De novo assembly and quality assessment of P. nigrum transcriptome
To investigate the molecular elements involved in immunity within P. nigrum, our study focused to establish a comparative transcriptome dataset with differentially expressed genes. This was accomplished by utilizing RNA-seq data acquired from leaves that were exposed to P. capsici. Our preliminary data analysis resulted in the generation of approximately 216 million paired end reads (Additional File 1). After initial pre-processing steps, we obtained high-quality data, with over 87% of the bases exhibiting an average base quality Q > 30 and a mean GC content of 43.81%. By utilizing a high-quality genome of P. nigrum as guidance, we generated a transcriptome assembly that comprised a total of 64,667 transcripts (Table 1). The transcript assembly exhibited a percent GC content of 44.04, and an N50 value of 2507 was observed, indicating the contiguity of the assembly. The cumulative length of the assembly was 129.43 Mb, with 50,696 contigs surpassing a length of 1000 base pairs. The largest contig identified in the assembly measured 27,925 base pairs. Notably, 84% of the reads were successfully mapped back to the assembly, indicating the comprehensive nature and accuracy of the transcriptome assembly. The assembly was further tested for completeness using BUSCO against Viridiplantae database and found a good 96.7% completeness for the transcripts.
Table 1
Reference based transcriptome assembly statistics of P. nigrum. The percentage value of BUSCO completeness shown in C: Complete; S: Complete and single-copy; D: Complete and duplicated; F: Fragmented; M: Missing; N: number of genes.
Assembly statistics |
Transcripts |
Number of sequences | 64667 |
Total length | 129429265 bp |
Longest sequence | 27925 bp |
Shortest sequence | 200 bp |
N50 | 2507 bp |
GC content | 44.40% |
BUSCO (N:425) | C:96.7% [S:32.7%, D:64.0%], F:0.7%, M:2.6% |
Unigenes |
Number of sequences | 74607 |
Total length | 25127195 aa |
Longest sequence | 5371 aa |
Shortest sequence | 86 aa |
BUSCO (N:425) | C:87.8% [S:44.7%, D:43.1%], F:8.9%, M:3.3% |
Functional annotation of P. nigrum transcriptome
The transcriptome assembly of P. nigrum predicted 74607 ORFs (unigenes) using TransDecoder program. We performed comprehensive homology searches against the Uniprot-Viridiplantae database for both transcripts and unigenes to functionally annotate the transcriptome. We used programs such as BLASTP for unigenes and BLASTX for transcripts, and 86.02% and 69.71% of them were effectively annotated, respectively. Notably, majority of these homologues had been found in cinnamon and lotus species, providing further insight into potential evolutionary relationships. Functionally, these homologues exhibited a diverse array of roles, with an abundance protein kinase domain-containing, ubiquitin-related, and pentatricopeptide repeat proteins in the homologous hits, indicating their importance in cellular regulation. We also noticed an abundance of RNA-binding proteins, such as RRM, and disease resistance proteins among the hits, which could be important in defense response. Furthermore, we used HMMScan to predict domains against the Pfam database, and 79.89% of the unigenes were allocated domains. PPR and LRR domains were found to be particularly prevalent, providing vital insights into the molecular machinery of the P. nigrum transcriptome. MSTRG.20872.1 (Pentatricopeptide repeat containing protein) with PPR domains and MSTRG.13016.1 (Disease resistance protein) with LRR domains were the most abundant transcripts after infection. These findings pave the way for intriguing future research in host resistance.
Comparative transcriptomics reveals the differentially expressed genes.
To elucidate the transcriptional changes related to innate immunity in P. nigrum during the early biotrophic colonization of P. capsici, we performed a comparative transcriptomics approach that generated differential gene expression between control and infected samples, namely P. capsici infection, sampled after 6 hpi and 12 hpi (Fig. 1). An analysis using edgeR, representing the logarithmic fold changes as a function of the mean of normalized counts revealed differentially expressed genes (DEGs). We found a total of 4,714 transcripts at 6 hpi and 9,416 transcripts at 12 hpi that showed significant differential expression compared to the control. These transcripts exhibited a log2 fold change of ± 1.5 in the TPM expression level and were statistically significant (FDR < 0.05). Among these DEGs, we observed 2,434 upregulated transcripts and 1,740 downregulated transcripts at 6 hpi respectively. At 12 hpi, there were 4,348 upregulated transcripts and 5,068 downregulated transcripts respectively. These DEGs likely represent the innate immune signature components of black pepper, indicating their involvement in perceiving and activating defense responses against P. capsici early in the infection process. Further, the hierarchical clustering of the DEGs across these conditions, along with the Volcano plots generated from edgeR analysis, clearly distinguished the coherence between the infected conditions (Fig. 2A and B). We analysed 15 top upregulated and downregulated transcripts from the differential expression analysis of control and infected (6 hpi and 12 hpi) samples (Fig. 2C and D, Additional File 2). We observed a significant upregulation of several methyltransferase genes in the infected samples, indicating their potential involvement in the response to the infection. Additionally, within the top upregulated transcripts, we identified several genes associated with defense responses, including a leaf rust resistance gene, an auxin response gene, and an amine oxidase gene. These findings suggest a robust activation of defense regulations in response to the infection. Conversely, among the downregulated transcripts, we observed the suppression of genes such as bHLH transcription factors, genes related to photosystem functions, and genes associated with cell wall processes. These downregulated genes may indicate a downscaling of certain cellular processes during the infection.
Analysis on key hormone signalling pathways in defense response.
Plants employ a complex defense mechanism to resist pathogenic agents. The SA, JA and ET mediated signalling pathways are recognized as an essential components of plant immune responses against pathogens. These signalling molecules play crucial roles in regulating plant defense pathways, enabling appropriate responses to various types of pathogens. SA initiates early defense-related gene expression in pathogen-infected plants, while JA induces late defense-related gene expression in pathogen-infected plants. JA is widely involved in regulating disease resistance against necrotrophic pathogens, while SA mediates broad-spectrum resistance against biotrophic and hemi biotrophic pathogens [10].
Salicylic acid signalling pathway
In our study, we investigated three key enzymes within the SA signalling pathway: Non-expressor of pathogenesis-related genes 1 (NPR1), WRKY transcription factor 70 (WRKY70), and Pathogenesis-related protein 1 (PR1) [11]. NPR1, serving as the central orchestrator for the SA signalling pathway, and acts as a transcriptional co-activator alongside TGA transcription factors, promoting the expression of defense-related genes like PR1. Our examination of the P. nigrum transcriptome assembly revealed the presence of two transcripts (MSTRG.29868.1 and MSTRG.29924.1) that contain the sequence signatures of NPR1 (Additional File 3). Intriguingly, we observed a noteworthy upregulation of both these transcripts at 6 hours post-infection (hpi) followed by their decline at 12 hpi (Table 2).
Table 2
Transcript expression of key enzymes involved in the SA, JA and ET hormone signalling pathways from three samples.
Pathway genes | Transcript hits | TPM values |
Control | 6 hpi | 12 hpi |
Salicyclic acid |
Non-expressor of pathogenesis-related genes 1 (NPR1) | MSTRG.29868.1 | 13.23 | 107.39 | 40.67 |
MSTRG.29924.1 | 11.53 | 76.94 | 24.88 |
WRKY transcription factor 70 (WRKY70) | MSTRG.8862.1 | 20.64 | 93.07 | 48.83 |
Pathogenesis-related protein 1 (PR1) | MSTRG.17950.1 | 0.84 | 143.17 | 9.07 |
MSTRG.18009.1 | 0.49 | 385.34 | 6.74 |
Glutaredoxin-C9 (GRX4) | MSTRG.5776.1 | 7.34 | 22.59 | 31.78 |
Transcription factor (TGA1) | MSTRG.1644.1 | 38.29 | 3.08 | 5.44 |
Transcription factor (TGA4) | MSTRG.4306.1 | 22.49 | 11.73 | 17.73 |
Jasmonic acid | | | | |
Coronatine Insensitive 1 (COI1) | MSTRG.25426.1 | 7.36 | 8.52 | 12.44 |
Jasmonate ZIM domain protein 1 (JAZ1) | MSTRG.20417.1 | 48.85 | 590.82 | 389.66 |
MSTRG.31785.1 | 39.23 | 1034.24 | 347.41 |
Transcription factor MYC2 (MYC2) | MSTRG.16496.1 | 10.34 | 4.79 | 32 |
MSTRG.4905.1 | 42.25 | 157.63 | 78.08 |
Mitogen-activated protein kinase 4 (MPK4) | MSTRG.830.2 | 59.38 | 26.18 | 47.95 |
Vegetative storage protein 2 (VSP2) | MSTRG.2523.1 | 31 | 2.05 | 7.33 |
MSTRG.3467.1 | 15.46 | 0.42 | 2.09 |
ET pathway |
Ethylene-insenstive 3-like (EIL1) | MSTRG.4336.1 | 13.69 | 1.17 | 22.23 |
MSTRG.3297.1 | 49.67 | 12.67 | 92.14 |
Ethylene-responsive transcription factor (ERF094) | MSTRG.15462.1 | 0.11 | 16.62 | 1.75 |
WRKY70, functioning as a positive regulator of SA-mediated defense while concurrently suppressing the JA response, was also a focus of our investigation. Notably, we identified a transcript (MSTRG.8862.1), corresponding to WRKY70 as per CAPS-protocol (Additional File 3), that exhibited an expression pattern with significant upregulation at 6 hpi, followed by a decline at 12 hpi (Table 2).
Pathogenesis-related protein 1 (PR-1) family members are renowned for their abundant production in plants upon encountering pathogenic threats. PR-1 gene expression has previously served as a reliable marker for evaluating salicylic acid-mediated disease resistance [12]. Our transcriptome analysis using CAPS_protocol identified two transcripts (MSTRG.17950.1 and MSTRG.18009.1) (Additional File 3). Both of which displayed a conspicuous early upregulation at 6 hpi, followed by a decrease at 12 hpi in response to P. capsici (Table 2). We also checked the expression of other enzymes such as GRX4 (MSTRG.5776.1), TGA1 (MSTRG.1644.1) and TGA4 (MSTRG.4306.1) and transcripts thus identified (Additional File 3). GRX4 showed an upregulated expression in infected sample, whereas a downregulation was observed for TGA transcription factors (Table 2).
Jasmonic acid signalling pathway
The major genes involved in JA signalling pathway are Coronatine Insensitive 1 (COI1), Jasmonate ZIM domain protein 1 (JAZ1), and Transcription factor MYC2 (MYC2). COI1, an essential F-box protein for all JA responses, plays a critical role in assembling the SCF (COI1) E3 ubiquitin ligase complex, which in turn, recruits Jasmonate ZIM-domain proteins for degradation via the 26S proteasome. In our study, we identified the COI1 transcript (MSTRG.25426.1) (Additional File 3), which showed a trivial increase in expression in the infected samples (Table 2). JAZ is a subfamily of plant-specific TIFY protein family that is characterized by a TIF[F/Y]XG motif within a larger (∼28 amino acids) conserved region known as the ZIM (or TIFY) domain. JAZ proteins were identified as the transcriptional repressors of JA signalling, and these proteins stimulated remarkable progress in understanding how JAs regulate large-scale changes in gene expression in model plants. In our investigation, we predicted two JAZ1 transcripts (MSTRG.20417.1 and MSTRG.31785.1) (Additional File 3) that exhibited higher expression at 6 hpi and decreased expression at 12 hpi (Table 2). MYC2 was the first transcription factor identified which could interact with JAZ proteins and initiate JA signalling. The JAZ proteins block the activity of MYC2 in the absence of bioactive JAs, demonstrated a mixed response in our study. One MYC2 transcript (MSTRG.16496.1) displayed increased expression at 12 hpi, while another (MSTRG.4905.1) exhibited higher expression at 6 hpi, compared to 12 hpi (Table 2, Additional File 3) suggesting complementary roles of these transcripts at different times post infection. Additionally, MPK4 was identified as a regulator, positively impacting GRX480 in the SA signalling pathway and negatively influencing MYC2 in the JA signalling pathway, thereby modulating JA-responsive genes. Notably, the transcript corresponding to MPK4 (MSTRG.830.2) exhibited consistent expression across all samples, while transcripts related to VSP2 (MSTRG.2523.1 and MSTRG.3467.1), regulated by MYC2, were downregulated in infected samples (Table 2, Additional File 3).
Ethylene signalling pathway
Key regulators of ethylene signalling are crucial for maintaining various plant responses to ethylene. In the ET signalling pathway, EIL1, in conjunction with JAZs-MYC2 from the JA signalling pathway, plays pivotal roles in facilitating the interaction between JA and ET pathways. In our investigation, we identified two transcripts (MSTRG.4336.1 and MSTRG.3297.1) encoding the EIL1 gene in the P. nigrum transcriptome (Additional File 3). Both transcripts exhibited reduced expression at 6 hpi compared to the control but displayed increased expression at 12 hpi (Table 2). The ethylene and JA pathways intersect in the transcriptional activation of Ethylene Response Factor 1 (ERF1), a gene encoding a transcription factor that regulates the expression of genes involved in pathogen responses to impede disease progression. Our study revealed that the ERF1 transcript (MSTRG15462.1) (Additional File 3) exhibited expression in the 6 hpi infected sample and decreased expression at 12 hpi (Table 2). The transcriptome expression for ethylene pathway genes were further validated using RT-qPCR. Our findings indicate that ERF1 transcript expression is temporally regulated during infection, with implications for disease progression. Our study reveals the vital collaboration between EIL1 and JAZ1-MYC2 in bridging the JA and ET pathways, evidenced by dynamic EIL1 expression patterns in P. nigrum. Additionally, our findings highlight the temporal regulation of ERF1 transcript expression during infection, highlighting its role in disease progression and underscoring the intricate interplay between ethylene and JA signalling pathways. In summary, our investigation underscores the critical role of key regulators in ethylene signalling for maintaining diverse plant responses to ethylene.
To confirm these observed expression patterns, we performed quantitative real-time polymerase chain reaction (qRT-PCR) analysis on selected transcripts (Fig. 3). We could observe a correlation between the expression levels derived from qRT-PCR and those determined by the transcriptome data for most of these transcripts in control, 6 hpi and 12 hpi. Overall, this study focused on the innate immunity of black pepper against P. capsici. Transcriptome analysis revealed the immune-related gene dynamics and allowed for the identification of differentially expressed transcripts. The significant activation of the SA and JA pathways early in infection, followed by the ET pathway involvement, shows black pepper complex defense mechanisms.