An optimised transformation protocol for Anthoceros agrestis and three more hornwort species

Land plants comprise two large monophyletic lineages, the vascular plants and the bryophytes, which diverged from their most recent common ancestor approximately 480 million years ago. Of the three lineages of bryophytes, only the mosses and the liverworts are systematically investigated, while the hornworts are understudied. Despite their importance for understanding fundamental questions of land plant evolution, they only recently became amenable to experimental investigation, with Anthoceros agrestis being developed as a hornwort model system. Availability of a high-quality genome assembly and a recently developed genetic transformation technique makes A. agrestis an attractive model species for hornworts. Here we describe an updated and optimised transformation protocol for A. agrestis which can be successfully used to genetically modify one more strain of A. agrestis and three more hornwort species, Anthoceros punctatus, Leiosporoceros dussi and Phaeoceros carolinianus. The new transformation method is less laborious, faster and results in the generation of greatly increased numbers of transformants compared to the previous method. We have also developed a new selection marker for transformation. Finally, we report the development of a set of different cellular localisation signal peptides for hornworts providing new tools to better understand hornwort cell biology

Towards a global interactomic map between the Ralstonia solanacearum species complex core T3Es and the tomato proteome

La stratégie de virulence du complexe d'espèces Ralstonia solanacearum (RSSC) repose sur des effecteurs de type III (T3E) injectés à l'intérieur des cellules végétales via un système de sécrétion de type III. Grâce aux séquences génomiques actuellement connues de plus de 150 souches phytopathogènes du RSSC, nous avons identifié différents ensembles de core-T3E selon qu'ils sont conservés parmi toutes les souches du RSSC ou dans des souches isolées à partir d'hôtes spécifiques. Comme ces core-T3E ont été conservés au sein des souches de RSSC affectant les Solanacées, nous émettons l'hypothèse que ces déterminants doivent être importants pour la virulence. Notre objectif principal est d'identifier les cibles putatives chez la tomate pour tous ces core-T3E en utilisant des criblages systématiques par double hybride sur une banque d'ADNc de racines de tomate. De nombreuses cibles pour la tomate, dont certaines sont des ''hubs'', ont été identifiées à partir de 14 criblages. Par des approches de double hybride matriciel, nous avons confirmé et étendu le nombre de cible "hub" à 17 candidats. La validation de certaines interactions in planta ainsi que la génétique de ces "hubs" sont poursuivies pour tester leur contribution au flétrissement bactérien. Des plantes de tomates CRISPR-Cas9 ont également été réalisés pour valider le rôle de certains de ces gènes.The Ralstonia solanacearum species complex (RSSC) virulence strategy relies on type III effectors (T3Es) injected inside the plant cells via a type III secretion system. Thanks to the currently known genomic sequences of more than 150 phytopathogenic strains of the RSSC, we have identified different sets of core-T3Es according of being conserved among all the RSSC strains or according to strains isolated from specific hosts. As these core-T3Es have been conserved through the evolution of the RSSC strains affecting Solanaceae, we hypothesize that they must be important for virulence in tomato. Our main objective is to identify putative tomato targets for all of these core-T3Es using systematic Y2H screening against a tomato root cDNA library. Many candidate tomato targets, some of which are "hubs", were identified from 14 screenings. These identified targets have been tested against all the core-T3Es and other effectors from different pathogens and thanks to the Y2H-pairwise-matrix, we were able to increase the number of the identified hubs up to 17 candidates. Validation of some interactions in planta as well as reverse genetics (insertion mutants in Petunia and Arabidopsis) for these "hubs" is carried on to test their contribution to the bacterial wilt. CRISPR-Cas9 tomato plants were also performed to validate the role of some of these genes

Vers une carte interactomique globale entre les effecteurs de type III conservés au sein du complexe d’espèce Ralstonia solanacearum et le protéome de la tomate

La stratégie de virulence du complexe d'espèces Ralstonia solanacearum (RSSC) repose sur des effecteurs de type III (T3E) injectés à l'intérieur des cellules végétales via un système de sécrétion de type III. Grâce aux séquences génomiques actuellement connues de plus de 150 souches phytopathogènes du RSSC, nous avons identifié différents ensembles de core-T3E selon qu'ils sont conservés parmi toutes les souches du RSSC ou dans des souches isolées à partir d'hôtes spécifiques. Comme ces core-T3E ont été conservés au sein des souches de RSSC affectant les Solanacées, nous émettons l'hypothèse que ces déterminants doivent être importants pour la virulence. Notre objectif principal est d'identifier les cibles putatives chez la tomate pour tous ces core-T3E en utilisant des criblages systématiques par double hybride sur une banque d'ADNc de racines de tomate. De nombreuses cibles pour la tomate, dont certaines sont des ‘‘hubs’’, ont été identifiées à partir de 14 criblages. Par des approches de double hybride matriciel, nous avons confirmé et étendu le nombre de cible ‘’hub’’ à 17 candidats. La validation de certaines interactions in planta ainsi que la génétique de ces "hubs" sont poursuivies pour tester leur contribution au flétrissement bactérien. Des plantes de tomates CRISPR-Cas9 ont également été réalisés pour valider le rôle de certains de ces gènes.The Ralstonia solanacearum species complex (RSSC) virulence strategy relies on type III effectors (T3Es) injected inside the plant cells via a type III secretion system. Thanks to the currently known genomic sequences of more than 150 phytopathogenic strains of the RSSC, we have identified different sets of core-T3Es according of being conserved among all the RSSC strains or according to strains isolated from specific hosts. As these core-T3Es have been conserved through the evolution of the RSSC strains affecting Solanaceae, we hypothesize that they must be important for virulence in tomato. Our main objective is to identify putative tomato targets for all of these core-T3Es using systematic Y2H screening against a tomato root cDNA library. Many candidate tomato targets, some of which are “hubs”, were identified from 14 screenings. These identified targets have been tested against all the core-T3Es and other effectors from different pathogens and thanks to the Y2H-pairwise-matrix, we were able to increase the number of the identified hubs up to 17 candidates. Validation of some interactions in planta as well as reverse genetics (insertion mutants in Petunia and Arabidopsis) for these “hubs” is carried on to test their contribution to the bacterial wilt. CRISPR-Cas9 tomato plants were also performed to validate the role of some of these genes

An optimized transformation protocol for Anthoceros agrestis and three more hornwort species.

Funder: Georges and Antoine Claraz Foundation grantFunder: University Research Priority Program “Evolution in Action” of the University of ZurichLand plants comprise two large monophyletic lineages, the vascular plants and the bryophytes, which diverged from their most recent common ancestor approximately 480 million years ago. Of the three lineages of bryophytes, only the mosses and the liverworts are systematically investigated, while the hornworts are understudied. Despite their importance for understanding fundamental questions of land plant evolution, they only recently became amenable to experimental investigation, with Anthoceros agrestis being developed as a hornwort model system. Availability of a high-quality genome assembly and a recently developed genetic transformation technique makes A. agrestis an attractive model species for hornworts. Here we describe an updated and optimized transformation protocol for A. agrestis, which can be successfully used to genetically modify one more strain of A. agrestis and three more hornwort species, Anthoceros punctatus, Leiosporoceros dussii, and Phaeoceros carolinianus. The new transformation method is less laborious, faster, and results in the generation of greatly increased numbers of transformants compared with the previous method. We have also developed a new selection marker for transformation. Finally, we report the development of a set of different cellular localization signal peptides for hornworts providing new tools to better understand the hornwort cell biology