Characterization of Xanthomonas oryzae pv.oryzae populations for improved resistance deployment in Africa : innovative strategies to control bacterial leaf blight of rice.

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 leurinduction. 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.Bacterial Leaf Blight (BLB) which is caused by Xanthomonas oryzae pv. oryzae (Xoo), is a major threat to rice production worldwide, leading up to 50% yield losses in Asia and Africa during severe infections. Xoo pathogenicity relies on the injection into the host cell of Transcription-Activator Like Effectors (TALEs) which are effective transcription factors specialized to hijack the plant transcriptional machinery. TALEs bind to Effector Binding Elements (EBE) in the promoter of susceptibility (S) genes and activate transcription, which is essential for disease development. Clade-III members of the family of SWEET sugar transporters are major BLB susceptibility genes. Compared to Asian Xoo, African strains have fewer TALEs and specifically targets SWEET14 as a major susceptibility gene. Besides, African Xoo use two distinct TAL effectors to induce SWEET14, highlighting the importance of this gene for bacterial proliferation. Naturally occurring polymorphism in the EBEs of SWEET promoters prevent TALE binding, resulting in loss of SWEET gene induction. These “unresponsive” alleles act as recessive resistance genes, preventing symptoms formation and reducing bacterial colonization in planta. Genetic engineering to gain resistance is an effective, economic and ecologic way to guarantee food and economic safety of rice producing countries. Poly-mutant edited lines at the level of the 5 major EBEs of the SWEET11a, SWEET13 and SWEET14 genes were generated via CRISPR-Cas9 technology. However, while our multi-edited lines were able to defeat Xoo strains of Asian origin, African Xoo strains still maintained some level of virulence.Here, we sought to unravel African Xoo dependence towards the use of SWEET genes. By inoculating a large collection of strains on knock-out lines for clade III SWEET genes, we show that SWEET13 is potentially involved in the residual virulence of African Xoo.We also invastigated the bacterial genetic bases for the virulence specificity of African Xoo. By phenotyping several bacterial mutants, we discovered a 35 kilobases genomic region conditionning the virulence of the model strain BAI3 from Burkina Faso, which appears promising to explain African particular virulence phenotype.Finally, while Asian strains of Xoo, first described in 1884, are widely studied, the later discovery of the pathogen on the African continent (1979) and the lesser importance of African rice cultivation have made these endemic strains less characterized. Previous results have shown that African difer strongly from Asian Xoo, and are genetically more closely related to Xanthomonas oryzae pv. oryzicola (Xoc). To better understand their evolutionary history and epidemic potential, and in order to promote the deployment of locally adapted sources of resistance, our objective was to characterize a large collection of African Xoo. Our analyses shed light on the genetic structure of Xoo populations on the African contient and reveal two different lineages : West African Xoo and East African Xoo. We show that East African Xoo strains are closely related to Asian strains, and that they also rely on the induction of SWEET11a to promote disease, using similar TAL effectors. East African strains, as their Asian relatives, do countain truncated TALEs that allow them to escape from the resistance mediated by Xa1 a rice nucleotide-binding domain leucine rich protein (NLR) that specifically recognize TALEs

The Complete Genome Resource of Xanthomonas oryzae pv. oryzae CIX2779 Includes the First Sequence of a Plasmid for an African Representative of This Rice Pathogen

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. [Graphic: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license

Iron-induced release of intracellular free calcium is involved in growth arrest of Arabidopsis thaliana primary root

Iron-induced release of intracellular free calcium is involved in growth arrest of Arabidopsis thaliana primary root. 11th national meeting of the Société Française de Biologie Végétale SFB

The Rice ILI2 Locus Is a Bidirectional Target of the African Xanthomonas oryzae pv. oryzae Major Transcription Activator-like Effector TalC but Does Not Contribute to Disease Susceptibility

International audienceXanthomonas 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

Transcriptomic dissection of Bradyrhizobium sp. strain ORS285 in symbiosis with Aeschynomene spp. inducing different bacteroid morphotypes with contrasted symbiotic efficiency

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. This article is protected by copyright. All rights reserved

An atypical class of non-coding small RNAs is produced in rice leaves upon bacterial infection

International audienceNon-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

Transcriptomic dissection of Bradyrhizobium sp. strain ORS285 in symbiosis with Aeschynomene spp. inducing different bacteroid morphotypes with contrasted symbiotic efficiency

International audienceTo 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

Broad-spectrum resistance to bacterial blight in rice using genome editing

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