Disease severity analysis in different potato cultivars
Potato leaves inoculated with A. solani spores started showing visual symptoms (necrosis) after 24 h. There was a clear difference in necrotic lesion diameter between the potato cultivars studied (Fig. 1). The infection efficiency was more than 90% (data not shown). The partially susceptible potato cultivar Kuras showed a maximum necrotic lesion of 0.61 cm followed by 0.35 cm for Désirée compared to 0.24 cm for the partially resistant cultivar Magnum Bonum (Fig. 1).
Transcriptome Sequencing, Mapping, And Principal Component Analysis
To study the changes in gene expression, we sequenced the total RNA from the three different potato cultivars at two-time points post A. solani inoculation. Overall, a total of 128.82 and 131.52 million reads were generated from control and A. solani inoculated leaf samples, respectively. The reads with adaptor contamination and low base quality were removed. An average of 89.13 and 87.63% of control and A. solani inoculated sequence reads were mapped to the potato genome (DM 1–3 516 R44 v6.1), respectively. The overall statistics are given in Table 1. PCA was carried out to estimate the similarity and differences between different potato cultivars, A. solani inoculation, and the biological replicates. There was a clear difference between the control and inoculated samples, forming four clear, discrete groupings. Irrespective of the potato cultivars, the samples were grouped based on control and A. solani inoculation, 18 and 36 h time points. The PC1 represented a high variance of 50% dividing the control and A. solani inoculated samples and the PC2 represented a 15% variance dividing the 18 and 36 h two-time points (Additional Fig S1).
Table 1
RNA sequencing statistics of different S. tuberosum during A. solani infection. Total reads (million), percentage of reads mapping to potato reference genome per time point. The data shown are the average of three biological replicates.
|
Potato cultivars and Time Point (Hours Post Inoculation)
|
|
Magnum Bonum
|
Désirée
|
Kuras
|
|
18
|
36
|
18
|
36
|
18
|
36
|
Total reads for control (Million)
|
26.10
|
21.40
|
18.77
|
22.17
|
20.43
|
19.93
|
Total reads for A. solani inoculated (Million)
|
25.27
|
21.57
|
23.70
|
18.73
|
20.13
|
22.13
|
Control sequence reads uniquely mapped to S. tuberosum reference genome (%)
|
89.17
|
88.80
|
89.89
|
89.49
|
87.89
|
89.52
|
A. solani inoculated sequence reads uniquely mapped to S. tuberosum reference genome (%)
|
90.48
|
86.33
|
88.92
|
87.57
|
86.42
|
86.38
|
Differential gene expression in response to A. solani infection, a global view
The differential gene expression analysis was carried out to understand the global changes in the transcripts when different potato cultivars were inoculated with A. solani. Significant differences in gene expression were noticed between the potato cultivars and at the two-time points studied. Magnum Bonum, a partially resistant early blight disease cultivar, had the least number of DEGs at 18 hpi (2052), followed by Désirée (3398), whereas the maximum numbers of DEGs were identified for the partially susceptible cultivar, Kuras (4046). At 36 hpi, the numbers were increased in Magnum Bonum (3753) and in Kuras (6261) but decreased in Désirée (2440) (Fig. 2A). Of the DEGs identified, interestingly, the number of up-regulated transcripts was significantly higher than the down-regulated ones in all the potato cultivars at 18 and 36 hpi. Several of the top 10 up-regulated DEGs were the same for Magnum Bonum and Kuras, but there was a higher expression in Kuras.
In contrast to the most up-regulated DEGs, no overlap was observed between Magnum Bonum and Kuras among the top down-regulated DEGs. The top 10 DEGs that are up- and down-regulated are provided in Table 2. The complete list of DEGs detected can be found in Additional files 1–3. To identify the common and unique DEGs for different potato cultivars and time points, Venn diagrams were generated (Fig. 2B-D). A total of 994 DEGs were common at 18 hpi of which 915 were up-regulated, and 77 were down-regulated. Similarly, at 36 hpi, 1210 transcripts were found to be in common, of which 1075 and 135 were up- and down-regulated, respectively (Additional Fig. S2A-F). We found 649 genes commonly expressed between the time points and different potato cultivars analyzed, of which, interestingly, 627 were up-regulated and 22 were down-regulated (Fig. 2B-D). Even though these are commonly expressed transcripts, there was a significant difference in the fold change values between the potato cultivars and time points. A significant up-regulation was observed for a peroxidase superfamily protein (Soltu.DM.10G019020.1) and allene oxide synthase (Soltu.DM.01G048780.1) in Kuras 18 hpi and fructose-bisphosphate aldolase (Soltu.DM.02G024280.2) was significantly down-regulated across the potato cultivars and time points analyzed. The top 20 up- and down-regulated common transcripts were displayed in a heat map (Fig. 3A&B). The complete details of the transcripts ID, gene function, corresponding PGSC_DM_V403 gene ID, PGSC functional gene annotation, and the corresponding log2 fold change were provided in Additional file 4.
Table 2
Top 10 up- and down-regulated DETs in Magnum Bonum, Désirée, and Kuras potato cultivars upon infection with A. solani at 18 and 36 hpi. Gene name. gene description. Log2 Fold change. and the adjusted P-value (Padj) of infected compared to control are shown.
Gene name
|
Description
|
Log2 fold change
|
18 hpi
|
Magnum Bonum
|
Up-regulated
|
Soltu.DM.10G018980.1
|
Peroxidase superfamily protein
|
10.68
|
Soltu.DM.08G011070.1
|
Ankyrin repeat family protein
|
10.67
|
Soltu.DM.01G040950.1
|
terpene synthase
|
10.14
|
Soltu.DM.05G021100.1
|
Rhamnogalacturonate lyase family protein
|
9.69
|
Soltu.DM.07G013650.1
|
cytochrome P450, family 716. subfamily A, polypeptide
|
9.68
|
Soltu.DM.12G027800.1
|
KNOTTED1-like homeobox gene
|
9.59
|
Soltu.DM.06G016360.1
|
terpene synthase
|
9.44
|
Soltu.DM.09G027720.3
|
MLP-like protein
|
9.41
|
Soltu.DM.10G019020.1
|
Peroxidase superfamily protein
|
9.33
|
Soltu.DM.01G035900.1
|
zinc induced facilitator-like
|
9.22
|
18 hpi
|
Magnum Bonum
|
Down-regulated
|
Soltu.DM.04G005660.1
|
glutamate-1-semialdehyde-2.1-aminomutase
|
-9.91
|
Soltu.DM.04G020260.3
|
chromatin remodeling
|
-8.66
|
Soltu.DM.08G021790.1
|
lectin protein kinase family protein
|
-8.64
|
Soltu.DM.11G016910.2
|
abscisic acid responsive elements-binding factor
|
-8.42
|
Soltu.DM.07G000240.4
|
response regulator
|
-8.38
|
Soltu.DM.12G029710.4
|
origin recognition complex subunit
|
-8.28
|
Soltu.DM.08G023390.2
|
pleckstrin homology (PH) domain-containing protein
|
-8.09
|
Soltu.DM.06G024580.1
|
respiratory burst oxidase homologue D
|
-8.02
|
Soltu.DM.02G029210.2
|
SAP domain-containing protein
|
-7.89
|
Soltu.DM.03G021350.1
|
Calcium-binding endonuclease/exonuclease/phosphatase family
|
-7.83
|
36 hpi
|
Magnum Bonum
|
Up-regulated
|
Soltu.DM.09G024040.1
|
carboxyesterase
|
10.97
|
Soltu.DM.01G048780.1
|
allene oxide synthase
|
10.95
|
Soltu.DM.02G032650.1
|
Peroxidase superfamily protein
|
10.31
|
Soltu.DM.05G021100.1
|
Rhamnogalacturonate lyase family protein
|
10.15
|
Soltu.DM.04G020660.1
|
cytochrome P450, family 71, subfamily B, polypeptide
|
10.13
|
Soltu.DM.01G040940.1
|
terpene synthase
|
9.95
|
Soltu.DM.02G006070.1
|
BURP domain-containing protein
|
9.68
|
Soltu.DM.09G026810.1
|
Protein kinase superfamily protein
|
9.60
|
Soltu.DM.06G018620.1
|
serine-type endopeptidase inhibitors
|
9.51
|
Soltu.DM.07G003530.1
|
copper ion binding;electron carriers
|
9.50
|
36 hpi
|
Magnum Bonum
|
Down-regulated
|
Soltu.DM.09G026790.2
|
S-adenosyl-L-methionine-dependent methyltransferases superfamily protein
|
-9.13
|
Soltu.DM.07G016780.3
|
ethylene-forming enzyme
|
-8.93
|
Soltu.DM.10G004560.2
|
diacylglycerol kinase
|
-8.73
|
Soltu.DM.08G021790.1
|
lectin protein kinase family protein
|
-8.68
|
Soltu.DM.12G029610.2
|
protochlorophyllide oxidoreductase B
|
-8.53
|
Soltu.DM.01G043110.1
|
Phosphatidylinositol-4-phosphate 5-kinase family protein
|
-8.43
|
Soltu.DM.05G009760.8
|
root hair specific
|
-8.13
|
Soltu.DM.04G034990.3
|
J-domain protein required for chloroplast accumulation response
|
-8.07
|
Soltu.DM.06G033730.2
|
Helicase/SANT-associated. DNA binding protein
|
-8.03
|
Soltu.DM.09G020140.2
|
methylcrotonyl-CoA carboxylase alpha chain. mitochondrial / 3-methylcrotonyl-CoA carboxylase 1 (MCCA)
|
-8.01
|
18 hpi
|
Désirée
|
Up-regulated
|
Soltu.DM.02G018060.1
|
Protein of unknown function (DUF_B2219) domain containing protein
|
10.13
|
Soltu.DM.01G036130.3
|
Transducin/WD40 repeat-like superfamily protein
|
10.12
|
Soltu.DM.10G000710.3
|
Protein kinase superfamily protein
|
9.24
|
Soltu.DM.04G028320.1
|
laccase
|
9.18
|
Soltu.DM.12G024440.1
|
Lactoylglutathione lyase / glyoxalase I family protein
|
9.10
|
Soltu.DM.06G016360.1
|
terpene synthase
|
9.04
|
Soltu.DM.10G005990.2
|
Protein BPS1, chloroplastic
|
8.77
|
Soltu.DM.08G024320.1
|
Protein of unknown function (DUF1639)
|
8.74
|
Soltu.DM.07G027860.3
|
amino acid permease
|
8.73
|
Soltu.DM.07G027440.1
|
emp24/gp25L/p24 family/GOLD family protein
|
8.67
|
18 hpi
|
Désirée
|
Down-regulated
|
Soltu.DM.01G043130.2
|
ankyrin repeat-containing 2B
|
-9.90
|
Soltu.DM.07G016780.3
|
ethylene-forming enzyme
|
-9.80
|
Soltu.DM.04G004430.2
|
histone acetyltransferase of the CBP family
|
-8.95
|
Soltu.DM.11G022970.4
|
auxin response factor
|
-8.75
|
Soltu.DM.01G034500.2
|
conserved hypothetical protein
|
-8.72
|
Soltu.DM.04G000750.3
|
Disease resistance protein (CC-NBS-LRR class) family
|
-8.53
|
Soltu.DM.06G020110.3
|
conserved hypothetical protein
|
-8.49
|
Soltu.DM.02G005200.3
|
Calcium-binding EF hand family protein
|
-8.39
|
Soltu.DM.02G005200.2
|
Calcium-binding EF hand family protein
|
-8.38
|
Soltu.DM.10G014560.2
|
TIP41-like family protein
|
-8.38
|
36 hpi
|
Désirée
|
Up-regulated
|
Soltu.DM.08G005960.3
|
O-acetylserine (thiol) lyase isoform C
|
10.82
|
Soltu.DM.02G006070.1
|
BURP domain-containing protein
|
9.74
|
Soltu.DM.12G024440.1
|
Lactoylglutathione lyase / glyoxalase I family protein
|
9.66
|
Soltu.DM.04G024810.3
|
UDP-sugar pyrophosphorylase
|
9.54
|
Soltu.DM.01G002240.1
|
2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily protein
|
9.49
|
Soltu.DM.12G021490.1
|
cellulose synthase like G3
|
9.40
|
Soltu.DM.12G026250.2
|
photosystem I light harvesting complex gene
|
9.03
|
Soltu.DM.09G018860.2
|
multidrug resistance-associated protein
|
8.98
|
Soltu.DM.01G047660.1
|
ARP protein (REF)
|
8.42
|
Soltu.DM.05G021100.1
|
Rhamnogalacturonate lyase family protein
|
8.37
|
36 hpi
|
Désirée
|
Down-regulated
|
Soltu.DM.07G016780.3
|
ethylene-forming enzyme
|
-9.98
|
Soltu.DM.12G023130.1
|
Zinc finger C-x8-C-x5-C-x3-H type family protein
|
-8.79
|
Soltu.DM.10G029600.1
|
UDP-Glycosyltransferase superfamily protein
|
-8.76
|
Soltu.DM.05G009830.1
|
cyclin-related
|
-8.76
|
Soltu.DM.03G007180.4
|
DUF4336 domain containing protein
|
-8.54
|
Soltu.DM.01G006350.2
|
BRI1 suppressor 1 (BSU1)-like
|
-8.48
|
Soltu.DM.01G043130.2
|
ankyrin repeat-containing 2B
|
-8.38
|
Soltu.DM.09G013230.4
|
splicing factor PWI domain-containing protein
|
-8.14
|
Soltu.DM.05G019260.1
|
DHHC-type zinc finger family protein
|
-8.09
|
Soltu.DM.05G009830.5
|
cyclin-related
|
-8.03
|
18 hpi
|
Kuras
|
Up-regulated
|
Soltu.DM.10G019020.1
|
Peroxidase superfamily protein
|
15.09
|
Soltu.DM.01G048780.1
|
allene oxide synthase
|
12.87
|
Soltu.DM.04G028320.1
|
laccase
|
12.43
|
Soltu.DM.08G017780.2
|
Enoyl-CoA hydratase/isomerase family
|
12.38
|
Soltu.DM.09G024040.1
|
carboxyesterase
|
12.27
|
Soltu.DM.01G040940.1
|
terpene synthase
|
12.24
|
Soltu.DM.01G040950.1
|
terpene synthase
|
11.81
|
Soltu.DM.01G040930.1
|
terpene synthase
|
11.33
|
Soltu.DM.02G013170.1
|
FAD-binding Berberine family protein
|
11.26
|
Soltu.DM.03G018200.1
|
detoxifying efflux carrier
|
11.25
|
18 hpi
|
Kuras
|
Down-regulated
|
Soltu.DM.08G021790.1
|
lectin protein kinase family protein
|
-9.67
|
Soltu.DM.05G009830.1
|
cyclin-related
|
-9.10
|
Soltu.DM.03G016800.2
|
RNA-binding (RRM/RBD/RNP motifs) family protein
|
-8.68
|
Soltu.DM.11G026620.2
|
myb domain protein
|
-8.60
|
Soltu.DM.03G022230.7
|
thylakoid-associated phosphatase
|
-8.60
|
Soltu.DM.01G026120.2
|
chromatin remodeling
|
-8.59
|
Soltu.DM.03G030790.2
|
fatty acid desaturase
|
-8.18
|
Soltu.DM.05G007470.2
|
Sterile alpha motif (SAM) domain-containing protein
|
-7.94
|
Soltu.DM.04G027060.3
|
Inositol monophosphatase family protein
|
-7.73
|
Soltu.DM.12G023400.2
|
WLM domain containing protein
|
-7.70
|
36 hpi
|
Kuras
|
Up-regulated
|
Soltu.DM.06G033990.1
|
Transcription factor jumonji (jmj) family protein / zinc finger (C5HC2 type) family protein
|
11.92
|
Soltu.DM.06G023620.2
|
BURP domain-containing protein
|
10.19
|
Soltu.DM.09G014180.1
|
terpene synthase
|
10.13
|
Soltu.DM.08G005570.1
|
auxin response factor
|
10.03
|
Soltu.DM.03G034140.1
|
Pectinacetylesterase family protein
|
9.99
|
Soltu.DM.01G003520.1
|
serine-type endopeptidase inhibitors
|
9.93
|
Soltu.DM.08G028070.1
|
Lactoylglutathione lyase / glyoxalase I family protein
|
9.88
|
Soltu.DM.10G000900.1
|
copper ion binding;electron carriers
|
9.66
|
Soltu.DM.07G013680.2
|
Pyridoxal phosphate (PLP)-dependent transferases superfamily protein
|
9.49
|
Soltu.DM.05G002820.1
|
Glutathione S-transferase family protein
|
9.48
|
36 hpi
|
Kuras
|
Down-regulated
|
Soltu.DM.02G033100.4
|
shaggy-like kinase
|
-9.70
|
Soltu.DM.09G030690.1
|
Auxin-responsive family protein
|
-9.04
|
Soltu.DM.06G003240.4
|
thiaminC
|
-8.74
|
Soltu.DM.01G038650.1
|
SGNH hydrolase-type esterase superfamily protein
|
-8.59
|
Soltu.DM.02G031500.6
|
EXS (ERD1/XPR1/SYG1) family protein
|
-8.55
|
Soltu.DM.01G034500.2
|
conserved hypothetical protein
|
-8.20
|
Soltu.DM.02G013810.2
|
chlorophyll A/B binding protein
|
-8.14
|
Soltu.DM.08G009420.1
|
Tetratricopeptide repeat (TPR)-like superfamily protein
|
-8.11
|
Soltu.DM.07G014240.2
|
Cysteine proteinases superfamily protein
|
-7.94
|
Soltu.DM.08G029010.2
|
Leucine-rich repeat (LRR) family protein
|
-7.86
|
Functional Classification Of Degs
The Gene Ontology (GO) enrichment analysis was carried out with total as well as up- and down-regulated DEGs from different potato cultivars and time points. Analysis showed that many top functional categories of the biological process (BP), cellular components (CC), and molecular function (MF) were commonly over-represented across the different potato cultivars and time points (Fig. 4A-F). The commonly over-represented (FDR < 0.05) top 10 GO term for BP at 18 and 36 hpi was the Organonitrogen compound biosynthetic process (GO:1901566). Other GO terms such as Cellular amide metabolic process (GO:0043603), Peptide metabolic process (GO:0006518), Peptide biosynthetic process (GO:0043043), Small molecule metabolic process (GO:0044281), cellular protein metabolic process (GO:0044267) were enriched in either 18 or 36 hpi (Fig. 4A&B). For CC at 18 hpi, ribosome (GO:0005840) and cytosol (GO:0005829) were common between the three potato cultivars and there were no GO terms in common at 36 hpi (Fig. 4C&D). For MF, oxidoreductase activity (GO:0016491), small molecule binding (GO:0036094), anion binding (GO:0043168), and nucleotide binding (GO:0000166) were common GO terms present across the potato cultivar and time points (Fig. 4E&F). Apart from the common GO terms, there were unique functional categories specific for individual potato cultivars and time points. To gain further insight into the biological significance, up- and down-regulated DEGs from different potato cultivars and time points were analyzed which revealed that many of the top enriched GO terms from the total DEGs fall under the up-regulated DEGs category. One of the highly enriched BP GO terms in the down-regulated DEGs across the potato cultivar and time points were Photosynthesis (GO:0015979), except for Désirée 36 and Magnum Bonum 18 hpi (Additional file 5).
The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was carried out to understand an overview of diverse pathways involved in the total as well as up- and down-regulated DEGs. The overall number of enriched pathways in different potato cultivars and time points are provided in Table 3. The metabolic pathways and biosynthesis of secondary metabolites were the commonly enriched pathways across the potato cultivars and time points. Pathways like the ribosome, carbon metabolism, protein processing in endoplasmic reticulum, biosynthesis of amino acids, citrate cycle (TCA cycle), and oxidative phosphorylation were significantly enriched at 18 hpi across the potato cultivars in the up-regulated DEGs. The number of transcripts in the enriched pathways was significantly higher in the up-regulated DEGs than in the down-regulated. The top 20 metabolic pathways enriched in each of the up- and down-regulated DEGs of different potato cultivars and at 18 and 36 hpi are shown in Fig. 5A&B and Fig. 5C&D, respectively. The complete list of GO and KEGG enrichment analyses of the DEGs is given in Additional file 5.
Table 3
The number of enriched pathways in KEGG analysis for the DEGs of different potato cultivars with A. solani infection. Different time points, Total DEGs, Up- and Down-regulated DEGs.
|
18 hpi
|
36 hpi
|
Potato cv.
|
Total DEGs
|
Up-regulated
|
Down-regulated
|
Total DEGs
|
Up-regulated
|
Down-regulated
|
Magnum Bonum
|
40
|
40
|
11
|
66
|
63
|
12
|
Désirée
|
64
|
64
|
8
|
57
|
52
|
8
|
Kuras
|
67
|
62
|
13
|
69
|
63
|
16
|
Transcription factors expressed in response to A. solani infection
Several transcripts encoding transcription factors (TFs) were identified from the commonly expressed and unique DEGs. From the 649 common expressed DEGs identified from all three potato cultivars and time points, a total of 20 TFs encoding transcripts falling in eight families were identified, of which 16 were up-regulated (six families) and four were down-regulated (three families) (Fig. 6A). The WRKY (five) and ERF (four) TF families had the maximum number of transcripts that all were up-regulated. The expression patterns of the 20 identified TFs are present as a heat map in Fig. 6B, and the complete list is provided in Additional file 6. In the case of unique DEGs, an increasing trend in the TFs number was observed with increased susceptibility of potato cultivar to early blight and an increase in infection time, except for Désirée 36 hpi. At 18 hpi a total number of 113, 145, and 436 TF transcripts were identified in Magnum Bonum, Désirée, and Kuras, respectively. Similarly, a high number of TFs was expressed in Kuras (160) followed by Magnum Bonum (76) at 36 hpi. There was a decrease in TF number in Désirée (35) at 36 hpi. These belong to approximately 42 families, and the dominant TF families identified are WRKY, ERF, bHLH, MYB, and C2H2 (Additional file 6).
To further understand the expression pattern of these TFs, we analyzed the up- and down-regulated DEGs separately. Among all the up-regulated DEGs there was a significantly higher percentage (4.38%) of TFs in Magnum Bonum compared to Désirée (2.12%) and Kuras (2.73%) at the early time point (18 hpi) of infection, even though the later cultivars have a higher number of DEGs. There were few differences in the TF percentage for the down-regulated DEGs between the 18 and 36 hpi in different potato cultivars (Additional file 6). We also observed significant differences in the number of TF transcripts expressed in each family between different potato cultivars, time points, and up- and down-regulated DEGs (Fig. 7A&B). In the up-regulated DEGs, the number of WRKY TFs identified in Magnum Bonum, Désirée, and Kuras at 18 hpi were 15, 10, and 11, respectively. The numbers were increased to 17, 9, and 26 for Magnum Bonum, Désirée, and Kuras, respectively, at 36 hpi. In the down-regulated DEGs, the WRKY transcripts were identified only in Kuras (three) at 18 hpi, and the numbers were three, six, and 13 for Magnum Bonum, Désirée, and Kuras, respectively, at 36 hpi. The other TF families with high numbers of genes at 18 and 36 hpi in the up-regulated DEGs were ERF, and NAC, which had fewer numbers in the down-regulated DEGs. Similarly, TF families like bHLH, HD-ZIP, GRAS, G2-like, MYB-related, and C3H were high in numbers for the down-regulated DEGs at 18 and 36 hpi. Also, a few TF families like SBP, ARF, TCP, Co-like, MIKC_MADS, TALE, and DBB were expressed only in the down-regulated DEGs at 18 and 36 hpi (Fig. 7A&B; Additional file 6).
Metabolism Pathway Analysis Of Degs
We mapped DEGs to different functional categories by functional annotation based on Mercator4 and divisions in MapMan bins to understand the A. solani affected pathways in different potato cultivars with time points. In general, many genes coding for multiple components of the photosynthesis machinery, starch biosynthesis and degradation pathway were down-regulated in the susceptible cultivar Kuras compared to Désirée and Magnum Bonum. The down-regulation was higher at 36 hpi compared to 18 hpi. On the other hand, genes involved in the Jasmonic acid (JA) and Ethylene (ET) biosynthesis pathways, Mevalonate (MVA) pathway, Isoprenyll-PP, and Terpenes were up-regulated across the potato cultivars and time points with few exceptions. All the transcript ID and the fold change values are given in Additional files 7–10.
Genes Involved In Photosynthesis
Photosynthesis is the key biochemical reaction occurring in all green plants. The genes involved in the light-harvesting complex (LHC), LHCa1, 2, 3, 4, 5, and 6 of the LHC1, PsaD, E, F, G, H, K, L, N, and O of the photosystem I (PS I) complex, and the high chlorophyll fluorescence (HCF) 101, PS I assembly 2 (PSA2), and the assembly factor PSA3 were highly down-regulated in Kuras at 18 and 36 hpi. Compared to Kuras, only a few of the above genes were down-regulated in Magnum Bonum 36 hpi and Désirée at 18 hpi; also, the extent of down-regulation was less (Fig. 8A; Additional file 7). Many genes involved in the LHCII, such as LHCb1/2/3, LHCb4, 5, 6, and LHCq were two-fold down-regulated in Kuras 36 hpi compared to Magnum Bonum 36 hpi (Fig. 8B; Additional file 7). Similarly, genes of the PS II assembly (LPA2, LPA3, HCF 136, 173, 243, Psb27, 28, 32, 33, PAM68, LHC related protein (OHP1), psbJ/psbN-translation activator (LPE1), Thioredoxin (TRX-M), LHCII-stabilizing factor (SEP3)) were down-regulated in Kuras at 18 and 36 hpi (Additional file 7). The components of the PS II oxygen-evolving center (PsbO/OEC33, PsbP, PsbQ, PsbR, PsbTn, PsbW, PsbX, PsbY), subunits and components of chlororespiration, components of cytochrome b6/f complex, plastocyanin electron carrier, PGRL1 of electron flow PGR5-PGR1 complex, Ferredoxin (Fd) targeted to NADP reduction, ATP synthase, and subunits of rubisco were down-regulated in Kuras at 18 and 36 hpi, as compared to Désirée and Magnum Bonum (Additional file 7).
Genes Involved In Starch Biosynthesis And Degradation
The genes involved in starch biosynthesis were down-regulated in Kuras at 18 and 36 hpi. The maximum of 1.5 and 2.2 fold down-regulation was observed with a large subunit of ADP-glucose pyrophosphorylase gene (soltu.dm.01g024440.1) in Kuras at 18 and 36 hpi, respectively. On the contrary, no starch biosynthesis genes were down-regulated at 18 hpi in Magnum Bonum, and starch synthase (SS) III (soltu.dm.02g020170.2) is the only gene down-regulated at 36 hpi in Magnum Bonum. At 18 hpi in Désirée, large subunit APL of ADP-glucose pyrophosphorylase (soltu.dm.01g024440.1), starch branching enzyme (soltu.dm.09g004100.1), and component ISA1 of ISA1-ISA2 isoamylase heterodimer (soltu.dm.07g005540.1) were down-regulated and starch synthase (SS) III (soltu.dm.02g020170.2) and scaffold protein of amylose biosynthesis (soltu.dm.02g026830.2) were down-regulated at 36 hpi. Similarly, genes such as alpha-amylase-binding scaffold protein (LSF1) (soltu.dm.12g016610.2) and beta-amylase (soltu.dm.07g018100.1) were down-regulated (1.6 fold) in Kuras at 18 hpi and a 1.85 fold down-regulation was observed for the plastidial alpha-glucan phosphorylase (PHS1) (soltu.dm.05g000570.1) gene at 36 hpi. Only a few genes were down-regulated in Magnum Bonum and Désirée at 18 and 36 hpi (Additional file 8).
Genes Involved In The Mevalonate Pathway, Isoprenyl-pp, And Terpenes
The genes involved in the mevalonate (MVA) pathway, Isoprenyl-PP, and Terpenes were up-regulated in all the potato cultivars and time points with a few exceptions (Additional file 9). All the six enzymes (acetyl-CoA C-acyltransferase, 3-hydroxy-3-methylglutaryl-CoA synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate diphosphate decarboxylase, and isopentenyl diphosphate isomerase) involved in the MVA pathway were up-regulated in different potato cultivars and time points. The maximum up-regulation of these transcripts was observed in Kuras at 36 and 18 hpi followed by Désirée and Magnum Bonum. One of the transcripts for the enzyme acetyl-CoA C-acyltransferase (soltu.dm.07g015120.1) was up-regulated in all the potato cultivars and another transcript soltu.dm.04g010070.3 for the same enzyme was expressed only in Désirée 36 hpi with a fold increase of 8.36. The 3-hydroxy-3-methylglutaryl-CoA synthase (soltu.dm.08g026810.1) was up-regulated 4.86 and 7.84 fold in Kuras at 18 and 36 hpi and 3.69 fold in Magnum Bonum 36 hpi (Additional file 9). The enzymes farnesyl diphosphate (FDP) synthase and isoprenyl diphosphate synthase (IDS) were up-regulated, and FDP synthase maximum fold-change(7.07) was observed at 36 hpi in Kuras, followed by Désirée at 18 hpi (4.07 fold). The IDS was up-regulated in all potato cultivars and time points, except Magnum Bonum at 36 hpi (Additional file 9). Many of the transcripts code for mono-/sesquiterpene-/diterpene synthases were up-regulated in multiple potato cultivars. The mono-/sesquiterpene-/diterpene synthases transcripts (soltu.dm.01g040930.1, soltu.dm.01g040950.1, soltu.dm.07g017540.1, soltu.dm.01g040960.1 and soltu.dm.07g017580.1) were expressed in all potato cultivars at 18 and 36 hpi. The highest fold-change expression of 11.81 and 11.33 was observed for soltu.dm.01g040950.1 and soltu.dm.01g040930.1 in Kuras 18 hpi. The down-regulation (7.90 fold) was noticed for the transcript soltu.dm.07g017230.1 in Désirée 36 hpi (Additional file 9).
Genes Involved In Ja And Et Biosynthesis Pathways
The JA and ET biosynthesis and signaling are shown to be very critical for imparting plant resistance against necrotrophic pathogen attacks [37, 38]. We identified multiple transcripts encoding the enzymes involved in the JA and ET biosynthesis pathways; as expected, all were up-regulated, except for two (Fig. 9; Additional file 10). The JA biosynthesis enzyme, Lipoxygenase (LOX; Soltu.DM.08G010990.1) was most up-regulated in Kuras 18 and 36 hpi (7.12 and 6.73 fold, respectively) followed by Magnum Bonum and Désirée. The Allene oxide synthase (AOS; Soltu.DM.01G048780.1) had the highest expression in Kuras 18 hpi (12.87 fold) and reduced to 8.92 fold at 36 hpi and for Magnum Bonum, the AOS levels were increased at 36 hpi (10.95 fold) compared to 18 hpi (6.93 fold). The 12-oxophytodienoate reductase (Soltu.DM.04G012240.1) levels increased at 36 hpi for Magnum Bonum and Kuras, but the maximum fold-change was seen in Désirée at 18 hpi (6.09 fold) (Additional file 10). Another transcript encodig a 12-oxophytodienoate reductase (Soltu.DM.04G012230.1) was 7.01, 7.29, and 5.56 times up-regulated in Kuras at 18 and 36 hpi and Désirée 36 hpi, respectively, and was not detected in Magnum Bonum at 18 and 36 hpi and Désirée at 18 hpi. Similarly, multiple transcripts were identified for 1-aminocyclopropane-1-carboxylate synthase (ACS) and 1-aminocyclopropane-1-carboxylate oxidase (ACO) part of the ET biosynthesis. Both ACS and ACO were significantly up-regulated in all potato cultivars and time points. One of the transcripts for ACS (Soltu.DM.01G034180.1) was highly up-regulated at 18 hpi in all potato cultivars and reduced at 36 hpi. The ACO transcript (Soltu.DM.07G016780.1) fold-change was maximum (4.98) in Magnum Bonum at 36 hpi followed by 18 hpi (4.79), and Kuras showed a 4.45 fold-increase at 18 hpi (Additional file 7).