Arabidopsis katanin binds microtubules using a multimeric microtubule-binding domain.

Katanin is a heterodimeric protein that mediates ATP-dependent destabilization of microtubules in animal cells. In plants, the catalytic subunit of Arabidopsis thaliana katanin (AtKSS, Arabidopsis thaliana Katanin Small Subunit) has been identified and its microtubule-severing activity has been demonstrated in vitro. In vivo, plant katanin plays a role in the organization of cortical microtubules, but the way it achieves this function is unknown. To go further in our understanding of the mechanisms by which katanin severs microtubules, we analyzed the functional domains of Arabidopsis katanin. We characterized the microtubule-binding domain of katanin both in vitro and in vivo. It corresponds to a poorly conserved sequence between plant and animal katanins that is located in the N-terminus of the protein. This domain interacts with cortical microtubules in vivo and has a low affinity for microtubules in vitro. We also observed that katanin microtubule-binding domain oligomerizes into trimers. These results show that, besides being involved in the interaction of katanin with microtubules, the microtubule-binding domain may also participate in the oligomerization of katanin. At the structural level, we observed that AtKSS forms ring-shaped oligomers

MtbHLH1, a bHLH transcription factor involved in Medicago truncatula nodule vascular patterning and nodule to plant metabolic exchanges

This study aimed at defining the role of a basic helix–loop–helix (bHLH) transcription factor gene from Medicago truncatula, MtbHLH1, whose expression is upregulated during the development of root nodules produced upon infection by rhizobia bacteria.We used MtbHLH1 promoter::GUS fusions and quantitative reverse-transcription polymerase chain reaction analyses to finely characterize the MtbHLH1 expression pattern. We altered MtbHLH1 function by expressing a dominantly repressed construct (CRES-T approach) and looked for possible MtbHLH1 target genes by transcriptomics.We found that MtbHLH1 is expressed in nodule primordia cells derived from pericycle divisions, in nodule vascular bundles (VBs) and in uninfected cells of the nitrogen (N) fixation zone. MtbHLH1 is also expressed in root tips, lateral root primordia cells and root VBs, and induced upon auxin treatment. Altering MtbHLH1 function led to an unusual phenotype, with a modified patterning of nodule VB development and a reduced growth of aerial parts of the plant, even though the nodules were able to fix atmospheric N. Several putative MtbHLH1 regulated genes were identified, including an asparagine synthase and a LOB (lateral organ boundary) transcription factor.Our results suggest that the MtbHLH1 gene is involved in the control of nodule vasculature patterning and nutrient exchanges between nodules and roots

Experimental Evolution of a Plant Pathogen into a Legume Symbiont

Following acquisition of a rhizobial symbiotic plasmid, adaptive mutations in the virulence pathway allowed pathogenic Ralstonia solanacearum to evolve into a legume symbiont under plant selection

Light microscopy of whole plant organs

International audiencePlants are ideal organisms for light microscopical studies of cellular mechanisms controlling cell organisation and cell functioning. However, most plant organs are not transparent to light which prevents high resolution imaging deep within plant tissues. Classically, access into plant organs is achieved by sectioning or whole-mount tissue clearing. Until recently, the protocols for clearing destroyed the signal from fluorescent markers which prevented the imaging of the distribution of fluorescent proteins and the three-dimensional reconstruction from optical slices of whole plant organs. From 2011, a number of protocols have been developed for whole brain and whole organism imaging for animal studies. Now, these protocols have been adapted for in-depth imaging of whole plant organs. Here, I present an overview of clearing techniques of plant organs and highlight the latest developments of plant tissue clearing in combination with high resolution fluorescence microscopy

Knr4 N-terminal domain controls its localization and function during sexual differentiation and vegetative growth

International audienceThe Saccharomyces cerevisiae protein Knr4 is composed of a globular central core flanked by two natively disordered regions. Although the central part of the protein holds most of its biological function, the N-terminal domain (amino acids 1-80) is essential in the absence of a functional CWI pathway. We show that this specific protein domain is required for the proper cellular localization of Knr4 at sites of polarized growth during vegetative growth and sexual differentiation (bud tip and 'shmoo' tip). Moreover, Knr4 N-terminal domain is also necessary for cell cycle arrest and shmoo formation in response to pheromone to occur at the correct speed. Thus, the presence of Knr4 at the incipient mating projection site seems important for the establishment of the following polarized growth. Cell wall integrity (CWI) and calcineurin pathways are known to share a common essential function, for which they can substitute for one another. Searching for Knr4 partners responsible for survival in a CWI-defective background, we found that the catalytic subunit of calcineurin Cna1 physically interacts with Knr4 in the yeast two-hybrid assay, in a manner dependent on the presence of the Knr4 N-terminal domain. In addition, we present evidence that Knr4 protein participates in the morphogenesis checkpoint, a safety mechanism that holds the cell cycle in response to bud formation defects or insults in cytoskeleton organization, and in which both the CWI pathway and calcineurin are involved

Localization and function analysis of Type III effector RipG7 of Ralstonia solanacearum

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Mechanism of Infection Thread Elongation in Root Hairs of Medicago truncatula and Dynamic Interplay with Associated Rhizobial Colonization

International audienceIn temperate legumes, endosymbiotic nitrogen-fixing rhizobia gain access to inner root tissues via a specialized transcellular apoplastic compartment known as the infection thread (IT). To study IT development in living root hairs, a protocol has been established for Medicago truncatula that allows confocal microscopic observations of the intracellular dynamics associated with IT growth. Fluorescent labeling of both the IT envelope (AtPIP2;1-green fluorescent protein) and the host endoplasmic reticulum (green fluorescent protein-HDEL) has revealed that IT growth is a fundamentally discontinuous process and that the variable rate of root hair invagination is reflected in changes in the host cell cytoarchitecture. The concomitant use of fluorescently labeled Sinorhizobium meliloti has further revealed that a bacteria-free zone is frequently present at the growing tip of the IT, thus indicating that bacterial contact is not essential for thread progression. Finally, these in vivo studies have shown that gaps within the bacterial file are a common feature during the early stages of IT development, and that segments of the file are able to slide collectively down the thread. Taken together, these observations lead us to propose that (1) IT growth involves a host-driven cellular mechanism analogous to that described for intracellular infection by arbuscular mycorrhizal fungi; (2) the non-regular growth of the thread is a consequence of the rate-limiting colonization by the infecting rhizobia; and (3) bacterial colonization involves a combination of bacterial cell division and sliding movement within the extracellular matrix of the apoplastic compartment