Papaya is a valuable crop that is widely cultivated, but it is vulnerable to Erwinia mallotivora, the pathogen that causes papaya dieback disease (PDD). Although effector and pathogenesis related genes have been identified, little is known about potential resistance genes and pathways involved in the PDD. A dual RNA sequencing (RNA-Seq) provided insights into host resistance or susceptibility to PDD and its pathogenesis. Host-pathogen interactions were evaluated in the Carica papaya varieties Eksotika (susceptible) and Viorica (highly tolerant) pre-inoculation, and at 6, 24, and 48-h post inoculation (HPI) with E. mallotivora using dual RNA-seq analysis. Comparative analysis between the three treatments in the host using DESeq2 revealed 420, 89, and 378 significantly differentially expressed genes (DEGs) at 6, 24 and 48 (HPIs), respectively. A total of 61 DEGs were identified in E. mallotivora at 6 HPI while 429 were identified at 48 HPI. Six putative effectors were found to be highly expressed at 48 HPI, indicating their involvement during late infection. In addition, three putative resistance gene families were highly expressed at 48 HPI, indicating their possible involvement in the resistance and defence mechanisms. The co-expression network analysis of effector (T3SS) genes and transcription factors related to the biotic stress response revealed an interaction between them, suggesting their involvement in the defence mechanism. The KEGG pathway enrichment analysis of DEGs in C. papaya suggests that the diterpenoid biosynthesis pathway and photosynthesis are correlated with defence strategies. The dual RNA-seq analysis enables the identification of candidate genes associated with the pathogenesis mechanism of E. mallotivora thus shedding some light on the discovery events in the stages of bacterial infection of C. papaya by E. mallotivora.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Price excludes VAT (USA)
Tax calculation will be finalised during checkout.
Abu Bakar, N., NormahfuzaHusna, S., Rafidah, B., Shaharuddin, N. A., & Rozeita, L. (2015). iTRAQ proteins analysis of early infected papaya plants with papaya dieback pathogen. Asian Journal of Plant Biology, 3(1), 1–7.
Abu Bakar, N., Barun, L., Rozano, L., Ahmad, M. F. R., Raih, M., & Tarmizi, A. A. (2017). Identification and validation of putative Erwinia mallotivora effectors via quantitative proteomics and real time analysis. Journal of Agricultureand Food Technology, 7(9), 10–21.
Agrofood Statistics (2018). Ministry of Agriculture and Agro-based Industry Malaysia. Retrieved January 14, 2020, from https://www.mafi.gov.my/documents/20182/29034/Perangkaan+Agromakanan+2018.pdf/56b191f9-1e19-4368-8497-b56cf6d7b538
Aliferis, K. A., Chamoun, R., & Jabaji, S. (2015). Metabolic responses of willow (Salix purpurea L.) leaves to mycorrhisation as revealed by mass spectrometry and 1H NMR spectroscopy metabolite profiling. Frontiers in Plant Science, 6, 344.
Ambawat, S., Sharma, P., Yadav, N. R., & Yadav, R. C. (2013). MYB transcription factor genes as regulators for plant responses: An overview. Physiology and Molecular Biology of Plants, 19(3), 307–321.
Anders, S., Pyl, P. T., & Huber, W. (2015). HTSeq—A Python framework to work with high-throughput sequencing data. Bioinformatics, 31(2), 166–169.
Antiabong, J. F., Ngoepe, M. G., & Abechi, A. S. (2016). Semi-quantitative digital analysis of polymerase chain reaction electrophoresis gel: Potential applications in low-income veterinary laboratories. Veterinary World, 9, 935–939.
Aoki, K., Ogata, Y., and Shibata, D. (2007). Approaches for extracting practical information from gene co-expression networks in plant biology. Plant and Cell Physiology, 48(3), 381-390.
Assenov, Y., Ramírez, F., Schelhorn, S. E., Lengauer, T., & Albrecht, M. (2008). Computing topological parameters of biological networks. Bioinformatics, 24(2), 282–284.
Azhar, H. M., Johari, S., Sulastri, J. N., Razali, M., Zulfa, M. M., Faimah, G. N., & Mariatulqabtiah, A. R. (2020). Field performance of selected papaya hybrids for tolerance to dieback disease. Journal of Tropical Agriculture and Food Science, 48(1), 25–33.
Bellezza, I., Peirce, M. J., and Minelli, A. (2014). Cyclic dipeptides: from bugs to brain. Trends in molecular medicine, 20(10), 551-558.
Bolton, M. D. (2009). Primary metabolism and plant defense—fuel for the fire. Molecular plant-microbe Interactions, 22(5), 487-497.
Boutet, E., Lieberherr, D., Tognolli, M., Schneider, M., & Bairoch, A. (2007). Uniprotkb/swiss-prot. In Plant bioinformatics (pp. 89–112). Humana Press.
Boyd, L. A., Ridout, C., O'Sullivan, D. M., Leach, J. E., & Leung, H. (2013). Plant–pathogen interactions: Disease resistance in modern agriculture. Trends in Genetics, 29(4), 233–240.
Cesarino, I. (2019). Structural features and regulation of lignin deposited upon biotic and abiotic stresses. Current Opinion in Biotechnology, 56, 209–214.
Chan, Y. K. (2009). Breeding papaya (Carica papaya L.). In Breeding plantation tree crops: Tropical species (pp. 121–159). Springer.
Dicko, M. H., Gruppen, H., Barro, C., Traoré, A. S., van Berkel, W. J., & Voragen, A. G. (2005). Impact of phenolic compounds and related enzymes in sorghum varieties for resistance and susceptibility to biotic and abiotic stresses. Journal of Chemical Ecology, 31(11), 2671–2688.
Dodds, P. N., & Rathjen, J. P. (2010). Plant immunity: Towards an integrated view of plant–pathogen interactions. Nature Reviews Genetics, 11(8), 539–548.
Dunning, J., Blankley, S., Hoang, L. T., Cox, M., Graham, C. M., James, P. L., & Openshaw, P. J. (2018). Progression of whole-blood transcriptional signatures from interferon-induced to neutrophil-associated patterns in severe influenza. Nature Immunology, 19(6), 625–635.
Fatima, U., and Senthil-Kumar, M. (2015). Plant and pathogen nutrient acquisition strategies. Frontiers in plant science, 6, 750.
FAO. (2019). Major tropical fruits - statistical compendium 2018. Rome. Retrievd January 14, 2020, from http://www.fao.org/3/ca5688en/ca5688en.pdf
Frank, M. R., Deyneka, J. M., & Schuler, M. A. (1996). Cloning of wound-induced cytochrome P450 monooxygenases expressed in pea. Plant Physiology, 110(3), 1035–1046.
Gazi, A. D., Sarris, P. F., Fadouloglou, V. E., Charova, S. N., Mathioudakis, N., Panopoulos, N. J., and Kokkinidis, M. (2012). Phylogenetic analysis of a gene cluster encoding an additional, rhizobial-like type III secretion system that is narrowly distributed among Pseudomonas syringae strains. BMC microbiology, 12(1), 1-15.
Graßmann, J. (2005). Terpenoids as plant antioxidants. Vitamins and Hormones, 72, 505–535.
Gupta, R., Lee, S. E., Agrawal, G. K., Rakwal, R., Park, S., Wang, Y., & Kim, S. T. (2015). Understanding the plant-pathogen interactions in the context of proteomics-generated apoplastic proteins inventory. Frontiers in Plant Science, 6, 352.
Hayden, K. J., Garbelotto, M., Knaus, B. J., Cronn, R. C., Rai, H., & Wright, J. W. (2014). Dual RNA-seq of the plant pathogen Phytophthora ramorum and its tanoak host. Tree Genetics and Genomes, 10(3), 489–502.
Jain, P., Singh, P. K., Kapoor, R., Khanna, A., Solanke, A. U., Krishnan, S. G., & Sharma, T. R. (2017). Understanding host-pathogen interactions with expression profiling of NILs carrying rice-blast resistance Pi9 gene. Frontiers in Plant Science, 8, 93.
Jones, J. D., & Dangl, J. L. (2006). The plant immune system. Nature, 444(7117), 323–329.
Juri, N. M., Samsuddin, A. F., Abdul-Murad, A. M., Tamizi, A. A., Shaharuddin, N. A., & Abu-Bakar, N. (2020). Discovery of pathogenesis related and effector genes of Erwinia mallotivora in Carica papaya (Eksotika I) seedlings via transcriptomic analysis. Internatinal Journal of Agriculture Biology,23, 1021–1032.
Kanehisa, M., Sato, Y., Kawashima, M., Furumichi, M., & Tanabe, M. (2016). KEGG as a reference resource for gene and proteinannotation. Nucleic Acids Research, 44(D1), D457–D462.
Khalaf, A. A., Gmitter, F. G., Conesa, A., Dopazo, J., and Moore, G. A. (2011). Fortunella margarita transcriptional reprogramming triggered by Xanthomonas citri subsp. citri. BMC plant biology, 11(1), 1-17.
Kim, D., Langmead, B., and Salzberg, S. L. (2015). HISAT: a fast spliced alignerwith low memory requirements. Nature Methods, 12, 357–360.
Kovalchuk, A., Zeng, Z., Ghimire, R. P., Kivimäenpää, M., Raffaello, T., & Liu, M.,& Asiegbu, F. O. (2019). Dual RNA-seq analysis provides new insights into interactions between Norway spruce and necrotrophic pathogen Heterobasidion annosum sl. BMC Plant Biology, 19(1), 1–17.
Krishnan, P., Bhat, R., Kush, A., & Ravikumar, P. (2012). Isolation and functional characterisation of bacterial endophytes from Carica papaya fruits. Journal of Applied Microbiology, 113(2), 308–317.
Kunjeti, S. G., Evans, T. A., Marsh, A. G., Gregory, N. F., Kunjeti, S., Meyers, B. C., & Donofrio, N. M. (2012). RNA-Seq reveals infection-related global gene changes in Phytophthora phaseoli, the causal agent of lima bean downy mildew. Molecular Plant Pathology, 13(5), 454–466.
Lee, M. H., Jeon, H. S., Kim, S. H., Chung, J. H., Roppolo, D., Lee, H. J., Cho, H. J., Tobimatsu, Y., Ralph, J., & Park, O. K. (2019). Lignin-based barrier restricts pathogens to the infection site and confers resistance in plants. The EMBO Journal, 38(23), e101948.
Li, H., Zhou, Y., & Zhang, Z. (2017). Network analysis reveals a common host–pathogen interaction pattern in Arabidopsis immune responses. Frontiers in Plant Science, 8, 893.
Liang, Q., Seo, G. J., Choi, Y. J., Kwak, M. J., Ge, J., Rodgers, M. A., & Jung, J. U. (2014). Crosstalk between the cGAS DNA sensor and Beclin-1 autophagy protein shapes innate antimicrobial immune responses. Cell Host and Microbe, 15(2), 228–238.
Liao, Z. X., Ni, Z., Wei, X. L., Chen, L., Li, J. Y., Yu, Y. H., & Huang, S. (2019). Dual RNA-seq of Xanthomonas oryzae pv. Oryzicola infecting rice reveals novel insights into bacterial-plant interaction. PLoS ONE, 14(4), e0215039.
Ling, A. C. K., Zaidan, M. W. A. M., Wee, C.-Y., Rozano, L., Supian, S., & Halim, M. M. A. (2016). A transcriptome next-generation sequencing of Carica papaya. In Proceedings of the 3rd Plant Genomics Congress, Kuala Lumpur (pp. 11–12).
Liu, J., Ding, P., Sun, T., Nitta, Y., Dong, O., Huang, X., & Zhang, Y. (2013). Heterotrimeric G proteins serve as a converging point in plant defense signaling activated by multiple receptor-like kinases. Plant Physiology, 161(4), 2146–2158.
Liu, L., Tang, X. F., Wu, Y. M., Huang, Y. B., and Tang, Y. X. (2008). The roles of MYB transcription factors on plant defense responses and its molecular mechanism. Yi Chuan Hereditas, 30(10), 1265-1271
Liu, W., Liu, J., Triplett, L., Leach, J. E., & Wang, G. L. (2014). Novel insights into rice innate immunity against bacterial and fungal pathogens. Annual Review of Phytopathology, 52, 213–241.
Lonjon, F., Turner, M., Henry, C., Rengel, D., Lohou, D., van de Kerkhove, Q.,and Vailleau, F. (2016). Comparative secretome analysis of Ralstonia solanacearum type 3 secretion-associated mutants reveals a fine control of effector delivery, essential for bacterial pathogenicity. Molecular and Cellular Proteomics, 15(2), 598-613.
Lowe, R. G., Cassin, A., Grandaubert, J., Clark, B. L., Van de Wouw, A. P., Rouxel, T., & Howlett, B. J. (2014). Genomes and transcriptomes of partners in plant-fungal-interactions between canola (Brassica napus) and two Leptosphaeria species. PLoS One, 9(7), e103098.
Lozovaya, V. V., Lygin, A. V., Zernova, O. V., Ulanov, A. V., Li, S., Hartman, G. L., & Widholm, J. M. (2007). Modification of phenolic metabolism in soybean hairy roots through down regulation of chalcone synthase or isoflavone synthase. Planta, 225(3), 665–679.
Maktar, N. H., Kamis, S., Mohd Yusof, F. Z., & Hussain, M. H. (2008). Erwinia papayae causing papaya dieback in Malaysia. Plant Pathology, 57(4), 774.
Malinovsky, F. G., Fangel, J. U., & Willats, W. G. (2014). The role of the cell wall in plant immunity. Frontiers in Plant Science, 5, 178.
Martin, M. (2011). Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet. Journal, 17(1), 10–12.
Mat Amin, N., Bunawan, H., Redzuan, R. A., & Jaganath, I. B. S. (2011). Erwinia mallotivora sp., a new pathogen of papaya (Carica papaya) in peninsular Malaysia. International Journal of Molecular Sciences, 12(1), 39–45.
Mata-Pérez, C., Sánchez-Calvo, B., Begara-Morales, J. C., Luque, F., Jiménez-Ruiz, J., Padilla, M. N., & Barroso, J. B. (2015). Transcriptomic profiling of linolenic acid-responsive genes in ROS signaling from RNA-seq data in Arabidopsis. Frontiers in Plant Science, 6, 122.
Miyamoto, T., Uemura, T., Nemoto, K., Daito, M., Nozawa, A., Sawasaki, T., and Arimura, G. I. (2019). Tyrosine kinase-dependent defense responses against herbivory in Arabidopsis. Frontiers in plant science, 776.
Mohd Taha, M. D., Mohd Jaini, M. F., Saidi, N. B., Abdul Rahim, R., Md Shah, U. K., & Mohd Hashim, A. (2019). Biological control of Erwinia mallotivora, the causal agent of papaya dieback disease by indigenous seed-borne endophytic lactic acid bacteria consortium. PLoS One, 14(12), e0224431.
Nguyen, Q. M., Iswanto, A. B. B., Son, G. H., & Kim, S. H. (2021). Recent advances in effector-triggered immunity in plants: New pieces in the puzzle create a different paradigm. International Journal of Molecular Sciences, 22(9), 4709.
O’Hare, T. J., & Williams, D. J. (2014). Papaya as a medicinal plant. In Genetics and genomics of papaya (pp. 391–407). Springer.
Pan, Q., Zhan, J., Liu, H., Zhang, J., Chen, J., Wen, P., & Huang, W. (2006). Salicylic acid synthesized by benzoic acid 2-hydroxylase participates in the development of thermotolerance in pea plants. Plant Science, 171(2), 226–233.
Persans, M. W., Wang, J., & Schuler, M. A. (2001). Characterization of maize cytochrome P450 monooxygenases induced in response to safeners and bacterial pathogens. Plant Physiology, 125(2), 1126–1138.
Pertea, M., Pertea, G. M., Antonescu, C. M., Chang, T. C., Mendell, J. T., & Salzberg, S. L. (2015). StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nature Biotechnology, 33(3), 290–295.
Rajesh, M. K., Rachana, K. E., Kulkarni, K., Sahu, B. B., Thomas, R. J., & Karun, A. (2018). Comparative transcriptome profiling of healthy and diseased Chowghat Green Dwarf coconut palms from root (wilt) disease hot spots. European Journal of Plant Pathology, 151(1), 173–193.
Redzuan, R. A., Abu Bakar, N., Rozano, L., Badrun, R., Mat Amin, N., & Mohd Raih, M. F. (2014). Draft genome sequence of Erwinia mallotivora BT-MARDI, causative agent of papaya dieback disease. Genome Announcements, 2(3), e00375–e00314.
Rojas, C. M., Senthil-Kumar, M., Tzin, V., & Mysore, K. (2014). Regulation of primary plant metabolism during plant-pathogen interactions and its contribution to plant defense. Frontiers in Plant Science, 5, 17.
Roshidi, A. S. (2010). Papaya disease alert. The Star Online.
Rozano, L., Abdullah-Zawawi, M. R., Supian, S., Wee, C.-Y., & Ling, A. C. K. (2015). In silico functional analysis of lincRNAs in Carica papaya and its role in plant defense. In Proceedings of the 2nd Plant Genomics Congress Asia, Kuala Lumpur (pp. 19–20).
Rozano, L., Ling, A. C. K., Supian, S., & Wee, C.-Y. (2016). Computational prediction on the role of hypothetical genes in papaya during dieback infection. In Proceedings of the 3rd Plant Genomics Congress, Kuala Lumpur (pp. 11–12).
Schenk, A., Weingart, H., & Ullrich, M. S. (2008). Extraction of high-quality bacterial RNA from infected leaf tissue for bacterial in planta gene expression analysis by multiplexed fluorescent northern hybridisation. Molecular Plant Pathology, 9, 227–235.
Schneider, C. A., Rasband, W. S., & Eliceiri, K. W. (2012). NIH image to ImageJ: 25 years of image analysis. Nature Methods, 9, 671–675.
Sekeli, R., Hamid, M. H., Razak, R. A., Wee, C. Y., & Ong-Abdullah, J. (2018). Malaysian Carica papaya L. var. Eksotika: Current research strategies fronting challenges. Frontiers in Plant Science, 9, 1380.
Seo, E., & Choi, D. (2015). Functional studies of transcription factors involved in plant defenses in the genomics era. Briefings in Functional Genomics, 14(4), 260–267.
Takafuji, K., Rim, H., Kawauchi, K., Mujiono, K., Shimokawa, S., Ando, Y., and Arimura, G. I. (2020). Evidence that ERF transcriptional regulators serve as possible key molecules for natural variation in defense against herbivores in tall goldenrod. Scientific reports, 10(1), 1-12.
Tang, D., Wang, G., & Zhou, J. M. (2017). Receptor kinases in plant-pathogen interactions: More than pattern recognition. The Plant Cell, 29(4), 618–637.
Tatusov, R. L., Fedorova, N. D., Jackson, J. D., Jacobs, A. R., Kiryutin, B., Koonin, E. V., & Natale, D. A. (2003). The COG database: An updated version includes eukaryotes. BMC Bioinformatics, 4(1), 1–14.
Teixeira, P. J. P. L., Thomazella, D. P. D. T., Reis, O., do Prado, P. F. V., do Rio, M. C. S., Fiorin, G. L., & Pereira, G. A. G. (2014). High-resolution transcript profiling of the atypical biotrophic interaction between Theobroma cacao and the fungal pathogen Moniliophthora perniciosa. The Plant Cell, 26(11), 4245–4269.
Teutsch, H. G., Hasenfratz, M. P., Lesot, A., Stoltz, C., Garnier, J. M., Jeltsch, J. M., Durst, F., & Werck-Reichhart, D. (1993). Isolation and sequence of a cDNA encoding the Jerusalem artichoke cinnamate 4-hydroxylase, a major plant cytochrome P450 involved in the general phenylpropanoid pathway. Proceedings of the National Academy of Sciences, 90(9), 4102–4106.
Toffolatti, S. L., De Lorenzis, G., Brilli, M., Moser, M., Shariati, V., Tavakol, E., et al. (2020). Novel aspects on the interaction between grapevine and Plasmopara viticola: Dual-RNA-Seq analysis highlights gene expression dynamics in the pathogen and the plant during the battle for infection. Genes, 11(3), 261.
Venegas-Molina, J., Proietti, S., Pollier, J., Orozco-Freire, W., Ramirez-Villacis, D., & Leon-Reyes, A. (2020). Induced tolerance to abiotic and biotic stresses of broccoli and Arabidopsis after treatment with elicitor molecules. Scientific Reports, 10(1), 1–17.
Wang, P., Zhao, Y., Li, Z., Hsu, C. C., Liu, X., Fu, L., & Zhu, J. K. (2018). Reciprocal regulation of the TOR kinase and ABA receptor balances plant growth and stress response. Molecular Cell, 69(1), 100–112.
Westermann, A. J., Barquist, L., & Vogel, J. (2017). Resolving host–pathogen interactions by dual RNA-seq. PLoS Pathogens, 13(2), e1006033.
Wu, J., Deng, Y., Hu, J., Jin, C., Zhu, X., & Li, D. (2020). Genome-wide analyses of direct target genes of an ERF11 transcription factor involved in plant defense against bacterial pathogens. Biochemical and Biophysical Research Communications, 532(1), 76–81.
Yahya, M., Rulli, M., Toivonen, L., Waris, M., & Peltola, V. (2017). Detection of host response to viral respiratory infection by measurement of messenger RNA for MxA, TRIM21, and viperin in nasal swabs. The Journal of Infectious Diseases, 216(9), 1099–1103.
Zainal Abidin, R., Norliza, A. B., Fairuz, Y. M., Norzihan, A., & Kalsom, A. U. (2016). Sequence information on single nucleotide polymorphism (SNP) through genome sequencing analysis of Carica papaya variety Eksotika and Sekaki. Journal of Tropical Agriculture and Food Science, 44(2), 219–228.
Zamora-Ballesteros, C., Pinto, G., Amaral, J., Valledor, L., Alves, A., Diez, J. J., & Martín-García, J. (2021). Dual RNA-sequencing analysis of resistant (Pinus pinea) and susceptible (Pinus radiata) hosts during fusarium circinatum challenge. International Journal of Molecular Sciences, 22(10), 5231.
Zipfel, C. (2014). Plant pattern-recognition receptors. Trends in Immunology, 35(7), 345–351.
This research was supported by the Malaysian Agricultural Research and Development Institute (MARDI) (PRB-405).
Conflict of interest
This manuscript has not been submitted to any other journal for simultaneous consideration. All authors declared that they have no conflicts of interest, consented to the manuscript submission, and have contributed sufficiently to the scientific work and therefore share collective responsibility and accountability for it.
Research involving human participants and/or animals
There was no research involving human participants and/or animals carried out for this submission.
Rights and permissions
About this article
Cite this article
Md Saad, M., Zainal-Abidin, RA., Hassan, M.A. et al. New insights into host-pathogen interactions in papaya dieback disease caused by Erwinia mallotivora in Carica papaya. Eur J Plant Pathol 163, 393–413 (2022). https://doi.org/10.1007/s10658-022-02484-z
- Erwinia mallotivora
- Carica papaya
- Dual RNA-seq
- Effector gene
- Papaya dieback disease
- Resistance gene