Mathematical Modeling of Photo- and Thermomorphogenesis in Plants.

Increased day lengths and warm conditions inversely affect plant growth by directly modulating nuclear phyB, ELF3, and COP1 levels. Quantitative measures of the hypocotyl length have been key to gaining a deeper understanding of this complex regulatory network, while similar quantitative data are the foundation for many studies in plant biology. Here, we explore the application of mathematical modeling, specifically ordinary differential equations (ODEs), to understand plant responses to these environmental cues. We provide a comprehensive guide to constructing, simulating, and fitting these models to data, using the law of mass action to study the evolution of molecular species. The fundamental principles of these models are introduced, highlighting their utility in deciphering complex plant physiological interactions and testing hypotheses. This brief introduction will not allow experimentalists without a mathematical background to run their own simulations overnight, but it will help them grasp modeling principles and communicate with more theory-inclined colleagues.

Spanish adaptation and validation of the child- and parent-report cystic fibrosis questionnaire-revised (CFQ-R).

OBJECTIVE: To evaluate the psychometric properties of the Spanish versions of the child- and parent-report cystic fibrosis questionnaire-revised (CFQ-R). METHODS: A Spanish adaptation of the CFQ-R was performed; 68 children with CF (6-13 years) and their parents completed the child- and parent-report CFQ-R, respectively, and the Revidierter KINDer Lebensqualitätsfragebogen (KINDL) questionnaire. The CFQ-R was completed twice, 7-10 days apart, and its psychometric properties were analyzed. RESULTS: The internal consistency of both CFQ-R versions was adequate (child-report version, Cronbach's α >.60 for all domains except "Treatment Burden" [α = .42] and "Social Functioning" [α = .57]; parent-report version, α > .60 for all domains except "Social Functioning" [α = .58]). For the child-report version, the lowest measurement error was for "Emotional Functioning" (standard error of measurement [SEM]: 8.3%; minimal detectable change [MDC90 ]: 19.3%), and the highest was for "Body Image" (SEM: 15%; MDC90 : 35%). For the parent-report version, the lowest measurement error was for "Physical Functioning" (SEM: 7.1%; MDC90 : 16.5%), and the highest was for "Weight" (SEM: 17.2%; MDC90 ; 40.1%). The correlation between the versions showed higher agreement for the domains related to observable signs ("Physical Functioning") and lower agreement for "Emotional Functioning." There was a significant correlation between the CFQ-R and KINDL. CONCLUSION: Both the child- and parent-report versions of the Spanish CFQ-R have adequate reliability and validity for clinical and research purposes. These versions can be administered before and after starting modulator therapy to assess its effect on daily functioning. The MDC90 can help identify, with a high probability, whether real changes have occurred in the quality-of-life subscales in children with CF.

COP1 dynamics integrate conflicting seasonal light and thermal cues in the control of Arabidopsis elongation.

As the summer approaches, plants experience enhanced light inputs and warm temperatures, two environmental cues with an opposite morphogenic impact. Key components of this response are PHYTOCHROME B (phyB), EARLY FLOWERING 3 (ELF3), and CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1). Here, we used single and double mutant/overexpression lines to fit a mathematical model incorporating known interactions of these regulators. The fitted model recapitulates thermal growth of all lines used and correctly predicts thermal behavior of others not used in the fit. While thermal COP1 function is accepted to be independent of diurnal timing, our model shows that it acts at temperature signaling only during daytime. Defective response of cop1-4 mutants is epistatic to phyB-9 and elf3-8, indicating that COP1 activity is essential to transduce phyB and ELF3 thermosensory function. Our thermal model provides a unique toolbox to identify best allelic combinations enhancing climate change resilience of crops adapted to different latitudes.

Hysteresis in PHYTOCHROME-INTERACTING FACTOR 4 and EARLY-FLOWERING 3 dynamics dominates warm daytime memory in Arabidopsis.

Despite the identification of temperature sensors and downstream components involved in promoting stem growth by warm temperatures, when and how previous temperatures affect current plant growth remain unclear. Here we show that hypocotyl growth in Arabidopsis thaliana during the night responds not only to the current temperature but also to preceding daytime temperatures, revealing a short-term memory of previous conditions. Daytime temperature affected the levels of PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) and LONG HYPOCOTYL 5 (HY5) in the nucleus during the next night. These factors jointly accounted for the observed growth kinetics, whereas nighttime memory of prior daytime temperature was impaired in pif4 and hy5 mutants. PIF4 promoter activity largely accounted for the temperature-dependent changes in PIF4 protein levels. Notably, the decrease in PIF4 promoter activity triggered by cooling required a stronger temperature shift than the increase caused by warming, representing a typical hysteretic effect; this hysteretic pattern required EARLY-FLOWERING 3 (ELF3). Warm temperatures promoted the formation of nuclear condensates of ELF3 in hypocotyl cells during the afternoon but not in the morning. These nuclear speckles showed poor sensitivity to subsequent cooling. We conclude that ELF3 achieves hysteresis and drives the PIF4 promoter into the same behavior, enabling a short-term memory of daytime temperature conditions.

Grupos de acogimiento: Sistematización de un modelo participativo para la formulación diagnóstica y terapéutica en un centro de salud mental comunitaria. Chorrillos-Perú, 2018 / Foster groups: Systematization of a participatory model for the diagnostic and therapeutic formulation in a community mental health center. Chorrillos-Peru, 2018

RESUMEN Objetivo: Sistematizar el proceso de formulación de diagnósticos y planes de tratamiento en grupos participativos de profesionales y usuarios, denominados Grupos de Acogimiento, como alternativa al modelo convencional de consultas psiquiátricas individuales y secuenciales, en el ámbito de la salud mental comunitaria. Material Métodos : El estudio se realizó en un centro de salud mental comunitaria (CSMC) de Lima durante el periodo enero-diciembre de 2018. Se siguió una metodología de investigación-acción en la que propuestas surgidas de la experiencia se implementaban para, luego, analizar críticamente sus resultados y ajustarlos sucesivamente hasta lograr un punto de estandarización o de consenso entre los participantes. Resultados: El modelo participativo se aplicó en 97% de los usuarios durante el periodo de observación y se llegó a la formulación de diagnósticos y planes de tratamiento durante la sesión del Grupo de Acogimiento, en el 85% de los pacientes. Las sesiones se estandarizaron en cuatro momentos concatenados y semi-estructurados: Presentación horizontal de participantes, comprensión empática mutua, exploración colectiva de causas, y decisiones terapéuticas compartidas. Conclusiones: El modelo participativo de Grupos de Acogimiento fue una alternativa viable y eficaz para la formulación inicial de diagnósticos y planes terapéuticos en el CSMC, con fases semiestructuradas convenientemente estandarizadas y replicables.

SUMMARY Objective: To systematize the process of diagnosis and treatment plan formulations in participatory groups of users and professionals, called Fostering Groups, as an alternative to the conventional model of individual and sequential psychiatric consults, in the field of community mental health. Material and Methods : The study was carried out in a community mental health center (CMHC) in Lima throughout the period January-December 2018. An action-research methodology was followed, in which proposals arising from the experiences were put into action to, then, critically analyze the and adjust them successively until reaching a point of standardization or consensus among the participants. Results : The participatory model was applied in 97% of the users during the observation period and the formulation of diagnoses and treatment plans during the Foster Group session was successfully reached in 85% of the patients. The sessions were standardized in four concatenated and semi-structured moments: Horizontal presentation of participants, mutual empathetic understanding, collective exploration of causes, and shared therapeutic decisions. Conclusions : The Fostering group model was a viable and effective alternative for the initial formulation of diagnoses and therapeutic plans in the CMHC, with semi-structured phases conveniently standardized and replicable.

Regulation of COP1 Function by Brassinosteroid Signaling.

Small increases in temperature result in enhanced elongation of the hypocotyl and petioles and hyponastic growth, in an adaptive response directed to the cooling of the leaves and to protect the shoot meristem from the warm soil. This response, collectively termed as thermomorphogenesis, relies on the faster reversion of phyB Pfr at warmer temperatures, which leads to enhanced activity of the basic-helix-loop-helix PHYTOCHROME INTERACTING FACTOR 4 (PIF4). PIF4 acts as a molecular hub integrating light and temperature cues with endogenous hormonal signaling, and drives thermoresponsive growth by directly activating auxin synthesis and signaling genes. Growth promotion by PIF4 depends on brassinosteroid (BR) signaling, as indicated by the impaired thermoresponse of BR-defective mutants and the partial restoration of pifq thermoresponsive defects by brassinolide (BL) application. Also, phyB limits thermomorphogenic elongation through negative regulation of the E3 ubiquitin ligase COP1 that triggers nuclear degradation of multiple photomorphogenesis-promoting factors acting antagonistically to PIF4. COP1 is indeed observed to accumulate in the nucleus in darkness, or in response to warm temperatures, with constitutive photomorphogenic cop1 mutants failing to respond to temperature. Here we explored the role of BR signaling on COP1 function, by growing cop1 seedlings on BL or the inhibitor brassinazole (BRZ), under different light and temperature regimes. We show that weak cop1 alleles exhibit a hyposensitive response to BL. Furthermore, while cop1-6 mutants display as described a wild-type response to temperature in continuous darkness, this response is abolished by BRZ. Application of this inhibitor likewise suppressed temperature-induced COP1 nuclear accumulation in N. benthamiana leaves. Overall these results demonstrate that cop1-6 is not a temperature-conditional allele, but this mutation allows for a partially active protein which unveils a pivotal role of active BR signaling in the control of COP1 activity.

Noble metal-based sorbents: A way to avoid new waste after mercury removal.

The development of technologies to control mercury emissions is now a legal requirement imposed by recent international agreements. The use of regenerable sorbents is consistent with this requirement as it allows mercury to be captured from industrial gases without generating new mercury-containing toxic waste. Because regenerable sorbents based on noble metals are often questioned due to the heavy investment they entail, this study assesses the viability of their use in terms of efficiency and cost. Its primary aim is to develop new regenerable sorbents based on an activated carbon support impregnated with Ag in order to compare their cost and behavior in a CO2 enriched gas stream with that of similar materials containing Au. Mercury retention efficiencies of 100 % can be achieved over several adsorption-desorption cycles depending on the type of noble metal used, particle size and impurities in the gas atmosphere. The results suggest that the Hg-Ag amalgamation process differs from the Hg-Au one, in that they show different kinetics of adsorption and temperatures of desorption. The Ag and Au regenerable sorbents developed in this study would be competitive given the environmental and health benefits they offer compared to the single-use activated carbons employed until now at industrial scale.

The dream school: Mind-changing perspectives of people with intellectual disabilities.

In this study, we analyse the characteristics that define an ideal school, from the perspective of people with intellectual disabilities. we carried out an inclusive research qualitative study with 36 Spanish adults with intellectual disabilities. Information from individual interviews about educational inclusion was summarized on to notecards, allowing participants to place messages into categories on a poster board. Identified themes included facilities and resources; values and rules; intimidation; what students learn; how students learn best; exams; teachers; and classmates. Results highlight the importance of inclusive values and the need for a curriculum that meets the needs of all students.

Convergent regulation of PIFs and the E3 ligase COP1/SPA1 mediates thermosensory hypocotyl elongation by plant phytochromes.

The ability of plants to sense and integrate daily and seasonal changes in light and temperature and to adjust their growth and development accordingly, is critical to withstand severe weather oscillations in a year. While molecular mechanisms controlling light responses are relatively well established, those involved in the perception and response to temperature are just beginning to be understood. Phytochromes emerged as major temperature sensors; due to warmer temperatures accelerate the dark reversal reaction to the Pr inactive state. Downstream of phytochromes, the bHLH Phytochrome Interacting Factors, and in particular PIF4, act as central signaling hubs to growth coordination in response to light and temperature cues, and to the gibberellin and brassinosteroid pathways. Here we discuss recent findings showing that phytochromes control PIFs activity not only by signaling their destruction in the light, but by modulating transcriptional repression of these factors by the circadian clock. Together with this repression, phytochromes inactivate the COP1/SPA ubiquitin ligase, which negatively regulates light signaling through degradation of a large set of nuclear photomorphogenesis-promoting factors that suppress PIFs activity.

Perception and signalling of light and temperature cues in plants.

Light and temperature patterns are often correlated under natural plant growth conditions. In this review, we analyse the perception and signalling mechanisms shared by both these environmental cues and discuss the functional implications of their convergence to control plant growth. The first point of integration is the phytochrome B (phyB) receptor, which senses light and temperature. Downstream of phyB, the signalling core comprises two branches, one involving PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and the other CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) and ELONGATED HYPOCOTYL 5 (HY5). The dynamics of accumulation and/or localization of each of these core signalling components depend on light and temperature conditions. These pathways are connected through COP1, which enhances the activity of PIF4. The circadian clock modulates this circuit, since EARLY FLOWERING 3 (ELF3), an essential component of the evening complex (EC), represses expression of the PIF4 gene and PIF4 transcriptional activity. Phytochromes are probably not the only entry point of temperature into this network, but other sensors remain to be established. The sharing of mechanisms of action for two distinct environmental cues is to some extent unexpected, as it renders these responses mutually dependent. There are nonetheless many ecological contexts in which such a mutual influence could be beneficial.

Sequential anaerobic-aerobic biodegradation of emerging insensitive munitions compound 3-nitro-1,2,4-triazol-5-one (NTO).

Insensitive munitions, such as 3-nitro-1,2,4-triazol-5-one (NTO), are being considered by the U.S. Army as replacements for conventional explosives. Environmental emissions of NTO are expected to increase as its use becomes widespread; but only a few studies have considered the remediation of NTO-contaminated sites. In this study, sequential anaerobic-aerobic biodegradation of NTO was investigated in bioreactors using soil as inoculum. Batch bioassays confirmed microbial reduction of NTO under anaerobic conditions to 3-amino-1,2,4-triazol-5-one (ATO) using pyruvate as electron-donating cosubstrate. However, ATO biodegradation was only observed after the redox condition was switched to aerobic. This study also demonstrated that the high-rate removal of NTO in contaminated water can be attained in a continuous-flow aerated bioreactor. The reactor was first fed ATO as sole energy and nitrogen source prior to NTO addition. After few days, ATO was removed in a sustained fashion by 100%. When NTO was introduced together with electron-donor (pyruvate), NTO degradation increased progressively, reaching a removal efficiency of 93.5%. Mineralization of NTO was evidenced by the partial release of inorganic nitrogen species in the effluent, and lack of ATO accumulation. A plausible hypothesis for these findings is that NTO reduction occurred in anaerobic zones of the biofilm whereas ATO was mineralized in the bulk aerobic zones of the reactor.

Microbial toxicity and characterization of DNAN (bio)transformation product mixtures.

2,4-Dinitroanisole (DNAN) is an emerging insensitive munitions compound. It undergoes rapid (bio)transformation in soils and anaerobic sludge. The primary transformation pathway catalyzed by a combination of biotic and abiotic factors is nitrogroup reduction followed by coupling of reactive intermediates to form azo-dimers. Additional pathways include N-acetylation and O-demethoxylation. Toxicity due to (bio)transformation products of DNAN has received little attention. In this study, the toxicity of DNAN (bio)transformation monomer products and azo-dimer and trimer surrogates to acetoclastic methanogens and the marine bioluminescent bacterium, Allivibrio fischeri, were evaluated. Methanogens were severely inhibited by 3-nitro-4-methoxyaniline (MENA), with a 50%-inhibiting concentration (IC50) of 25 µM, which is more toxic than DNAN with the same assay, but posed a lower toxicity to Allivibrio fischeri (IC50 = 219 µM). On the other hand, N-(5-amino-2-methoxyphenyl) acetamide (Ac-DAAN) was the least inhibitory test-compound for both microbial targets. Azo-dimer and trimer surrogates were very highly toxic to both microbial systems, with a toxicity similar or stronger than that of DNAN. A semi-quantitative LC-QTOF-MS method was employed to determine product mixture profiles at different stages of biotransformation, and compared with the microbial toxicity of the product-mixtures formed. Methanogenic toxicity increased due to putative reactive nitroso-intermediates as DNAN was reduced. However, the inhibition later attenuated as dimers became the predominant products in the mixtures. In contrast, A. fischeri tolerated the initial biotransformation products but were highly inhibited by the predominant azo-dimer products formed at longer incubation times, suggesting these ultimate products are more toxic than DNAN.

New Rimocidin/CE-108 Derivatives Obtained by a Crotonyl-CoA Carboxylase/Reductase Gene Disruption in Streptomyces diastaticus var. 108: Substrates for the Polyene Carboxamide Synthase PcsA.

The rimJ gene, which codes for a crotonyl-CoA carboxylase/reductase, lies within the biosynthetic gene cluster for two polyketides belonging to the polyene macrolide group (CE-108 and rimocidin) produced by Streptomyces diastaticus var. 108. Disruption of rimJ by insertional inactivation gave rise to a recombinant strain overproducing new polyene derivatives besides the parental CE-108 (2a) and rimocidin (4a). The structure elucidation of one of them, CE-108D (3a), confirmed the incorporation of an alternative extender unit for elongation step 13. Other compounds were also overproduced in the fermentation broth of rimJ disruptant. The new compounds are in vivo substrates for the previously described polyene carboxamide synthase PcsA. The rimJ disruptant strain, constitutively expressing the pcsA gene, allowed the overproduction of CE-108E (3b), the corresponding carboxamide derivative of CE-108D (3a), with improved pharmacological properties.

ELF3-PIF4 interaction regulates plant growth independently of the Evening Complex.

The circadian clock plays a pivotal role in the control of Arabidopsis hypocotyl elongation by regulating rhythmic expression of the bHLH factors PHYTOCHROME INTERACTING FACTOR 4 and 5 (PIF4 and 5). Coincidence of increased PIF4/PIF5 transcript levels with the dark period allows nuclear accumulation of these factors, and in short days it phases maximal hypocotyl growth at dawn. During early night, PIF4 and PIF5 transcription is repressed by the Evening Complex (EC) proteins EARLY FLOWERING3 (ELF3), EARLY FLOWERING4 (ELF4), and LUX ARRHYTHMO (LUX). While ELF3 has an essential role in EC complex assembly, several lines of evidence indicate that this protein controls plant growth via other mechanisms that are presently unknown. Here, we show that the ELF3 and PIF4 proteins interact in an EC-independent manner, and that this interaction prevents PIF4 from activating its transcriptional targets. We also show that PIF4 overexpression leads to ELF3 protein destabilization, and that this effect is mediated indirectly by negative feedback regulation of photoactive PHYTOCHROME B (phyB). Physical interaction of the phyB photoreceptor with ELF3 has been reported, but its functional relevance remains poorly understood. Our findings establish that phyB is needed for ELF3 accumulation in the light, most likely by competing for CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)-mediated ubiquitination and the proteasomal degradation of ELF3. Our results explain the short hypocotyl phenotype of ELF3 overexpressors, despite their normal clock function, and provide a molecular framework for understanding how warm temperatures promote hypocotyl elongation and affect the endogenous clock.

Comparación de la efectividad de la dosis única frente a la dosis triple de cefazolina como profilaxis antibiótica en cesáreas / Comparison of single versus triple dose of cefazolin in antibiotic prophylaxis for caesarean section

Determinar la efectividad de la dosis única comparada con la dosis triple de Cefazolina como profilaxis antibiótica para reducir las infecciones post-cesárea. Material y Métodos: Estudio observacional, analítico, retrospectivo, transversal y comparativo. Se incluyó 82 historias clínicas de pacientes sometidas a cesárea en el Hospital Nacional Guillermo Almenara Irigoyen que recibieron dosis única o dosis triple de Cefazolina en el año 2011. De acuerdo al esquema recibido, se evaluaron las complicaciones infecciosas. Resultados: No hubo diferencia estadísticamente significativa entre la dosis única y la dosis triple en la incidencia de infecciones totales (9,3% vs 16,4%, p= 0,422), infección del sitio operatorio (7% vs 12,8%, p= 0,793), endometritis (0% vs 2,6%, p= 1,116) e infección del tracto urinario (2,3% vs 0%, p= 0,918). Conclusión: La dosis única de Cefazolina como profilaxis antibiótica fue igual de efectiva como la dosis triple en la prevención de la morbilidad infecciosa post-cesárea...

To compare the effectiveness of single dose versus triple dose of Cefazolinas prophylactic antibiotics in women undergoing cesarean section. Material and Methods: An observational, analytic, retrospective, cross sectional comparative study. Eighty two medical records of women undergoing caesarean section who received either single dose or triple dose in the Almenara Irigoyen Hospital were included. Women were compared on the basis of development of postoperative infections. Results: The study groups did not have statistically significant differences in the incidence of total infections (9,3% vs 16,4%, p= 0,422), wound infections(7% vs 12,8%, p= 0,793), endometritis (0% vs 2,6%, p= 1,116) and urinary tract infection(2,3% vs 0%, p= 0,918). Conclusion: A single prophylaxis dose of Cefazolin was as effective as triple dose in preventing post-caesarean infectious morbidity...

Nicotiana benthamiana resistance to non-adapted Melon necrotic spot virus results from an incompatible interaction between virus RNA and translation initiation factor 4E.

Nicotiana benthamiana has been described as non-host for Melon necrotic spot virus (MNSV). We investigated the basis of this resistance using the unique opportunity provided by strain MNSV-264, a recombinant virus that is able to overcome the resistance. Analysis of chimeric MNSV mutants showed that virulence in N. benthamiana is conferred by a 49 nucleotide section of the MNSV-264 3'-UTR, which acts in this host as a cap-independent translational enhancer (3'-CITE). Although the 3'-CITE of non-adapted MNSV-Mα5 is active in susceptible melon, it does not promote efficient translation in N. benthamiana, thus preventing expression of proteins required for virus replication. However, MNSV-Mα5 gains the ability to multiply in N. benthamiana cells if eIF4E from a susceptible melon variety (Cm-eIF4E-S) is supplied in trans. These data show that N. benthamiana resistance to MNSV-Mα5 results from incompatibility between the MNSV-Mα5 3'-CITE and N. benthamiana eIF4E in initiating efficient translation of the viral genome. Therefore, non-host resistance conferred by the inability of a host susceptibility factor to support viral multiplication may be a possible mechanism for this type of resistance to viruses.

ccz-1 mediates the digestion of apoptotic corpses in C. elegans.

During development, the processes of cell division, differentiation and apoptosis must be precisely coordinated in order to maintain tissue homeostasis. The nematode C. elegans is a powerful model system in which to study cell death and its control. C. elegans apoptotic cells condense and form refractile corpses under differential interference contrast (DIC) microscopy. Activation of the GTPase CED-10 (Rac) in a neighbouring cell mediates the recognition and engulfment of the cell corpse. After inclusion of the engulfed corpse in a phagosome, different proteins are sequentially recruited onto this organelle to promote its acidification and fusion with lysosomes, leading to the enzymatic degradation of the cell corpse. We show that CCZ-1, a protein conserved from yeasts to humans, mediates the digestion of these apoptotic corpses. CCZ-1 seems to act in lysosome biogenesis and phagosome maturation by recruiting the GTPase RAB-7 over the phagosome.

The pcsA gene from Streptomyces diastaticus var. 108 encodes a polyene carboxamide synthase with broad substrate specificity for polyene amides biosynthesis.

Two structurally related polyene macrolides are produced by Streptomyces diastaticus var. 108: rimocidin (3a) and CE-108 (2a). Both bioactive metabolites are biosynthesized from the same pathway through type I polyketide synthases by choosing a starter unit either acetate or butyrate, resulting in 2a or 3a formation, respectively. Two additional polyene amides, CE-108B (2b) and rimocidin B (3b), are also produced "in vivo" when this strain was genetically modified by transformation with engineered SCP2*-derived vectors carrying the ermE gene. The two polyene amides, 2b and 3b, showed improved pharmacological properties, and are generated by a tailoring activity involved in the conversion of the exocyclic carboxylic group of 2a and 3a into their amide derivatives. The improvement on some biological properties of the resulting polyenes, compared with that of the parental compounds, encourages our interest for isolating the tailoring gene responsible for the polyene carboxamide biosynthesis, aimed to use it as tool for generating new bioactive compounds. In this work, we describe the isolation from S. diastaticus var. 108 the corresponding gene, pcsA, encoding a polyene carboxamide synthase, belonging to the Class II glutamine amidotransferases and responsible for "in vivo" and "in vitro" formation of CE-108B (2b) and rimocidin B (3b). The fermentation broth from S. diastaticus var. 108 engineered with the appropriate pcsA gene construction, showed the polyene amides to be the major bioactive compounds.

Mechanism of plant eIF4E-mediated resistance against a Carmovirus (Tombusviridae): cap-independent translation of a viral RNA controlled in cis by an (a)virulence determinant.

Translation initiation factors are universal determinants of plant susceptibility to RNA viruses, but the underlying mechanisms are poorly understood. Here, we show that a sequence in the 3' untranslated region (3'-UTR) of a viral genome that is responsible for overcoming plant eIF4E-mediated resistance (virulence determinant) functions as a 3' cap-independent translational enhancer (3'-CITE). The virus/plant pair studied here is Melon necrotic spot virus (MNSV) and melon, for which a recessive resistance controlled by melon eIF4E was previously described. Chimeric viruses between virulent and avirulent isolates enabled us to map the virulence and avirulence determinants to 49 and 26 nucleotides, respectively. The translational efficiency of a luc reporter gene flanked by 5'- and 3'-UTRs from virulent, avirulent and chimeric viruses was analysed in vitro, in wheatgerm extract, and in vivo, in melon protoplasts, showing that: (i) the virulence determinant mediates the efficient cap-independent translation in vitro and in vivo; (ii) the avirulence determinant was able to promote efficient cap-independent translation in vitro, but only when eIF4E from susceptible melon was added in trans, and, coherently, only in protoplasts of susceptible melon, but not in the protoplasts of resistant melon; (iii) these activities required the 5'-UTR of MNSV in cis. Thus, the virulence and avirulence determinants function as 3'-CITEs. The activity of these 3'-CITEs was host specific, suggesting that an inefficient interaction between the viral 3'-CITE of the avirulent isolate and eIF4E of resistant melon impedes the correct formation of the translation initiation complex at the viral RNA ends, thereby leading to resistance.

EcoTILLING for the identification of allelic variants of melon eIF4E, a factor that controls virus susceptibility.

BACKGROUND: Translation initiation factors of the 4E and 4G protein families mediate resistance to several RNA plant viruses in the natural diversity of crops. Particularly, a single point mutation in melon eukaryotic translation initiation factor 4E (eIF4E) controls resistance to Melon necrotic spot virus (MNSV) in melon. Identification of allelic variants within natural populations by EcoTILLING has become a rapid genotype discovery method. RESULTS: A collection of Cucumis spp. was characterised for susceptibility to MNSV and Cucumber vein yellowing virus (CVYV) and used for the implementation of EcoTILLING to identify new allelic variants of eIF4E. A high conservation of eIF4E exonic regions was found, with six polymorphic sites identified out of EcoTILLING 113 accessions. Sequencing of regions surrounding polymorphisms revealed that all of them corresponded to silent nucleotide changes and just one to a non-silent change correlating with MNSV resistance. Except for the MNSV case, no correlation was found between variation of eIF4E and virus resistance, suggesting the implication of different and/or additional genes in previously identified resistance phenotypes. We have also characterized a new allele of eIF4E from Cucumis zeyheri, a wild relative of melon. Functional analyses suggested that this new eIF4E allele might be responsible for resistance to MNSV. CONCLUSION: This study shows the applicability of EcoTILLING in Cucumis spp., but given the conservation of eIF4E, new candidate genes should probably be considered to identify new sources of resistance to plant viruses. Part of the methodology described here could alternatively be used in TILLING experiments that serve to generate new eIF4E alleles.