Agrobacterium-mediated infection of whole plants by yellow dwarf viruses
ABSTRACT Barley yellow dwarf virus-PAV (BYDV-PAV) and cereal yellow dwarf virus-RPV (CYDV-RPV) are only transmitted between host plants by aphid vectors and not by mechanical transmission. This presents a severe limitation for the use of a reverse genetics approach to analyze the effects of mutations in these viruses on plant infection and aphid transmission. Here we describe the use of agroinfection to infect plants with BYDV-PAV and CYDV-RPV. The cDNAs corresponding to the complete RNA genomes of BYDV-PAV and CYDV-RPV were cloned into a binary vector under the control of the cauliflower mosaic virus 35S promoter and the nopaline synthase transcription termination signal. The self-cleaving ribozyme from hepatitis virus D was included to produce a transcript in planta with a 3' terminus identical to the natural viral RNA. ELISA and RT-PCR analysis showed that the replicons of BYDV-PAV and CYDV-RPV introduced by Agrobacterium into Nicotiana benthamiana and N. clevelandii gave rise to a local infection in the infiltrated mesophyll cells. After several weeks systemic infection of phloem tissue was detected, although no systemic symptoms were observed. Three heterologous virus silencing suppressors increased the efficiency of agroinfection and accumulation of BYDV-PAV and CYDV-RPV in the two Nicotiana species. The progeny viruses purified from infiltrated tissues were successfully transmitted to oat plants by aphids, and typical yellow dwarf symptoms were observed. This study reports the first agroinfection of eudicot plants using BYDV-PAV and CYDV-RPV.
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ABSTRACT: RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in eukaryotes, including higher plants. To counteract this, several plant viruses express silencing suppressors that inhibit RNA silencing in host plants. Here, we show that both 2b protein from peanut stunt virus (PSV) and a hairpin construct (designated hp-RDR6) that silences endogenous RNA-dependent RNA polymerase 6 (RDR6) strongly suppress RNA silencing. The Agrobacterium infiltration system was used to demonstrate that both PSV 2b and hp-RDR6 suppressed local RNA silencing as strongly as helper component (HC-Pro) from potato virus Y (PVY) and P19 from tomato bush stunt virus (TBSV). The 2b protein from PSV eliminated the small-interfering RNAs (siRNAs) associated with RNA silencing and prevented systemic silencing, similar to 2b protein from cucumber mosaic virus (CMV). On the other hand, hp-RDR6 suppressed RNA silencing by inhibiting the generation of secondary siRNAs. The small coat protein (SCP) of squash mosaic virus (SqMV) also displayed weak suppression activity of RNA silencing. Agrobacterium-mediated gene transfer was used to investigate whether viral silencing suppressors or hp-RDR6 enhanced accumulations of green fluorescence protein (GFP) and β-glucuronidase (GUS) as markers of expression in leaf tissues of Nicotina benthamiana. Expression of both GFP and GUS was significantly enhanced in the presence of PSV 2b or CMV 2b, compared to no suppression or the weak SqMV SCP suppressor. Co-expression with hp-RDR6 also significantly increased the expression of GFP and GUS to levels similar to those induced by PVY HC-Pro and TBSV P19.Virus Genes 02/2012; 44(3):495-504. DOI:10.1007/s11262-012-0725-x
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ABSTRACT: Lettuce chlorosis virus (LCV) is a single stranded, positive strand RNA virus that is solely transmitted by specific whitefly vectors (Bemisia tabaci biotypes A and B) but not by mechanical leaf-rub inoculation. The roles of viral encoded proteins involved in the infection cycle of LCV have not yet been characterized due to the lack of reverse genetic tools. We present here a report of the successful development of an Agrobacterium-mediated inoculation system for the cloned cDNA constructs of LCV. The cDNAs of both LCV RNAs 1 and 2 were engineered into binary vectors in which the expression of LCV RNAs was regulated under a Cauliflower mosaic virus (CaMV) 35S promoter. In addition, by engineering the sequence elements of the Hepatitis delta virus ribozyme and the nopaline synthase 3' untranslated region immediately downstream of the last nucleotide of LCV RNAs 1 and 2 in the binary vector constructs, the in planta produced LCV transcripts were expected to bear authentic 3' termini. Both constructs were transformed into Agrobacterium tumefaciens cells and infiltrated in Nicotiana benthamiana plants. Three to four weeks post-agroinoculation, the N. benthamiana plants developed typical interveinal chlorosis and LCV infection was detected in the systemic leaves by reverse transcription-PCR. Virions purified from the LCV-infected N. benthamiana plants were flexuous rod-shaped and were transmissible by both B. tabaci biotypes A and B following membrane feeding. These results support the conclusion that Agrobacterium-mediated inoculation of LCV binary vectors in N. benthamiana plants results in LCV infection and the production of biologically active, whitefly transmissible virions. This system represents an important tool for use with reverse genetics designed for the study of LCV gene functions.Virus Research 08/2012; 169(1):310-5. DOI:10.1016/j.virusres.2012.08.010
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ABSTRACT: Cotton blue disease is the most important viral disease of cotton in the southern part of South America. Its etiological agent, cotton leafroll dwarf virus (CLRDV), is specifically transmitted to host plants by the aphid vector (Aphis gossypii) and any attempt to perform mechanical inoculations of this virus into its host has failed. This limitation has held back the study of this virus and the disease it causes. In this study, a full-length cDNA of CLRDV was constructed and expressed in vivo under the control of cauliflower mosaic virus 35S promoter. An agrobacterium-mediated inoculation system for the cloned cDNA construct of CLRDV was developed. Northern and immunoblot analyses showed that after several weeks the replicon of CLRDV delivered by Agrobacterium tumefaciens in Gossypium hirsutum plants gave rise to a systemic infection and typical blue disease symptoms correlated to the presence of viral RNA and P3 capsid protein. We also demonstrated that the virus that accumulated in the agroinfected plants was transmissible by the vector A. gossypii. This result confirms the production of biologically active transmissible virions. In addition, the clone was infectious in Nicotiana benthamiana plants which developed interveinal chlorosis three weeks postinoculation and CLRDV was detected both in the inoculated and systemic leaves. Attempts to agroinfect Arabidopsis thaliana plants were irregularly successful. Although no symptoms were observed, the P3 capsid protein as well as the genomic and subgenomic RNA were irregularly detected in systemic leaves of some agroinfiltrated plants. The inefficient infection rate infers that A. thaliana is a poor host for CLRDV. This is the first report on the construction of a biologically-active infectious full-length clone of a cotton RNA virus showing successful agroinfection of host and non-host plants. The system herein developed will be useful to study CLRDV viral functions and plant-virus interactions using a reverse genetic approach.Virus Research 04/2013; DOI:10.1016/j.virusres.2013.04.007