Callose-Regulated Symplastic Communication Coordinates Symbiotic Root Nodule Development

The formation of nitrogen-fixing nodules in legumes involves the initiation of synchronized programs in the root epidermis and cortex to allow rhizobial infection and nodule development. In this study, we provide evidence that symplastic communication, regulated by callose turnover at plasmodesmata (PD), is important for coordinating nodule development and infection in Medicago truncatula. Here, we show that rhizobia promote a reduction in callose levels in inner tissues where nodules initiate. This downregulation coincides with the localized expression of M. truncatula β-1,3-glucanase 2 (MtBG2), encoding a novel PD-associated callose-degrading enzyme. Spatiotemporal analyses revealed that MtBG2 expression expands from dividing nodule initials to rhizobia-colonized cortical and epidermal tissues. As shown by the transport of fluorescent molecules in vivo, symplastic-connected domains are created in rhizobia-colonized tissues and enhanced in roots constitutively expressing MtBG2. MtBG2-overexpressing roots additionally displayed reduced levels of PD-associated callose. Together, these findings suggest an active role for MtBG2 in callose degradation and in the formation of symplastic domains during sequential nodule developmental stages. Interfering with symplastic connectivity led to drastic nodulation phenotypes. Roots ectopically expressing β-1,3-glucanases (including MtBG2) exhibited increased nodule number, and those expressing MtBG2 RNAi constructs or a hyperactive callose synthase (under symbiotic promoters) showed defective nodulation phenotypes. Obstructing symplastic connectivity appears to block a signaling pathway required for the expression of NODULE INCEPTION (NIN) and its target NUCLEAR FACTOR-YA1 (NF-YA1) in the cortex. We conclude that symplastic intercellular communication is proactively enhanced by rhizobia, and this is necessary for appropriate coordination of bacterial infection and nodule development

Developmentally early and late onset of Rps10 silencing in Arabidopsis thaliana: genetic and environmental regulation

Transgene dosage, silencing competence of the transgene loci, and photoperiod conditions were found to regulate the onset and efficiency of Rps10 silencing in two independent transgenic lines of Arabidopsis thaliana. The Rps10 gene encodes the S10 protein which is part of the small subunit of mitochondrial ribosomes. Homozygous plants presented developmentally early onset of silencing, a very efficient decrease in the level of Rps10 transcripts, as well as a severe and uniform phenotype called P1. P1 plants either died during the vegetative growth phase or were rescued by reversion resulting from inactivation of silencing. A wide variety of morphological and developmental abnormalities observed within the hemizygous transformants allowed their classification into three categories P2, P3, and P4. The most severe and early was the P2 phenotype found in only one transgenic line and most probably resulting from high competence of the transgene loci. Developmentally late onset of silencing occurred only in the short day photoperiod and was characteristic for the P3 and P4 plants. This phenomenon was attributed to conditions favourable to silencing achieved in the short day photoperiod, e.g. a greatly prolonged vegetative phase accompanied by a gradual increase of the level of Rps10 transcripts. To the best of our knowledge, this is the first report indicating that the onset of silencing depends on the photoperiod conditions in A. thaliana

A protein complex required for polar growth of rhizobial infection threads

Abstract: During root nodule symbiosis, intracellular accommodation of rhizobia by legumes is a prerequisite for nitrogen fixation. For many legumes, rhizobial colonization initiates in root hairs through transcellular infection threads. In Medicago truncatula, VAPYRIN (VPY) and a putative E3 ligase LUMPY INFECTIONS (LIN) are required for infection thread development but their cellular and molecular roles are obscure. Here we show that LIN and its homolog LIN-LIKE interact with VPY and VPY-LIKE in a subcellular complex localized to puncta both at the tip of the growing infection thread and at the nuclear periphery in root hairs and that the punctate accumulation of VPY is positively regulated by LIN. We also show that an otherwise nuclear and cytoplasmic exocyst subunit, EXO70H4, systematically co-localizes with VPY and LIN during rhizobial infection. Genetic analysis shows that defective rhizobial infection in exo70h4 is similar to that in vpy and lin. Our results indicate that VPY, LIN and EXO70H4 are part of the symbiosis-specific machinery required for polar growth of infection threads

A protein complex required for polar growth of rhizobial infection threads

Abstract: During root nodule symbiosis, intracellular accommodation of rhizobia by legumes is a prerequisite for nitrogen fixation. For many legumes, rhizobial colonization initiates in root hairs through transcellular infection threads. In Medicago truncatula, VAPYRIN (VPY) and a putative E3 ligase LUMPY INFECTIONS (LIN) are required for infection thread development but their cellular and molecular roles are obscure. Here we show that LIN and its homolog LIN-LIKE interact with VPY and VPY-LIKE in a subcellular complex localized to puncta both at the tip of the growing infection thread and at the nuclear periphery in root hairs and that the punctate accumulation of VPY is positively regulated by LIN. We also show that an otherwise nuclear and cytoplasmic exocyst subunit, EXO70H4, systematically co-localizes with VPY and LIN during rhizobial infection. Genetic analysis shows that defective rhizobial infection in exo70h4 is similar to that in vpy and lin. Our results indicate that VPY, LIN and EXO70H4 are part of the symbiosis-specific machinery required for polar growth of infection threads

Transgene Silencing and Transgene-Derived siRNA Production in Tobacco Plants Homozygous for an Introduced AtMYB90 Construct

Transgenic tobacco (Nicotiana tabacum) lines were engineered to ectopically over-express AtMYB90 (PAP2), an R2–R3 Myb gene associated with regulation of anthocyanin production in Arabidopsis thaliana. Independently transformed transgenic lines, Myb27 and Myb237, accumulated large quantities of anthocyanin, generating a dark purple phenotype in nearly all tissues. After self-fertilization, some progeny of the Myb27 line displayed an unexpected pigmentation pattern, with most leaves displaying large sectors of dramatically reduced anthocyanin production. The green-sectored 27Hmo plants were all found to be homozygous for the transgene and, despite a doubled transgene dosage, to have reduced levels of AtMYB90 mRNA. The observed reduction in anthocyanin pigmentation and AtMYB90 mRNA was phenotypically identical to the patterns seen in leaves systemically silenced for the AtMYB90 transgene, and was associated with the presence of AtMYB90-derived siRNA homologous to both strands of a portion of the AtMYB90 transcribed region. Activation of transgene silencing in the Myb27 line was triggered when the 35S::AtMYB90 transgene dosage was doubled, in both Myb27 homozygotes, and in plants containing one copy of each of the independently segregating Myb27 and Myb237 transgene loci. Mapping of sequenced siRNA molecules to the Myb27 TDNA (including flanking tobacco sequences) indicated that the 3′ half of the AtMYB90 transcript is the primary target for siRNA associated silencing in both homozygous Myb27 plants and in systemically silenced tissues. The transgene within the Myb27 line was found to consist of a single, fully intact, copy of the AtMYB90 construct. Silencing appears to initiate in response to elevated levels of transgene mRNA (or an aberrant product thereof) present within a subset of leaf cells, followed by spread of the resulting small RNA to adjacent leaf tissues and subsequent amplification of siRNA production

Caractérisation de deux déterminants moléculaires impliqués dans le processus d'infection lors de l'interaction symbiotique entre la légumineuse modèle Medicago truncatula et sinorhizobium meliloti

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

La régulation transcriptionnelle du gène MtENOD11 au cours des endosymbioses racinaires (caractérisation des éléments cis-régulateurs spécifiques de la réponse aux facteurs Nod)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

La régulation transcriptionnelle du gène MtENOD11 au cours des endosymbioses racinaires (caractérisation des éléments cis-régulateurs spécifiques de la réponse aux facteurs Nod)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

A simple Agrobacterium tumefaciens-mediated transformation method for rapid transgene expression in Medicago truncatula root hairs

International audienceMedicago truncatula is widely used as a model legume for symbiotic and pathogenic microbial interaction studies. Although a number of Agrobacterium-mediated transformation methods have been developed for M. truncatula, a rapid root transformation system was not yet available for this model plant. Here, we describe an easy method for rapid transgene expression in root hairs of M. truncatula, using young seedlings co-cultivated with the disarmed hypervirulent A. tumefaciens strain AGL1. This method leads to efficient expression of various GUS and fluorescent reporters in M. truncatula root hairs. We showed that transgene expression is detected as soon as 2 days following co-culture, in root hairs of a particular responsive zone lying 0.5–2 cm behind the root tip. This method can be used with a variety of M. truncatula genotypes, and is particularly useful for rapid investigation of the sub-cellular localization of fluorescent fusion proteins. Moreover, combining distinct Agrobacterium strains during the initial co-culture step efficiently generates co-transformed root hairs, suitable for co-localization of different fluorescent fusion proteins in the same cell