bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.05.27.656240

Comparative Transcriptomics Reveals Context- and Strain-Specific Regulatory Programs of Agrobacterium During Plant Colonization

Wu, Y.·Chang, H.-Y.·Wu, C.-H.·Lai, E.-M.·Kuo, C.-H.
View original source ↗Export metadata ↓

Abstract

Agrobacterium is a genus of plant-associated bacteria capable of transferring DNA into host genomes to induce tumorigenesis. The process has been primarily studied in a few model strains, particularly C58, and developed into Agrobacterium-mediated transformation (AMT) for genetic manipulation. However, the diversity of wild-type strains and their context-specific regulatory responses remain poorly characterized. Here, we evaluated five wild-type strains and identified 1D1108 as superior in tumorigenesis on legumes and transient transformation in Nicotiana benthamiana. Under in vitro virulence induction with acetosyringone (AS), we identified 126 differentially expressed genes (DEGs) in 1D1108. Although the number of DEGs was comparable to those in C58 and the legume isolate 1D1609 under the same condition, only 22 DEGs, primarily within the vir regulon, were conserved, indicating extensive divergence among these Agrobacterium strains. Leaf infiltration of N. benthamiana revealed 1,134 DEGs specifically regulated in planta for 1D1108. These included genes involved in attachment, virulence regulation, type IV pilus, succinoglycan biosynthesis, and diverse nutrient transporters, providing new evidence on expression regulation during colonization. Comparative analyses of in planta transcriptomes with C58 and Pseudomonas syringae DC3000 revealed distinct secretion systems required for pathogenesis, namely type IV for Agrobacterium and type III for Pseudomonas, and only approximately 5-19% of DEGs were conserved. These limited transcriptomic overlaps underscore the importance of studying gene expression in strains and conditions directly relevant to the biological context, rather than relying on model systems. Together, this work reveals how environmental and host-associated cues shape transcriptional responses in plant-associated bacteria. IMPORTANCEAgrobacterium is a key tool for plant genetic engineering, yet much of our knowledge about its biology comes from a few strains studied under artificial conditions. This work combines tumor formation assays on legumes and transient transformation in Nicotiana benthamiana with transcriptomic analyses to explore how diverse Agrobacterium strains, particularly the high-performing wild-type strain 1D1108, respond to host and environmental cues. By comparing gene expression under in vitro and in planta conditions, we found that the bacterial response within plant tissue is more complex than previously appreciated. Cross-strain comparisons further revealed limited conservation in gene expression regulation, even under similar conditions. Comparative analysis with Pseudomonas syringae revealed the activation of distinct secretion systems for pathogenesis and differences in regulatory programs used during plant colonization. These findings underscore the importance of studying bacteria in biologically relevant contexts and highlight the limitations of relying solely on model strains to infer regulatory responses.

Source connections

Explore related subjects

microbiologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Wu, Y., Chang, H.-Y., Wu, C.-H., Lai, E.-M., Kuo, C.-H.. 2025-05-27. Comparative Transcriptomics Reveals Context- and Strain-Specific Regulatory Programs of Agrobacterium During Plant Colonization. https://doi.org/10.1101/2025.05.27.656240

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Matrix-controlled emergence of biofilm architecture shapes antimicrobial survival

Biofilms are structured microbial communities whose extracellular matrix is widely regarded as a basis of their protection against antimicrobial compounds. Yet how matrix production by individual bacteria gives rise to collective architecture and antimicrobial protection remains poorly understood. Here, we systematically varied expression of the master biofilm regulator csgD in Salmonella enterica and found that increasing matrix production reorganizes biofilms from dense, isotropic packings into sparse, nematically aligned communities by altering cell-cell interactions. By combining experimentally measured biofilm architectures with reaction-diffusion modeling, we show that these structural changes produce distinct patterns of antimicrobial killing, ranging from preferential killing near the liquid-biofilm interface to more uniform killing throughout the community. Consequently, increasing matrix production unexpectedly reduces antimicrobial survival by shifting the biofilm into different transport regimes, while strain-specific physiological differences further modulate antimicrobial depletion. Rather than acting as a passive barrier, EPS therefore shapes antimicrobial susceptibility by reorganizing biofilm architecture and its transport properties. EPS thus provides a physical link between molecular regulation, collective architecture and antimicrobial survival, providing a quantitative framework for understanding how cellular matrix production generates emergent biofilm function.

microbiology↗
bioRxiv2026

Mapping virulence-associated protein interaction networks reveals regulators of thermotolerance in Cryptococcus neoformans

Protein-protein interactions (PPIs) influence critical biological processes in pathogenic microorganisms, such as the human fungal pathogen, Cryptococcus neoformans. Fungal thermotolerance and stress response pathways are key virulence determinants that directly impact pathogen adaptation and survival and the infection process. To establish a comprehensive baseline of PPIs in C. neoformans and explore these interactions to infer functional roles for uncharacterized proteins, we applied size exclusion chromatography coupled with mass spectrometry to the secreted and cellular proteomes of the fungi. As a result, 216 and 1699 unique proteins were identified across 24 secretome and proteome fractions, respectively. The predicted secretome networks included expected proteins associated with vesicles and virulence, indicating a role in extracellular defense. Whereas the cryptococcal proteome highlighted interactions among proteins with defined roles in fungal virulence for protein stability and thermotolerance, including two previously uncharacterized proteins, CNAG_00287 and CNAG_05199, putatively involved in complex formation with heat-shock proteins (HSP). Based on sequence and structure homology, we propose that CNAG_00287 is a tetratricopeptide repeat-containing co-chaperone that modulates Hsp 70 activity and CNAG_05199 functions as a Hsp70. We validated the thermotolerance role of CNAG_00287 in heat-related stress, as its absence significantly impaired fungal growth in nutrient-limited media at 37 {degrees}C. Together, this work resolves virulence-associated PPIs within C. neoformans and reveals new molecular regulators of thermotolerance that underpin fungal pathogenicity.

microbiology↗
bioRxiv2026

Environmental filtering and host identity collectively shape root-associated microbiomes of Ericaceae and ectomycorrhizal plants in fumarole fields

Background Symbiosis with microbes is a key strategy that has enabled plants to colonize extreme environments. Since the benefits conferred by root-associated microbes depend on both environmental conditions and host-microbe combinations, plant adaptation to harsh environments is closely linked to the assembly of root microbial communities. Understanding how environmental and host filtering jointly shape these communities is therefore fundamental to elucidating the mechanisms underlying plant adaptation to extreme environments. Results In this study, we investigated the differentiation of root-associated prokaryotic and fungal communities and individual operational taxonomic units (OTUs) across two contrasting habitats surrounding fumaroles, solfatara-field and forest-edge habitats, and six dominant Ericaceae and ectomycorrhizal plant taxa. Prokaryotic and fungal OTUs rarely exhibited strong preferences for both habitat and host identity. Instead, many of prokaryotic and fungal OTUs specialized to one of these niches, collectively generating root microbial communities differentiated by both factors. Nonetheless, striking specializations in habitat and host niches were observed in the fungal family Hyaloscyphaceae (Helotiales). To gain insight into the evolutionary basis of microbial specialization, we examined phylogenetic signals in preference phenotypes. The resulting weak phylogenetic signals in these preference phenotypes further suggest that this fungal clade has undergone substantial ecological divergence. Conclusion Overall, our findings indicate that root-associated microbial communities in extreme environments are assembled through the accumulation of microbial taxa specialized to either habitat or host, and that strong ecological specialization in fungi can arise with little phylogenetic constraint.

microbiology↗