Brachypodium: A Monocot Grass Model Genus for Plant Biology

The genus Brachypodium represents a model system that is advancing our knowledge of the biology of grasses, including small grains, in the postgenomics era. The most widely used species, Brachypodium distachyon, is a C-3 plant that is distributed worldwide. B. distachyon has a small genome, short life cycle, and small stature and is amenable to genetic transformation. Due to the intensive and thoughtful development of this grass as a model organism, it is well-suited for laboratory and field experimentation. The intent of this review is to introduce this model system genus and describe some key outcomes of nearly a decade of research since the first draft genome sequence of the flagship species, B. distachyon, was completed. We discuss characteristics and features of B. distachyon and its congeners that make the genus a valuable model system for studies in ecology, evolution, genetics, and genomics in the grasses, review current hot topics in Brachypodium research, and highlight the potential for future analysis using this system in the coming years

Comparative Study of Monoclonal and Recombinant Antibody-Based Immunoassays for Fungicide Analysis in Fruit juices

[EN] A comparative study of the analytical performance of enzyme-linked immunosorbent assays (ELISAs), based on monoclonal and recombinant antibodies, for the determination of fungicide residues in fruit juices has been carried out. To this aim, three murine hybridoma cell lines secreting specific monoclonal antibodies against (RS)-2-(2,4-dichlorophenyl)-3-(1H-1,2,4-triazol-1-yl)propyl-1,1,2,2-tetrafluoroethyl ether (tetraconazole), 2-(4-triazolyl)benzimidazole (thiabendazole), and (RS)-1-(beta-allyloxy-2,4-dichlorophenylethyl)imidazole (imazalil) were used as a source of immunoglobulin gene fragments for the production of single-chain variable fragment (scFv) and fusion scFv-pIII recombinant antibodies in Escherichia coli. Selected recombinant antibodies displayed cross-reactivity profiles very similar to those of the parent monoclonal antibodies. Imazalil and tetraconazole recombinant antibodies showed one order of magnitude lower affinity than their respective monoclonal antibodies, whereas the thiabendazole recombinant antibodies showed an affinity similar to that of their parent monoclonal antibody. On the other hand, scFv-pIII fusion fragments showed similar analytical properties as, and occasionally better than, scFv recombinant antibodies. Finally, ELISAs developed from each antibody type showed similar analytical performance when applied to the analysis of the target fungicides in fruit juices.This work was funded by Ministerio de Educacion y Ciencia (MEC, Spain, Project AGL2002-03266). E. P. was the recipient of a doctoral fellowship from Conselleria d'Educacio (Generalitat Valenciana, Spain).Moreno Tamarit, MJ.; Plana Andani, E.; Manclus Ciscar, JJ.; Montoya Baides, Á. (2014). Comparative Study of Monoclonal and Recombinant Antibody-Based Immunoassays for Fungicide Analysis in Fruit juices. Food Analytical Methods. 7(2):481-489. https://doi.org/10.1007/s12161-013-9655-zS48148972Abad A, Manclús JJ, Moreno M, Montoya A (2001) J AOAC Int 84:1–6Alcocer MJC, Doyen C, Lee HA, Morgan MRA (2000) J Agric Food Chem 48:4053–4059Brichta J, Vesela H, Franek M (2003) Vet Med 48:237–247Brichta J, Hnilova M, Viskovic T (2005) Vet Med 50:231–252Charlton K, Harris WJ, Potter AJ (2001) Biosens Bioelec 16:639–646EU Pesticide Database (2013) Pesticide EU-MRLs. http://ec.europa.eu/sanco_pesticides/public/index.cfm . Accessed Jan 2013Ferrer C, Martínez-Bueno MJ, Lozano A, Fernández-Alba AR (2011) Talanta 83:1552–1561Garret SD, Appleford DJA, Wyatt GM, Lee HA, Morgan MRA (1997) J Agric Food Chem 45:4183–4189Graham BM, Porter AJ, Harris WJ (1995) J Chem Technol Biotech 63:279–289Hiemstra M, de Kok A (2007) J Chromatog A 1154:3–25Kipriyanov SM, Moldenhauer G, Little M (1997) J Immunol Meth 200:69–77Kramer K, Hock B (2007) Recombinant antibodies for agrochemicals: Evolutionary optimization. In: Kennedy IR, Solomon KR, Gee SJ, Crossan AN, Wang S, Sánchez-Bayo F (eds) Rational environmental management of agrochemicals: Risk assessment, monitoring, and remedial action. ACS Symposium Series, vol. 966, pp 155−170Krebber A, Bornhauser S, Burmester J, Honegger A, Willuda J, Bosshard HR, Plückthun A (1997) J Immunol Meth 201:35–55Leong SSJ, Chen WN (2008) Chem Engin Sci 63:1401–1414Li T, Zhang Q, Liu Y, Chen D, Hu B, Blake DA, Liu F (2006) J Agric Food Chem 54:9085–9091Manclús JJ, Moreno M, Plana E, Montoya A (2008) J Agric Food Chem 56:8790–8800Markus V, Janne L, Urpo L (2011) Trends Anal Chem 30:219–226Mersmann M, Schmidt A, Tesar M, Schöneberg A, Welschof M, Kipriyanov S, Terness P, Little M, Pfizenmaier K, Moosmayer D (1998) J Immunol Meth 220:51–58Moreno M, Plana E, Montoya A, Caputo P, Manclús JJ (2007) Food Addit Contam 24:704–712Morozova VS, Levashova AI, Eremin SA (2005) J Anal Chem 60:202–217Nishi K, Imajuku Y, Nakata M, Ohde K, Miyake S, Morimune K, Kawata M, Ohkawa H (2003) J Pest Sci 28:301–309Nishi K, Ishiuchi M, Morimune K, Ohkawa H (2005) J Agric Food Chem 53:5096–5104Scholthof KB, Whang G, Karu AE (1997) J Agric Food Chem 45:1509–1517Sheedy C, MacKenzie CR, Hall JC (2007) Biotech Adv 25:25333–25352Tout NL, Yau KYF, Trevors JT, Lee H, Hall JC (2001) J Agric Food Chem 49:3628–3637Webb SR, Lee H, Hall JC (1997) J Agric Food Chem 45:535–541Yau KYF, Tout NL, Trevors JT, Lee H, Hall JC (1998) J Agric Food Chem 46:4457–4463Yoshioka N, Akiyama Y, Matsuoka T, Mitsuhashi T (2010) Food Control 21:212–21

AGO1 and AGO2 Act Redundantly in miR408-Mediated Plantacyanin Regulation

Background: In Arabidopsis, AGO1 and AGO2 associate with small RNAs that exhibit a Uridine and an Adenosine at their 59 end, respectively. Because most plant miRNAs have a 59U, AGO1 plays many essential roles in miRNA-mediated regulation of development and stress responses. In contrast, AGO2 has only been implicated in antibacterial defense in association with miR393*, which has a 59A. AGO2 also participates in antiviral defense in association with viral siRNAs. Principal Findings: This study reveals that miR408, which has a 59A, regulates its target Plantacyanin through either AGO1 or AGO2. Indeed, neither ago1 nor ago2 single mutations abolish miR408-mediated regulation of Plantacyanin. Only an ago1 ago2 double mutant appears compromised in miR408-mediated regulation of Plantacyanin, suggesting that AGO1 and AGO2 have redundant roles in this regulation. Moreover, the nature of the 59 nucleotide of miR408 does not appear essential for its regulatory role because both a wildtype 59A-MIR408 and a mutant 59U-MIR408 gene complement a mir408 mutant. Conclusions/Significance: These results suggest that miR408 associates with both AGO1 and AGO2 based on criteria that differ from the 59 end rule, reminiscent of miR390-AGO7 and miR165/166-AGO10 associations, which are not based on the nature of the 59 nucleotide

Identification of Pns6, a putative movement protein of RRSV, as a silencing suppressor

RNA silencing is a potent antiviral response in plants. As a counterdefense, most plant and some animal viruses encode RNA silencing suppressors. In this study, we showed that Pns6, a putative movement protein of Rice ragged stunt virus (RRSV), exhibited silencing suppressor activity in coinfiltration assays with the reporter green fluorescent protein (GFP) in transgenic Nicotiana benthamiana line 16c. Pns6 of RRSV suppressed local silencing induced by sense RNA but had no effect on that induced by dsRNA. Deletion of a region involved in RNA binding abolished the silencing suppressor activity of Pns6. Further, expression of Pns6 enhanced Potato virus × pathogenicity in N. benthamiana. Collectively, these results suggested that RRSV Pns6 functions as a virus suppressor of RNA silencing that targets an upstream step of the dsRNA formation in the RNA silencing pathway. This is the first silencing suppressor to be identified from the genus Oryzavirus

Transient Expression of Hemagglutinin Antigen from Low Pathogenic Avian Influenza A (H7N7) in Nicotiana benthamiana

The influenza A virus is of global concern for the poultry industry, especially the H5 and H7 subtypes as they have the potential to become highly pathogenic for poultry. In this study, the hemagglutinin (HA) of a low pathogenic avian influenza virus of the H7N7 subtype isolated from a Swedish mallard Anas platyrhynchos was sequenced, characterized and transiently expressed in Nicotiana benthamiana. Recently, plant expression systems have gained interest as an alternative for the production of vaccine antigens. To examine the possibility of expressing the HA protein in N. benthamiana, a cDNA fragment encoding the HA gene was synthesized de novo, modified with a Kozak sequence, a PR1a signal peptide, a C-terminal hexahistidine (6×His) tag, and an endoplasmic retention signal (SEKDEL). The construct was cloned into a Cowpea mosaic virus (CPMV)-based vector (pEAQ-HT) and the resulting pEAQ-HT-HA plasmid, along with a vector (pJL3:p19) containing the viral gene-silencing suppressor p19 from Tomato bushy stunt virus, was agro-infiltrated into N. benthamiana. The highest gene expression of recombinant plant-produced, uncleaved HA (rHA0), as measured by quantitative real-time PCR was detected at 6 days post infiltration (dpi). Guided by the gene expression profile, rHA0 protein was extracted at 6 dpi and subsequently purified utilizing the 6×His tag and immobilized metal ion adsorption chromatography. The yield was 0.2 g purified protein per kg fresh weight of leaves. Further molecular characterizations showed that the purified rHA0 protein was N-glycosylated and its identity confirmed by liquid chromatography-tandem mass spectrometry. In addition, the purified rHA0 exhibited hemagglutination and hemagglutination inhibition activity indicating that the rHA0 shares structural and functional properties with native HA protein of H7 influenza virus. Our results indicate that rHA0 maintained its native antigenicity and specificity, providing a good source of vaccine antigen to induce immune response in poultry species

Advances in dissecting mosquito innate immune responses to arbovirus infection

Arthropod-borne viruses – arboviruses – are a significant threat to public health. Whilst there is considerable knowledge about arbovirus interactions with vertebrate immunity, relatively little is known about how vectors such as mosquitoes control arbovirus infections. In this review, we discuss novel findings in the field of mosquito antiviral responses to arboviruses, in particular RNA interference, the up-and-coming field of general immune-signalling pathways, and cell death/apoptosis

Distinct Effects of p19 RNA Silencing Suppressor on Small RNA Mediated Pathways in Plants

RNA silencing is one of the main defense mechanisms employed by plants to fight viruses. In change, viruses have evolved silencing suppressor proteins to neutralize antiviral silencing. Since the endogenous and antiviral functions of RNA silencing pathway rely on common components, it was suggested that viral suppressors interfere with endogenous silencing pathway contributing to viral symptom development. In this work, we aimed to understand the effects of the tombusviral p19 suppressor on endogenous and antiviral silencing during genuine virus infection. We showed that ectopically expressed p19 sequesters endogenous small RNAs (sRNAs) in the absence, but not in the presence of virus infection. Our presented data question the generalized model in which the sequestration of endogenous sRNAs by the viral suppressor contributes to the viral symptom development. We further showed that p19 preferentially binds the perfectly paired ds-viral small interfering RNAs (vsiRNAs) but does not select based on their sequence or the type of the 5’ nucleotide. Finally, co-immunoprecipitation of sRNAs with AGO1 or AGO2 from virus-infected plants revealed that p19 specifically impairs vsiRNA loading into AGO1 but not AGO2. Our findings, coupled with the fact that p19-expressing wild type Cymbidium ringspot virus (CymRSV) overcomes the Nicotiana benthamiana silencing based defense killing the host, suggest that AGO1 is the main effector of antiviral silencing in this host-virus combination