Biology subjects

Aoun, N.

Publications and source records attributed to Aoun, N..

5 recordsLinked to original sources

bioRxiv2026

Evolutionary and functional genomics reveal that Ralstonia wilt pathogens actively deploy antimicrobial warfare while leveraging physiological adaptations during plant infection

The xylem pathogens in the Ralstonia solanacearum species complex (RSSC) cause wilt diseases that threaten global food security. These diverse pathogens are highly adapted to the in planta environment where they proliferate and cause rapid, aggressive diseases. To understand the genetic underpinnings of these pathogens in planta fitness, we performed forward genetic screens in three RSSC species, using high-throughput random barcode transposon sequencing (RB-TnSeq). We quantified the competitive fitness of hundreds of thousands of mutants during stem colonization of susceptible tomato plants. We compared this in planta forward genetic screen with a prior forward genetic screen performed in a reductionist, plant-like environment: ex vivo xylem sap harvest from healthy tomato plants. The comparative genetic screens revealed both conserved and lineage-specific orthologs that enable RSSCs pathogenic success. Consistently, these diverse pathogens navigated the opportunities and stressors in planta by maintaining a resilient cell envelope barrier, complementing the nutritional availability in xylem sap, fine-tuning their utilization of the host-manipulating type III secretion system, and dynamically regulating their gene expression through a suite of environment sensing and transcriptional regulatory proteins. Surprisingly, the strain-specific fitness factors shed light on ecological interactions beyond pathogen dominance of their susceptible host, even during the single-strain infections, the pathogens wielded arsenals of anti-microbial weapons. Specifically during growth in planta, each RSSC strain required a unique repertoire of type VI secretion system immunity proteins that provide self-protection to their own toxins. We contextualized the in planta fitness factors through evolutionary genomic comparisons of the RSSC wilt pathogens to their non-pathogenic neighbors. Together, these analyses reveal how conserved and lineage-specific fitness determinants have evolved to support pathogenic success in the plant vascular niche.

genetics↗

bioRxiv2025

Natural variation in an NLR pair confers thermostable resistance to a devastating bacterial pathogen

Climate change reshapes host-pathogen interactions by increasing pathogen aggressiveness and weakening plant immune responses, particularly under heat stress. Nucleotide-binding leucine-rich repeat (NLR) immune receptors, key players in pathogen recognition, are altered under elevated temperatures in both plants and mammals, posing a major challenge to disease resistance. In Arabidopsis thaliana, the well-described RPS4/RRS1 NLR pair confers resistance to the worldwide devastating phytopathogenic Ralstonia pseudosolanacearum. Here, by combining the exploration of natural genetic variation with genetic mapping, polymorphism analysis, structural modeling, and functional complementation, we demonstrated that the paucity of full thermostable resistance is primarily associated with a unique RPS4/RRS1 haplotype carrying key substitutions in leucine-rich repeat domains. These findings reveal natural genetic diversity as a source of thermostable resistance, highlighting promising opportunities to engineer climate-resilient plants.

plant biology↗

bioRxiv2025

The EPS-I exopolysaccharide transforms Ralstonia wilt pathogen biofilms into viscoelastic fluids for rapid dissemination in planta

Ralstonia solanacearum species complex (RSSC) pathogens cause destructive plant wilt diseases of a wide variety of crops, leading to significant agricultural losses worldwide. These bacteria rapidly spread through the water-transporting xylem where they grow prolifically and produce abundant biofilm that clogs xylem vessels. To understand RSSC biofilm behavior in planta, we examined their complex fluid mechanics. Rheological analyses revealed that unlike all previously analyzed microbial biofilms, RSSC biofilms are shear-thinning, viscoelastic fluids at physiologically relevant shear forces. To determine which factors confer these unique mechanics, we analyzed biofilms of bacterial mutants with altered biofilm components. Genetic analysis demonstrated that development of the viscous-dominant biofilms required production of EPS-I, an amphiphilic exopolysaccharide that is a major virulence factor for all RSSC pathogens. We show that EPS-I confers "biofilm mobility", which allows wildtype RSSC colonies to passively expand when deformed. Despite its high metabolic cost, bioassays demonstrated that EPS-I production conferred a net fitness benefit where biofilm mobility allowed the pathogen to spread and access more nutrients in complex environments like xylem vessels. The RSSC are a monophyletic lineage of aggressive plant wilt pathogens, and our evolutionary hypothesis testing suggests the origin of the eps biosynthetic gene cluster coincides with the emergence of wilt pathogenesis in the RSSC ancestor. Furthermore, comparative physiological assays demonstrated that biofilm mobility is unique to the RSSC within the genus Ralstonia. In summary, EPS-I production is a key evolutionary innovation that enables RSSC dispersal and virulence by conferring unique biofilm mechanics. Significance StatementRalstonia solanacearum species complex (RSSC) pathogens threaten global food security by fatally wilting plants. A soft matter physics lens demystified the cryptic role of a major virulence factor, the EPS-I exopolysaccharide. EPS-I transforms RSSC biofilms into viscoelastic fluids, a mechanical behavior not previously described for other microbial biofilms that are almost always viscoelastic solids. We demonstrate that the development of fluid biofilms was a key evolutionary innovation that enabled pathogenic success of these aggressive pathogens that rapidly wilt plants.

microbiology↗

bioRxiv2024

Gene editing of the E3 ligase PIRE1 fine-tunes ROS production for enhanced bacterial disease resistance in tomato

Reactive oxygen species (ROS) accumulation is required for effective plant defense. Accumulation of the Arabidopsis NADPH oxidase RBOHD is regulated by phosphorylation of a conserved C-terminal residue (T912) leading to ubiquitination by the RING E3 ligase PIRE. Arabidopsis PIRE knockouts exhibit enhanced ROS production and resistance to the foliar pathogen Pseudomonas syringae. Here, we identified 170 PIRE homologs, which emerged in Tracheophytes and expanded in Angiosperms. We investigated the role of Solanum lycopersicum (tomato) PIRE homologs in regulating ROS production, RBOH stability, and disease resistance. Mutational analyses of residues corresponding to T912 in the tomato RBOHD ortholog, SlRBOHB, affected protein accumulation and ROS production in a PIRE-dependent manner. Using CRISPR-cas9, we generated mutants in two S. lycopersicum PIRE homologs (SlPIRE). SlPIRE1 edited lines (Slpire1) in the tomato cultivar M82 displayed enhanced ROS production upon treatment with flg22, an immunogenic epitope of flagellin. Furthermore, Slpire1 exhibited decreased disease symptoms and bacterial accumulation when inoculated with foliar bacterial pathogens Pseudomonas syringae and Xanthomonas campestris. However, Slpire1 exhibited similar levels of colonization as wild type upon inoculation with diverse soilborne pathogens. These results indicate that phosphorylation and ubiquitination crosstalk regulate RBOHs in multiple plant species, and PIRE is a promising target for foliar disease control. This study also highlights the pathogen-specific role of PIRE, indicating its potential for targeted manipulation to enhance foliar disease resistance without affecting root-associated interactions, positioning PIRE as a promising target for improving overall plant health.

plant biology↗

bioRxiv2023

A pangenomic atlas reveals that eco-evolutionary dynamics shape plant pathogen type VI secretion systems

Soil-borne Ralstonia solanacearum species complex (RSSC) bacteria disrupt rhizosphere and endophytic microbial communities as they invade roots and fatally wilt plants. RSSC pathogens secrete antimicrobial toxins using a type VI secretion system (T6SS). To investigate how evolution and ecology have shaped pathogen T6SS biology, we analyzed the T6SS gene content and architecture across the RSSC pangenome and their evolutionarily relatives. Our analysis reveals that two ecologically similar Burkholderiaceae taxa, xylem pathogenic RSSC bacteria and Acidovorax, have convergently evolved to wield large arsenals of T6SS toxins. To understand the mechanisms underlying genomic enrichment of T6SS toxins, we compiled an atlas of 1,069 auxiliary ("aux") T6SS toxin clusters across 99 high-quality RSSC genomes. We classified 25 types of aux clusters with toxins that predominantly target lipids, nucleic acids, or unknown cellular substrates. The aux clusters were in diverse genetic neighborhoods and had complex phylogenetic distributions, suggesting frequent horizontal gene flow. Phages and other mobile genetic elements account for most of the aux cluster acquisition on the chromosome but very little on the megaplasmid. Nevertheless, RSSC genomes were more enriched in aux clusters on the megaplasmid. Secondary replicons like megaplasmids often evolve more rapidly than the more evolutionarily stable chromosome. Although the single ancestral T6SS was broadly conserved in the RSSC, the T6SS was convergently lost in atypical lineages with vectored transmission. Overall, our data suggest dynamic interplay between the lifestyle of soil-transmitted RSSC lineages and the evolution of T6SSs with robust arsenals of toxins. This pangenomic atlas poises the RSSC as an emerging, tractable model to understand the role of the T6SS in shaping pathogen populations.

evolutionary biology↗