Rhizobia transform legume nodules via native T-DNA transfer systems

S.-C. Hsieh; E.-M. Lai; C.-H. Kuo

doi:10.64898/2026.11.03.763973

AI summary · DOI: 10.64898/2026.11.03.763973 · bioRxiv: Microbiology

Rhizobia transform legume nodules via native T-DNA transfer systems

S.-C. Hsieh, E.-M. Lai, C.-H. Kuo

Original paper Published on 04 Nov. 2026 bioRxiv preprint License: CC BY 4.0 Also in: fr

How to read this summary? Illustrated guide

The essentials

The study identifies rhizobial T-DNA systems that transfer DNA into native legume root nodules and suggests these systems represent a distinct branch of a broader, ancient transfer capability.

Question

Do non-oncogenic T-DNA transfer systems occur beyond agrobacteria, and can they function in plants? What do their distribution and transfer machinery reveal about the evolution of bacteria-to-plant DNA transfer?

Method

The authors screened 63,759 alphaproteobacterial genomes for T-DNA borders, identifying candidate loci and characterizing their cargo genes and transfer-system components. They tested six mini-T-DNAs from three rhizobial strains in Agrobacterium-mediated leaf assays, then assayed transfer by the rhizobia in their native plant hosts; they also disrupted vir genes and analyzed regulation and transfer-system phylogeny.

Findings

Result The genome-wide screen identified 157 compact mini-T-DNAs, including 80 with a rolB family member, and found that most apparently empty loci contained unannotated rolB-like genes.
See the passage in the paper
We identified 157 such loci, of which 80 contain a rolB family member, 12 encode short ORFs with no detectable homologues, and 54 lack annotated coding sequences.
Result All six tested rhizobial mini-T-DNAs were transferred by Agrobacterium and drove expression of their rolB or rolB-like cargos in plant cells.
See the passage in the paper
All six constructs produced GUS signals in plant cells, indicating that the rhizobial T-DNAs were transferred and that their rolB/rolB-like cargos were expressed.
Result Three rhizobial strains transferred T-DNA into nodules of their native hosts, and disruption of virD2 abolished the detected transfer.
See the passage in the paper
All three strains showed rhizobial T-DNA-dependent GUS expression in nodules, and the binary vector carrying the GUS gene under the 35S promoter did not produce the signal (Fig. 4).
Authors’ interpretation The tested rhizobial systems could activate core transfer genes through VirG-dependent regulation, but their upstream regulatory inputs differed from those described for agrobacteria.
See the passage in the paper
Rhizobial T-DNA transfer systems therefore appear to retain conserved, VirG-dependent activation of the core transfer genes while being wired to distinct upstream signals.
Authors’ interpretation Phylogenetic patterns support a model in which many rhizobial systems retain an ancestral-like VirB configuration, while canonical AMT systems acquired a different VirB module through replacement.
See the passage in the paper
Together, the VirB4 and VirD4 phylogenies support a modular-replacement model in which plasmid-borne VirB4 clade B-associated systems resemble an ancestral-like configuration, whereas clade A modules were recruited into canonical AMT and related systems.

Limitations

The study tested transfer in only three rhizobial strains and their host pairs, and the role of transfer in symbiosis remains unresolved.
See the passage in the paper
Only a minority of rhizobia carry RMT systems, and in the three strains tested here RMT was dispensable for nodulation under laboratory conditions, leaving its role in the symbiosis, the functions of the rhizobial RolB/RolB-like cargos, and the signals that trigger transfer to be defined.
The evolutionary direction between rhizobial and agrobacterial systems cannot be established from the analyses; horizontal transfer and recombination complicate ancestry claims.
See the passage in the paper
But taken together, these signals suggest that the extant RMT and AMT systems are better understood as co-descendants of a shared pool of ancestral systems, shaped by ongoing HGT and selection, than as one having given rise to the other.
The study does not establish whether transfer is confined to nodules or quantify transfer efficiency and host range.
See the passage in the paper
Realizing these applications will require defining transfer efficiency, host range, and tissue specificity directly, but the distinct properties of RMT make it a promising complement to established transformation systems.

Why it matters

The authors argue that bacteria-to-plant DNA transfer is not exclusive to pathogenic agrobacteria: rhizobial transformation is a symbiosis-associated counterpart, and additional transfer systems may occur across plant-associated bacteria. They present RMT as a possible complement to existing plant transformation approaches, while emphasizing that its efficiency, host range, and tissue specificity remain to be determined.

Written by AI from the paper. Passages are checked word for word against the text we analysed; the rest may contain errors.

AI-generated keywords: Rhizobial DNA transfer; T-DNA; RolB-like proteins; plant transformation; symbiosis

Read further

Comprehensive Summary
Layman's Summary
Blog article
Original abstract

The study asks whether bacteria-to-plant DNA transfer is limited to pathogenic agrobacteria or whether nonpathogenic rhizobia also possess functional T-DNA transfer systems. The authors argue that searches relying on tumor or root-growth symptoms may overlook systems that transfer DNA without causing disease. To investigate this possibility, they combined comparative genomics with experiments testing whether candidate DNA regions could enter plant cells and be expressed. The researchers screened 63,759 alphaproteobacterial genomes for sequences resembling T-DNA border repeats. Of 1,706 initial candidates, they applied filters for coding-sequence density and GC content, calibrated against known T-DNAs. This yielded 157 compact “mini-T-DNAs”: 80 contained a recognized RolB-family gene, 12 contained short open reading frames without detectable homologues, and 54 had no annotated coding sequence. Profile-based searches indicated that 53 of these 54 apparently empty regions encode previously unannotated RolB-like proteins, generally only 100–150 amino acids long. Most candidates occurred in genomes containing the rhizobial nodABC gene cluster, which the study used as a marker for rhizobia. The authors estimate that these systems occur in about 2% of nodABC-positive genomes. The candidate regions also had features consistent with T-DNAs, including characteristic border sequences and compact cargo organization. To test whether the DNA could be transferred and expressed, the team placed six mini-T-DNAs from *Rhizobium etli* CFN42, *Rhizobium johnstonii* 3841, and *Mesorhizobium mediterraneum* USDA3392 into a reporter system. When *Agrobacterium* delivered them into *Nicotiana benthamiana* leaves, all six produced GUS reporter signals. Disrupting the predicted cargo start codons eliminated those signals. The researchers then tested transfer by the rhizobia themselves. Leaf infiltration did not produce detectable transformation, but inoculating each strain onto its compatible legume host did: reporter expression appeared in root nodules, but not in adjacent root tissue. It was detected in all examined nodules for *R. etli* on common bean and *R. johnstonii* on pea. Approximately one-third of *M. mediterraneum* nodules on chickpea showed a signal. In pea nodules, expression was confined to the tips. Disrupting virD2 abolished transfer in all three strains without preventing nodule formation. This supports a requirement for the T-DNA transfer machinery and indicates that transfer was not needed for nodulation under the tested conditions. The study also found that two tested RolB-like proteins caused severe epinasty and stunted growth when expressed in Arabidopsis, suggesting that at least some cargos can alter plant development. Regulatory assays showed that the three systems differ from the familiar agrobacterial induction pathway: none of their virB promoters responded detectably to acetosyringone or the symbiotic flavonoid naringenin. *R. etli* virB was activated by low pH and certain sugars, while the divergent regulators in the other strains could activate vir genes when experimentally expressed. Comparative analyses of VirB4 and VirD4 proteins suggest that many rhizobial systems retain a distinct, possibly ancestral-like secretion-system architecture, whereas canonical agrobacterial systems may have acquired a different virB module through replacement. The authors conclude that plant-directed T-DNA transfer is not unique to pathogenic agrobacteria and propose rhizobia-mediated transformation as a symbiosis-associated counterpart.

Rhizobia Can Deliver DNA to Plants Without Causing Disease

For decades, bacteria-to-plant DNA transfer has been closely associated with pathogenic agrobacteria—the microbes famous for causing tumors or abnormal root growth. But those visible symptoms may have shaped what researchers thought to look for. Could bacteria transfer DNA into plants without producing disease?

In “Rhizobia transform legume nodules via native T-DNA transfer systems,” S.-C. Hsieh, E.-M. Lai, and C.-H. Kuo investigate that possibility. Their findings point to a surprising answer: some nonpathogenic rhizobia possess functional DNA-transfer systems, and can deliver bacterial DNA into the plant cells of root nodules during symbiosis.

Looking beyond symptoms

The researchers reasoned that a search focused on tumors or unusual root growth could miss bacteria that transfer DNA without causing obvious disease. They instead looked for genetic signatures associated with T-DNA: short DNA regions bounded by characteristic repeats and carrying compact cargo.

They screened 63,759 alphaproteobacterial genomes for sequences resembling T-DNA border repeats. An initial search found 1,706 candidates. The team then used coding-sequence density and GC content, calibrated against known T-DNAs, to narrow the list to 157 compact regions they called “mini-T-DNAs.”

Some candidates carried recognizable RolB-family genes, while others contained short, unfamiliar open reading frames—or appeared to have no annotated coding sequence at all. That last group proved especially interesting. Profile-based searches suggested that 53 of 54 apparently empty regions encode previously unrecognized RolB-like proteins, typically just 100–150 amino acids long.

Most of the candidate regions appeared in genomes with the nodABC gene cluster, a marker used to identify rhizobia. The authors estimate that these systems occur in about 2% of nodABC-positive genomes. Their compact organization and border sequences further supported the idea that the regions could be T-DNAs.

Testing whether the DNA works

Genomic clues alone cannot show that DNA actually enters plant cells. To test function, the researchers placed six mini-T-DNAs from Rhizobium etli CFN42, Rhizobium johnstonii 3841, and Mesorhizobium mediterraneum USDA3392 into a reporter system. When Agrobacterium delivered the sequences into Nicotiana benthamiana leaves, all six produced a detectable GUS reporter signal.

The team also altered the predicted cargo start codons. These changes eliminated the signals, supporting the conclusion that the reporter expression depended on the mini-T-DNA cargo rather than simply on the surrounding experimental setup.

Next came the key test: could the rhizobia deliver their own DNA? Infiltrating leaves with the bacteria did not produce detectable transformation. But when each strain was inoculated onto its compatible legume host, reporter expression appeared in root nodules—not in nearby root tissue.

All examined common-bean nodules associated with R. etli showed reporter expression.
All examined pea nodules associated with R. johnstonii showed expression, concentrated at the nodule tips.
About one-third of chickpea nodules associated with M. mediterraneum showed a signal.

When the researchers disrupted virD2, a gene required for T-DNA transfer, reporter expression disappeared in all three strains. The bacteria could still form nodules, indicating that transfer was not necessary for nodulation under the conditions tested. Instead, the results support a distinct process: DNA delivery occurring in the context of a successful symbiotic relationship.

What might the transferred genes do?

The study also explored whether T-DNA cargo could influence plant development. Two tested RolB-like proteins caused severe leaf bending, or epinasty, and stunted growth when expressed in Arabidopsis. This suggests that at least some transferred genes can alter plant development, although the experiments do not establish the natural effects of every cargo in its host.

The regulation of these systems also differs from the familiar pattern in pathogenic agrobacteria. None of the three tested rhizobial virB promoters responded detectably to acetosyringone or the symbiotic flavonoid naringenin. In R. etli, low pH and certain sugars activated virB. The other strains had divergent regulators that could activate vir genes when experimentally expressed.

Comparisons of VirB4 and VirD4 proteins suggest that many rhizobial systems retain a distinct, possibly ancestral-like secretion-system architecture. The authors propose that canonical agrobacterial systems may have acquired a different virB module through replacement—an evolutionary scenario that could help explain why the systems differ.

A new perspective on plant–bacteria interactions

The central message is that plant-directed T-DNA transfer is not exclusive to pathogenic agrobacteria. Rhizobia, better known for their beneficial partnerships with legumes, can transfer DNA into cells within root nodules. The authors describe this as a symbiosis-associated counterpart to the well-known pathogenic system.

Important questions remain: how widespread are these transfers, what benefits or costs do they bring, and what do the different T-DNA cargo genes do in nature? By looking beyond disease symptoms, this research opens a new way to investigate how bacteria and plants exchange genetic material—and how such exchanges may have evolved.

Created on 09 Nov. 2026

Was this synthesis useful?

Score: 0

Ask questions about this paper to our AI assistant

How to chat with this paper? Illustrated guide

You can also chat with multiple papers at once here.

AI assistant instructions?

Tools for this paper

Explain it differently 1 free rewrite left today

Save to a collection Group papers in collections to compare them, chat with them and export them. Continue with Google Create a free account Log in

Export BibTeX · RIS (Zotero, Mendeley, EndNote) · Obsidian (Markdown) · Notion (Markdown)

Advanced tools Premium

For researchers who follow a field closely: citation alerts, journal-club slides, version comparison and Zotero export. Discover Premium

Zotero

Citation alerts Citation alerts guide

Who works on it? The people and institutions who publish on its topic

Weekly letter Presented in "Biology and medicine this week" of 9 November 2026. Biology and medicine this week: Inherited protection, bacterial clocks and a revised cattle family tree

Journal club Premium+ Slides to present it Propose for the journal club

Talk about it in public Premium+ Press kit: release, questions, hype check Press kit guide

Papers around this one

See them on a map

Ranked by how close their title and abstract are to this paper’s. Select some to read them together.

Closest papers

Close Another approach to the problem
A broad-host-range Rhizobium rhizogenes strain enables transient expression across diverse crop species
Freddie J. King, Juan Carlos Lopez-Agudelo, Christopher Stephens et al. · 2026 · PLoS Biology · Not summarized yet

AS109 enables transient gene expression across diverse crop species, whereas this paper tests native rhizobial T-DNA transfer into legume nodules.

Abstract
Agrobacterium-mediated transient expression has revolutionised plant research, enabling numerous landmark discoveries across diverse areas of plant biology. Yet this powerful approach remains largely confined to solanaceous species, leaving most economically important crop families without a comparable rapid assay platform. Here, we show that an engineered Rhizobium rhizogenes strain, AS109, mediates efficient transient expression across diverse dicot species spanning multiple taxonomic families, consistently outperforming commonly used laboratory agrobacterial strains. Leveraging the broad host range of AS109, we establish a suite of functional assays in faba bean (Vicia faba), including pr…
Close Broader context
Transcriptional silencing of a conjugative Sinorhizobium megaplasmid upon transfer to different rhizobial species
Cristina Robles de la Cruz, L. Cervantes, G. Torres Tejerizo et al. · 2026 · Frontiers in Microbiology · Not summarized yet

This study examines host-dependent silencing after conjugative megaplasmid transfer between rhizobia, rather than bacteria-to-plant DNA transfer.

Abstract
Horizontal gene transfer of large conjugative megaplasmids drives bacterial evolution, however, it forces new hosts to rapidly adapt to a massive influx of exogenous DNA; even the acquisition of small plasmids can have variable effects on host genome activity and fitness. In Sinorhizobium fredii GR64 (symbiont of the common bean) the megaplasmid pSfr64a (191 kb) displays a high conjugative transfer rate. Interestingly, when pSfr64a is transferred to Rhizobium etli (another bean symbiont) conjugative transfer from this host became nearly undetectable, whereas in Agrobacterium tumefaciens it is maintained at a high rate. To unravel the host-plasmid dynamics driving this host-specific response,…
Close Another approach to the problem
DipTRANS: an improved method for in planta transformation and genome engineering in Nicotiana benthamiana.
Fa-Zhan Qi, Wei-Wei Chen, Jon P. Cody et al. · 2026 · Trends in Biotechnology · Not summarized yet

DipTRANS uses Agrobacterium to engineer Nicotiana plants through induced meristems, whereas this paper examines natural rhizobial T-DNA delivery.

Abstract
Plant transformation remains constrained by labor-intensive tissue culture. Our previous work showed that direct delivery of developmental regulators (DRs) can induce de novo meristems on plants, offering a promising transformation approach. In this resource article, we introduced DipTRANS (Direct in planta Transformation), an optimized, soil-based heritable transformation platform for Nicotiana benthamiana that bypasses sterile culture entirely. DipTRANS is built on DR-induced de novo meristem formation. After optimizing parameters, including regulator combinations, Agrobacterium strain, and infiltration density, DipTRANS yielded transformation efficiencies to 46.7%. Developmental abnormali…
Close Broader context
Type III effectors of symbiotic Rhizobia include diverse predicted transcriptional and post-transcriptional modulators
Albin Teulet, S. Schornack · 2026 · PLoS Biology · Not summarized yet

This study characterizes rhizobial type III effectors that modulate host gene regulation, not the T-DNA cargo and transfer systems examined here.

Abstract
Rhizobia are soil bacteria that establish nitrogen-fixing symbioses with legumes. While many rhizobia use a Type III Secretion System to deliver “Nodulation Outer Protein” (Nop) effectors, some uniquely use these proteins to initiate nodule organogenesis, bypassing classical signalling. The molecular functions of these effectors remain largely unknown due to extreme sequence divergence. Using AlphaFold2-mediated structural proteomics, we identified a modular architecture in rhizobial effectors composed of 22 distinct structural units. We reveal that many Nop effectors are cryptic transcriptional or post-transcriptional regulators, harbouring unrecognised nucleic acid–binding modules and RNA-…
Close Broader context
Rhizobial type III effectors as regulators of root-nodule symbiosis in legumes.
Alicia Camuel, Albin Teulet, P. del Cerro · 2026 · Journal of Experimental Botany · Not summarized yet

This review discusses how rhizobial type III effectors regulate legume nodule symbiosis, rather than rhizobial T-DNA transfer.

Abstract
The rhizobia-legume nitrogen-fixing symbiosis begins when plants perceive rhizobial Nod factors (NF), activating the Common Symbiosis Signalling Pathway (CSSP) that drives nodule organogenesis. Rhizobia also inject Type III effectors (T3E) into host cells through a Type III Secretion System (T3SS) related to those of pathogenic bacteria. These effectors were long viewed only as suppressors of plant immunity and determinants of host compatibility, but this picture has changed. Recent studies revealed that rhizobial effectors (known as Nodulation Outer Proteins, Nops) such as NopT, NopM, NopL, NopP and NopZ directly target NF receptors or associated kinases, thereby regulating CSSP activation.…
Close Broader context
Genetic code expansion enables plant-directed control of bacterial activity.
Vivian Zhong, Michaela A. Jones, Avaniek Cabales et al. · 2026 · Nature Biotechnology · Not summarized yet

This study engineers bacterial protein synthesis to control microbial activity near plants, rather than transferring bacterial DNA into plant cells.

Abstract
Microorganisms have the potential to deliver growth-promoting benefits to plants and crops; however, to be useful in a field setting, their gene expression should be programmable and restricted to specific plant hosts and conditions. Here, using O-methyl-L-tyrosine (OMY) as a model compound, we establish noncanonical amino acids (ncAAs) as a tool for plant-based control of associated microbial activity. We use genetic code expansion to engineer OMY-dependent control of protein synthesis in the soil bacterium Bacillus subtilis. Engineered B. subtilis strains express an orthogonal aminoacyl-tRNA synthetase and tRNA pair that, upon provision of OMY, results in suppression of in-frame amber stop…
Close Another approach to the problem
Establishment of an efficient Agrobacterium rhizogenes-mediated hairy root transformation system for functional analysis in tomato
Si-Yi Zhang, Long-Zhu Huang, Jun-Jie Liu et al. · 2026 · Frontiers in Plant Science · Not summarized yet

This study optimizes Agrobacterium rhizogenes-mediated tomato hairy-root transformation, whereas this paper tests native rhizobial DNA transfer in legume nodules.

Abstract
Tomato is an important model for functional genomics in Solanaceae, and elucidating its gene functions provides valuable insights into conserved genetic regulatory networks across dicotyledonous plants. Hairy root transformation is a key technology for functional studies in tomato. However, systematic optimization of its parameters remains limited. Here, we established an efficient Agrobacterium rhizogenes-mediated hairy root transformation system for tomato cv. Micro-Tom. Using the RUBY visual reporter gene, we systematically evaluated multiple parameters, including A. rhizogenes strain, bacterial optical density (OD600), inoculation duration, acetosyringone (AS) concentration, and the type…
Close Broader context
Suppression of plant immune responses enhances Agrobacterium-mediated transient expression in Nicotiana benthamiana.
Yun-Jee Kang, Fen Xie, Ge Bai et al. · 2026 · Plant Science · Not summarized yet

This study investigates plant immune responses during Agrobacterium transient expression, while this paper focuses on the diversity and function of T-DNA transfer systems.

Abstract
Agrobacterium tumefaciens-mediated transient expression in Nicotiana benthamiana is widely used for recombinant protein production and gene-function studies. However, agroinfiltration can activate host stress and defense responses, thereby limiting heterologous protein accumulation. To distinguish Agrobacterium-associated transcriptional responses from effects of the infiltration procedure, we performed RNA sequencing (RNA-seq) using an infection group (GV3101 carrying empty p1300), a buffer-only mock-infiltration group (Mock), and untreated wild-type (WT) plants. Relative to the mock group, the infection group contained 3,529 upregulated and 2,487 downregulated differentially expressed gene…

Go further

How are these papers found?

We compare the title and abstract of this paper with those of every paper summarized on this site, and with the papers Semantic Scholar recommends, those it cites and those citing it (for a much-cited paper, its most recent citations and, from OpenAlex, the most cited ones). Among the prior work and the follow-ups, the papers that use each other in depth, a method it uses and the much-cited follow-ups come a little higher. The levels (very close, close, related) come from the similarity of the texts, not from a judgment of quality; * marks a paper without an abstract, compared by its title. The sentence under a paper is written by an AI from the two abstracts: check it in the papers. A paper marked not relevant by three readers leaves the list.

Updated on 9 November 2026.

Disclaimer: The AI-based summarization tool and virtual assistant provided on this website may not always provide accurate and complete summaries or responses. We encourage you to carefully review and evaluate the generated content to ensure its quality and relevance to your needs.

Rhizobia transform legume nodules via native T-DNA transfer systems

The essentials

Question

Method

Findings

Limitations

Why it matters

Read further

Definitions

Rhizobia Can Deliver DNA to Plants Without Causing Disease

Looking beyond symptoms

Testing whether the DNA works

What might the transferred genes do?

A new perspective on plant–bacteria interactions

Ask questions about this paper to our AI assistant

Tools for this paper

Advanced tools Premium

Papers around this one

Closest papers

Go further

Cite this paper (BibTeX)