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Molecular Pathogens (online), 2024, Vol. 15 ISSN 1925-1998 http://microbescipublisher.com/index.php/mp © 2024 MicroSci Publisher, an online publishing platform of Sophia Publishing Group. All Rights Reserved. Sophia Publishing Group (SPG), founded in British Columbia of Canada, is a multilingual publisher. Latest Content 2024, Vol.15, No.4 【Review Article】 Microbial Warriors: Using Predatory Bacteria to Combat Pathogens 170-178 JimMason DOI: 10.5376/mp.2024.15.0016 【Research Insight】 Transcriptomic Insights into Wheat Disease Resistance 179-188 Xinguang Cai, Qiangsheng Qian DOI: 10.5376/mp.2024.15.0017 Emerging Viral and Mycoviral Threats to Rice Cultivation 189-199 Danyan Ding DOI: 10.5376/mp.2024.15.0018 【Feature Review】 FusariumBoll Rot in Cotton: Pathogen Dynamics and Control Options 200-208 ZhenLi DOI: 10.5376/mp.2024.15.0019 【Review and Progress】 Mechanisms of Immune Evasion by African Swine Fever Virus: An Integrated Review 209-218 Xiaofang Lin DOI: 10.5376/mp.2024.15.0020
Molecular Pathogens 2024, Vol.15, No.4, 170-178 http://microbescipublisher.com/index.php/mp 170 Review Article Open Access Microbial Warriors: Using Predatory Bacteria to Combat Pathogens JimMason The HITAR Institute Canada, British Columbia, Canada Corresponding email: jim.mason@hitar.org Molecular Pathogens, 2024, Vol.15, No.4 doi: 10.5376/mp.2024.15.0016 Received: 16 May, 2024 Accepted: 22 Jun., 2024 Published: 08 Jul., 2024 Copyright © 2024 Mason, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Mason J., 2024, Microbial warriors: using predatory bacteria to combat pathogens, Molecular Pathogens, 15(4): 170-178 (doi: 10.5376/mp.2024.15.0016) Abstract Predatory bacteria have garnered increasing attention in pathogen control research due to their unique predatory mechanisms. This study provides an overview of the historical background of microbial predation and the discovery of predatory bacteria, focusing on the mechanisms of bacterial predation, including predator-prey cellular interactions and metabolic adaptations. Both in vitro and in vivo studies have demonstrated the significant effectiveness of these predatory bacteria in eliminating multidrug-resistant pathogens, particularly highlighting their potential in biofilm-related infections. Although predatory bacteria show promise for clinical applications, challenges such as prey resistance, environmental factors, and safety concerns still require further investigation and resolution. In the future, genetic engineering, applications in agriculture and veterinary medicine, and the integration with bioengineering and nanotechnology will pave new pathways for the application of predatory bacteria. This study aims to enhance the potential of predatory bacteria through these innovative approaches, ultimately providing a basis for their clinical use as therapeutic agents. Keywords Predatory bacteria; Pathogen control; Multidrug resistance; Biofilm infections; Genetic engineering 1 Introduction Microbial predation is a fundamental ecological process that significantly influences the structure and dynamics of microbial communities. Predatory bacteria, such as myxobacteria and Bdellovibrio bacteriovorus, employ various strategies to hunt and consume other microorganisms, including bacteria and fungi. These predators can secrete antibiotic metabolites and hydrolytic enzymes to lyse their prey, releasing nutrients into the environment (Korp et al., 2016; Sydney et al., 2021). Predatory bacteria are found in diverse environments, from soil and water to marine ecosystems and even within host-associated microbiomes, where they can regulate community structure and potentially protect hosts from pathogenic bacteria. The study of bacterial predation dates back over 75 years, beginning with the investigation of myxobacteria. Since then, numerous predatory strains and their hunting strategies have been identified, revealing the widespread distribution and ecological significance of these organisms (Pérez et al., 2016). Bdellovibrio bacteriovorus, for example, was discovered to invade and kill Gram-negative bacteria, including antibiotic-resistant pathogens, making it a potential candidate for therapeutic applications (Negus et al., 2017; Madhusoodanan, 2019). Recent discoveries have also highlighted novel predatory groups, such as Bradymonabacteria, which exhibit unique survival strategies in saline environments (Mu et al., 2020). This study aims to provide a comprehensive overview of the current understanding of predatory bacteria and their potential applications in combating pathogenic microorganisms. We will explore the mechanisms of predation, the ecological roles of predatory bacteria, and their interactions with prey and other microbial community members. Additionally, we will discuss the potential of using predatory bacteria as an alternative to traditional antibiotics in the fight against antibiotic-resistant pathogens. 2 Mechanisms of Predation by Bacteria 2.1 Classification of predatory bacteria Predatory bacteria can be classified into three main groups based on their dependency on prey for survival: obligate predators, facultative predators, and opportunistic predators. Obligate predators, such as Bdellovibrio
Molecular Pathogens 2024, Vol.15, No.4, 170-178 http://microbescipublisher.com/index.php/mp 171 bacteriovorus, are completely dependent on prey for their growth and reproduction. Facultative predators, like Myxococcus xanthus, can survive on prey but also thrive on other nutrient sources. Opportunistic predators, such as Bradymonabacteria, can live independently of prey but will exploit prey when available (Mu et al., 2020). 2.2 Predator-prey interactions at the cellular level Predatory bacteria employ various strategies to interact with and kill their prey. For instance, Bdellovibrio bacteriovorus attaches to the exterior of Gram-negative prey cells, enters the periplasm, and consumes the host's resources before lysing the cell to find new prey. Myxococcus xanthus, on the other hand, secretes antibiotic metabolites and hydrolytic enzymes that lyse prey organisms, releasing nutrients into the extracellular environment (Sydney et al., 2021). These interactions often lead to significant changes in the prey's genome and phenotypic traits, as seen in coevolving communities of Myxococcus xanthus and Escherichia coli (Nair et al., 2019). 2.3 Metabolic adaptations for predation Predatory bacteria have evolved various metabolic adaptations to facilitate their predatory lifestyle. For example, Bradymonabacteria can synthesize polymers like polyphosphate and polyhydroxyalkanoates, which may aid in their survival and predation in saline environments. Myxococcus xanthus produces a range of secondary metabolites, including antibiotics, which are used as predatory weapons. These metabolic capabilities not only support their predatory activities but also allow them to adapt to different environmental conditions. 2.4 Ecological role of predatory bacteria in natural environments Predatory bacteria play a crucial role in shaping microbial community structures and dynamics. They influence the composition and diversity of microbial ecosystems by selectively preying on specific bacteria, thereby controlling bacterial populations and promoting biodiversity. In marine environments, predatory bacteria like Halobacteriovorax are prevalent on coral surfaces and help regulate the microbiome by preying on potential pathogens. Predatory bacteria can transform the landscape of biofilms, affecting the spatial community ecology and assembly processes (Wucher et al., 2021). Their presence and activity are essential for maintaining the balance and health of various ecosystems (Welsh et al., 2015; Pérez et al., 2016). 3 Key Predatory Bacterial Species 3.1 Bdellovibrio bacteriovorus Bdellovibrio bacteriovorus is a small Deltaproteobacterium known for its unique ability to prey on other Gram-negative bacteria. This predatory bacterium has garnered significant attention due to its potential application as a "living antibiotic" to combat antibiotic-resistant pathogens. B. bacteriovorus invades the periplasmic space of its prey, where it digests host resources and proliferates, eventually releasing multiple daughter cells to continue the predation cycle (Figure 1) (Laloux, 2020; Cavallo et al., 2021). Studies have shown that B. bacteriovorus can significantly reduce the viability of microbial communities, such as those found in activated sludge, by altering their composition and reducing biomass (Feng et al., 2017). The bacterium's ability to secrete nucleases during its predatory cycle helps degrade prey DNA, potentially reducing the spread of antibiotic resistance genes (Bukowska-Faniband et al., 2020). The broad host range and the ability to kill many antibiotic-resistant pathogens make B. bacteriovorus a promising candidate for therapeutic applications. 3.2 Myxococcus xanthus Myxococcus xanthus is a well-characterized myxobacterium that preys on a wide range of Gram-negative and Gram-positive bacteria, as well as fungi. This predatory bacterium employs a generalist predatory mechanism involving the secretion of antibiotic metabolites and hydrolytic enzymes, which lyse prey organisms and release nutrients into the extracellular environment (Negus et al., 2017; Findlay et al., 2019). M. xanthus has been studied extensively for its predation strategies and the molecular responses of prey organisms. Research has identified several genes in prey bacteria, such as Pseudomonas aeruginosa, that contribute to resistance against M. xanthus predation. These genes are involved in metal/oxidative stress response, motility, and detoxification of antimicrobial peptides. The broad prey range and the ability to overcome various resistance mechanisms make M. xanthus an important model organism for studying bacterial predation.
Molecular Pathogens 2024, Vol.15, No.4, 170-178 http://microbescipublisher.com/index.php/mp 172 Figure 1 TEM images of various stages of predation (Adopted from Cavallo et al., 2021) Image caption: Images I, II and III showB. bacteriovorus HD100 (indicated with arrows) attached to the outer surface of a prey cell or in its immediate surroundings. Image IV shows a late stage of predation where the new-born predators are in the bdelloplast, prior to its disruption (Adopted from Cavallo et al., 2021) 3.3 Other emerging predatory bacterial species In addition to Bdellovibrio bacteriovorus and Myxococcus xanthus, other predatory bacteria are emerging as potential biocontrol agents. For instance, Bacteriovorax stolpii HI3 and Myxococcus sp. MH1 have been isolated from freshwater environments and characterized for their predation capabilities. B. stolpii HI3 exhibits rapid and extensive predation on a wide spectrum of Gram-negative bacteria, although prey bacteria can regrow through phenotypic resistance. In contrast, Myxococcus sp. MH1 shows lower predation efficiency but longer-lasting effects (Osińska et al., 2020; Inoue et al., 2022). These findings highlight the diverse predation strategies and environmental preferences of different predatory bacteria, suggesting their potential for biotechnological applications in various settings. 4 Predatory Bacteria as a Tool Against Pathogens 4.1 Mechanisms of action against pathogens Predatory bacteria, such as Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus, exhibit unique mechanisms to combat pathogens. These bacteria prey on Gram-negative bacteria by attaching to their prey, penetrating their outer membrane, and consuming their cellular contents. This process involves a free-living, invasive attack phase followed by an intracellular reproductive phase, where the predator degrades the host's macromolecules and reuses them for its own growth (Figure 2) (Madhusoodanan, 2019; Makowski et al., 2019). Mathematical models have been instrumental in understanding these mechanisms, allowing researchers to predict the dynamics of predator-prey interactions and the potential effectiveness of predatory bacteria in various environmental conditions (Summers and Kreft, 2022). The study by Makowski et al. (2019) revealed the unique mechanism by which carnivorous Bdellovibrio grow and reproduce through the degradation and utilization of host resources, providing new insights for the development of novel antimicrobial therapies, particularly in addressing multidrug-resistant pathogens. These predatory bacteria hold promise as "living antibiotics" for combating various pathogenic infections.
Molecular Pathogens 2024, Vol.15, No.4, 170-178 http://microbescipublisher.com/index.php/mp 173 Figure 2 Spatiotemporal analysis of chromosome replication in a B. bacteriovorus cell growing in a bdelloplast (Adopted from Makowski et al., 2019) Image caption: (A) Free-living predatory and host cell. (B) Attachment of B. bacteriovorus to an E. coli cell. (C) Bdelloplast formation. (D) Appearance of the first replisome focus at pilus pole of B. bacteriovorus cell-the start of chromosome replication. (E and F) Further growth and chromosome replication. (G) Termination of predatory chromosome replication. (H) The beginning of B. bacteriovorus filament septation. (E) The release of progeny cells from the bdelloplast (Adopted from Makowski et al., 2019) 4.2 Effectiveness in preclinical studies 4.2.1 In vitro studies: targeting multidrug-resistant bacteria In vitro studies have demonstrated the potential of predatory bacteria to target and kill multidrug-resistant pathogens. For instance, Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus have been shown to effectively reduce bacterial populations in controlled environments, highlighting their potential as a tool against antibiotic-resistant bacteria. These studies underline the non-cytotoxic nature of predatory bacteria on human cell lines, further supporting their safety and efficacy as therapeutic agents (Gupta et al., 2016).
Molecular Pathogens 2024, Vol.15, No.4, 170-178 http://microbescipublisher.com/index.php/mp 174 4.2.2 In vivo studies: animal models demonstrating pathogen clearance In vivo studies have provided promising results regarding the safety and efficacy of predatory bacteria. For example, the intravenous administration of Bdellovibrio bacteriovorus in rats demonstrated that these bacteria are non-toxic and do not cause adverse histopathological effects. Although an initial increase in pro-inflammatory cytokines was observed, levels returned to baseline within 18 hours, indicating a transient immune response (Shatzkes et al., 2017). However, while predatory bacteria were able to reduce bacterial burden in the lungs, they were less effective in systemic infections, such as those caused byKlebsiella pneumoniae in the bloodstream. 4.3 Therapeutic applications and clinical potential 4.3.1 Potential use in human infectious diseases The potential use of predatory bacteria in treating human infectious diseases is gaining traction. Studies have shown that these bacteria are non-pathogenic to human cells and can effectively target and kill multidrug-resistant pathogens (Gupta et al., 2016; Mitchell et al., 2020). This positions predatory bacteria as a promising alternative or adjunct to traditional antibiotics, especially in cases where conventional treatments fail due to resistance. 4.3.2 Applications in biofilm-related infections Biofilm-related infections pose a significant challenge due to their resistance to antibiotics. Predatory bacteria have shown potential in disrupting biofilms and reducing bacterial load within these structures. By penetrating and consuming the bacteria within biofilms, predatory bacteria could offer a novel approach to treating these persistent infections (Madhusoodanan, 2019). 4.4 Synergistic effects with traditional antibiotics Combining predatory bacteria with traditional antibiotics could enhance the overall effectiveness of treatment. This synergistic approach may help in reducing bacterial resistance and improving patient outcomes. Studies suggest that while predatory bacteria alone are effective, their combination with antibiotics could provide a more comprehensive strategy to combat multidrug-resistant infections (Tyson and Sockett, 2017; Liu et al., 2024). 5 Challenges and Limitations 5.1 Resistance development by prey organisms One of the primary concerns with the use of predatory bacteria as a therapeutic tool is the potential for prey organisms to develop resistance. Although predatory bacteria like Bdellovibrio bacteriovorus have co-evolved with their prey, making it difficult for pathogens to resist through simple mutations, the possibility of resistance development cannot be entirely ruled out. Predatory bacteria encode diverse predatory enzymes that are hard for pathogens to resist by simple mutation (Negus et al., 2017). However, the rapid appearance of mutations that confer resistance to other antibacterial agents, such as colicins, suggests that similar mechanisms could potentially arise against predatory bacteria (Upatissa et al., 2023). Therefore, continuous monitoring and research are essential to understand and mitigate the risk of resistance development. 5.2 Environmental factors affecting predatory efficacy The efficacy of predatory bacteria can be significantly influenced by environmental factors. For instance, the presence of certain nutrients, pH levels, and temperature can affect the predatory activity of Bdellovibrio bacteriovorus. Studies have shown that predatory bacteria are effective in vitro, but their performance in vivo can vary due to the complex interactions within a host's body (Shatzkes et al., 2016). Mathematical models have been used to predict the dynamics of predator-prey systems under various environmental conditions, highlighting the importance of understanding these factors to optimize the use of predatory bacteria. Therefore, further research is needed to identify and control environmental variables that could impact the effectiveness of predatory bacteria in different settings. 5.3 Regulatory and safety concerns for clinical applications The introduction of live predatory bacteria as a therapeutic agent raises several regulatory and safety concerns. Although studies have demonstrated that predatory bacteria are non-toxic and non-immunogenic in rodent models
Molecular Pathogens 2024, Vol.15, No.4, 170-178 http://microbescipublisher.com/index.php/mp 175 (Gupta et al., 2016), the long-term effects and safety in humans remain to be fully assessed. Regulatory bodies will require extensive data on the safety, efficacy, and potential side effects of using live bacteria in clinical settings. Additionally, the potential for horizontal gene transfer and the impact on the native microbiome are critical factors that need thorough investigation (Bukowska-Faniband et al., 2020). Ensuring that predatory bacteria do not disrupt the host's normal microbial flora or cause unintended consequences is paramount for their acceptance and use in clinical applications. 6 Future Directions and Innovations 6.1 Genetic engineering to enhance predatory capabilities The genetic engineering of predatory bacteria, such as Bdellovibrio bacteriovorus, holds significant promise for enhancing their predatory capabilities. Recent studies have identified and characterized numerous genes essential for the predation process, which opens the door to genetic modifications aimed at improving the efficiency and specificity of these microbial warriors. For instance, high-throughput genetic screens have revealed over 100 genes specifically required for predative growth on human pathogens like Vibrio cholerae and Escherichia coli in both planktonic and biofilm states (Duncan et al., 2019). By targeting these genes, researchers can potentially engineer B. bacteriovorus strains with enhanced killing rates and the ability to target specific bacterial species or states more effectively. Additionally, the sequential release of nucleases during the predatory cycle, as characterized in other studies, provides further molecular targets for genetic enhancement (Livingstone et al., 2018; Song et al., 2024). These advancements could lead to the development of more potent and precise predatory bacteria, offering a viable alternative to traditional antibiotics in the fight against antibiotic-resistant pathogens. 6.2 Application in agriculture and veterinary medicine The application of predatory bacteria in agriculture and veterinary medicine represents a promising avenue for reducing the reliance on chemical antibiotics and mitigating the spread of antibiotic resistance. Bdellovibrio bacteriovorus has demonstrated the ability to kill a broad range of Gram-negative bacteria, including many that are pathogenic to plants and animals (Negus et al., 2017). By integrating these predatory bacteria into agricultural practices, it may be possible to control bacterial infections in crops and livestock more sustainably. This approach not only helps in managing diseases but also reduces the overall pool of antibiotic resistance genes in the environment, as predatory bacteria can degrade exogenous DNA through the secretion of nucleases (Bukowska-Faniband et al., 2020). Future research should focus on optimizing the delivery and efficacy of predatory bacteria in various agricultural and veterinary settings, ensuring that they can be effectively deployed to protect plant and animal health. 6.3 Integration with bioengineering and nanotechnology The integration of predatory bacteria with bioengineering and nanotechnology offers innovative solutions to combat multidrug-resistant (MDR) bacteria. Nanotechnology, in particular, has shown great potential in enhancing the delivery and effectiveness of antimicrobial agents. The development of nanomaterial-based therapeutics can overcome current pathways linked to acquired drug resistance and target biofilms, which are notoriously difficult to treat with conventional antibiotics (Hetta et al., 2023). By combining the predatory capabilities of Bdellovibrio bacteriovorus with nanotechnology, it may be possible to create synergistic treatments that are more effective against MDR infections. For example, nanoparticles can be engineered to deliver predatory bacteria directly to infection sites, enhancing their ability to target and kill pathogenic bacteria (Johnke et al., 2017). This multidisciplinary approach could lead to the development of next-generation antimicrobial therapies that leverage the strengths of both biological and nanotechnological innovations. 7 Concluding Remarks The exploration of predatory bacteria, particularly Bdellovibrio bacteriovorus, as potential therapeutic agents against antibiotic-resistant pathogens has yielded promising results. These bacteria have demonstrated the ability to effectively prey on a wide range of Gram-negative bacteria, including multi-drug-resistant strains such as Salmonella, Escherichia coli, and Yersinia pestis. Studies have shown that B. bacteriovorus can persist within
Molecular Pathogens 2024, Vol.15, No.4, 170-178 http://microbescipublisher.com/index.php/mp 176 human phagocytic cells without affecting host cell viability, suggesting a potential for safe therapeutic use. Additionally, predatory bacteria have been found to be non-toxic and non-immunogenic in human cell lines and animal models, further supporting their safety profile. Importantly, these bacteria have shown efficacy in reducing bacterial burdens in vivo, as evidenced by their ability to protect mice from lethal bacterial challenges and reduce Klebsiella pneumoniae burden in rat lungs. While the current findings are encouraging, several areas require further investigation to fully realize the therapeutic potential of predatory bacteria. Detailed molecular studies are needed to understand the predatory mechanisms and the sequential release of enzymes during the predatory cycle. This knowledge will be crucial for optimizing the use of predatory bacteria as therapeutic agents. Investigating the interactions between predatory bacteria and host immune cells will provide insights into their persistence, immune evasion, and potential immunomodulatory effects. Long-term studies on the genetic stability of predatory bacteria are essential to ensure that they do not acquire pathogenic traits through horizontal gene transfer. Rigorous clinical trials are necessary to evaluate the safety, efficacy, and optimal dosing regimens of predatory bacteria in human subjects. Expanding the range of pathogens tested, including those with different resistance mechanisms, will help establish the broad-spectrum efficacy of predatory bacteria. The potential of predatory bacteria as therapeutic agents represents a novel and promising approach to combating antibiotic-resistant infections. Their unique mode of action, which involves the direct predation and elimination of pathogenic bacteria, offers a complementary strategy to traditional antibiotics. The ability of predatory bacteria to reduce bacterial burdens in vivo without causing significant adverse effects highlights their potential as "living antibiotics". However, the transition from experimental models to clinical application will require comprehensive research to address safety, efficacy, and regulatory challenges. If these hurdles can be overcome, predatory bacteria could become a valuable addition to the arsenal against antibiotic-resistant pathogens, offering hope in the fight against one of the most pressing global health threats of our time. Acknowledgments I would like to express my gratitude to the reviewers for their valuable feedback, which helped improve the manuscript. Conflict of Interest Disclosure The author affirms that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest. References Bukowska-Faniband E., Andersson T., and Lood R., 2020, Studies on Bd0934 and Bd3507, two secreted nucleases fromBdellovibrio bacteriovorus, reveal sequential release of nucleases during the predatory cycle, Journal of Bacteriology, 202(18): 1-14. https://doi.org/10.1128/JB.00150-20 Cavallo F., Jordana L., Friedrich A., Glasner C., and Dijl J., 2021, Bdellovibrio bacteriovorus: a potential ‘living antibiotic’ to control bacterial pathogens, Critical Reviews in Microbiology, 47: 630-646. https://doi.org/10.1080/1040841X.2021.1908956 Duncan M., Gillette R., Maglasang M., Corn E., Tai A., Lazinski D., Shanks R., Kadouri D., and Camilli A., 2019, High-throughput analysis of gene function in the bacterial predator Bdellovibrio bacteriovorus, mBio, 10(3): 1-12. https://doi.org/10.1128/mBio.01040-19 Feng S., Tan C., Constancias F., Kohli G., Cohen Y., and Rice S., 2017, Predation by Bdellovibrio bacteriovorus significantly reduces viability and alters the microbial community composition of activated sludge flocs and granules, FEMS Microbiology Ecology, 93(4): fix020. https://doi.org/10.1093/femsec/fix020 Findlay J., Flick-Smith H., Keyser E., Cooper I., Williamson E., and Oyston P., 2019, Predatory bacteria can protect SKH-1 mice from a lethal plague challenge, Scientific Reports, 9: 7225. https://doi.org/10.1038/s41598-019-43467-1 Gupta S., Tang C., Tran M., and Kadouri D.,2016, Effect of predatory bacteria on human cell lines, PLoS ONE, 11(8): e0161242. https://doi.org/10.1371/journal.pone.0161242 Hetta H., Ramadan Y., Al-Harbi A., Ahmed E., Battah B., Ellah N., Zanetti S., abd Donadu M., 2023, Nanotechnology as a promising approach to combat multidrug resistant bacteria: a comprehensive review and future perspectives, Biomedicines, 11(2): 413. https://doi.org/10.3390/biomedicines11020413
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Molecular Pathogens 2024, Vol.15, No.4, 179-188 http://microbescipublisher.com/index.php/mp 179 Research Insight Open Access Transcriptomic Insights into Wheat Disease Resistance Xinguang Cai, Qiangsheng Qian Modern Agricultural Research Center of Cuixi Academy of Biotechology, Zhuji, 311800, Zhejiang, China Corresponding author: qiangsheng.qian@cuixi.org Molecular Pathogens, 2024, Vol.15, No.4 doi: 10.5376/mp.2024.15.0017 Received: 20 May, 2024 Accepted: 30 Jun., 2024 Published: 12 Jul., 2024 Copyright © 2024 Cai and Qian, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Cai X.G., and Qian Q.S., 2024, Transcriptomic insights into wheat disease resistance, Molecular Pathogens, 15(4): 179-188 (doi: 10.5376/mp.2024.15.0017) Abstract Wheat is one of the most important staple crops globally, but its production is threatened by various diseases, resulting in significant economic losses and yield reduction. Understanding the molecular mechanisms underlying wheat's disease resistance is crucial for enhancing its resilience. This study utilizes transcriptomics to systematically analyze gene expression patterns in wheat under different biotic stresses. Transcriptomics reveals key resistance genes, transcription factors regulating resistance responses, and specific molecular pathways involved in wheat-pathogen interactions, offering valuable tools and data for improving disease resistance in wheat. The aim of this research is to explore their potential applications in wheat breeding by summarizing current transcriptomic insights into wheat disease resistance. Keywords Wheat resistance; Transcriptomics; Pathogen interaction; Gene expression; Breeding improvement 1 Introduction Wheat, a staple food crop, is cultivated globally. However, wheat production is significantly threatened by various diseases, including rusts (leaf rust, stem rust, and stripe rust), powdery mildew, fusariumhead blight, and wheat blast. These diseases can cause substantial yield losses, with recent estimates indicating global wheat yield losses of up to 21% due to these pathogens (Singh et al., 2016; Hafeez et al., 2021). The continuous evolution of virulent pathogen strains exacerbates the problem, making it challenging to maintain effective disease control (Singh et al., 2016; Mapuranga et al., 2022). Additionally, climate change is expected to alter disease dynamics, potentially increasing the prevalence and severity of certain diseases in regions like Northwestern Europe (Miedaner and Juroszek, 2021). Resistance (R) genes play a critical role in recognizing and responding to pathogen attacks, and recent advances in genomic technologies have accelerated the identification and functional characterization of these genes (Deng et al., 2020). There are three primary resistance mechanisms in cereals: plasma membrane-localized receptor proteins, intracellular immune receptors, and quantitative disease resistance, each contributing uniquely to the plant's defense system (Krattinger and Keller, 2016). The integration of molecular breeding techniques, such as genome-wide association studies (GWAS) and CRISPR/Cas-9, has further enhanced our ability to develop wheat varieties with broad-spectrum and durable resistance (Jabran et al., 2023). This study aims to summarize the latest advances in understanding the molecular basis of disease resistance, highlight the importance of creating resources such as the Wheat Resistance Gene Atlas to rapidly deploy the R gene, and discuss the potential for integrating advanced molecular techniques into breeding programs to enhance disease resistance in wheat. 2 Transcriptomics in Wheat Research 2.1 Overview of transcriptomics and its role in plant research Transcriptomics, the study of the complete set of RNA transcripts produced by the genome under specific circumstances, has become a pivotal tool in plant research. This high-resolution, sensitive, and high-throughput next-generation sequencing (NGS) approach, commonly known as RNA-Sequencing (RNA-Seq), allows researchers to identify gene predictions and perform functional analyses to understand biological processes,
Molecular Pathogens 2024, Vol.15, No.4, 179-188 http://microbescipublisher.com/index.php/mp 180 molecular functions, and cellular components (Tyagi et al., 2022). By examining the transcriptome, scientists can gain insights into gene expression patterns, regulatory mechanisms, and the functional roles of genes in various developmental stages and environmental conditions. This knowledge is crucial for improving breeding selection and cultivation practices, ultimately enhancing crop yield and resilience. 2.2 Advances in wheat transcriptomic technologies Recent advancements in transcriptomic technologies have significantly enhanced our ability to study wheat at the molecular level. Techniques such as RNA-Seq have been employed to characterize the transcriptomes of distinct cell types in biological tissues efficiently (Saini et al., 2021). For instance, the development of methods like Simplified Poly (A) Anchored Sequencing (SiPAS) has enabled large-scale gene expression quantification with high sensitivity, accuracy, and reproducibility, making transcriptomics a more powerful tool for deciphering genome function (Wang et al., 2021). Additionally, integrative analyses combining mRNA, noncoding RNA (ncRNA), and DNA methylation data have provided deeper insights into complex regulatory networks, such as those involved in preharvest sprouting resistance in wheat (Zhang et al., 2021). These technological advancements have paved the way for more comprehensive and detailed studies of wheat transcriptomes, facilitating the identification of key genes and pathways involved in disease resistance and other important traits. 2.3 Transcriptome analysis in the study of biotic stress Transcriptome analysis has been instrumental in understanding wheat's response to biotic stress, such as pathogen infections. For example, studies have utilized RNA-Seq to investigate the transcriptomic changes in wheat varieties with different resistances to pathogens like wheat yellow mosaic virus (WYMV) and wheat dwarf virus (WDV) (Sharaf et al., 2023). These analyses have revealed significant changes in gene expression and RNA modifications (Figure 1), such as N6-methyladenosine (m6A) methylation, which play crucial roles in plant defense responses. Furthermore, alternative splicing (AS) events have been shown to enhance transcript and protein diversity, contributing to stress adaptation in wheat during pathogen interactions (Zhang et al., 2019). By identifying differentially expressed transcripts and splicing variants, researchers can pinpoint specific genes and pathways that are activated or suppressed in response to biotic stress, providing valuable targets for breeding disease-resistant wheat varieties. Integrating transcriptomic data with genomic and phenotypic information has also proven effective in predicting resistance to diseases like fusarium head blight (FHB), demonstrating the potential of transcriptomics in enhancing predictive breeding strategies (Michel et al., 2021). 3 Key Pathogen-Responsive Genes in Wheat 3.1 Identification of major resistance genes The identification of major resistance genes in wheat has been significantly advanced through various genomic and transcriptomic studies. For instance, a meta-analysis of quantitative trait loci (QTL) mapping identified 63 meta-QTLs (MQTLs) associated with resistance to multiple diseases such as septoria tritici blotch, fusariumhead blight, and karnal bunt. This study also identified 194 differentially expressed genes (DEGs) linked to disease resistance, providing a valuable resource for marker-assisted breeding (Saini et al., 2021). Additionally, the wheat resistance gene Lr34, which encodes an ABCG-type transporter, has been shown to confer durable resistance against multiple pathogens. This gene's mode of action involves the constitutive activation of multiple defense pathways, including the induction of jasmonic acid and salicylic acid, which are crucial for plant defense (Chauhan et al., 2015). 3.2 Gene families involved in disease resistance Several gene families play pivotal roles in wheat disease resistance. The NB-ARC-encoding gene family, which includes nucleotide-binding site-leucine-rich repeat (NBS-LRR) genes, is particularly noteworthy. A genome-wide identification study revealed 2151 NB-ARC-encoding genes in wheat, many of which are organized into clusters formed by tandem duplications. These genes are essential for recognizing pathogenic effectors and initiating immune responses (Andersen et al., 2020). Another significant gene family includes the pathogenesis-related (PR) proteins, such as PR-1, TLP, Chitinase, and β-1, 3-glucanase, which were found to be highly expressed in resistant wheat varieties, suggesting their crucial role in defense mechanisms against pathogens like fusarium equiseti (Manghwar et al., 2021).
Molecular Pathogens 2024, Vol.15, No.4, 179-188 http://microbescipublisher.com/index.php/mp 181 Figure 1 Summary of differential expression in WDV-infected and non-inoculated genotypes (Adopted from Sharaf et al., 2023) Image caption: (A) Heatmap of transcript expression and hierarchical clustering analysis of the WDV-infected and non-inoculated genotypes. (B) A bar chart showing the number of differentially expressed transcripts (DETs) in the three genotypes after WDV infection. (C) A Venn diagram showing the overlaps of the DETs of the three genotypes after WDV infection (Adopted from Sharaf et al., 2023) 3.3 Role of transcription factors in regulating resistance responses Transcription factors (TFs) are key regulators of gene expression in response to pathogen attacks. In wheat, the MYB transcription factor family has been implicated in resistance to the Wheat Dwarf Virus (WDV). Studies have shown that MYB TFs are differentially expressed in resistant and susceptible wheat genotypes, indicating their role in modulating resistance responses (Sharaf et al., 2023). Furthermore, the involvement of N6-methyladenosine (m6A) methylation in the regulation of TFs has been highlighted in the context of wheat resistance to wheat yellow mosaic virus (WYMV). This epigenetic modification affects the expression of genes involved in plant defense, underscoring the complex regulatory networks that govern disease resistance in wheat (Zhang et al., 2021).
Molecular Pathogens 2024, Vol.15, No.4, 179-188 http://microbescipublisher.com/index.php/mp 182 4 Mechanisms of Wheat Disease Resistance 4.1 Molecular pathways activated by pathogen attack Wheat plants activate a variety of molecular pathways in response to pathogen attacks. These pathways often involve the production of defense-related molecules such as flavonoids and terpenes, which are synthesized through pathogen-induced biosynthetic pathways encoded by biosynthetic gene clusters (BGCs) (Saini et al., 2021). Additionally, resistance (R) genes play a crucial role in recognizing pathogen-derived molecules either directly or indirectly, triggering a cascade of defense responses. These R genes encode proteins that function as cell surface or intracellular receptors (Figure 2), which can detect pathogen molecules and initiate defense mechanisms (Krattinger and Keller, 2016). The wheat resistance gene Lr34, for example, induces multiple defense pathways, including the production of lignin and hordatines, which contribute to both basal and inducible disease resistance (Chauhan et al., 2015). Figure 2 Using the R gene atlas and pathogen diversity information to determine appropriate stacks (Adopted from Hafeez et al., 2021) 4.2 Crosstalk between different signaling pathways Crosstalk between different signaling pathways is essential for a coordinated defense response in wheat. Hormonal signaling pathways, such as those involving jasmonic acid (JA) and salicylic acid (SA), are often
Molecular Pathogens 2024, Vol.15, No.4, 179-188 http://microbescipublisher.com/index.php/mp 183 interconnected and can influence each other to fine-tune the plant's defense mechanisms (Wang and Li, 2024). For instance, the wheat gene Lr34 not only activates multiple defense pathways but also induces high levels of JA and SA, demonstrating the interplay between these hormonal pathways in enhancing disease resistance (Polturak et al., 2022). Additionally, systemic acquired resistance (SAR) in wheat involves the activation of different gene pathways, including acquired resistance (AR) and systemic immunity (SI), which are regulated by NPR1 homologs and other downstream genes (Wang et al., 2018). 4.3 Hormonal regulation of disease resistance Hormonal regulation plays a pivotal role in modulating wheat's defense responses against pathogens. Key hormones such as jasmonic acid (JA) and salicylic acid (SA) are central to the plant's immune system. The wheat resistance gene Lr34, for example, leads to the constitutive induction of JA and SA, which are crucial for activating various defense pathways (Poretti et al., 2021). Additionally, the TIME FOR COFFEE (TIC) gene in Brachypodium distachyon, a model grass, is involved in jasmonate signaling and contributes to nonhost resistance to wheat stem rust, highlighting the importance of hormonal regulation in disease resistance (Coletta et al., 2021). The interplay between these hormones and their signaling pathways ensures a robust and effective defense response. 4.4 Role of secondary metabolites in defense Secondary metabolites, these compounds, which include flavonoids and terpenes, are produced through pathogen-induced biosynthetic pathways and serve as phytoalexins or signaling molecules that enhance the plant's resistance to diseases. The production of these metabolites is often regulated by specific gene clusters, and their accumulation can deter pathogen growth and spread (Li etal., 2024). For instance, the wheat resistance gene Lr34 induces the production of lignin and hordatines, which are secondary metabolites that contribute to the plant's structural defense and antimicrobial activity (Chauhan et al., 2015). The strategic deployment of these metabolites is a crucial aspect of wheat's overall defense strategy. 5 Transcriptomic Signatures of Wheat-Pest Interactions 5.1 Specific transcriptomic responses to fungal pathogens Wheat's interaction with fungal pathogens, particularly Puccinia striiformis f. sp. tritici (Pst), involves complex transcriptomic changes. The identification of microRNAs (miRNAs) and their targets has revealed their significant roles in wheat's defense mechanisms. For instance, the miRNA-like RNA 1 (Pst-milR1) from Pst suppresses wheat defenses by targeting the pathogenesis-related 2 (PR2) gene, thereby impairing the plant's immune response (Wang et al., 2017). Additionally, the AP2/ERF transcription factor TaAP2-15 has been shown to enhance wheat resistance to Pst by regulating the expression of pathogenesis-related genes and reactive oxygen species (ROS)-scavenging genes (Hawku et al., 2021). Another study highlighted the role of the R2R3 MYB transcription factor TaMYB391, which positively regulates hypersensitive response (HR)-associated cell death and resistance to Pst through the induction of PR genes and ROS accumulation (Hawku et al., 2022). These findings underscore the importance of specific transcription factors and miRNAs in modulating wheat's transcriptomic responses to fungal pathogens. 5.2 Responses to bacterial and viral pathogens Wheat's transcriptomic responses to bacterial and viral pathogens involve distinct signaling pathways and regulatory mechanisms. The interaction between wheat and bacterial pathogens often triggers systemic acquired resistance (SAR), mediated by key transcriptional regulators such as NPR1. A conserved protein from Pst, PNPi, interacts with wheat NPR1, reducing the induction of pathogenesis-related genes and thereby manipulating the plant's defense response (Wu et al., 2022). In the context of viral pathogens, host-induced gene silencing (HIGS) has emerged as a promising strategy. For example, silencing the PsCPK1 gene in Pst through HIGS significantly enhances wheat resistance to stripe rust by reducing the pathogen's growth and development (Qi et al., 2017). These studies highlight the diverse transcriptomic responses of wheat to bacterial and viral pathogens, involving both transcriptional regulators and gene silencing mechanisms.
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