Study of the functional domains of the PTGS suppressor V2 from geminivirus Beet curly top virus (BCTV)

Geminiviruses constitute a group of plant viruses that infect vegetable crops all over the world. Among the Geminiviridae family, the genera Mastrevirus, Begomovirus and Curtovirus are the most abundant. Suppression of gene silencing is a key mechanism for viral infection in plants. In begomovirus, V2 is a strong posttranscriptional gene silencing suppressor. We recently showed that V2 from curtovirus Beet curly top virus (BCTV) is a PTGS suppressor by impairing the RDR6/SGS3 pathway, as V2 from begomovirus. In order to identify the domains involved in the suppression activity and viral pathogenicity, we performed an alignment of several begomovirus and curtovirus V2 proteins. A protein kinase C (PKC) phosphorylation motif essential for suppression activity in begomovirus (P1) was found in all analysed sequences. We also found similar hydrophobic profiles, with two hydrophobic domains (H1 and H2) followed by a long hydrophilic domain. Then we generated BCTV V2 mutant proteins and performed transient assays in Nicotiana benthamiana plants to test their suppression activity. We also expressed them from a Potato virus X-derived vector to check the symptoms produced. Additionally, their subcellular localization was determined. Finally, we produced BCTV viruses mutated in the different domains and N. benthamiana plants were infected, analysing virus levels and symptoms produced. The results showed that P1, H1 and H2 are involved in the suppression activity and viral pathogenicity.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

miRNA/phasiRNA mediated regulation of plant defense response against P. syringae

Gene silencing is a mechanism of regulation of gene expression where the small RNAs (sRNAs) are key components for giving specificity to the system. In plants, two main types of noncoding small RNA molecules have been found: microRNAs (miRNAs) and small interfering RNAs (siRNAs). DCL proteins acting on large RNA precursors produce the mature forms of sRNAs (20-24nt) that can act as negative regulators of gene expression. In recent years, the role of miRNAs in regulation of gene expression in plant responses against bacterial pathogens is becoming clearer. Comparisons carried out in our lab between expression profiles of different Arabidopsis thaliana mutants affected in gene silencing, and plants challenged with Pseudomonas syringae pathovar tomato DC3000, led us to identify a set of uncharacterized R genes, belonging to the TIR-NBS-LRR gene family, as differentially expressed in these conditions. Through the use of bioinformatics tools, we found a miRNA* of 22 nt putatively responsible for down-regulating expression of these R genes. We have validated this regulation, and have also established that the corresponding pri-miRNA is down-regulated upon PAMPs or bacteria perception. Using GUS reporters, we have characterized the expression pattern of both pri-miRNA and its best target R genes. We demonstrate that plants with altered levels of miRNA* (knockdown or overexpression lines) exhibit altered PTI-associated phenotypes, supporting a role for this miRNA* in the defence response against this bacterial pathogen. Finally, we identify phasiRNAs that arise from the transcript of one of the R target genes in a miRNA*-RDR6-DCL4-dependent manner.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

V2 from a curtovirus is a suppressor of post-transcriptional gene silencing

The suppression of gene silencing is a key mechanism for the success of viral infection in plants. DNA viruses from the Geminiviridae family encode several proteins that suppress transcriptional and post-transcriptional gene silencing (TGS/PTGS). In Begomovirus, the most abundant genus of this family, three out of six genome-encoded proteins, namely C2, C4 and V2, have been shown to suppress PTGS, with V2 being the strongest PTGS suppressor in transient assays. Beet curly top virus (BCTV), the model species for the Curtovirus genus, is able to infect the widest range of plants among geminiviruses. In this genus, only one protein, C2/L2, has been described as inhibiting PTGS. We show here that, despite the lack of sequence homology with its begomoviral counterpart, BCTV V2 acts as a potent PTGS suppressor, possibly by impairing the RDR6 (RNA-dependent RNA polymerase 6)/suppressor of gene silencing 3 (SGS3) pathway

Characterization of Curtoviral V2 Protein

Geminiviruses are single-stranded DNA plant viruses with circular genomes packaged within geminate particles. Among the Geminiviridae family, Begomovirus and Curtovirus comprise the two best characterized genera. Curtovirus and Old World begomovirus possess similar genome structures with six to seven open-reading frames (ORF). Among them, begomovirus and curtovirus V2 ORFs share the same location in the viral genome, encode proteins of similar size, but show extremely poor sequence homology between the genera. V2 from Beet curly top virus (BCTV), the model species for the Curtovirus genus, as it begomoviral counterpart, suppresses post-transcriptional gene silencing (PTGS) by impairing the RDR6/SGS3 pathway and localizes in the nucleus spanning from the perinuclear region to the cell periphery. By aminoacid sequence comparison we have identified that curtoviral and begomoviral V2 proteins shared two hydrophobic domains and a putative phosphorylation motif. These three domains are essential for BCTV V2 silencing suppression activity, for the proper nuclear localization of the protein and for systemic infection. The lack of suppression activity in the mutated versions of V2 is complemented by the impaired function of RDR6 in Nicotiana benthamiana but the ability of the viral mutants to produce a systemic infection is not recovered in gene silencing mutant backgrounds. We have also demonstrated that, as its begomoviral homolog, V2 from BCTV is able to induce systemic symptoms and necrosis associated with a hypersensitive response-like (HR-like) when expressed from Potato virus X vector in N. benthamiana, and that this pathogenicity activity does not dependent of its ability to supress PTG

R gene regulation mediated by miRNA/phasiRNA during plant defense response against P. syringae

In plants, two main types of noncoding small RNA molecules have been found: microRNAs (miRNAs) and small interfering RNAs (siRNAs), differing these in their biogenesis and mode of action, but sharing similar sizes (20-24 nt). In plants, their mature forms are products of the activity of DCL proteins and can act as negative regulators of gene expression. In recent years, the role of miRNAs in regulation of gene expression in plant responses against bacterial pathogens is becoming clearer. Comparisons carried out in our lab between expression profiles of different Arabidopsis thaliana mutants affected in gene silencing, and plants challenged with Pseudomonas syringae pathovar tomato DC3000, led us to identify a set of uncharacterized R genes, belonging to the TIR-NBS-LRR gene family, as differentially expressed in these conditions. By bioinformatics tools, we found a miRNA* of 22 nt putatively responsible for down-regulating expression of these R genes. We have also found that the corresponding pri-miRNA is down-regulated after PAMP-perception. We demonstrate that plants with altered levels of this miRNA* (knockdown lines or overexpression lines) exhibit altered PTI-associated phenotypes, suggesting a role for this miRNA* in this defence response against bacteria. We have characterized the expression pattern of both primiRNA and its best target R genes. Finally, we identify phasiRNAs that arise from the transcript of this R gen in a miRNA*-RDR6-DCL4-dependent mannerUniversidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Regulación de genes de resistencia de la familia TIR-NBS-LRR mediada por miRNA/phasiRNA durante la interacción con P. syringae

Durante un estrés biótico, las plantas modulan la expresión de una batería de genes involucrados en la respuesta de defensa, proceso donde recientemente se ha determinado el papel esencial que desempeña el silenciamiento génico. El silenciamiento génico es un mecanismo de regulación de la expresión génica, donde destacan como principales moléculas efectoras los pequeños RNAs (sRNAs). En plantas, estos sRNAs, son clasificados en pequeños RNAs interferentes (siRNAs) o microRNAs (miRNAs), presentando tamaños similares (20-24 nt) pero difiriendo en su biogénesis y modo de acción. Los miRNAs son pequeños RNAs de cadena sencilla que actúan regulando negativamente la expresión de genes, mediante su unión al complejo RISC (Rna Induced Silencing Complex) y en una forma dependiente de secuencia. En nuestro laboratorio, mediante el análisis de datos transcriptómicos, y el uso de herramientas bioinformáticas, identificamos un miRNA* de 22 nt como potencial regulador de la expresión de genes de resistencia (“R”) del tipo TIR-NBS-LRR. Posteriormente hemos validado dicha regulación y caracterizado los patrones de expresión tanto del Pri-miRNA como de un gen “R” regulado por este, en diferentes tejidos y estadios del desarrollo, así como durante la interacción con P. syringae. Por otro lado, hemos generado plantas transgénicas que presentan niveles alterados del miRNA* (incremento y reducción) y hemos observado que muestran fenotipos alterados de PTI y una mayor/menor colonización de P. syringae. Finalmente hemos identificado la producción de sRNAs (phasiRNAs) a partir del gen de resistencia, en una forma dependiente de miRNA*-RDR6-DCL4, pudiendo estos sRNAs secundarios regular otros transcritos de la misma familia de genes de resistencia.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Impacts of the Tropical Pacific/Indian Oceans on the Seasonal Cycle of the West African Monsoon

The current consensus is that drought has developed in the Sahel during the second half of the twentieth century as a result of remote effects of oceanic anomalies amplified by local land–atmosphere interactions. This paper focuses on the impacts of oceanic anomalies upon West African climate and specifically aims to identify those from SST anomalies in the Pacific/Indian Oceans during spring and summer seasons, when they were significant. Idealized sensitivity experiments are performed with four atmospheric general circulation models (AGCMs). The prescribed SST patterns used in the AGCMs are based on the leading mode of covariability between SST anomalies over the Pacific/Indian Oceans and summer rainfall over West Africa. The results show that such oceanic anomalies in the Pacific/Indian Ocean lead to a northward shift of an anomalous dry belt from the Gulf of Guinea to the Sahel as the season advances. In the Sahel, the magnitude of rainfall anomalies is comparable to that obtained by other authors using SST anomalies confined to the proximity of the Atlantic Ocean. The mechanism connecting the Pacific/Indian SST anomalies with West African rainfall has a strong seasonal cycle. In spring (May and June), anomalous subsidence develops over both the Maritime Continent and the equatorial Atlantic in response to the enhanced equatorial heating. Precipitation increases over continental West Africa in association with stronger zonal convergence of moisture. In addition, precipitation decreases over the Gulf of Guinea. During the monsoon peak (July and August), the SST anomalies move westward over the equatorial Pacific and the two regions where subsidence occurred earlier in the seasons merge over West Africa. The monsoon weakens and rainfall decreases over the Sahel, especially in August.Peer reviewe

Search for new particles in events with energetic jets and large missing transverse momentum in proton-proton collisions at root s=13 TeV

A search is presented for new particles produced at the LHC in proton-proton collisions at root s = 13 TeV, using events with energetic jets and large missing transverse momentum. The analysis is based on a data sample corresponding to an integrated luminosity of 101 fb(-1), collected in 2017-2018 with the CMS detector. Machine learning techniques are used to define separate categories for events with narrow jets from initial-state radiation and events with large-radius jets consistent with a hadronic decay of a W or Z boson. A statistical combination is made with an earlier search based on a data sample of 36 fb(-1), collected in 2016. No significant excess of events is observed with respect to the standard model background expectation determined from control samples in data. The results are interpreted in terms of limits on the branching fraction of an invisible decay of the Higgs boson, as well as constraints on simplified models of dark matter, on first-generation scalar leptoquarks decaying to quarks and neutrinos, and on models with large extra dimensions. Several of the new limits, specifically for spin-1 dark matter mediators, pseudoscalar mediators, colored mediators, and leptoquarks, are the most restrictive to date.Peer reviewe

Development and validation of HERWIG 7 tunes from CMS underlying-event measurements

This paper presents new sets of parameters (“tunes”) for the underlying-event model of the HERWIG7 event generator. These parameters control the description of multiple-parton interactions (MPI) and colour reconnection in HERWIG7, and are obtained from a fit to minimum-bias data collected by the CMS experiment at s=0.9, 7, and 13Te. The tunes are based on the NNPDF 3.1 next-to-next-to-leading-order parton distribution function (PDF) set for the parton shower, and either a leading-order or next-to-next-to-leading-order PDF set for the simulation of MPI and the beam remnants. Predictions utilizing the tunes are produced for event shape observables in electron-positron collisions, and for minimum-bias, inclusive jet, top quark pair, and Z and W boson events in proton-proton collisions, and are compared with data. Each of the new tunes describes the data at a reasonable level, and the tunes using a leading-order PDF for the simulation of MPI provide the best description of the dat

Probing effective field theory operators in the associated production of top quarks with a Z boson in multilepton final states at root s=13 TeV

Peer reviewe