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      VvSWEET7 Is a Mono- and Disaccharide Transporter Up-Regulated in Response to Botrytis cinerea Infection in Grape Berries

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          Abstract

          The newly-identified SWEETs are high-capacity, low-affinity sugar transporters with important roles in numerous physiological mechanisms where sugar efflux is critical. SWEETs are desirable targets for manipulation by pathogens and their expression may be transcriptionally reprogrammed during infection. So far, few plant SWEET transporters have been functionally characterized, especially in grapevine. In this study, in the Botrytis-susceptible variety “Trincadeira,” we thoroughly analyzed modifications in the gene expression profile of key SWEET genes in Botrytis cinerea-infected grape berries. VvSWEET7 and VvSWEET15 are likely to play an important role during fruit development and Botrytis infection as they are strongly expressed at the green and mature stage, respectively, and were clearly up-regulated in response to infection. Also, B. cinerea infection down-regulated VvSWEET17a expression at the green stage, VvSWEET10 and VvSWEET17d expression at the veraison stage, and VvSWEET11 expression at the mature stage. VvSWEET7 was functionally characterized by heterologous expression in Saccharomyces cerevisiae as a low-affinity, high-capacity glucose and sucrose transporter with a K m of 15.42 mM for glucose and a K m of 40.08 mM for sucrose. VvSWEET7-GFP and VvSWEET15-GFP fusion proteins were transiently expressed in Nicotiana benthamiana epidermal cells and confocal microscopy allowed to observe that both proteins clearly localize to the plasma membrane. In sum, VvSWEETs transporters are important players in sugar mobilization during grape berry development and their expression is transcriptionally reprogrammed in response to Botrytis infection.

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          A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants.

          Genome sequencing has resulted in the identification of a large number of uncharacterized genes with unknown functions. It is widely recognized that determination of the intracellular localization of the encoded proteins may aid in identifying their functions. To facilitate these localization experiments, we have generated a series of fluorescent organelle markers based on well-established targeting sequences that can be used for co-localization studies. In particular, this organelle marker set contains indicators for the endoplasmic reticulum, the Golgi apparatus, the tonoplast, peroxisomes, mitochondria, plastids and the plasma membrane. All markers were generated with four different fluorescent proteins (FP) (green, cyan, yellow or red FPs) in two different binary plasmids for kanamycin or glufosinate selection, respectively, to allow for flexible combinations. The labeled organelles displayed characteristic morphologies consistent with previous descriptions that could be used for their positive identification. Determination of the intracellular distribution of three previously uncharacterized proteins demonstrated the usefulness of the markers in testing predicted subcellular localizations. This organelle marker set should be a valuable resource for the plant community for such co-localization studies. In addition, the Arabidopsis organelle marker lines can also be employed in plant cell biology teaching labs to demonstrate the distribution and dynamics of these organelles.
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            Sugar transporters for intercellular exchange and nutrition of pathogens.

            Sugar efflux transporters are essential for the maintenance of animal blood glucose levels, plant nectar production, and plant seed and pollen development. Despite broad biological importance, the identity of sugar efflux transporters has remained elusive. Using optical glucose sensors, we identified a new class of sugar transporters, named SWEETs, and show that at least six out of seventeen Arabidopsis, two out of over twenty rice and two out of seven homologues in Caenorhabditis elegans, and the single copy human protein, mediate glucose transport. Arabidopsis SWEET8 is essential for pollen viability, and the rice homologues SWEET11 and SWEET14 are specifically exploited by bacterial pathogens for virulence by means of direct binding of a bacterial effector to the SWEET promoter. Bacterial symbionts and fungal and bacterial pathogens induce the expression of different SWEET genes, indicating that the sugar efflux function of SWEET transporters is probably targeted by pathogens and symbionts for nutritional gain. The metazoan homologues may be involved in sugar efflux from intestinal, liver, epididymis and mammary cells.
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              Fungal small RNAs suppress plant immunity by hijacking host RNA interference pathways.

              Botrytis cinerea, the causative agent of gray mold disease, is an aggressive fungal pathogen that infects more than 200 plant species. Here, we show that some B. cinerea small RNAs (Bc-sRNAs) can silence Arabidopsis and tomato genes involved in immunity. These Bc-sRNAs hijack the host RNA interference (RNAi) machinery by binding to Arabidopsis Argonaute 1 (AGO1) and selectively silencing host immunity genes. The Arabidopsis ago1 mutant exhibits reduced susceptibility to B. cinerea, and the B. cinerea dcl1 dcl2 double mutant that can no longer produce these Bc-sRNAs displays reduced pathogenicity on Arabidopsis and tomato. Thus, this fungal pathogen transfers "virulent" sRNA effectors into host plant cells to suppress host immunity and achieve infection, which demonstrates a naturally occurring cross-kingdom RNAi as an advanced virulence mechanism.
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                Author and article information

                Contributors
                Journal
                Front Plant Sci
                Front Plant Sci
                Front. Plant Sci.
                Frontiers in Plant Science
                Frontiers Media S.A.
                1664-462X
                27 January 2020
                2019
                : 10
                : 1753
                Affiliations
                [1] 1 Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho , Braga, Portugal
                [2] 2 Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), University of Trás-os-Montes e Alto Douro , Vila Real, Portugal
                [3] 3 University of Lisbon, Lisbon Science Faculty, BioISI, Campo Grande , Lisbon, Portugal
                [4] 4 i3S-Institute of Research and Innovation in Health, University of Porto , Porto, Portugal
                [5] 5 IBMC-Institute for Molecular and Cell Biology, University of Porto , Porto, Portugal
                [6] 6 Institute of Molecular and Cellular Biology of Plants, Spanish National Research Council (CSIC), Polytechnic University of Valencia , Valencia, Spain
                Author notes

                Edited by: Aziz Aziz, Université de Reims Champagne-Ardenne, France

                Reviewed by: Patricio Arce, Pontifical Catholic University of Chile, Chile; Michele Perazzolli, University of Trento, Italy

                *Correspondence: Artur Conde, arturconde@ 123456bio.uminho.pt

                This article was submitted to Plant Microbe Interactions, a section of the journal Frontiers in Plant Science

                Article
                10.3389/fpls.2019.01753
                6996298
                32047506
                e3196e0d-a72f-4994-9b37-b7348bfbc218
                Copyright © 2020 Breia, Conde, Pimentel, Conde, Fortes, Granell and Gerós

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 26 June 2019
                : 13 December 2019
                Page count
                Figures: 5, Tables: 0, Equations: 0, References: 103, Pages: 13, Words: 7064
                Funding
                Funded by: Fundação para a Ciência e a Tecnologia 10.13039/501100001871
                Award ID: UID/AGR/04033/2019, UID/BIA/04050/2019, PTDC/BIA-FBT/30341/2017, PTDC/BIA-FBT/28165/2017, PTDC/ASP-HOR/28485/2017, PD/BD/113616/2015, PD/00122/2012
                Funded by: Programa Operacional Temático Factores de Competitividade 10.13039/501100011929
                Award ID: POCI-01-0145-FEDER-030341, POCI-01-0145-FEDER-028165 , NORTE-01-0145-FEDER-000017 , BPD/UTAD/INTERACT/VW/218/2016
                Categories
                Plant Science
                Original Research

                Plant science & Botany
                sugar transporter,sweet,biotic stress,grey mold,botrytis cinerea,plant pathogens,grape berry,grapevine

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