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Transgenic resistance against Citrus tristeza virus (CTV) and analysis of the viral p23 protein as pathogenicity determinant in citrusSoler Calvo, Nuria 02 September 2013 (has links)
El virus de la tristeza de los cítricos (Citrus tristeza virus; CTV) es el agente causal de unas de
las enfermedades virales de los árboles cítricos más devastadoras en el mundo. CTV está restringido al
floema en su huésped cítrico natural, y ha desarrollado tres proteínas supresoras de silenciamiento que
actúan a nivel intra-(p23 y p20) e intercelular (p20 y p25) para superar la fuerte defensa antiviral del
huésped. La interferencia de RNA, una aproximación basada en el uso de dsRNA para desencadenar el
silenciamiento de RNA, ha sido utilizada ampliamente para generar plantas transgénicas resistentes a
virus. Considerando el importante papel de p23, p20 y p25 en la patogénesis de CTV, hemos
transformado plantas de lima Mexicana con un vector intrón-horquilla que porta la secuencia completa en
versión no traducible de los genes p25, p20, p23 y el extremo 3¿-UTR de la cepa T36 de CTV, para
intentar silenciar su expresión en células infectadas.
Se ha observado resistencia completa a la infección viral en tres líneas transgénicas,
manteniéndose todas sus propagaciones asintomáticas y libres de virus tras ser inoculadas mediante
injerto con CTV-T36, tanto en el portainjertos no transgénico como directamente sobre la variedad
transgénica. La acumulación de siRNA derivados del transgén fue necesaria pero no suficiente para lograr
resistencia frente a CTV en las plantas. Al inocular propagaciones de las líneas transgénicas inmunes con
una cepa de CTV divergente, la resistencia fue parcialmente superada, destacando la importancia de la
identidad de secuencia en el mecanismo subyacente a la interferencia de RNA. Este trabajo es el primero
en que se consigue resistencia completa a CTV en un huésped cítrico muy sensible, actuando
simultáneamente sobre los tres supresores virales de silenciamiento mediante interferencia de RNA. La
proteína p23 codificada por el virus es además un importante factor de patogenicidad. La expresión
ectópica de p23 en plantas de cítricos induce aberraciones fenológicas semejantes a síntomas de CTV.
Para estudiar en más detalle el papel de p23 en la patogénesis de CTV, se ha sobre-expresado en lima
Mexicana el gen p23 de CTV T36 y tres versiones truncadas del mismo bajo el control del promotor 35S
del virus del mosaico de la coliflor (Cauliflower mosaic virus). Solo la versión truncada, que expresa los
aminoácidos del 1 al 157 (p23-¿157) indujo síntomas similares a los producidos por CTV, aunque más
suaves que los inducidos por la expresión de la proteína p23 entera (209 aminoácidos), permitiendo
delimitar la región responsable de la patogénesis de p23 en cítricos a un fragmento de 157 aminoácidos
que incluye el dedo de zinc y los motivos básicos flanqueantes de la proteína. La actividad de p23 como
supresor de silenciamiento de RNA en N. benthamiana se perdía en todos los mutantes de p23 probados,
lo cual indica que la supresión de silenciamiento implica a la mayoría de las regiones de la proteína. Para
profundizar más en el papel de p23 en la patogénesis, en un siguiente paso hemos restringido la expresión
de transgenes derivados de p23 a células asociadas al floema de lima Mexicana mediante el uso del
promotor especifico de floema del virus del moteado amarillo de la comelina (Commelina yellow mottle
virus, CoYMV). Se transformó lima Mexicana con construcciones que portaban el gen p23 completo, ya
sea de la cepa agresiva de CTV T36 o de la suave T317, o con un fragmento que comprende el dedo de
zinc y los motivos básicos flanqueantes de la primera, todas ellas bajo el control bien del promotor de
CoYMV o bien del promotor constitutivo 35S. La expresión de estas construcciones en el floema dio
lugar a aberraciones semejantes a los síntomas específicos de CTV, pero no a los síntomas inespecíficos
observados cuando se expresaba p23 de forma constitutiva. Por otra parte, la apariencia e intensidad de
las aberraciones fenotípicas más notorias similares a síntomas inducidos por CTV generadas por la
expresión específica en floema del gen p23 se relacionó positivamente con la agresividad de la cepa
origen utilizada. Además, la expresión en tejidos floemáticos del fragmento de p23 que comprende el
dominio de dedo de zinc y los motivos básicos flanqueantes fue suficiente para inducir síntomas
semejantes a los producidos por la infección con CTV, confirmando así que la región N-terminal
delimitada por los aminoácidos 1 y 157 podría determinar, al menos en parte, la patogénesis de CTV en
lima Mexicana. / Citrus tristeza virus (CTV) is the causal agent of one of the most devastating viral diseases of citrus trees in the world. CTV is phloem-restricted in natural citrus hosts, and has evolved three silencing suppressor proteins acting at intra- (p23 and p20) and inter-cellular level (p20 and p25) to overcome strong host antiviral defense in citrus. RNA interference (RNAi), an approach based on using dsRNA to trigger RNA silencing, has been widely used for generating transgenic plants resistant against viruses. Considering the important role of p23, p20 and p25 in CTV pathogenesis, we have transformed Mexican lime plants with an intron-hairpin vector carrying full untranslatable versions of genes p25, p20, p23 and the 3¿-UTR from the CTV strain T36, to attempt silencing their expression in CTV-infected cells. Complete resistance to viral infection was observed in three transgenic lines, with all their propagations remaining symptomless and virus-free after graft-inoculation with CTV-T36, either in the non-transgenic rootstock or directly in the transgenic scion. Accumulation of transgene-derived siRNAs was necessary but not sufficient for CTV resistance. Challenging immune transformants with a divergent CTV strain resulted in partial breakage of the resistance, stressing the importance of sequence identity in the underlying RNAi mechanism. This is the first evidence that it is possible to achieve full resistance to CTV in a highly sensitive citrus host by targeting simultaneously its three viral silencing suppressors through RNAi. The p23 protein encoded by the virus is additionally an important pathogenicity factor. Ectopic expression of p23 in
transgenic citrus plants induces developmental aberrations resembling CTV symptoms. To explore in more detail the role of p23 in CTV pathogenesis, the p23 gene from CTV T36 and three truncated versions thereof under the control of the Cauliflower mosaic virus 35S promoter were used to transform Mexican lime. Only the truncated version expressing amino acids 1 to 157 (p23¿158-209) elicited CTV-like symptoms, similar to, albeit milder than, those incited by expressing the whole p23 protein (209 amino acids), thus delimiting the region responsible for p23 pathogenesis in citrus to a 157 amino acid fragment including the Zn finger and flanking basic motifs of the protein. RNA silencing suppressor activity of p23 in N. benthamiana was abolished by all mutants tested, indicating that silencing suppression involves most p23 regions. To better define the role of p23 in CTV pathogenesis, we next restricted the expression of p23-derived transgenes to phloem-associated cells in Mexican lime plants by means of using the phloem-specific promoter from Commelina yellow mottle virus (CoYMV). Constructions carrying the complete gene p23 from either the severe T36 or the mild T317 CTV strains, or a fragment comprising the zinc-finger and flanking basic motifs from the former, either under the control of the CoYMV promoter or the constitutive 35S promoter were used for genetic transformation of Mexican lime. Expression of these constructs in the phloem incited aberrations resembling CTV-specific symptoms, but not the unspecific symptoms observed when p23 was constitutively expressed. Moreover, appearance and intensity of the most notorious CTV-like phenotypic aberrations induced by the phloem-specific expression of
the p23 gene were positively related with the aggressiveness of the source CTV strain used. Additionally, expression in phloem-tissues of the p23 fragment comprising the zinc-finger domain and flanking basic motifs was sufficient to induce CTV-like symptoms, corroborating that the N-terminal region (delimited by amino acids 1 and 157) determines, at least in part, CTV pathogenesis in Mexican lime. / Soler Calvo, N. (2013). Transgenic resistance against Citrus tristeza virus (CTV) and analysis of the viral p23 protein as pathogenicity determinant in citrus [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/31631 / TESIS
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Towards the development of transgenic banana bunchy top virus (BBTV)-resistant banana plants : interference with replicationTsao, Theresa Tsun-Hui January 2008 (has links)
Banana bunchy top virus (BBTV) causes one of the most devastating diseases of banana. Transgenic virus resistance is now considered one of the most promising strategies to control BBTV. Pathogen-derived resistance (PDR) strategies have been applied successfully to generate plants that are resistant to numerous different viruses, primarily against those viruses with RNA genomes. BBTV is a circular, single-stranded (css) DNA virus of the family Nanoviridae, which is closely related to the family Geminiviridae. Although there are some successful examples of PDR against geminiviruses, PDR against the nanoviruses has not been reported. Therefore, the aim of this thesis was to investigate the potential of BBTV genes to interfere with virus replication when used as transgenes for engineering banana plants resistance to BBTV. The replication initiation protein (Rep) of nanoviruses is the only viral protein essential for viral replication and represents an ideal target for PDR. Therefore, this thesis focused on the effect of wild-type or mutated Rep genes from BBTV satellite DNAs or the BBTV integral genome on the replication of BBTV in banana embryogenic cell suspensions.
A new Rep-encoding satellite DNA, designated BBTV DNA-S4, was isolated from a Vietnamese BBTV isolate and characterised. When the effect of DNA-S4 on the replication of BBTV was examined, it was found that DNA-S4 enhanced the replication of BBTV. When the replicative capabilities of DNA-S4 and the previously characterised Rep-encoding BBTV satellite, DNA-S1, were compared, it was found that the amount of DNA-S4 accumulated to higher levels than DNA-S1. The interaction between BBTV and DNA-S1 was also examined. It was found that over-expression of the Rep encoded by DNA-S1 using ubi1 maize polyubiquitin promoter enhanced replication of BBTV. However, when the Rep-encoded by DNA-S1 was expressed by the native S1 promoter (in plasmid pBT1.1-S1), it suppressed the replication of BBTV. Based on this result, the use of DNA-S1 as a possible transgene to generate PDR against BBTV was investigated.
The roles of the Rep-encoding and U5 genes of BBTV DNA-R, and the effects of over-expression of these two genes on BBTV replication were also investigated. Three mutants of BBTV DNA-R were constructed; plasmid pUbi-RepOnly-nos contained the ubi1 promoter driving Rep expression from DNA-R, plasmid pUbi-IntOnly-nos contained the ubi1 promoter driving expression of the DNA-R internal gene product (U5), while plasmid pUbi-R.ORF-nos contained the ubi1 promoter driving the expression of both Rep and the internal U5 gene product. The replication of BBTV was found to be significantly suppressed by pUbi-RepOnly-nos, weakly suppressed by pUbi-IntOnly-nos, but strongly enhanced by pUbi-R.ORF-nos.
The effect of mutations in three conserved residues within the BBTV Rep on BBTV replication was also assessed. These mutations were all made in the regions in the ATPase motifs and resulted in changes from hydrophilic to hydrophobic residues (i.e. K187→M, D224→I and N268→L). None of these Rep mutants was able to initiate BBTV replication. However, over-expression of Reps containing the K187→M or N268→L mutations significantly suppressed the replication of BBTV.
In summary, the Rep constructs that significantly suppressed replication of DNA-R and -C in banana embryogenic cell suspensions have the potential to confer resistance against BBTV by interfering with virus replication. It may be concluded that BBTV satellite DNAs are not ideal for conferring PDR because they did not suppress BBTV replication consistently. Wild-type Rep transcripts and mutated (i.e. K187→M and N248→L) Rep proteins of BBTV DNA-R, however, when over-expressed by a strong promoter, are all promising candidates for generating BBTV-resistant banana plants.
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