Weirdo19ES is a novel singleton mycobacteriophage that selects for glycolipid deficient phage-resistant M. Smegmatis mutants

The sequencing and bioinformatics analysis of bacteriophages infecting mycobacteria has yielded a large amount of information on their evolution, including that on their environmental propagation on other genera such as Gordonia, closely related to Mycobacterium. However, little is known on mycobacteriophages cell biology such as the nature of their receptor (s) or their replication cycle. As part of our on-going screening for novel mycobacteriophages, we herein report the isolation and genome bioinformatics analysis of Weirdo19ES, a singleton Siphoviridae temperate mycobacteriophage with a 70.19% GC content. Nucleotide and protein sequence comparison to actinobacteriophage databases revealed that Weirdo19ES shows low homology to Gordonia phage Ruthy and mycobacteriophages falling in clusters Q and G and to singleton DS6A.Weirdo19ES also displays uncommon features such as a very short Lysin A gene (with only one enzymatic domain) and two putative HNH endonucleases. Mycobacterium smegmatis mutants resistant to Weirdo19ES are cross- resistant to I3. In agreement with that phenotype, analysis of cell envelope of those mutants showed that Weirdo19ES shares receptors with the transducing mycobacteriophage I3.This singleton mycobacteriophage adds up to the uncommonness of local mycobacteriophages previously isolated by our group and helps understanding the nature of mycobacteriophage receptors.Fil: Suárez, Cristian Alejandro. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Franceschelli, Jorgelina Judith. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Tasselli, Sabrina Emilse. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Morbidoni, Héctor Ricardo. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; Argentin

Analysis of Novel Mycobacteriophages Indicates the Existence of Different Strategies for Phage Inheritance in Mycobacteria

Mycobacteriophages have been essential in the development of mycobacterial genetics through their use in the construction of tools for genetic manipulation. Due to the simplicity of their isolation and variety of exploitable molecular features, we searched for and isolated 18 novel mycobacteriophages from environmental samples collected from several geographic locations. Characterization of these phages did not differ from most of the previously described ones in the predominant physical features (virion size in the 100?400 nm, genome size in the 50?70 kbp, morphological features compatible with those corresponding to the Siphoviridae family), however novel characteristics for propagation were noticed. Although all the mycobacteriophages propagated at 30uC, eight of them failed to propagate at 37uC. Since some of our phages yielded pinpoint plaques, we improved plaque detection by including sub-inhibitory concentrations of isoniazid or ampicillin-sulbactam in the culture medium. Thus, searches for novel mycobacteriophages at low temperature and in the presence of these drugs would allow for the isolation of novel members that would otherwise not be detected. Importantly, while eight phages lysogenized Mycobacterium smegmatis, four of them were also capable of lysogenizing Mycobacterium tuberculosis. Analysis of the complete genome sequence obtained for twelve mycobacteriophages (the remaining six rendered partial genomic sequences) allowed for the identification of a new singleton. Surprisingly, sequence analysis revealed the presence of parA or parA/parB genes in 7/18 phages including four that behaved as temperate in M. tuberculosis. In summary, we report here the isolation and preliminary characterization of mycobacteriophages that bring new information to the field.Fil: Stella, Emma Julieta. Universidad Nacional de Rosario. Facultad de Cs.medicas. Escuela de Cs.medicas. Cat.de Microbiologia,parasitologia y Virologia;Fil: Franceschelli, Jorgelina Judith. Universidad Nacional de Rosario. Facultad de Cs.medicas. Escuela de Cs.medicas. Cat.de Microbiologia,parasitologia y Virologia;Fil: Tasselli, Sabrina Emilse. Universidad Nacional de Rosario. Facultad de Cs.medicas. Escuela de Cs.medicas. Cat.de Microbiologia,parasitologia y Virologia;Fil: Morbidoni, Héctor Ricardo. Universidad Nacional de Rosario. Facultad de Cs.medicas. Escuela de Cs.medicas. Cat.de Microbiologia,parasitologia y Virologia

PthA4AT, a 7.5-repeats transcription activator-like (TAL) effector from Xanthomonas citri ssp. citri, triggers citrus canker resistance

Transcription activator-like effectors (TALEs) are important effectors of Xanthomonas spp. that manipulate the transcriptome of the host plant, conferring susceptibility or resistance to bacterial infection. Xanthomonas citri ssp. citri variant AT (X. citri AT) triggers a host-specific hypersensitive response (HR) that suppresses citrus canker development. However, the bacterial effector that elicits this process is unknown. In this study, we show that a 7.5-repeat TALE is responsible for triggering the HR. PthA4AT was identified within the pthA repertoire of X. citri AT followed by assay of the effects on different hosts. The mode of action of PthA4AT was characterized using protein-binding microarrays and testing the effects of deletion of the nuclear localization signals and activation domain on plant responses. PthA4AT is able to bind DNA and activate transcription in an effector binding element-dependent manner. Moreover, HR requires PthA4AT nuclear localization, suggesting the activation of executor resistance (R) genes in host and non-host plants. This is the first case where a TALE of unusually short length performs a biological function by means of its repeat domain, indicating that the action of these effectors to reprogramme the host transcriptome following nuclear localization is not limited to ‘classical’ TALEs.Fil: Roeschlin, Roxana Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Uviedo, Facundo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: García, Lucila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Molina, María Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Favaro, María Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Chiesa, Maria Amalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; ArgentinaFil: Tasselli, Sabrina Emilse. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Franco Zorrilla, José Manuel. Instituto de Bioquímica y Biología Molecular; ArgentinaFil: Forment, Javier. Universidad Politécnica de Valencia; EspañaFil: Gadea, José. Universidad Politécnica de Valencia; EspañaFil: Marano, María Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentin

TuMV Infection Alters the Regulation of miR168/AGO1 and miR403/AGO2 Systems in Arabidopsis

Plant argonaute (AGO) proteins—chiefly AGO1 and 2—restrict viral infections. AGO1/2 also participate in developmental processes and are tightly regulated by microRNAs. Researchers have conducted extensive studies on the regulatory loop involving miR168/AGO1 in viral infections, though comparatively less attention has been given to the miR403/AGO2 system. Here, we simultaneously studied both regulatory systems in Arabidopsis plants infected with turnip mosaic virus (TuMV). TuMV simultaneously altered both miR168 and miR403 precursors as well as their mature forms at medium to late stages of infection. While TuMV decreased miRNA precursor molecules, it induced the overaccumulation of mature miRNA forms, without evidence of concomitant transcriptional alteration. The AGO1 protein remained at basal levels, whereas the AGO2 protein overaccumulated. The application of exogenous salicylic acid (SA) in healthy plants resulted in elevated AGO2 mRNA levels. Conversely, this hormone did not induce any significant changes in either AGO1 mRNA levels or those of miRs 168 and 403. This response is coherent with previous results, which showed enhanced levels of SA under TuMV infection and the partially differential sensitivity that AGO proteins have against this defense hormone. Our results also highlight the key role of AGO2 in leaves as an antiviral molecule and demonstrate the different responsiveness of the AGO1/miR168 and AGO2/miR403 systems regarding TuMV infection and SA response. Taken together, the results presented here are in line with previous reports studying abiotic and biotic impacts on microRNA biogenesis and AGO-dependent antiviral defense and further expand the knowledge of the miR403/AGO2 regulatory system.Fil: Manacorda, Carlos Augusto. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Tasselli, Sabrina Emilse. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Marano, María Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Asurmendi, Sebastian. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentin