Transgenic resistance against Citrus tristeza virus (CTV) and analysis of the viral p23 protein as pathogenicity determinant in citrus.

Abstract

AgradecimientosMe gustaría agradecer en primer lugar a Leandro Peña y al Instituto Valenciano de Investigaciones Agrarias por darme la oportunidad de realizar la Tesis doctoral en el Laboratorio de Transformación genética.Quiero agradecer a todo el equipo de Cultivo In vitro, a Pepe, Juana Mari, Cloti, Carmen, Pablo, Toni, Vio, etc., por todo el apoyo, sus consejos y sus enseñanzas.A todo el laboratorio de Leandras: los postdoc Magda, Mamen y Juan, por toda la ayuda y enseñanzas que me disteis; a Joseph, por cuidar tan bien de nuestras plantitas; y como no, agradecer a mis chicas: Ana, Ali, Elsa, Berta, Montse y Ana cubana; además de todos los compañeros de pasillo ivias/ex-ivias: Jesús, Vero, Andrés, Frank, Marta, Jose, Diana, Ezequiel; los que ya se fueron: Inma, Lucía, Nubi, Ague, Marta, Rosa, Esther, Giovanni, Jorge, María, Mari Cruz… por todo el apoyo, el cariño, las risas, los consejos, los cafés, las alquerías, las escapadas, los lunes al sol, … mil gracias, porque ahora son grandes amigos con los que he compartido tantos y tantos buenos momentos.A los que he olvidado nombrar, gracias también.Y por último, gracias a mi familia, porque sin ellos nada de esto sería posible. ABSTRACTCitrus 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 intercellular 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 CTVinfected 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 RESUMEN 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 es...

Grapefruit: History, Use, and Breeding

“…CTV has several intra and intercellular silencing suppressors that affect host resistance and facilitate viral colonization. Calvo (2013) targeted these suppressors with small interfering RNA and obtained transgenic plants resistant to the virus; however, the resistance was compromised when CTV strains with divergent suppressors were used to challenge the transgenic plants. Several groups are now conducting research to produce transgenic citrus resistant to HLB and citrus canker, two of the most economically significant citrus diseases.…”