Identification and analysis of seven effector protein families with different adaptive and evolutionary histories in plant-associated members of the Xanthomonadaceae.

The Xanthomonadaceae family consists of species of non-pathogenic and pathogenic γ-proteobacteria that infect different hosts, including humans and plants. In this study, we performed a comparative analysis using 69 fully sequenced genomes belonging to this family, with a focus on identifying proteins enriched in phytopathogens that could explain the lifestyle and the ability to infect plants. Using a computational approach, we identified seven phytopathogen-enriched protein families putatively secreted by type II secretory system: PheA (CM-sec), LipA/LesA, VirK, and four families involved in N-glycan degradation, NixE, NixF, NixL, and FucA1. In silico and phylogenetic analyses of these protein families revealed they all have orthologs in other phytopathogenic or symbiotic bacteria, and are involved in the modulation and evasion of the immune system. As a proof of concept, we performed a biochemical characterization of LipA from Xac306 and verified that the mutant strain lost most of its lipase and esterase activities and displayed reduced virulence in citrus. Since this study includes closely related organisms with distinct lifestyles and highlights proteins directly related to adaptation inside plant tissues, novel approaches might use these proteins as biotechnological targets for disease control, and contribute to our understanding of the coevolution of plant-associated bacteria

Proteomics-based identification of differentially abundant proteins reveals adaptation mechanisms of Xanthomonas citri subsp citri during Citrus sinensis infection

Background: Xanthomonas citri subsp. citri (Xac) is the causal agent of citrus canker. A proteomic analysis under in planta infectious and non-infectious conditions was conducted in order to increase our knowledge about the adaptive process of Xac during infection. Results: For that, a 2D-based proteomic analysis of Xac at 1, 3 and 5 days after inoculation, in comparison to Xac growth in NB media was carried out and followed by MALDI-TOF-TOF identification of 124 unique differentially abundant proteins. Among them, 79 correspond to up-regulated proteins in at least one of the three stages of infection. Our results indicate an important role of proteins related to biofilm synthesis, lipopolysaccharides biosynthesis, and iron uptake and metabolism as possible modulators of plant innate immunity, and revealed an intricate network of proteins involved in reactive oxygen species adaptation during Plants'Oxidative Burst response. We also identified proteins previously unknown to be involved in Xac-Citrus interaction, including the hypothetical protein XAC3981. A mutant strain for this gene has proved to be non-pathogenic in respect to classical symptoms of citrus canker induced in compatible plants. Conclusions: This is the first time that a protein repertoire is shown to be active and working in an integrated manner during the infection process in a compatible host, pointing to an elaborate mechanism for adaptation of Xac once inside the plant.Fundacao de Amparo a Pesquisa do Estado de Sao PauloFundacao de Amparo a Pesquisa do Estado de Minas GeraisBIGA grant-CAPESUniv Fed Ouro Preto, Inst Ciencias Exatas & Biol, Dept Ciencias Biol DECBI, Ouro Preto, MG, BrazilUniv Fed Ouro Preto, Nucleo Pesquisas Ciencias Biol NUPEB, Ouro Preto, MG, BrazilUniv Fed Rio de Janeiro, Inst Quim, Dept Bioquim DBq, Rio De Janeiro, RJ, BrazilUniv Estadual Paulista, UNESP, Dept Tecnol, Fac Ciencias Agr & Vet Jaboticabal, Jaboticabal, SP, BrazilUniv Estadual Campinas, UNICAMP, Inst Quim, Campinas, SP, BrazilUniv Sao Paulo, Inst Quim, Dept Bioquim, Sao Paulo, SP, BrazilUniv Fed Sao Paulo UNIFESP, Dept Ciencias Biol, Diadema, SP, BrazilVirginia Tech, Biocomplex Inst, Blacksburg, VA USAUniv Sao Paulo, Inst Quim, Dept Bioquim, Sao Paulo, SP, BrazilUniv Fed Sao Paulo UNIFESP, Dept Ciencias Biol, Diadema, SP, BrazilCAPESFAPESP: 04/02006-7Fundacao de Amparo a Pesquisa do Estado de Minas Gerais: CBB-APQ-04425-10BIGA grant-CAPES: 3385/2013Web of Scienc

Detection and identification of Xanthomonas pathotypes associated with citrus diseases using comparative genomics and multiplex PCR

Background. In Citrus cultures, three species of Xanthomonas are known to cause distinct diseases. X. citri subsp. citri patothype A, X. fuscans subsp. aurantifolii pathotypes B and C, and X. alfalfae subsp. citrumelonis, are the causative agents of cancrosis A, B, C, and citrus bacterial spots, respectively. Although these species exhibit different levels of virulence and aggressiveness, only limited alternatives are currently available for proper and early detection of these diseases in the fields. The present study aimed to develop a new molecular diagnostic method based on genomic sequences derived from the four species of Xanthomonas. Results. Using comparative genomics approaches, primers were synthesized for the identification of the four causative agents of citrus diseases. These primers were validated for their specificity to their target DNA by both conventional and multiplex PCR. Upon evaluation, their sensitivity was found to be 0.02 ng/mu l in vitro and 1.5 x 10(4) CFU ml(-1) in infected leaves. Additionally, none of the primers were able to generate amplicons in 19 other genomes of Xanthomonas not associated with Citrus and one species of Xylella, the causal agent of citrus variegated chlorosis (CVC). This denotes strong specificity of the primers for the different species of Xanthomonas investigated in this study. Conclusions. We demonstrated that these markers can be used as potential candidates for performing in vivo molecular diagnosis exclusively for citrus-associated Xanthomonas. The bioinformatics pipeline developed in this study to design specific genomic regions is capable of generating specific primers. It is freely available and can be utilized for any other model organism.7CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE MINAS GERAIS - FAPEMIG481226/2013-3CFP 51/2013; 3385/2013APQ-02387-1

Brazilian Ironstone Plant Communities as Reservoirs of Culturable Bacteria With Diverse Biotechnological Potential

Extensive mineral extractivism in the Brazilian Iron Quadrangle (IQ) region has destroyed large areas of land, decimating plant species, and their associated microbiota. Very little is known about the microbiota of the region; hence, cultivable bacteria associated with plants of its soils were investigated for their biotechnological potential. Samples were collected from nine plant species and six soils, and 65 cultivable bacterial isolates were obtained. These represent predominantly gram-positive bacilli (70%) capable of producing amylases (55%), proteases (63%), cellulases (47%), indole acetic acid (IAA) (46%), siderophores (26%), and to solubilize phosphate (9%). In addition, 65% of these were resistant to ampicillin, 100% were sensitive to tetracycline, and 97% were tolerant to high arsenic concentrations. Three isolates were studied further: the isolate FOB3 (Rosenbergiella sp.) produced high concentrations of IAA in vitro in the absence of tryptophan – shown by the significant improvement in plant germination and growth rate where the isolate was present. For isolates C25 (Acinetobacter sp.) and FG3 (Serratia sp.), plasmids were purified and inserted into Escherichia coli cells where they modified the physiological profile of the transformed strains. The E. coli::pFG3B strain showed the highest capacity for biofilm production, as well as an increase in the replication rate, arsenic tolerance and catalase activity. Moreover, this strain increased DNA integrity in the presence of arsenic, compared to the wild-type strain. These results help to explain the importance of bacteria in maintaining plant survival in ferruginous, rocky soils, acting as plant growth promoters, and to highlight the biotechnological potential of these bacteria.IMPORTANCE The Iron Quadrangle region is responsible for ∼60% of all Brazilian iron production and, at the same time, is responsible for housing a wide diversity of landscapes, and consequently, a series of endemic plant species and dozens of rare species – all of which have been poorly studied. Studies exploring the microbiota associated with these plant species are limited and in the face of the continuous pressure of extractive action, some species along with their microbiota are being decimated. To understand the potential of this microbiota, we discovered that cultivable bacterial isolates obtained from plants in the ferruginous rocky soil of the Iron Quadrangle region have diverse biotechnological potential, revealing a genetic ancestry still unknown

Toward sustainable environmental quality: Identifying priority research questions for Latin America

The Global Horizon Scanning Project (GHSP) is an innovative initiative that aims to identify important global environmental quality research needs. Here we report 20 key research questionsfrom LatinAmerica (LA).Members of the Society of Environmental Toxicology and Chemistry (SETAC) LA and other scientistsfrom LA were asked to submit research questions that would represent priority needs to address in the region. One hundred questions were received, then partitioned among categories, examined, and some rearranged during a workshop in Buenos Aires,Argentina. Twenty priority research questions were subsequently identified. These research questions included developing, improving, and harmonizing across LA countries methods for 1) identifying contaminants and degradation products in complex matrices (including biota); 2) advancing prediction of contaminant risks and effects in ecosystems, addressing lab-to-field extrapolation challenges, and understanding complexities of multiple stressors (including chemicals and climate change); and 3) improving management and regulatory tools toward achieving sustainable development. Whereas environmental contaminants frequently identified in these key questions were pesticides, pharmaceuticals, endocrine disruptors or modulators, plastics, and nanomaterials, commonly identified environmental challenges were related to agriculture, urban effluents, solid wastes, pulp and paper mills, and natural extraction activities. Several interesting research topics included assessing and preventing pollution impacts on conservation protected areas, integrating environment and health assessments, and developing strategiesfor identification, substitution, and design of less hazardous chemicals (e.g., green chemistry). Finally, a recurrent research need included developing an understanding of differential sensitivity of regional species and ecosystems to environmental contaminants and other stressors. Addressing these critical questions will support development of long-term strategic research efforts to advance more sustainable environmental quality and protect public health and the environment in LA.Centro de Investigaciones del Medioambient

Protected areas: A focus on Brazilian freshwater biodiversity

Brazil has a variety of aquatic ecosystems and rich freshwater biodiversity, but these components have been constantly damaged by the expansion of unsustainable activities. An array of different conservation strategies is needed, especially the creation of protected areas (PAs, hereafter). However, Brazil's PAs are biased towards terrestrial ecosystems and we argue that current PAs have limited efficacy in the protection of freshwater biodiversity. New PAs should better consider aquatic environments, covering entire basins, rivers and other freshwater habitats. We recommend ways to implement these PAs and provide guidance to avoid social impacts. Freshwater systems in Brazil provide essential goods and services but these ecosystems are being rapidly degraded and will be lost if not adequately protected. © 2018 The Authors. Diversity and Distributions Published by John Wiley & Sons Lt