Cacao, belonging to the genus Theobroma, is a crop of significant economic importance12. Understanding the molecular defense mechanisms of cacao plants and their ability to suppress pathogen development is crucial in tackling black pod disease caused by P. palmivora. Nonetheless, no previous analysis has compared the occurrence of miRNAs in regulating genes between resistant and susceptible species affected by BPD caused by P. palmivora. In this study, the identification of miRNAs that potentially target important mRNAs involved in plant defense and pathogen suppression was successfully conducted.
The present study identified a total of 54 known miRNAs, consisting of 40 distinct miRNA families. Among these families, miR160, miR396, miR399, and miR159 families exhibited a higher representation, each consisting of 3 miRNA species. The remaining families consisted of either 1 or 2 miRNAs. In another study, Cheng et al. 13 successfully identified 31 miRNA families in banana roots infected with Fusarium oxysporum f. sp. Cubense. Meanwhile, Rabuma et al. 7 also succeeded in identifying 24 miRNA families in chili leaves infected with Phytophthora capsica. Xin et al. 14 also conducted a study on miRNA identification of wheat infected with Erysiphe graminis, pathogen that causes powdery mildew disease in wheat, where the result showed that there were 132 miRNA families identified.
Subsequently, we discovered a set of 67 miRNAs classified as novel, predicted by using miRDeep2. The accuracy of miRDeep2 in identifying miRNAs ranged from 98.6–99.9%, and it successfully reported hundreds of novel miRNAs. The prediction process involved utilizing high throughput sequencing data, mapped reads, and regenerating miRNA precursors using the miRDeep2 program. To differentiate novel miRNAs from known ones, the generated miRNAs were compared to the known miRNA database15. In a similar study by Cheng et al. 13 that employed the same program, 28 novel miRNAs responsive to Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) were successfully identified in banana roots. However, only 5 novel miRNAs were found to have putative target genes. Another study by Liu et al. 16 analyzed putative novel miRNAs and successfully identified 310 novel miRNAs associated with the ripening process of hot pepper (Capsicum annum L.).
In our study, the identified miRNAs exhibited a length range of 18 to 27 nucleotides, with the majority (60 miRNAs) having a length of 21 nucleotides. This finding agrees with Zhang et al. 17 research on Peony flowers with identified miRNAs lengths ranging from 18 to 24 nucleotides. Guo et al. 18 also identified soybean miRNAs with lengths varying from 18 to 25 nucleotides. Another study that aligns with these findings is Liu et al. 16, which observed that miRNAs vary in length from 18 to 28 nucleotides during the stage of chili fruit ripening. Most miRNAs fall within the range of 20 to 24 nucleotides, with 24 nucleotides being the most abundant, additionally, 23 nucleotides are the typical length observed for miRNAs. Through numerous comparisons of miRNA lengths derived from numerous studies, it becomes evident that the length of miRNAs varies depending on the organism type and the specific role of miRNA in regulating processes within certain plants or growth phases.
We also predicted the pre-miRNAs structure of some known and novel pre-miRNA with the highest abundance (Fig. 4). pre-miRNAs must form a loop structure to be processed into mature miRNA. The stability of the pre-miRNA structure is determined by its MFE (minimum free energy) value, where a lower MFE indicates a more stable structure. The MFE of some miRNA with the highest abundance ranging from − 39.70 to -25.60 kcal/mol for known miRNA and − 42.60 to -14.30 kcal/mol for novel miRNA. In another study, Tiwari et al. 19 showed that the MFE of known and novel pre-miRNA of potato plant with low nitrogen stress were ranging between − 114.9 to -19.9 kcal/mol for known miRNAs and − 166.4 to -15.4 kcal/mol for novel miRNAs. Hence, the stability of pre-miRNA secondary structure will also determine the abundance of miRNAs in response to pathogen.
Upon pathogen infection, plants activate their initial line of defense by initiating pattern-triggered immunity (PTI) as a first defense barrier and effector-triggered immunity (ETI) as a second barrier20,21. Varied responses to pathogen attacks are observed in resistant and susceptible plant varieties, influenced by miRNA expression towards target genes22. miRNAs regulate gene expression during plant-pathogen interactions13. For instance, in susceptible chili pepper plants, the excessive expression of miRNA398, which targets receptor protein kinase, enhances susceptibility to rot disease22. This indicates that plant susceptibility arises from the regulation of gene expression, could be mediated by miRNAs suppressing their disease-resistance genes.
The PsRNA analysis of the T. cacao genome identified 9 upregulated miRNAs predicted to target 1,112 genes. miR165b has been identified as a potential regulator of the putative Theobroma cacao gene that encodes the disease resistance RPP13-like protein through mRNA cleavage. Similarly, Novel-miR34 is predicted to target the same gene by inhibiting its translation at the mRNA level. Lewis et al. 23 postulated in their research that the ZAR1 protein, which shares homology with RPP13, serves as a recognition factor for type III bacterial pathogen effectors (T3Es) like Pseudomonas syringae. ZAR1 belongs to the coiled-coil (CC) nucleotide-binding site and leucine-rich repeat (NBS-LRR) protein class, which includes the R protein.
Furthermore, miR167a, Novel-miR34, and Novel-miR6 have been predicted to target disease-resistance genes, specifically RGA, At4g19060, and At4g27220, through mRNA cleavage. The RGA (Resistance Gene Analog) protein provides plant protection against pathogens by interacting directly or indirectly with avirulence proteins found in the pathogens. This interaction triggers a defense response that inhibits pathogen growth24. The proteins At4g19060 and At4g27220 belong to the NLR (nucleotide-binding, leucine-rich repeat) protein family, which detect pathogens' presence and activity. NLR proteins can directly recognize pathogen effectors by binding to them through the LRR domain or indirectly detect modifications in host proteins caused by effector actions. In certain cases, host protein domains targeted by pathogen effectors have become integrated or fused into NLR proteins25,26.
In addition to their targeting of plant resistance genes, the four upregulated miRNAs, miR3627a, Novel-miR6, miR3954a, and Novel-miR34, are also predicted to target seven L-type lectin-domain containing receptor kinases. Wang et al. 27 have reported the involvement of L-type lectin receptor kinases (LecRK) in the resistance response against oomycete pathogens such as Phytophthora infestans and Phytophthora capsici.
Several miRNAs, including Novel-miR34, Novel-miR6, miR3954a, Novel-miR18, Novel-miR59, and miR156b, are predicted to target cellulose synthase-like protein genes and expansin genes, which are essential for cell wall synthesis and elongation. These processes play a crucial role in plant defense against pathogen infection. Specifically, cellulose synthase-like proteins are necessary for synthesizing cell wall polysaccharides required for root hair elongation in rice. They are considered functional orthologs of CSLD3/KOJAK in the Arabidopsis28. On the other hand, expansin genes contribute to the loosening and extension of plant cell walls by disrupting non-covalent bonds between cellulose microfibrils and the glucan matrix.
In addition to the upregulated miRNAs, the downregulated miRNAs identified in this study also exert significant regulatory roles in modulating the expression of various genes associated with plant defense against pathogens. Notably, seven downregulated miRNAs, namely Novel-miR2, Novel-miR39, Novel-miR9, Tca-MIR472a, Tca-MIR482a, Tca-MIRN9, tcc-miR399a, and tcc-miR396c, were predicted to target disease resistance genes, receptor protein kinases, and cell wall structural components. The downregulation of these miRNAs leads to decreased repression activity, enabling their target transcripts to be regulated normally without any specific treatment. This finding suggests that the reduced expression levels of these miRNAs may contribute to the maintenance of proper gene expression patterns associated with plant resistance mechanisms.
Identifying miRNAs that target their mRNA within the genome provides potential sources of plant resistance by eliminating specific miRNAs, leading to the continuous improvement of resistance genes. Silencing miRNAs using methods such as employing long noncoding RNA (lncRNA) represents an approach to weaken the repressive effects of miRNAs on their target genes7,8. For example, the elimination of miR482/2118s by lncRNAs acting in a 'zig-zag-zig' model of the plant immune system increases the levels of the target gene NBS-LRR which is an important component of ETI29.
Another strategy to inhibit miRNA expression and alleviate gene repression is the utilization of CRISPR/Cas9-based genome editing. In a study conducted by Hong et al. 30, the MIR482 family, which targets the NBS-LRR protein, was successfully knocked out, resulting in enhanced plant resistance to Phytophthora infestans in tomato plants. Interestingly, his study also observed a decrease in reactive oxygen species (ROS) activity in response to plant stress. These findings are consistent with previous report by Hong et al. 31, which demonstrated that upregulation of MIR482c in tomato led to an increase in ROS levels and a decrease in the expression of POD and SOD genes, indicating the involvement of miRNA-mediated regulation in plant stress responses.
Another form of plant defense against pathogen infection is Cross Kingdom RNAi. This defense mechanism involves the transfer of small regulatory RNA molecules into the invading pathogen, subsequently initiating the silencing of virulence genes within the pathogen's genome32. The silencing of these virulence genes critically undermines the pathogen's ability to establish infection, thereby suppressing infection32.
Schaefer et al. 33 also showed that suppression of SvrPm3a1/f1, Bgt_Bcg-6, and Bgt_Bcg-7 effectors in wheat with RNAi can reduce effector expression and increase plant resistance to infection of powdery mildew. Wang et al. 34 also found that the Botrytis can uptake exogenous sRNA which in this study is an sRNA specifically targeting the Bc-DCL1 and Bc-DCL2 genes involved in synthesizing pathogenic effectors. The uptake will then cause repression of effector production thereby suppressing infection by the pathogen. On the Contrary, pathogens do the same by sending sRNAs to plants in self-defense. Wang et al. 35 showed that Botyris cinerea sends Bc-siR37 sRNA into Arabidopsis plants to suppress the immunity. Bc-siR37 is predicted to target several important Arabidopsis genes, such as WRKY transcription factors, receptor-like kinases, and cell wall-modifying enzymes.
Target analysis on P. palmivora transcripts revealed that 9 upregulated miRNAs were predicted to regulate 694 genes. Among them, 12 were identified as RxLR effector genes, which regulated by Novel-miR59, miR3627a, Novel-miR6, miR156b, and miR167a. RXLR effectors represent a group of proteins characterized by RXLR (Arginine-any amino acid-leucine-arginine) motifs highly conserved at the N-terminal while displaying greater diversity at the C-terminal region. The N-terminal segment facilitates the translocation of the effector into the host cell, whereas the C-terminal region is involved in the effector's activity and function36. Pathogens release effectors to counter the plant PTI response. Within the host cell, RxLR effectors will target diverse components within the plant defense system. This can be achieved by disrupting signal transduction pathways or modifying specific proteins functionality37. Upon recognition by host R proteins, RxLR proteins elicit host effector-triggered immunity (ETI) by triggering a hypersensitive response (HR).
Novel-miR6 and Novel-miR18 are also predicted to target Crinkler (CRN) family protein effector genes. CRN effectors are characterized by an N terminus containing a highly conserved LxLFLAK (leucine-any amino acid-phenylalanine-leucine-alanine-lysine) motif, while their C terminus exhibits more sequence diversity. These CRN effectors mainly induce necrosis in host cells38,39. Silencing of these effector genes can suppres P. palmivora infection. Hou et al. 40 demonstrated that targeting effector PSR2 of Phytophthora capsici using siRNA can effectively suppress the infection of P. capsici in Arabidopsis thaliana.
Other miRNAs were also predicted to target important genes in P. palmivora, including genes involved in respiration, glycolysis, protein transport, protein phosphorylation, transcription factors, transmembrane proteins and mRNA processing. These genes play critical roles in various metabolic activities of P. palmivora. Silencing these genes can disrupt the growth of P. palmivora. The study conducted by Hu et al. 41 demonstrated that targeting genes encoding acetyl-CoA, 40S ribosomal protein, and glycine cleavage system in Phakopsora pachyrhizi using dsRNA can effectively suppress the growth of P. pachyrhizi.
miRNAs possess notable environmentally friendly characteristics and exhibit high specificity towards targets, making them a promising candidate to be used in developing plant protection methods with the RNAi approach42. Currently, many RNAi approaches rely on transgenic plants expressing double-stranded RNA43, However, public reaction towards transgenic plants has been unfavorable. Koch et al. 11 demonstrate that applying CYP3-dsRNA that targets Fusarium graminearum CYP3 gene through spraying or Spray-Induced Gene Silencing (SIGS) can effectively repress the infection of F. graminearum on barley. This finding highlights the potential of SIGS as an environmentally friendly alternative for plant protection methods. Therefore, identifying miRNAs that target pathogen effectors provides potential sources of plant resistance by repressing the expression of effector gene, thereby suppressing infection and improving plant resistance against pathogen.
In general, this study also holds potential in contributing to miRNA-based breeding strategies aimed at improving crop traits. miRNAs are known to play a role in various functions in plants, including response to stress, disease resistance regulation, flower development, and grain yield 44. Identification and elimination of disease- and pathogen-associated miRNAs, as well as identification and overexpression of miRNAs associated with crop yield and quality, can serve as effective strategies in molecular breeding. Hong et al. 30 showed that elimination of miR482b and miR482c, which are known to contribute to tomato susceptibility to P. infestans, resulted in increased resistance of tomato plants to P. infestans infection. Zhang et al. 45 also showed that overexpression of miRNA OsmiR397 could increase grain size and promote panicle branching, hence promoting rice yield.
Additionally, miRNAs can also serve as molecular markers in plant breeding programs. Due to their tissue and developmentally-specifics expression patterns, miRNAs can be used as reliable markers for trait selection and identification of elite germplasms. Sihag et al. 46 succeed in developing heat responsive miRNA-SSE markers for characterization of wheat germplasm for terminal heat tolerance breeding. Mondal et al. 47 also successfully identified salt responsive miRNA-SSR markers in rice. The development of miRNA-based molecular markers allows for efficient and precise selection of desired traits, reducing the time and resources required for traditional breeding methods.
In our study, we also identified seven promising miRNAs (Table 3) with potential for further research and development. Among them, five miRNAs, namely Tca-MIR3954a, Novel-miR34, tcc-miR396c, Tca-MIR472a, and Tca-MIR482a, were identified as targets for elimination in T. cacao transcripts. These miRNAs play important roles in pathogen resistance and cell wall formation in cacao plants. By eliminating the suppressive effects on their target genes, it is expected that deletion of these five miRNAs in cacao plants will improve their resistance to pathogens. In addition, two miRNAs, Novel-miR6 and tcc-miR399a, were identified as potential candidates for biocontrol agents against P. palmivora. These miRNAs are predicted to target effector proteins in the pathogen. By applying these synthetic miRNAs to cacao plants, the expression of effector genes in P. palmivora can be targeted and repressed, thus suppressing the infection caused by P. palmivora in cacao plants.
Table 3
Differentially expressed miRNAs that target both their own transcripts and genes in P. palmivora without contradicting each other.
miRNA ID
|
Regulation
|
Target on Theobroma cacao transcripts
|
Target on Phytopthora palmivora transcripts
|
|
Target prediction
|
Protein Function
|
Target Prediction
|
Protein Function
|
Tca-MIR3954a
|
Up
|
Protein chromatin remodeling 19 (LOC18609933) mRNA
|
Plant pathogen resistance
|
CAMK/CDPK protein kinase complete cds
|
Protein phosphorilation
|
Novel-miR34
|
Up
|
Cellulose synthase-like protein D3 (LOC18599512) mRNA
|
Synthesis of a cell wall polysaccharide essential
|
CMGC/DYRK protein kinase complete cds
|
Protein phosphorilation
|
tcc-miR396c
|
Down
|
G-type lectin S-receptor-like serine/threonine-protein kinase LECRK1 (LOC18611782) mRNA
|
Involved in innate immunity.
|
Pre-mRNA-splicing factor SYF1-like protein (PITG_11158) mRNA, complete cds
|
Pre-mRNA splicing
|
Tca-MIR472a
|
Down
|
Putative disease resistance protein At3g14460 (LOC108662475) mRNA
|
Potential disease resistance protein.
|
Transmembrane protein, putative (PITG_19640) complete cds
|
Transmembrane protein
|
Tca-MIR482a
|
Down
|
Probable disease resistance protein At1g12280 (LOC18595262) mRNA
|
Potential disease resistance protein.
|
Cytochrome oxidase subunit I (coxi) gene, partial cds; mitochondrial
|
Cellular respiration
|
Novel-miR6
|
Up
|
RING finger protein 5 (LOC18591332) mRNA
|
Protein ubiquitination
|
Secreted RxLR effector peptide protein, putative (PITG_13593) complete cds
|
Effector that acts as an elicitor of hypersensitive response (HR)
|
tcc-miR399a
|
Down
|
Probable ubiquitin-conjugating enzyme E2 24 (LOC18596849) mRNA
|
Protein ubiquitination
|
Secreted RxLR effector peptide protein, putative (PITG_14984) complete cds
|
Effector that acts as an elicitor of hypersensitive response (HR)
|
The selection of these seven miRNAs from a total of 17 differentially expressed miRNAs was based on their targets in T. cacao transcripts and genes in P. palmivora that are not contradictory in enhancing resistance. This discovery potentially advances our understanding of the molecular mechanisms at the miRNA level in the interactions between cacao plants and the pathogen P. palmivora.