The evolutionary origin of nitrogen-fixing symbiosis has been a long-standing question. To address this, we focused on Bradyrhizobium, a globally abundant bacterial genus that includes classic symbiotic lineages, which rely on the common Nod factor signaling pathway to form nodules, and close relatives capable of fixing nitrogen in a free-living state. We isolated 88 strains carrying the key genes for nitrogen fixation (nif) from nonlegume environments and analyzed them alongside 586 public Bradyrhizobium genomes harboring these genes to reconstruct a robust phylogeny of nif genes. Analysis suggests that the earliest-diverging nif lineages are members capable of free-living nitrogen fixation, supporting the interpretation that this lifestyle is ancestral. The Nod factor-dependent symbiotic lineages are polyphyletic, with our data supporting at least three independent origins via horizontal acquisition of symbiosis islands. This evolutionary history is reflected in a genomic dichotomy: lineages capable of free-living nitrogen fixation possess a conserved nif island architecture that consistently includes the oxygen-protective gene glbO, whereas the symbiotic nif-associated regions are highly variable and universally lack glbO. Using both loss-of-function and gain-of-function genetic approaches, we show that glbO contributes significantly to nitrogenase activity under free-living conditions, whereas it is dispensable within the protected nodule environment. This work provides a framework for the evolution of nitrogen-fixing symbiosis, supporting the view that free-living nitrogen-fixing ancestors gave rise repeatedly and independently to symbiotic lineages in Bradyrhizobium.

African rice ( Oryza glaberrima ) was independently domesticated in West Africa around 3000 years ago, and has long been intertwined in the history of the region. The gradual replacement of African rice by Asian rice ( Oryza sativa ), which was introduced when European settlers arrived, has since dominated rice cultivation in Africa. Domesticated rice species are affected by bacterial leaf blight (BLB), which is caused by the pathogen Xanthomonas oryzae pv. oryzae (Xoo). Here we provide evidence that the bacterial leaf blight pathogen in Africa (AfXoo) belongs to a distinct phylogroup from the one circulating in Asia (AsXoo), and has a different evolutionary history. Analysis of 88 AfXoo genomes identified five groups, one of which is a highly diverse population that might have probably given rise to three independent clonal populations based on multiple genetic tests. Tip-dating analysis revealed that the emergence and expansion of AfXoo coincided with the rise and fall of African rice nearly a thousand years ago, and O. sativa served as a bottleneck in the evolution of AfXoo over time. Although the type III effectors (T3E), proteins that are secreted by the pathogen to evade host resistance or seize control of host nutrients, are highly conserved in AfXoo, we observed some variation in effector families. Different evolutionary modifications in the transcription activator-like effectors (TALEs), especially in repeat variable di-residues (RVDs), likely enabled adaptation to both host species. Previous analyses carried out on samples collected in Burkina Faso have shown that there could be more than one TALE repertoire combination in the field, and genome sequencing data revealed potential TALE evolutionary mechanisms that could happen. Our research provides a comprehensive genetic history of bacterial blight in West Africa, and its past and present impact on rice cultivation in the region. For thousands of years, rice cultivation has been an integral part of African agriculture. However, the cultivation of the locally domesticated African rice cultivar ( Oryza glaberrima ) has been gradually shifted towards Asian rice varieties ( Oryza sativa ), which has affected the adaptation of the native pathogen population. One of these pathogens is the causal agent of bacterial leaf blight, Xanthomonas oryzae pv. oryzae ( Xoo ). Here we performed a population genomics approach to understand the evolutionary history and virulence spectrum of African Xoo (AfXoo), a unique phylogroup within the Xanthomonas oryzae species. Our results suggest that AfXoo were first adapted to African rice at least a thousand years ago. The introduction of O. sativa has shaped the recent population dynamics of AfXoo. TALEs are tightly conserved in AfXoo with multiple sequence variations unique to different populations, which could be explained by different evolutionary forces acting upon both domesticated rice. Our results highlight the interplay between crop domestication and cultivation and pathogen evolution.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight (BLB), a major rice disease causing up to 70% yield loss in Asia and West Africa. First described in Japan in 1884 and later reported in West Africa in the 1970s, BLB recently emerged in East Africa, with an epidemic reported in Tanzania in 2019. Remarkably, the disease was detected for the first time in Madagascar the same year, representing a serious threat to food security. To investigate the origin of BLB in Madagascar, we isolated 73 Xoo strains from symptomatic rice leaves collected between 2019 and 2023. Multilocus variable-number of tandem-repeats analysis genotyping revealed 19 haplotypes forming a single clonal complex, indicating low diversity and a likely recent introduction. To come up with disease control strategies, IRBB-based race-typing was achieved and identified four resistance genes (Xa8, xa13, Xa21, and Xa23) that confer resistance to all Malagasy strains tested, whereas the 19 Malagasy varieties assessed were susceptible. The analysis of SWEET knockout lines confirmed that Malagasy strains rely on the susceptibility gene OsSWEET11 for full virulence. Whole-genome sequencing and transcription activator-like effector repertoire analyses of two strains allowed for the identification of a PthXo1 ortholog predicted to induce OsSWEET11. Single-nucleotide polymorphism-based phylogenetic analyses clustered Malagasy strains within Asian lineages, most closely related to strains originated from India. Malagasy strains did not cluster with recently reported Tanzanian Xoo, suggesting independent introductions. Overall, our study demonstrates that BLB in Madagascar results from a recent and single introduction from Asia and identifies effective resistance genes for deployment. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The authors have withdrawn this manuscript owing to finding inconsistencies in genotyping of kitaake rice eds1 pad4 double mutant lines and to establishing that combined loss of EDS1 and PAD4 does not underlie the stunted ‘autoimmune-like’ phenotype reported in this preprint. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding authors: Jane Parker, Thomas Kroj and Haitao Cui (co-corresponding) ### Competing Interest Statement The authors have declared no competing interest.

Bacterial leaf blight (BB) of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), threatens global food security and the livelihood of small-scale rice producers. Analyses of Xoo collections from Asia, Africa and the Americas demonstrated complete continental segregation, despite robust global rice trade. Here, we report unprecedented BB outbreaks in Tanzania. The causative strains, unlike endemic African Xoo, carry Asian-type TAL effectors targeting the sucrose transporter SWEET11a and iTALes suppressing Xa1. Phylogenomics clustered these strains with Xoo from Southern-China. African rice varieties do not carry effective resistance. To protect African rice production against this emerging threat, we developed a hybrid CRISPR-Cas9/Cpf1 system to edit all known TALe-binding elements in three SWEET promoters of the East African elite variety Komboka. The edited lines show broad-spectrum resistance against Asian and African strains of Xoo, including strains recently discovered in Tanzania. The strategy could help to protect global rice crops from BB pandemics.

Xanthomonas oryzae pv. oryzae (Xoo) strains that cause bacterial leaf blight (BLB) limit rice (Oryza sativa) production and require breeding more resistant varieties. Transcription activator-like effectors (TALEs) activate transcription to promote leaf colonization by binding to specific plant host DNA sequences termed effector binding elements (EBEs). Xoo major TALEs universally target susceptibility genes of the SWEET transporter family. TALE-unresponsive alleles of clade III OsSWEET susceptibility gene promoter created with genome editing confer broad resistance on Asian Xoo strains. African Xoo strains rely primarily on the major TALE TalC, which targets OsSWEET14. Although the virulence of a talC mutant strain is severely impaired, abrogating OsSWEET14 induction with genome editing does not confer equivalent resistance on African Xoo. To address this contradiction, we postulated the existence of a TalC target susceptibility gene redundant with OsSWEET14. Bioinformatics analysis identified a rice locus named ATAC composed of the INCREASED LEAF INCLINATION 2 (ILI2) gene and a putative lncRNA that are shown to be bidirectionally upregulated in a TalC-dependent fashion. Gain-of-function approaches with designer TALEs inducing ATAC sequences did not complement the virulence of a Xoo strain defective for SWEET gene activation. While editing the TalC EBE at the ATAC loci compromised TalC-mediated induction, multiplex edited lines with mutations at the OsSWEET14 and ATAC loci remained essentially susceptible to African Xoo strains. Overall, this work indicates that ATAC is a probable TalC off-target locus but nonetheless documents the first example of divergent transcription activation by a native TALE during infection.

The bacterial plant pathogen Xanthomonas oryzae pv. oryzae is responsible for the foliar rice bacterial blight disease. Genetically contrasted, continent-specific, sublineages of this species can cause important damages to rice production both in Asia and Africa. We report on the genome of the CIX2779 strain of this pathogen, previously named NAI1 and originating from Niger. Oxford Nanopore long reads assembly and Illumina short reads polishing produced a genome sequence composed of a 4,725,792-bp circular chromosome and a 39,798-bp-long circular plasmid designated pCIX2779_1. The chromosome structure and base-level sequence are highly related to reference strains of African X. oryzae pv. oryzae and encode identical transcription activator-like effectors for virulence. Importantly, our in silico analysis strongly indicates that pCIX2779_1 is a genuine conjugative plasmid, the first indigenous one sequenced from an African strain of the X. oryzae species. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Non-coding small RNAs (sRNA) act as mediators of gene silencing and regulate plant growth, development and stress responses. Early insights into plant sRNAs established a role in antiviral defense and they are now extensively studied across plant–microbe interactions. Here, sRNA sequencing discovered a class of sRNA in rice (Oryza sativa) specifically associated with foliar diseases caused by Xanthomonas oryzae bacteria. Xanthomonas-induced small RNAs (xisRNAs) loci were distinctively upregulated in response to diverse virulent strains at an early stage of infection producing a single duplex of 20–22 nt sRNAs. xisRNAs production was dependent on the Type III secretion system, a major bacterial virulence factor for host colonization. xisRNA loci overlap with annotated transcripts sequences, with about half of them encoding protein kinase domain proteins. A number of the corresponding rice cis-genes have documented functions in immune signaling and xisRNA loci predominantly coincide with the coding sequence of a conserved kinase motif. xisRNAs exhibit features of small interfering RNAs and their biosynthesis depend on canonical components OsDCL1 and OsHEN1. xisRNA induction possibly mediates post-transcriptional gene silencing but they do not broadly suppress cis-genes expression on the basis of mRNA-seq data. Overall, our results identify a group of unusual sRNAs with a potential role in plant–microbe interactions.

Bacterial blight of rice is an important disease in Asia and Africa. The pathogen, Xanthomonas oryzae pv. oryzae ( Xoo ), secretes one or more of six known transcription-activator-like effectors (TALes) that bind specific promoter sequences and induce, at minimum, one of the three host sucrose transporter genes SWEET11 , SWEET13 and SWEET14 , the expression of which is required for disease susceptibility. We used CRISPR–Cas9-mediated genome editing to introduce mutations in all three SWEET gene promoters. Editing was further informed by sequence analyses of TALe genes in 63 Xoo strains, which revealed multiple TALe variants for SWEET13 alleles. Mutations were also created in SWEET14 , which is also targeted by two TALes from an African Xoo lineage. A total of five promoter mutations were simultaneously introduced into the rice line Kitaake and the elite mega varieties IR64 and Ciherang-Sub1. Paddy trials showed that genome-edited SWEET promoters endow rice lines with robust, broad-spectrum resistance.

To circumvent the paucity of nitrogen sources in the soil legume plants establish a symbiotic interaction with nitrogen-fixing soil bacteria called rhizobia. During symbiosis, the plants form root organs called nodules, where bacteria are housed intracellularly and become active nitrogen fixers known as bacteroids. Depending on their host plant, bacteroids can adopt different morphotypes, being either unmodified (U), elongated (E) or spherical (S). E- and S-type bacteroids undergo a terminal differentiation leading to irreversible morphological changes and DNA endoreduplication. Previous studies suggest that differentiated bacteroids display an increased symbiotic efficiency (E > U and S > U). In this study, we used a combination of Aeschynomene species inducing E- or S-type bacteroids in symbiosis with Bradyrhizobium sp. ORS285 to show that S-type bacteroids present a better symbiotic efficiency than E-type bacteroids. We performed a transcriptomic analysis on E- and S-type bacteroids formed by Aeschynomene afraspera and Aeschynomene indica nodules and identified the bacterial functions activated in bacteroids and specific to each bacteroid type. Extending the expression analysis in E- and S-type bacteroids in other Aeschynomene species by qRT-PCR on selected genes from the transcriptome analysis narrowed down the set of bacteroid morphotype-specific genes. Functional analysis of a selected subset of 31 bacteroid-induced or morphotype-specific genes revealed no symbiotic phenotypes in the mutants. This highlights the robustness of the symbiotic program but could also indicate that the bacterial response to the plant environment is partially anticipatory or even maladaptive. Our analysis confirms the correlation between differentiation and efficiency of the bacteroids and provides a framework for the identification of bacterial functions that affect the efficiency of bacteroids.© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

La bactériose vasculaire du riz (BLB), causée par Xanthomonas oryzae pv. oryzae (Xoo) est une menace majeure pour la production mondiale de riz, entraînant chaque année des pertes de rendement atteignant jusque 50% en Asie et en Afrique. La pathogénie de Xoo repose sur l'injection dans la cellule hôte d’effecteurs particuliers, appelés Transcription-Activator Like Effectors (TALEs). Ces effecteurs agissent comme des facteurs de transcription qui détournent la machinerie transcriptionnelle de la plante. Les TALEs se lient à des séquences spécifiques appelées Effector Binding Elements (EBE) dans le promoteur de gènes dit de sensibilité (S) et activent leur transcription, qui est essentielle au développement de la maladie. Les membres du clade III de la famille des transporteurs de sucres SWEET sont les principaux gènes de sensibilité à la BLB. Les Xoo Africaines ciblent SWEET14 via deux TALEs distincts, soulignant l'importance de ce gène pour leur virulence. Des mutations naturelles dans les EBEs des promoteurs des gènes SWEET empêchent la fixation des TALE correspondants, et annule leur induction. Ces allèles " non-réceptifs " donnent des gènes de résistance récessifs, réduisant la formation de symptômes et la prolifération bactérienne in planta. La création de résistance par ingénierie génétique est un moyen efficace, économique et écologique de garantir la sécurité alimentaire et financière des pays producteurs de riz. Des lignées poly-mutantes éditées au niveau des 6 EBEs majeurs des gènes SWEET11a, SWEET13 et SWEET14 ont été généré via la technologie CRISPR. Bien que ces lignées éditées soient résistantes aux souches d’origine Asiatique, les souches Africaines de Xoo conservent un certain niveau de virulence.Nous avons alors cherché à comprendre la dépendance des Xoo Africaines vis-à-vis de l'utilisation des gènes SWEET. Par l’inoculation d’une importante collection d’isolats Africains sur des lignées mutantes pour les gènes SWEET du clade III, nous montrons que SWEET13 est potentiellement impliqué dans la virulence résiduelle des Xoo Africaines.Nous avons également prospecté les bases génétiques spécifiant la virulence des Xoo Africaines. La caractérisation phénotypique de plusieurs mutants nous a permis de découvrir une région génomique de 35 kilobases, conditionnant la virulence de la souche modèle BAI3 du Burkina Faso et se révélant prometteuse dans l’explication du phénotype Africain.Un autre volet de nos travaux a consisté en l’étude des populations de Xoo Africaines. Bien que les souches Asiatiques de Xoo, décrites pour la première fois en 1884, aient largement été étudiées, la découverte ultérieure de l'agent pathogène sur le continent Africain (1979) et l’importance plus réduite de la riziculture en Afrique font que ces populations endémiques ont été étudiées plus tardivement. Des résultats antérieurs ont montré que les Xoo Africaines sont très éloignées des Xoo Asiatiques et génétiquement plus proches de Xanthomonas oryzae pv. oryzicola (Xoc). Pour mieux comprendre leur histoire évolutive et leur potentiel épidémique, ainsi que pour promouvoir le déploiement local de sources de résistance adaptées, notre objectif était de caractériser une importante collection de Xoo Africaines. Nos premières analyses via l’utilisation de marqueurs moléculaires ont mis en évidence la structure génétique des populations de Xoo sur le continent Africain et ont révélé que les isolats de l'Ouest sont clairement distincts des isolats de l'Est, ces derniers se révélant très proches des Xoo Asiatiques. Un deuxième volet d’analyses, par séquençages complets de génomes, a confirmé le caractère invasif de ces populations et pointé leur origine vers le Yunnan et la Thaïlande. A l’instar des souches Asiatiques, ces souches dépendent de SWEET11a selon le même modèle d’induction et possèdent des TALEs tronqués qui leur permettent d'échapper à la résistance médiée par le gène hôte Xa1.