysis
GO term and KEGG pathway enrichment analysis
Quantitative real-time PCR validation
Results
Plants with typical viral symptoms in Y35A and Y35AB treatments
Overview of transcriptome sequencing
Sample name | Y35AB-1 | Y35AB-2 | Y35AB-3 | Y35A-1 | Y35A-2 | Y35A-3 | CK-1 | CK-2 | CK-3 | mean |
---|---|---|---|---|---|---|---|---|---|---|
Raw reads | 12166453 | 11965470 | 13272880 | 15527372 | 12560210 | 14214740 | 12646000 | 11302705 | 11794017 | 12827761 |
Clean reads | 12032720 | 11834496 | 13118731 | 15503750 | 12537134 | 14192135 | 12633203 | 11290015 | 11782562 | 12769416 |
Clean bases | 0.60G | 0.59G | 0.66G | 0.78G | 0.63G | 0.71G | 0.63G | 0.56G | 0.59G | 0.64 |
Total mapped | 11620770 | 11438690 | 12605297 | 14934455 | 12075773 | 13732342 | 12346163 | 11139553 | 11524248 | 12379699 |
96.58% | 96.66% | 96.09% | 96.33% | 96.32% | 96.76% | 97.73% | 98.67% | 97.81% | 96.99% | |
Uniquely mapped | 8961324 | 8847537 | 9609766 | 11389720 | 9176512 | 10320807 | 9238644 | 8316046 | 8564617 | 9380553 |
74.47% | 74.76% | 73.25% | 73.46% | 73.19% | 72.72% | 73.13% | 73.66% | 72.69% | 73.48% | |
Q30 (%) | 95.74 | 95.68 | 95.58 | 96.20 | 96.08 | 96.14 | 95.76 | 95.70 | 95.80 | 95.85 |
GC contents (%) | 43.58 | 43.75 | 44.11 | 44.15 | 44.23 | 44.38 | 43.93 | 43.80 | 43.92 | 43.98 |
Analysis of DEGs
GO enrichment analysis
KEGG pathway enrichment analysis
Screening of Y35AB vs CK and Y35A vs CK common highly expressed DEGs
Gene symbol | Unigene ID | Protein properties | log2FCa | log2FCb | pathway |
---|---|---|---|---|---|
PsaFa
| Niben101Scf 00271g04024 | photosystem I reaction center subunitIII, chloroplastic-like | -1.4180 | -1.1769 | Photosynthesis |
PsaFb
| Niben101Scf 02156g05001 | photosystem I reaction center subunit III, chloroplastic-like | -1.5190 | -1.6335 | Photosynthesis |
PsaFc
| Niben101Scf 04964g00002 | photosystem I reaction center subunit III, chloroplastic-like | -1.5257 | -1.2865 | Photosynthesis |
PsaNa
| Niben101Scf 17701g01020 | photosystem I reaction center subunit N, chloroplastic-like | -1.6702 | -1.5537 | Photosynthesis |
PsaNb
| Niben101Scf 35628g00001 | photosystem I reaction center subunit N, chloroplastic-like | -1.3129 | -1.0396 | Photosynthesis |
ChlHa
| Niben101Scf 04388g00011 | magnesium-chelatase subunit ChlH, chloroplastic-like | -1.6623 | -2.1794 | Porphyrin and chlorophyll metabolism |
ChlHb
| Niben101Scf 07214g00015 | magnesium-chelatase subunit ChlH, chloroplastic-like | -1.4638 | -1.8585 | Porphyrin and chlorophyll metabolism |
BchP
| Niben101Scf 06249g03002 | geranylgeranyl diphosphate reductase, chloroplastic-like | -1.8576 | -1.0521 | Porphyrin and chlorophyll metabolism |
HemA
| Niben101Scf 10063g00003 | glutamyl-tRNA reductase 1,chloroplastic-like | -1.4101 | -2.0903 | Porphyrin and chlorophyll metabolism |
DXS
| Niben101Scf 00246g04005 | probable 1-deoxy-D-xylulose-5-phosphate synthase, chloroplastic-like | -1.0040 | -1.0126 | Metabolic pathways |
GME1-X1
| Niben101Scf 01696g06050 | GDP-mannose 3,5-epimerase 1-like,transcript variant X1 | -1.6022 | -1.1137 | Metabolic pathways |
CAB3A
| Niben101Scf 03961g00004 | chlorophyll a-b binding protein 50, chloroplastic-like | -1.1418 | -1.0719 | Metabolic pathways |
GDCSa
| Niben101Scf 02480g02012 | glycine dehydrogenase (decarboxylating), mitochondrial-like | -1.5732 | -1.1698 | Carbon metabolism |
GDCSb
| Niben101Scf 03839g04019 | glycine dehydrogenase (decarboxylating), mitochondrial-like | -1.1991 | -1.0395 | Carbon metabolism |
rbcS
| Niben101Scf 02381g04022 | ribulose bisphosphate carboxylase small chain S41, chloroplastic-like | -1.2691 | 0.7603 | Carbon fixation in photosynthetic organisms |
FBA1
| Niben101Scf 02864g04008 | fructose-bisphosphate aldolase 1,chloroplastic-like | -1.3167 | -1.0072 | Carbon fixation in photosynthetic organisms |
glsF
| Niben101Scf 04198g01002 | ferredoxin-dependent glutamate synthase, chloroplastic-like | -1.1161 | -1.0704 | Glyoxylate and dicarboxylate metabolism |
GRP
| Niben101Scf 03052g00011 | glycine-rich protein-like | 2.1379 | 1.4647 | No pathway enriched |
GRP3a
| Niben101Scf 01084g05013 | glycine-rich protein 3-like | 3.9423 | 3.1735 | No pathway enriched |
GRP3b
| Niben101Scf 01084g03004 | glycine-rich protein 3-like | 2.0529 | 1.8836 | No pathway enriched |
CAX3
| Niben101Scf 01329g00002 | vacuolar cation/proton exchanger 3-like | -2.2799 | -1.2535 | No pathway enriched |
FSD1
| Niben101Scf 03679g03009 | Fe superoxide dismutas ,chloroplastic-like | -2.1527 | -1.9924 | No pathway enriched |
PU
| Niben101Scf 10524g02015 | putative uncharacterized protein | 2.0616 | 0.9243 | No pathway enriched |
Validation of the selected genes by RT-qPCR
Analysis of pathogenesis-related (PR) genes
PR family | Unigene ID | Protein properties | log2FCa | qvaluea | log2FCb | qvalueb |
---|---|---|---|---|---|---|
PR1 | Niben101Scf13926g01014 | Cysteine-rich secretory protein, allergen V5/Tpx-1-related | ||||
Niben101Scf03376g03004 | Cysteine-rich secretory protein, allergen V5/Tpx-1-related | |||||
Niben101Scf00107g03008 | Cysteine-rich secretory protein, allergen V5/Tpx-1-related | |||||
Niben101Scf01999g07002 | Cysteine-rich secretory protein, allergen V5/Tpx-1-related | |||||
PR2 | Niben101Scf01001g00005 | Glucan endo-1,3-beta-glucosidase-like, Glycoside hydrolase, family 17 | ||||
Niben101Scf01001g00004 | Glucan endo-1,3-beta-glucosidase-like, Glycoside hydrolase, family 17 | |||||
Niben101Scf01001g00003 | Glucan endo-1,3-beta-glucosidase-like, Glycoside hydrolase, family 17 | Inf | 0.023496 | |||
Niben101Ctg13736g00004 | Glucan endo-1,3-beta-glucosidase-like, Glycoside hydrolase, family 17 | Inf | 1.94E-04 | |||
Niben101Scf04869g03002 | Glucan endo-1,3-beta-glucosidase-like, Glycoside hydrolase, family 17 | 3.9490 | 0.038646 | 5.4381 | 2.14E-04 | |
Niben101Scf01001g00006 | Glucan endo-1,3-beta-glucosidase-like, Glycoside hydrolase, family 17 | |||||
PR3 | Niben101Scf02041g00002 | Chitinase 8, Glycoside hydrolase, family 19 | 5.1058 | 1.99E-05 | ||
PR4 | Niben101Scf01400g00014 | Thaumatin-like protein | ||||
Niben101Scf03436g01016 | Thaumatin-like protein | |||||
PR5 | Niben101Scf00126g00008 | Pathogenesis-related thaumatin superfamily protein | 0.9949 | 0.027102 | ||
Niben101Scf05554g05006 | Pathogenesis-related thaumatin superfamily protein | 1.4585 | 3.24E-08 | |||
PR6 | Niben101Scf00953g05001 | Cysteine-rich secretory protein, allergen V5/Tpx-1-related | ||||
Niben101Scf04053g01004 | Cysteine-rich secretory protein, allergen V5/Tpx-1-related | |||||
PR9 | Niben101Scf03460g04004 | Peroxidase 53, Haem peroxidase | ||||
Niben101Scf07182g05012 | Peroxidase 53, Haem peroxidase | |||||
PR10 | Niben101Scf03526g00006 | Major pollen allergen Bet v 1-M/N, Bet v I type allergen | ||||
Niben101Scf10735g00016 | Major pollen allergen Bet v 1-M/N, Bet v I type allergen | |||||
Niben101Scf02474g01024 | Major pollen allergen Bet v 1-M/N, Bet v I type allergen | |||||
Niben101Scf01938g04007 | Major pollen allergen Bet v 1-M/N, Bet v I type allergen | |||||
PR11 | Niben101Scf06295g04023 | Chitinase-3-like protein 2, Glycoside hydrolase superfamily | 4.4843 | 0.007133 | ||
Niben101Scf01789g04010 | Chitinase-3-like protein 2, Glycoside hydrolase superfamily | |||||
PR17 | Niben101Scf03385g02011 | Plant basic secretory protein family protein, uncharacterised protein family | 2.7696 | 6.69E-04 | ||
Niben101Scf03385g01006 | Plant basic secretory protein family protein, uncharacterised protein family | |||||
Niben101Scf01341g01002 | Plant basic secretory protein family protein, uncharacterised protein family | |||||
Niben101Ctg10643g00004 | Plant basic secretory protein family protein, uncharacterised protein family |
Analysis of LRR-RLKs genes
Unigene ID | Protein properties | log2FCa | qvaluea | log2FCb | qvalueb |
---|---|---|---|---|---|
Niben101Scf13404g00002 | Brassinosteroid LRR receptor kinase-like; BRI1 | -0.8361 | 0.000272 | ||
Niben101Scf07123g01015 | Elicitor-inducible leucine-rich repeats receptor-like protein | 5.5080 | 0.000289 | ||
Niben101Scf00104g02013 | F-box/LRR-repeat protein 17-like | 0.8664 | 0.021066 | ||
Niben101Scf04252g00009 | F-box/LRR-repeat protein 17-like | 1.8551 | 0.003213 | ||
Niben101Scf09559g00005 | F-box/LRR-repeat MAX2 homolog A-like | 0.8581 | 0.018415 | ||
Niben101Scf15394g00006 | F-box/LRR-repeat protein 14-like | -0.8927 | 0.013552 | ||
Niben101Scf20268g00001 | LRR receptor-like serine/threonine-protein kinase ERECTA | -0.7116 | 0.011214 | ||
Niben101Scf05301g03002 | LRR receptor-like serine/threonine-protein kinase ERL1 | -0.7516 | 0.013687 | -0.7589 | 0.013627 |
Niben101Scf00247g01007 | LRR receptor-like serine/threonine-protein kinase ERL1 | -0.5633 | 0.024694 | ||
Niben101Scf11723g02003 | LRR receptor-like serine/threonine-protein kinase ERL1 | -0.6679 | 0.026512 | -0.7100 | 0.017084 |
Niben101Scf03619g03004 | LRR receptor-like serine/threonine-protein kinase FEI2 | -0.5426 | 0.018415 | ||
Niben101Scf03925g01010 | LRR receptor-like serine/threonine-protein kinase GSO1 | 2.3343 | 0.007479 | 3.1551 | 0.041202 |
Niben101Scf02203g04006 | Plant intracellular Ras-group-related LRR protein 4-like | -0.6640 | 0.048845 | ||
Niben101Scf00206g00014 | putative F-box/LRR-repeat protein At5g02700 | 1.5307 | 0.020135 | ||
Niben101Scf00073g04006 | probable LRR receptor-like serine/threonine-protein kinase At1g07650 | -1.4546 | 0.005161 | ||
Niben101Scf00244g03004 | probable LRR receptor-like serine/threonine-protein kinase At1g06840 | -0.6603 | 0.021347 | ||
Niben101Scf00418g02007 | probable LRR receptor-like serine/threonine-protein kinase At4g37250 | -0.5934 | 0.047527 | ||
Niben101Scf00920g09027 | probable LRR receptor-like serine/threonine-protein kinase At5g48740 | -1.1084 | 0.000766 | ||
Niben101Scf01061g08014 | probable LRR receptor-like serine/threonine-protein kinase At4g20940 | -1.0192 | 0.001468 | ||
Niben101Scf01278g09008 | probable LRR receptor-like serine/threonine-protein kinase At1g56140 | 0.9239 | 0.023952 | ||
Niben101Scf02357g08010 | probable LRR receptor-like serine/threonine-protein kinase At1g07650 | -0.6424 | 0.001803 | ||
Niben101Scf02665g15003 | probable LRR receptor-like serine/threonine-protein kinase At1g06840 | -0.5769 | 0.044996 | ||
Niben101Scf02827g07005 | probable LRR receptor-like serine/threonine-protein kinase At3g47570 | 2.3756 | 0.027087 | ||
Niben101Scf03098g00011 | probable LRR receptor-like serine/threonine-protein kinase At1g34110 | -1.2224 | 3.35E-06 | ||
Niben101Scf03262g02006 | probable LRR receptor-like serine/threonine-protein kinase At2g24230 | -0.6865 | 0.030882 | ||
Niben101Scf03445g05005 | probable LRR receptor-like serine/threonine-protein kinase At4g26540 | -0.8802 | 0.000669 | ||
Niben101Scf03735g07013 | probable LRR receptor-like serine/threonine-protein kinase At4g36180 | 2.0409 | 0.002356 | ||
Niben101Scf05348g01026 | probable LRR receptor-like serine/threonine-protein kinase At1g12460 | -1.3373 | 3.02E-05 | ||
Niben101Scf05405g07003 | probable LRR receptor-like serine/threonine-protein kinase At3g47570 | 2.0599 | 0.003076 | ||
Niben101Scf05405g07006 | probable LRR receptor-like serine/threonine-protein kinase At3g47570 | 1.5589 | 0.022376 | ||
Niben101Scf05767g05006 | probable LRR receptor-like serine/threonine-protein kinase At4g20940 | -0.8478 | 0.000497 | ||
Niben101Scf05977g01008 | probable LRR receptor-like serine/threonine-protein kinase At1g12460 | -1.2826 | 8.11E-05 | ||
Niben101Scf06144g00013 | probable LRR receptor-like serine/threonine-protein kinase At4g20940 | -0.9557 | 7.81E-05 | ||
Niben101Scf06151g02014 | probable LRR receptor-like serine/threonine-protein kinase At1g67720 | 0.7683 | 0.046840 | ||
Niben101Scf06562g02013 | probable LRR receptor-like serine/threonine-protein kinase At4g26540 | -0.5968 | 0.021640 | -0.9943 | 2.01E-05 |
Niben101Scf06789g03005 | probable LRR receptor-like serine/threonine-protein kinase At1g67720 | 0.9483 | 0.042093 | 0.9711 | 0.042650 |
Niben101Scf07034g06018 | probable LRR receptor-like serine/threonine-protein kinase At4g36180 | -0.8501 | 0.034812 | ||
Niben101Scf07515g02006 | probable LRR receptor-like serine/threonine-protein kinase At1g56140 | 0.6846 | 0.005558 | ||
Niben101Scf07681g01013 | probable LRR receptor-like serine/threonine-protein kinase At4g20940 | -0.7450 | 0.014076 | ||
Niben101Scf07695g01023 | probable LRR receptor-like serine/threonine-protein kinase At4g30520 | 0.7628 | 0.001146 | ||
Niben101Scf07736g01007 | probable LRR receptor-like serine/threonine-protein kinase At1g63430 | 0.6791 | 0.006612 | ||
Niben101Scf08134g02008 | probable LRR receptor-like serine/threonine-protein kinase At4g37250 | -0.5193 | 0.047055 | -0.7817 | 0.000797 |
Niben101Scf10101g00023 | probable LRR receptor-like serine/threonine-protein kinase At4g30520 | 0.6651 | 0.002463 | ||
Niben101Scf11047g00001 | probable LRR receptor-like serine/threonine-protein kinase At2g16250 | 0.7640 | 0.047280 |
Analysis of brassinosteroid and jasmonic acid pathway
Gene symbol | Unigene ID | Protein properties | log2FCa | qvaluea | log2FCb | qvalueb | |
---|---|---|---|---|---|---|---|
BR |
CYP90C1/D1
| Niben101Scf05764g04013 | 3-epi-6-deoxocathasterone 23-monooxygenase | -0.8193 | 0.019172 | ||
BRI1
| Niben101Scf13404g00002 | Brassinosteroid LRR receptor kinase-like, BRI1 | -0.8361 | 2.72E-04 | |||
BZR1
| Niben101Scf03110g05009 | Brassinosteroid resistant 1 | 0.5592 | 0.025611 | |||
BZR2
| Niben101Scf06112g01006 | Brassinosteroid resistant 2 | -1.2497 | 7.36E-07 | |||
CYCD3-X1
| Niben101Scf05643g01012 | Cyclin D3, 3 protein, plant | 1.3753 | 1.44E-07 | |||
CYCD3-X1
| Niben101Scf02445g10017 | Cyclin D3, 3 protein, plant | 1.2863 | 0.002511 | |||
CYCD3-X2
| Niben101Scf00107g02002 | Cyclin D3, 2 protein, plant | -0.8767 | 0.017675 | -1.2925 | 2.43E-04 | |
JA |
PLA1/A2
| Niben101Scf00711g02004 | Triacylglycerol lipase SDP1-like; Phospholipase A1/A2 | 1.8227 | 0.003036 | ||
DOX1
| Niben101Scf07070g02002 | Alpha-dioxygenase | -3.8126 | 7.60E-13 | -3.9498 | 7.95E-14 | |
loxF
| Niben101Scf05407g00001 | Linoleate 9S-lipoxygenase-like | 0.9407 | 0.021540 | |||
HPL1
| Niben101Scf00313g08016 | Fatty acid hydroperoxide lyase, HPL1 | -1.2170 | 1.14E-09 | -0.9629 | 7.37E-07 | |
HPL1
| Niben101Scf02207g11009 | Fatty acid hydroperoxide lyase, HPL1 | -1.4966 | 1.06E-05 | |||
KAT2
| Niben101Scf10189g02009 | 3-ketoacyl-CoA thiolase 2, peroxisomal | 0.6201 | 0.007053 | |||
SAMT-X1
| Niben101Scf05122g00005 | Salicylate carboxymethyltransferase-like | -1.4545 | 2.97E-11 | -0.8505 | 2.96E-04 | |
SAMT-X2
| Niben101Scf01146g12006 | Salicylate carboxymethyltransferase-like | 1.6487 | 0.010833 | |||
JAR1
| Niben101Scf01076g00005 | Jasmonic acid-amino synthetase | -0.6477 | 0.003456 | -0.8340 | 0.001705 | |
COI1
| Niben101Scf02280g08005 | Coronatine-insensitive 1, COI1 | -0.6123 | 0.024317 | -0.8990 | 2.81E-04 | |
JAZ1
| Niben101Scf00298g01006 | Jasmonate ZIM-domain protein 1 | 0.7577 | 0.039355 | |||
JAZ2
| Niben101Scf07798g03026 | Jasmonate ZIM domain-containing protein | 0.7728 | 3.95E-04 | |||
MYC2 | Niben101Scf15224g00002 | Transcription factor MYC2 | -0.6238 | 0.042856 |