Genome wide identification and analysis of P. nigrum PR-1 genes
Eleven potential PnPR-1 gene candidates were obtained from the genome-wide analysis in comparison to the A. thaliana PR genes. The number of exons harboring in each PnPR-1 gene is one, expect in Pn2.357 which has two exons. The length of PnPR-1 proteins ranged from 127 to 357 amino acid residues with molecular weight ranging from 14.38 to 38.49 kDa. The theoretical isoelectric point (pI) data categorized PnPR-1 proteins into acidic (Pn21.1032 and Pn36.35) and others as basic in nature (Table 1). Furthermore, the signal peptide regions and cleavage sites were also identified in all the PnPR-1 protein sequences except Pn11_1637 and Pn31.171 (Table 2).
Table 1
Sequence characteristics and physio-chemical properties of the PR-1 proteins in Piper nigrum.
Genome CDS Id
|
Genome Scaffold No.
|
Exon No.
|
Start
|
Stop
|
Strand
|
Protein length (AA)
|
Molecular weight (kDa)
|
Theoretical pI
|
Pn2.460
|
Pn23
|
1
|
1315655
|
1316146
|
-
|
155
|
17.09488
|
4.82
|
Acidic
|
Pn2.459
|
Pn23
|
1
|
1320928
|
1321419
|
-
|
192
|
21.91361
|
5.8
|
Pn2.433
|
Pn23
|
1
|
1493563
|
1494054
|
-
|
168
|
18.35074
|
7.58
|
Basic
|
Pn2.357
|
Pn23
|
2
|
2226595
|
2229193
|
-
|
127
|
14.37881
|
7.62
|
Pn2.340
|
Pn23
|
1
|
2402850
|
2403341
|
-
|
185
|
19.80756
|
9.1
|
Pn21.1032
|
Pn3
|
1
|
3202437
|
3202904
|
+
|
163
|
17.85002
|
9.15
|
Pn31.171
|
Pn15
|
1
|
26854949
|
26855332
|
-
|
163
|
17.87807
|
9.3
|
Pn36.35
|
Pn25
|
1
|
5754975
|
5755553
|
-
|
163
|
17.88005
|
9.3
|
Pn11.1637
|
Pn4
|
1
|
33322945
|
33323502
|
-
|
176
|
19.63337
|
9.37
|
Pn8.549
|
Pn8
|
1
|
25779763
|
25780269
|
-
|
163
|
17.96421
|
9.44
|
Pn14.1312
|
Pn14
|
1
|
5932334
|
5932864
|
+
|
357
|
38.49596
|
11.3
|
Table 2
Signal peptide region detected from the PnPR-1 proteins
Genome Id
|
Cleavage site position
|
Sequence position
|
Probability
|
Protein type
|
Signal Peptide (Sec/SPI)
|
Other
|
Pn21.1032
|
16 and 17
|
CNA-QN
|
0.9541
|
Likelihood
|
0.9981
|
0.0019
|
Pn2.340
|
24 and 25
|
AQA-QN
|
0.8849
|
Likelihood
|
0.9984
|
0.0016
|
Pn2.357
|
24 and 25
|
AQA-QN
|
0.8898
|
Likelihood
|
0.9986
|
0.0014
|
Pn2.433
|
24 and 25
|
AQA-QN
|
0.8892
|
Likelihood
|
0.9987
|
0.0013
|
Pn2.459
|
24 and 25
|
AQA-QN
|
0.8898
|
Likelihood
|
0.9986
|
0.0014
|
Pn2.460
|
24 and 25
|
AQA-QN
|
0.8892
|
Likelihood
|
0.9987
|
0.0013
|
Pn36.35
|
29 and 30
|
ASS-SP
|
0.6292
|
Likelihood
|
0.9813
|
0.0187
|
Pn14.1312
|
31 and 32
|
TNA-AL
|
0.4399
|
Likelihood
|
0.7324
|
0.2676
|
Pn8.549
|
30 and 31
|
TLA-QN
|
0.3454
|
Likelihood
|
0.5106
|
0.4894
|
Pn31.171
|
-
|
-
|
-
|
Likelihood
|
0.0029
|
0.9971
|
Pn11.1637
|
-
|
-
|
-
|
Likelihood
|
0.2691
|
0.7309
|
All the eleven PnPR-1 protein sequences were identified as potential members of CAP superfamily with CAP domain structure indicated in green line, along with caveolin-binding motif (CBM; marked in yellow) and CAP-derived peptide (CAPE; marked in blue) motif (Fig. 1). Comparison of PnPR-1 sequences with various other monocot and dicot plants showed distinct CAP domain with CBM and CAPE (Fig. S1). The signal peptide regions (Pink colored boxes) of PnPR-1, were also represented in Fig. 1 and the cleavage site predicted was indicated by the arrowhead.
Sequence conservation of PnPR-1 genes
The nucleotide and protein sequence conservation of all the PnPR-1 candidates were checked using Mega 7. The subsequent phylogenetic analysis of all eleven PR-1 nucleotide and protein sequences through the maximum-likelihood method with 1000 bootstraps (Fig. S2A & S2B) revealed two main clusters leaving one outnumbered group (Pn21.1032). A total of ten conserved motifs were identified using MEME server. Motif 1 and 2 were conserved in all the deduced PR-1 proteins whereas motif 3 and motif 6 are conserved in all PnPR-1 proteins except in Pn31.171 and Pn2.357 respectively. Motif 8, motif 9 and motif 10 were preserved in Pn36.35 along with Pn14.1312, Pn31.171, Pn1.1637 respectively (Fig. 2).
GO and KEGG pathway analysis of PnPR-1 genes
Gene ontology (GO) analysis carried out using PANNZER2 online server yielded five biological processes, four molecular functions and four cellular components. Based on the GO enrichment analysis, predicted PR proteins have their roles in all the three classes. Most of the PR-1 genes have their role in defense responses and response to biotic stimulus in biological function. In terms of molecular function, it has protein kinase activity, adenyl nucleotide and purine ribonucleoside binding activities. Cellular component showed its role in extracellular region (Table 3). Kegg pathways analysis categorized its role in Environmental Information Processing- Signal transduction pathways like MAPK signaling pathway - plant (04016), Plant hormone signal transduction (04075) and Plant-pathogen interaction (04626).
Table 3
The gene ontology (GO) term distribution of PnPR-1 proteins
GO Id
|
GO Domain
|
Function Description
|
GO:0006952
|
Biological process
|
Defense response
|
GO:0009607
|
Biological process
|
Response to biotic stimulus
|
GO:0048544
|
Biological process
|
Recognition of pollen
|
GO:0006468
|
Biological process
|
Protein phosphorylation
|
GO:0010274
|
Biological process
|
Hydrotropism
|
GO:0004672
|
Molecular function
|
Protein kinase activity
|
GO:0030554
|
Molecular function
|
Adenyl nucleotide binding
|
GO:0035639
|
Molecular function
|
Purine ribonucleoside triphosphate binding
|
GO:0032555
|
Molecular function
|
Purine ribonucleotide binding
|
GO:0005576
|
Cellular component
|
Extracellular region
|
GO:0016020
|
Cellular component
|
Membrane
|
GO:0031224
|
Cellular component
|
Intrinsic component of membrane
|
Secondary and 3D structure of PnPR-1 protein
A varied percentage of α-helices (15.6–36.88%), extended strand (10.64–23.78%), beta turn (3.65–5.88%) and random coil (37.42–67.79%) were found in the Pn-PR1 proteins (Fig. S3). 3D-strucures of the Pn-PR1 genes were generated using Phyre2online server and pocket structures were predicted (Fig. 3). The distinct structural variations were detected among the PnPR-1 proteins. At the same time, proportion of disordered regions of PR-1 proteins ranged from 4.5–28.1%.
Cis-regulatory elements of PnPR-1 genes
The cis-elements were found to be distributed over the 2.0 kb upstream promoter region of PnPR-1 genes (Fig. 4). The length of these cis-elements varied from 9 to 42 bp in Softberry database whereas 4 to 24 bp in NewPlace database. Among the ten PR-1 gene loci, the typical TGA binding site LS7, WBSI, G-box and C- motif were found in the promoter regions of Pn36.35. Meanwhile, GT motif and Zc2A/T-2 were found in the Pn2.340, Pn2.433 and Pn2.460. The hormone signalling elements such as ABI4 and GCC-box were present in Pn2.459 and Pn2.340, Pn2.433 respectively. The stress responsive MYB was also detected in the promoter regions of PnPR1 genes such as Pn21.1032, Pn31.171 and Pn36.35. Among 154 cis-elements detected from New Place database, CAAT box (CAAT), E-box (CANNTG), DOFCOREZM (AAAG) regions were found to be widely distributed across the PnPR-1 promoter regions (Fig. S4).
Expression of PR-1 genes during P. capsici infection in P. nigrum
The assembled transcriptome of RNA-seq data from control and P. capsici infected P. nigrum plants revealed 60437 transcripts (Communicated). From the assembled data seven transcripts of PnPR-1 genes were mapped to the P. capsici - P. nigrum interaction pathway. The transcripts length ranges from 391-1015bps. Differential expression of these transcripts was assessed from their corresponding FPKM values among control leaf (CL), infected leaf (IL) and infected root (IR). CL10113.C1/2 and Unigene17664 (Fig. 5A-C) that respectively mapped to the Pn23 and Pn8 scaffolds were highly expressed (4.57 to 5.7-fold) in the infected leaf during pathogen infection. Meanwhile Unigene11116, Unigene15555, Unigene26912 and Unigene693 were moderately expressed in IL (Fig. 5D-G). All the transcripts, except Unigene693 were downregulated drastically (2 to 7-fold) in IR compared to IL (Fig. 5 main figure).
RT-qPCR validation of PnPR-1 genes
The expression of PnPR-1 gene was further validated using RT-qPCR from P. capsici infected P. nigrum plants. In agreement with the transcriptome results, a high upregulation of PnPR-1 gene CL10113.C2 was detected from the infected leaf. Furthermore, the temporal expression at 6 hrs, 12 hrs and 24hrs of P. capsici infection revealed sequential increase in expression of PnPR-1 genes from 6 to 12 hours of infection (Fig. 6A). A drastic upregulation of PnPR-1 expression was observed in the leaves at 24hours after infection compared to the mock control plant (ML). The trypan blue staining has enabled us to discrimination between intact-viable and damaged regions on leaves. Viable cells were colorless whereas infected tissue was stained in blue (Fig. 6B).