1,721,099 research outputs found
Molecular mechanisms involved in the pathogenesis of beet soil-borne viruses.
No full textThe genus Benyvirus includes the most important and widespread sugar beet viruses transmitted through the soil by the plasmodiophorid Polymyxa betae. In particular Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, causes an abnormal rootlet proliferation known as rhizomania. Beet soil-borne mosaic virus (BSBMV) is widely distributed in the United States and, up to date has not been reported in others countries.
My PhD project aims to investigate molecular interactions between BNYVV and BSBMV and the mechanisms involved in the pathogenesis of these viruses.
BNYVV full-length infectious cDNA clones were available as well as full-length cDNA clones of BSBMV RNA-1, -2, -3 and -4. Handling of these cDNA clones in order to produce in vitro infectious transcripts need sensitive and expensive steps, so I developed agroclones of BNYVV and BSBMV RNAs, as well as viral replicons allowing the expression of different proteins.
Chenopodium quinoa and Nicotiana benthamiana plants have been infected with in vitro transcripts and agroclones to investigate the interaction between BNYVV and BSBMV RNA-1 and -2 and the behavior of artificial viral chimeras. Simultaneously I characterized BSBMV p14 and demonstrated that it is a suppressor of post-transcriptional gene silencing sharing common features with BNYVV p14
Produzione di virus sintetici per lo studio dei meccanismi di interazione coinvolti nell'induzione di resistenza
No full textBeet soil-borne mosaic virus (BSBMV) and Beet necrotic yellow vein virus
(BNYVV) are members of Benyvirus genus. BSBMV has been reported only in
the United States while BNYVV has a worldwide distribution. Both viruses are
vectored by Polymyxa betae, possess similar host ranges, particles number and
morphology. Both viruses are not serologically related but have similar genomic
organizations. Field isolates consist of four RNA species but some BNYVV
isolates contain a fifth RNA. RNAs 1 and 2 are essential for infection and
replication while RNAs 3 and 4 play important roles on plant and vector
interactions, respectively. Nucleotide and amino acid analyses revealed BSBMV
and BNYVV are different enough to be classified in two different species.
Additionally in BNYVV/BSBMV mixed infections, a competition was
previous described in sugar beet, where BNYVV infection reduces BSBMV
accumulation in both susceptible and resistant cultivars.
Considering all this observations we hypothesized that BNYVV and
BSBMV crossed study, exploiting their similarities and divergences, can improve
investigation of molecular interactions between sugar beets and Benyviruses.
The main achievement of our research is the production of a cDNA
biologically active clones collection of BNYVV and BSBMV RNAs, from which
synthetic copies of both Benyviruses can be transcribed.
Moreover, through recombination experiments we demonstrated, for the
first time, the BNYVV RNA 1 and 2 capability to trans-replicate and encapsidate
BSBMV RNA 3 and 4, either the BSBMV RNA 1 and 2 capability to replicate
BNYVV RNA2 in planta. We also demonstrated that BSBMV RNA3 support
long-distance movement of BNYVV RNA 1 and 2 in B. macrocarpa and that
85
foreign sequence as p29HA, GFP and RFP, are successfully expressed, in C.
quinoa, by BSBMV RNA3 based replicon (RepIII) also produced by our
research. These results confirm the close correlation among the two viruses.
Interestingly, the symptoms induced by BSBMV RNA-3 on C. quinoa
leaves are more similar to necrotic local lesions caused by BNYVV RNA-5 p26
than to strongly chlorotic local lesions or yellow spot induced by BNYVV RNA-
3 encoded p25. As previous reported BSBMV p29 share 23% of amino acid
sequence identity with BNYVV p25 but identity increase to 43% when compared
with sequence of BNYVV RNA-5 p26.
Based on our results the essential sequence (Core region) for the longdistance
movement of BSBMV and BNYVV in B. macrocarpa, is not only
carried by RNA3s species but other regions, perhaps located on the RNA 1 and
2, could play a fundamental role in this matter.
Finally a chimeric RNA, composed by the 5’ region of RNA4 and 3’ region
of RNA3 of BSBMV, has been produced after 21 serial mechanically inoculation
of wild type BSBMV on C. quinoa plants. Chimera seems unable to express any
protein, but it is replicated and transcript in planta. It could represent an
important tool to study the interactions between Benyvirus and plant host.
In conclusion different tools, comprising a method to study synthetic
viruses under natural conditions of inoculum through P. Betae, have been
produced and new knowledge are been acquired that will allow to perform future
investigation of the molecular interactions between sugar beets and Benyviruses
Reverse genetic studies of Benyvirus - Polymyxa betae molecular interaction: Role of the RNA4-encoded protein in virus transmission
No full textBeet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, is included within viruses transmitted through the soil from plasmodiophorid as Polymyxa betae. BNYVV is the causal agent of Rhizomania, which induces abnormal rootlet proliferation and is widespread in the sugar beet growing areas in Europe, Asia and America; for review see (Peltier et al., 2008). In this latter continent, Beet soil-borne mosaic virus (BSBMV) has been identified (Lee et al., 2001) and belongs to the benyvirus genus together with BNYVV, both vectored by P. betae. BSBMV is widely distributed only in the United States and it has not been reported yet in others countries. It was first identified in Texas as a sugar beet virus morphologically similar but serologically distinct to BNYVV. Subsequent sequence analysis of BSBMV RNAs evidenced similar genomic organization to that of BNYVV but sufficient molecular differences to distinct BSBMV and BNYVV in two different species (Rush et al., 2003). Benyviruses field isolates usually consist of four RNA species but some BNYVV isolates contain a fifth RNA. RNAs -1 contains a single long ORF encoding polypeptide that shares amino acid homology with known viral RNA-dependent RNA polymerases (RdRp) and helicases. RNAs -2 contains six ORFs: capsid protein (CP), one readthrough protein, triple gene block proteins (TGB) that are required for cell-to-cell virus movement and the sixth 14 kDa ORF is a post-translation gene silencing suppressor. RNAs -3 is involved on disease symptoms and is essential for virus systemic movement. BSBMV RNA-3 can be trans-replicated, trans-encapsidated by the BNYVV helper strain (RNA-1 and -2) (Ratti et al., 2009). BNYVV RNA-4 encoded one 31 kDa protein and is essential for vector interactions and virus transmission by P. betae (Rahim et al., 2007). BNYVV RNA-5 encoded 26 kDa protein that improve virus infections and accumulation in the hosts.
We are interest on BSBMV effect on Rhizomania studies using powerful tools as full-length infectious cDNA clones. B-type full-length infectious cDNA clones are available (Quillet et al., 1989) as well as A/P-type RNA-3, -4 and -5 from BNYVV (unpublished). A-type BNYVV full-length clones are also available, but RNA-1 cDNA clone still need to be modified. During the PhD program, we start production of BSBMV full-length cDNA clones and we investigate molecular interactions between plant and Benyviruses exploiting biological, epidemiological and molecular similarities/divergences between BSBMV and BNYVV.
During my PhD researchrs we obtained full length infectious cDNA clones of BSBMV RNA-1 and -2 and we demonstrate that they transcripts are replicated and packaged in planta and able to substitute BNYVV RNA-1 or RNA-2 in a chimeric viral progeny (BSBMV RNA-1 + BNYVV RNA-2 or BNYVV RNA-1 + BSBMV RNA-2). During BSBMV full-length cDNA clones production, unexpected 1,730 nts long form of BSBMV RNA-4 has been detected from sugar beet roots grown on BSBMV infected soil. Sequence analysis of the new BSBMV RNA-4 form revealed high identity (~100%) with published version of BSBMV RNA-4 sequence (NC_003508) between nucleotides 1-608 and 1,138-1,730, however the new form shows 528 additionally nucleotides between positions 608-1,138 (FJ424610). Two putative ORFs has been identified, the first one (nucleotides 383 to 1,234), encode a protein with predicted mass of 32 kDa (p32) and the second one (nucleotides 885 to 1,244) express an expected product of 13 kDa (p13). As for BSBMV RNA-3 (Ratti et al., 2009), full-length BSBMV RNA-4 cDNA clone permitted to obtain infectious transcripts that BNYVV viral machinery (Stras12) is able to replicate and to encapsidate in planta. Moreover, we demonstrated that BSBMV RNA-4 can substitute BNYVV RNA-4 for an efficient transmission through the vector P. betae in Beta vulgaris plants, demonstrating a very high correlation between BNYVV and BSBMV. At the same time, using BNYVV helper strain, we studied BSBMV RNA-4’s protein expression in planta. We associated a local necrotic lesions phenotype to the p32 protein expression onto mechanically inoculated C. quinoa. Flag or GFP-tagged sequences of p32 and p13 have been expressed in viral context, using Rep3 replicons, based on BNYVV RNA-3. Western blot analyses of local lesions contents, using FLAG-specific antibody, revealed a high molecular weight protein, which suggest either a strong interaction of BSBMV RNA4’s protein with host protein(s) or post translational modifications. GFP-fusion sequences permitted the subcellular localization of BSBMV RNA4’s proteins. Moreover we demonstrated the absence of self-activation domains on p32 by yeast two hybrid system approaches. We also confirmed that p32 protein is essential for virus transmission by P. betae using BNYVV helper strain and BNYVV RNA-3 and we investigated its role by the use of different deleted forms of p32 protein. Serial mechanical inoculation of wild-type BSBMV on C. quinoa plants were performed every 7 days. Deleted form of BSBMV RNA-4 (1298 bp) appeared after 14 passages and its sequence analysis shows deletion of 433 nucleotides between positions 611 and 1044 of RNA-4 new form. We demonstrated that this deleted form can’t support transmission by P. betae using BNYVV helper strain and BNYVV RNA-3, moreover we confirmed our hypothesis that BSBMV RNA-4 described by Lee et al. (2001) is a deleted form. Interesting after 21 passages we identifed one chimeric form of BSBMV RNA-4 and BSBMV RNA-3 (1146 bp). Two putative ORFs has been identified on its sequence, the first one (nucleotides 383 to 562), encode a protein with predicted mass of 7 kDa (p7), corresponding to the N-terminal of p32 protein encoded by BSBMV RNA-4; the second one (nucleotides 562 to 789) express an expected product of 9 kDa (p9) corresponding to the C-terminal of p29 encoded by BSBMV RNA-3.
Results obtained by our research in this topic opened new research lines that our laboratories will develop in a closely future. In particular BSBMV p32 and its mutated forms will be used to identify factors, as host or vector protein(s), involved in the virus transmission through P. betae. The new results could allow selection or production of sugar beet plants able to prevent virus transmission then able to reduce viral inoculum in the soil
" Valutazione di metodi diagnostici molecolari per la certificazione virus esente del materiale vegetale da propagazione in Egitto" supportato dal Ministero degli Affari Esteri Italiano nell'ambito del protocollo bilaterale tra Italia ed Egitto,
No full textL’attività svolta durante il primo anno del programma esecutivo Italia/ Egitto ha permesso alle due unità di ricerca di defininire un quadro preciso delle principali necessità di cui la certificazione virus esente del materiale da propagazione abbisogna nella frutticoltura Egiziana. L’insieme di tali informazioni, delle metodologie diagnostiche molecolari messe a punto e dell’esperineza acquisita dal laboratorio di Virologia del Botany and Microbiology Department (Faculty of Science, Alexandria University) rappresenta la garanzia per un fruttuoso proseguimento del progetto. Nell’ambito della collaborazione bilaterale, durante il primo anno di attività, le due unità di ricerca hanno organizzato incontri con i rappresentati di alcune tra le più importanti aziende agricole pubbliche e private e di alcuni centri di ricerca (dell’Università e del Ministero dell’Agricoltura) che operano nell’area di Alexandria
Étude des interactions Benyvirus-Polymyxa betae par génétique inverse,rôle de la protéine codée par l'ARN4 dans la transmission
No full textLes virus Beet soil-borne mosaic virus (BSBMV) et Beet necrotic yellow vein virus (BNYVV, agent de la Rhizomanie) font partie du genre Benyvirus. Des clones d'ADNc infectieux de BSBMV ont été obtenus et ont permis d'entreprendre l'étude des interactions moléculaires entre plante et Benyvirus sur les bases des différences biologiques des deux virus. Les transcrits infectieux de BSBMV peuvent substituer ceux du BNYVV et ainsi produire des recombinants chimériques.La transmission par Polymyxa betae est liée à la présence de l'ARN4 et un ARN4 de 1730 nts de BSBMV, non encore décrit, a été caractérisé. Nous avons démontré que cet ARN4 pouvait remplacer celui du BNYVV lors de la transmission et que seule l'expression de la protéine p32 suffit à la transmission du virus. La mutagenèse de la protéine p32 étiquetée a été réalisée et a permis de rechercher les domaines essentiels à ses fonctions.Beet soil-borne mosaic virus (BSBMV) and Beet necrotic yellow vein virus (BNYVV, responsible for Rhizomania disease) belong to the Benyvirus genus. BSBMV full-length cDNA clones were produced to investigate molecular interactions between plant and benyviruses exploiting BSBMV/BNYVV biological and molecular divergences. BSBMV Full-length infectious cDNA clones can substitute BNYVV RNA-1 or -2 to produce chimeric viral progenies. Virus transmission by Polymyxa betae is directly linked to the viral RNA4 and a 1,730 nts long BSBMV RNA4, not previously described, has been molecularly characterized. We demonstrated that BSBMV RNA4 could substitute BNYVV RNA4 for an efficient transmission. Replicon mediated expression of the BSBMV p32 protein complemented RNA4 defective strain demonstrating for the first time that the p32 protein but not full-length RNA4 is essential for Benyvirus transmission. Mutagenesis or Flag or GFP-tagged p32 has been to investigate the domains of BSBMV p32 protein essential for its properties
Sixth Symposium of the International Working Group on Plant Viruses with Fungal Vectors
No full textDal 5 al 7 Settembre presso l’Aula Prodi (P.zza S. Giovanni in Monte) si è svolto il sesto congresso internazionale del Working Group on Plant Viruses with Fungal Vectors (IWGPVFV). IWGPVFV è un gruppo internazionale di ricerca sui virus delle piante trasmessi dal terreno mediante vettori fungini e protozoi. Virus questi ultimi che sono responsabili di numerose malattie nelle colture cerealicole, industriali e orticole a livello mondiale.
Durante i tre giorni del convegno, a cui hanno partecipato circa 130 relatori provenienti dall’Europa, dall’Asia, dall’America, dall’Africa e dall’Australia, sono state discusse una settantina di ricerche riguardanti la caratterizzazione e tassonomia dei virus delle piante trasmessi da funghi e protozoi, la caratterizzazione biologica e molecolare dei vettori di tali virus, l’epidemiologia e il controllo delle malattie con particolare riguardo allo studio dei meccanismi di interazione ospite - patogeno e le fonti di germoplasma nell' ambito della resistenza alle virosi.
Il Convegno, patrocinato dall’Università di Bologna e sponsorizzato da UniboCultura e Promega Italia, è organizzato dalla Dott. ssa Concepcion Rubies Autonell, del DiSTA della Facoltà di Agraria dell’Università di Bologna e dal Dr Victor Vallega, C.R.A. Istituto Sperimentale per la Ricerca Cerealicola di Roma. All’organizzazione del convegno hanno collaborato la Dott.ssa Annamaria Pisi e il Dr. Claudio Ratti del DiSTA della Facoltà di Agraria dell’Università di Bologna.
Ulteriori informazioni possono riscontrarsi nei siti:
www.agrsci.unibo.it/iwgpvfv/
e
www.rothamsted.bbsrc.ac.uk/ppi/Iwgpvfv/index.htm
Mécanismes moléculaires à l'origine de la pathogenicité de phytovirus de betterave sucrière transmis par un vecteur tellurique
No full textLe virus des nervures jaunes et nécrotiques de la betterave (Beet necrotic yellow vein virus, BNYVV) est l agent infectieux responsable de la rhizomanie de la betterave sucrière, une maladie caractérisée par une prolifération anarchique du chevelu racinaire. Le Beet soil-borne mosaic virus (BSBMV) appartient également au genre Benyvirus mais n est retrouvé qu en Amérique du Nord. Ce virus, identifié pour la première fois au Texas, est morphologiquement et génétiquement semblable au BNYVV mais sérologiquement éloigné. Compte tenu des différences moléculaires existant, le BSBMV et BNYVV correspondent à deux espèces virales distinctes. Mon projet de thèse a consisté à étudier les interactions moléculaires entre le BNYVV et le BSBMV et rechercher les mécanismes impliqués dans la pathogénicité de ces deux virus. Des clones complets cDNA infectieux du BNYVV étaient disponibles, tout comme ceux de BSBMV. Compte tenu de l aspect versatile de l obtention de transcrits infectieux de ces différents clones, j ai entrepris de produire des clones cDNA de chacun des ARN viraux sous contrôle d un promoteur constitutive végétal pour initier l infection par agroinfiltration. Les plantes hôtes Chenopodium quinoa et Nicotiana benthamiana ont été inoculées par des transcrits et agroinfiltrées pour initier l infection virale et étudier l interaction entre les ARN génomiques 1 et 2 des deux virus et étudier les propriétés de constructions chimères. En parallèle à ce travail, j ai réalisé la caractérisation du suppresseur de RNA silencing du BSBMV en le comparant à celui du BNYVV.The genus Benyvirus includes the most important and widespread sugar beet viruses transmitted through the soil by the plasmodiophorid Polymyxa betae. In particular Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, causes an abnormal rootlet proliferation known as rhizomania. Beet soil-borne mosaic virus (BSBMV) is widely distributed in the United States and, up to date has not been reported in others countries. My PhD project aims to investigate molecular interactions between BNYVV and BSBMV and the mechanisms involved in the pathogenesis of these viruses.BNYVV full-length infectious cDNA clones were available as well as full-length cDNA clones of BSBMV RNA-1, -2, -3 and -4. Handling of these cDNA clones in order to produce in vitro infectious transcripts need sensitive and expensive steps, so Ideveloped agroclones of BNYVV and BSBMV RNAs, as well as viral replicons allowing the expression of different proteins.Chenopodium quinoa and Nicotiana benthamiana plants have been infected with in vitro transcripts and agroclones to investigate the interaction between BNYVV and BSBMV RNA-1 and -2 and the behavior of artificial viral chimeras. Simultaneously I characterized BSBMV p14 and demonstrated that it is a suppressor of posttranscriptional gene silencing sharing common features with BNYVV p14.STRASBOURG-Bib.electronique 063 (674829902) / SudocSudocFranceF
Indexed data for comparing the reaction of durum wheat cultivars to Soil-borne cereal mosaic virus assayed in different seasons.
No full textEvaluating the reaction to Cereal soilborne mosaic virus (CSBMV) of wheat cultivars in replicated, large-plot, field trials in which genotypes are compared in terms of symptom severity and ELISA values, as well as on the basis of agronomical performance has furnished reliable data, useful for both wheat growers and plant breeders.
Indexed data for fifteen field trials carried out in eight seasons and involving a total of 215 cultivars of durum and hexaploid wheat are presented. As might be expected, use of indexes did not appreciably modify the reaction thus far attributed to single cultivars. On the other hand, use of such indexed data has furnished more objective data for ranking cultivar reactions to CSBMV in Italy, and will enable more precise comparisons with data produced by research groups in other countries
Reaction of 81 cultivars of common wheat in northern Italy to wheat soilborne mosaic virus during five seasons.
No full textInduzione di resistenza in barbabietola da zucchero a Cercospora beticola Sacc. mediante applicazioni di acido salicilico e BTH (BION®).
No full textStudio dell'induzione di resistenza in barbabietola da zucchero a Cercospora beticola Sacc. mediante applicazioni di acido salicilico e BTH (BION®)
- …
