Disrupting insect-mediated transmission of plant viruses

Many plant viruses are vectored by aphids in the non-persistent mode, in which virus particles are transported on these insects’ specialised piercing mouthparts (stylets). Virus infection can change plant-vector interactions and it is thought that this may accelerate virus transmission. To better understand how to inhibit virus transmission, I studied vectoring of cucumber mosaic virus (CMV) and turnip mosaic virus (TuMV) between Arabidopsis thaliana plants by the aphid Myzus persicae. Among other things, I investigated if there were differences in the extent to which the viruses modified aphid-plant interactions in different Arabidopsis accessions, as well as the intrinsic susceptibility of these accessions to aphid infestation. A range of Arabidopsis accessions showed differences in attractiveness and susceptibility to aphids and variations in virus-induced changes in plant-aphid interactions. I found that aphids were attracted to plant volatiles emitted by CMV-infected Col-0 and CMV-infected Ei-2 plants. Interestingly, aphids settle more readily on CMV-infected Ei-2 plants than CMV-infected Col-0 plants. However, aphids did not prefer to settle on CMV-infected plants of either accession. Thus, I used these accessions to determine how to manipulate aphid behaviour to inhibit virus transmission using two types of microcosm: simple lines of plants (with various mixtures of the two accessions) and two-dimensional arrays (‘fields’) of plants. The simple line experiments showed that aphid-mediated transmission could be disrupted using mixtures of accessions with differences in intrinsic aphid attractiveness and susceptibility to aphid infestation. In the two-dimensional ‘field’ experiments, two approaches, which included CMV- and TuMV-resistant plants were most effective in inhibiting virus transmission. Resistant plants in both Arabidopsis backgrounds were either mixed in various proportions (resistant v. susceptible and Col-0 v. Ei-2) in randomised planting layouts or with plants arranged as barriers. A 33% proportion of virus-resistant plants randomly distributed was sufficient to inhibit virus transmission. A barrier of Ei-2 CMV-resistant plants or TuMV-resistant plants in a population of Col-0 plants reduced virus transmission of CMV and TuMV. The latter approach retained more aphids than populations of solely Col-0 plants. Thus, Ei-2 plants can be used as trap plants to attract and ‘sanitise’ viruliferous aphids by inducing them to deposit virus particles in resistant plants, thus inhibiting onward transmission. The results show that there is potential for using intraspecific variation in host plants to inhibit aphid-mediated transmission.Studentship provided by the Ecuadorian Government through SENESCYT (Secretaria de Educación Superior, Ciencia y Tecnología e Innovación

Caracterización morfológica y molecular de accesiones de maíz negro (Zea mays L.) mediante análisis de secuencias simples

Molecular and morphological characterization of maize lines from a breeding program has an important application in the analysis of genetic diversity in order to generate hybrids lines. In the present study, 10 microsatellites (SSR) and 4 morphological traits were used to estimate the genetic relationship among 24 inbred lines of purple maize (Zea mays L.) from Ecuador and CIMMYT. Genetic distance was estimated using the Simple Matching coefficient. As a result, the 24 accessions were grouped in 5 clusters for the morphological traits and in 7 clusters for the molecular analysis using the UPGMA clustering.La caracterización molecular y morfológica de accesiones de maíz dentro de un programa de mejoramiento vegetal es de importante aplicación en la estimación de relaciones genéticas para la generación de híbridos. En este trabajo se emplearon 10 microsatélites y 4 características morfológicas para analizar y estimar el grado de relación genética entre 24 accesiones endocriadas de maíz morado (Zea mays L.) procedentes del Ecuador y del CIMMYT

Prevalência da má- nutrição em pacientes maiores de 7 anos no Hospital Vicente Corral Moscoso Cuenca-Equador nutritionDay 2019

Hospital malnutrition represents a global health problem. It is associated with increased complications, morbidity and mortality, days of hospital stay, and increased costs. nutritionDay (nDay) is a project to fight against hospital malnutrition. It is an audit carried out simultaneously in all participating countries on a single day, using standard questionnaires that generate a national report.La malnutrición hospitalaria representa un problema de salud global. Esta se asocia con aumento de complicaciones, morbimortalidad, días de estancia hospitalaria e incremento de costos. El nutritionDay (nDay) es un proyecto creado para luchar contra la malnutrición hospitalaria, que consta de una auditoría que se realiza simultáneamente en todos los países participantes en un solo día, utilizando cuestionarios estándares que generan un informe nacional.A má nutrição hospitalar representa um problema de saúde global. Isso está associado ao aumento de complicações, morbimortalidade, dias de internação hospitalar e aumento de custos. O nutritionDay (nDay) é um projeto para lutar contra a má nutrição hospitalar. É uma auditoria que é realizada simultaneamente em todos os países participantes em um único dia, utilizando questionários padronizados que geram um informe nacional

Bioengineering secreted proteases converts divergent Rcr3 orthologs and paralogs into extracellular immune co-receptors

Secreted immune proteases “Required for Cladosporium resistance-3” (Rcr3) and “Phytophthora-inhibited protease-1” (Pip1) of tomato (Solanum lycopersicum) are both inhibited by Avirulence-2 (Avr2) from the fungal plant pathogen Cladosporium fulvum. However, only Rcr3 acts as a decoy co-receptor that detects Avr2 in the presence of the Cf-2 immune receptor. Here, we identified crucial residues in tomato Rcr3 that are required for Cf-2-mediated signaling and bioengineered various proteases to trigger Avr2/Cf-2-dependent immunity. Despite substantial divergence in Rcr3 orthologs from eggplant (Solanum melongena) and tobacco (Nicotiana spp.), minimal alterations were sufficient to trigger Avr2/Cf-2-mediated immune signaling. By contrast, tomato Pip1 was bioengineered with 16 Rcr3-specific residues to initiate Avr2/Cf-2-triggered immune signaling. These residues cluster on one side of the protein next to the substrate-binding groove, indicating a potential Cf-2 interaction site. Our findings also revealed that Rcr3 and Pip1 have distinct substrate preferences determined by two variant residues and that both are suboptimal for binding Avr2. This study advances our understanding of Avr2 perception and opens avenues to bioengineer proteases to broaden pathogen recognition in other crops

Effects of the cucumber mosaic virus 2a protein on aphid-plant interactions in Arabidopsis thaliana.

The cucumber mosaic virus (CMV) 2a RNA-dependent RNA polymerase protein has an additional function in Arabidopsis thaliana, which is to stimulate feeding deterrence (antixenosis) against aphids. Antixenosis is thought to increase the probability that aphids, after acquiring CMV particles from brief probes of an infected plant's epidermal cells, will be discouraged from settling and instead will spread inoculum to neighbouring plants. The amino acid sequences of 2a proteins encoded by a CMV strain that induces antixenosis in A. thaliana (Fny-CMV) and one that does not (LS-CMV) were compared to identify residues that might determine the triggering of antixenosis. These data were used to design reassortant viruses comprising Fny-CMV RNAs 1 and 3, and recombinant CMV RNA 2 molecules encoding chimeric 2a proteins containing sequences derived from LS-CMV and Fny-CMV. Antixenosis induction was detected by measuring the mean relative growth rate and fecundity of aphids (Myzus persicae) confined on infected and on mock-inoculated plants. An amino acid sequence determining antixenosis induction by CMV was found to reside between 2a protein residues 200 and 300. Subsequent mutant analysis delineated this to residue 237. We conjecture that the Fny-CMV 2a protein valine-237 plays some role in 2a protein-induced antixenosis

Exogenous Application of RNAi-Inducing Double-Stranded RNA Inhibits Aphid-Mediated Transmission of a Plant Virus.

Plant viruses are difficult to control, and they decrease both the quality and yield of crops, thus threatening global food security. A new approach that uses topical application of double-stranded RNA (dsRNA) to induce antiviral RNA-interference has been shown to be effective at preventing virus infection in a range of plants following mechanical inoculation. In this study, topical application of dsRNA was effective against mechanical inoculation and aphid-mediated inoculation with the potyvirus bean common mosaic virus (BCMV). Topical application of dsRNAs targeting either the coding region of the potyviral nuclear inclusion b (NIb) protein (BCMVNIb-dsRNA) or the coat protein (CP) coding region (BCMVCP-dsRNA) protected Nicotiana benthamiana and cowpea (Vigna unguiculata) plants against mechanical inoculation with BCMV. BCMVCP-dsRNA was selected for subsequent aphid transmission experiments. BCMVCP-dsRNA was loaded onto layered double hydroxide nanoparticles to form BCMVCP-BioClay which is a more stable formulation for delivering dsRNA than naked dsRNA. BCMVCP-BioClay was shown to be successful in protecting plants against BCMV transmission by the aphid Myzus persicae. Spraying detached N. benthamiana leaves with BCMVCP-BioClay 5 days prior to exposure to viruliferous aphids protected the leaves from infection by BCMV. Importantly, spraying of intact N. benthamiana and cowpea plants with BCMVCP-BioClay 5 days prior to exposure to viruliferous aphids protected plants of both species from BCMV infection. This study demonstrates that topical application of dsRNA using BioClay protects plants from aphid-mediated virus transmission, which is an important first step toward developing practical application of this approach in crop protection.Secretaria Nacional de Educación Superior, Ciencia y Technologí e Innovación, Ecuador Accelerated Partnership Grant, Queensland Government (2014000652), awarded to NM with Nufarm Australia Limited as the industry partner. EW PhD programme with NM is supported by a scholarship from The University of Queenslan

Exogenous application of RNAi-inducing double-stranded RNA inhibits aphid-mediated transmission of a plant virus

Plant viruses are difficult to control, and they decrease both the quality and yield of crops, thus threatening global food security. A new approach that uses topical application of double-stranded RNA (dsRNA) to induce antiviral RNA-interference has been shown to be effective at preventing virus infection in a range of plants following mechanical inoculation. In this study, topical application of dsRNA was effective against mechanical inoculation and aphid-mediated inoculation with the potyvirus bean common mosaic virus (BCMV). Topical application of dsRNAs targeting either the coding region of the potyviral nuclear inclusion b (Nib) protein (BCMVNIb-dsRNA) or the coat protein (CP) coding region (BCMVCP-dsRNA) protected Nicotiana benthamiana and cowpea (Vigna unguiculata) plants against mechanical inoculation with BCMV. BCMVCP-dsRNA was selected for subsequent aphid transmission experiments. BCMVCP-dsRNA was loaded onto layered double hydroxide nanoparticles to form BCMVCP-BioClay which is a more stable formulation for delivering dsRNA than naked dsRNA. BCMVCP-BioClay was shown to be successful in protecting plants against BCMV transmission by the aphid Myzus persicae. Spraying detached N. benthamiana leaves with BCMVCP-BioClay 5 days prior to exposure to viruliferous aphids protected the leaves from infection by BCMV. Importantly, spraying of intact N. benthamiana and cowpea plants with BCMVCP-BioClay 5 days prior to exposure to viruliferous aphids protected plants of both species from BCMV infection. This study demonstrates that topical application of dsRNA using BioClay protects plants from aphid-mediated virus transmission, which is an important first step toward developing practical application of this approach in crop protection

Modelling and manipulation of aphid-mediated spread of non-persistently transmitted viruses.

Aphids vector many plant viruses in a non-persistent manner i.e., virus particles bind loosely to the insect mouthparts (stylet). This means that acquisition of virus particles from infected plants, and inoculation of uninfected plants by viruliferous aphids, are rapid processes that require only brief probes of the plant's epidermal cells. Virus infection alters plant biochemistry, which causes changes in emission of volatile organic compounds and altered accumulation of nutrients and defence compounds in host tissues. These virus-induced biochemical changes can influence the migration, settling and feeding behaviours of aphids. Working mainly with cucumber mosaic virus and several potyviruses, a number of research groups have noted that in some plants, virus infection engenders resistance to aphid settling (sometimes accompanied by emission of deceptively attractive volatiles, that can lead to exploratory penetration by aphids without settling). However, in certain other hosts, virus infection renders plants more susceptible to aphid colonisation. It has been suggested that induction of resistance to aphid settling encourages transmission of non-persistently transmitted viruses, while induction of susceptibility to settling retards transmission. However, recent mathematical modelling indicates that both virus-induced effects contribute to epidemic development at different scales. We have also investigated at the molecular level the processes leading to induction, by cucumber mosaic virus, of feeding deterrence versus susceptibility to aphid infestation. Both processes involve complex interactions between specific viral proteins and host factors, resulting in manipulation or suppression of the plant's immune networks.JPC gratefully acknowledges funding from the U.K. Biotechnological and Biological Sciences Research Council (BBSRC: SCPRID Grant Number BB/J011762/1; GCRF Grant Number BB/P023223/1). JMM receives support from the BBSRC BB/R005397/1 GCRF-CONNECTED Network (https://www.connectedvirus.net/). RD and CAG are funded by the Bill and Melinda Gates Foundation. LGW is supported by a PhD studentship from the BBSRC-Cambridge University Doctoral Training Programme (BB/M011194/1). AB-C was funded by a PhD studentship from the Secretaria Nacional de Educación Superior, Ciencia y Technologí e Innovación, Republic of Ecuador. WA is supported by a Cambridge-Africa PhD studentship. SJR was funded by a Postdoctoral Fellowship from the National Research Foundation of the Republic of Korea. FOW is funded by a Royal Society-FLAIR Fellowship (Grant number FLR\R1\190462)