Effectiveness of gene silencing induced by viral vectors based on Citrus leaf blotch virus is different in Nicotiana benthamiana and citrus plants

Agüero, Jesús; Vives, María del Carmen; Velázquez, Karelia; Pina, J. A.; Navarro, Luís; Moreno, Pedro; Guerri, José

doi:10.1016/j.virol.2014.04.017

Cited by 46 publications

(36 citation statements)

References 51 publications

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“…Similar photo-bleaching phenotypes caused by PDS gene silencing have been reported in N. benthamiana and Brachypodium distachyon plants inoculated with Bamboo mosaic virus and its satellite RNA vectors (Liou et al 2014) and in citrus plants infected with Citrus leaf blotch virus vector (Agüero et al 2014). One possible explanation for the vein-restricted photo-bleaching phenotype in white lupin plants is that PSV was hard to spread to whole leaflets and existed in or around veins.…”

Section: Discussionsupporting

confidence: 68%

Peanut stunt virus-induced gene silencing in white lupin (Lupinus albus)

Yamagishi

Masuta

Suzuki

et al. 2015

Plant Biotechnology

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White lupin (Lupinus albus L.) plants develop cluster roots and have strong resistance to phosphorus starvation. Although many expressed sequences have been identified to explain the mechanisms used by white lupin to acquire phosphorus, the lack of a stable transformation technique has made it challenging to evaluate the functions of these expressed sequences. Virus-induced gene silencing (VIGS) is an attractive method for assaying gene function in species that are difficult to stably transform. Here, we show that the Peanut stunt virus (PSV) vector effectively induces silencing of endogenous genes in white lupin. It is unknown whether PSV is useful for VIGS; therefore, we first inoculated Nicotiana benthamiana plants with PSV harbouring fragments of the N. benthamiana phytoene desaturase gene (NbPDS). Two out of four distinct sequence fragments of NbPDS induced photo-bleaching in N. benthamiana, indicating that PSV can be used to knockdown endogenous gene sequences in a sequence-dependent manner. White lupin plants inoculated with PSV harbouring fragments of the L. albus PDS gene (LaPDS) developed photo-bleaching that was associated with a significant reduction in LaPDS mRNA accumulation. PSV spread systemically in leaves, roots, and cluster roots, and small interfering RNA of LaPDS was detected in these organs. This is the first study to demonstrate the use of VIGS by PSV, suggesting that this vector can be applied to supress endogenous gene expression in shoots and roots of white lupin and to clarify the mechanisms of phosphorus starvation resistance.

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Section: Discussionsupporting

confidence: 68%

Peanut stunt virus-induced gene silencing in white lupin (Lupinus albus)

Yamagishi

Masuta

Suzuki

et al. 2015

Plant Biotechnology

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“…a Rough lemon ( C. jambhiri Lush)), simultaneously graft inoculated with two bark patches from the infected C. excelsa plants (Figure b), and (iii) the root of small hybrid plantlets was cut‐off at the crown and the aerial portion was top‐worked onto a rootstock, simultaneously graft inoculated with two bark patches from the infected C. excelsa plants (Figure c). All plants inoculated by either procedure with the clbvINpr‐AtFT or the clbvINpr‐CiFT constructs became systemically infected, as detected by conventional reverse transcription PCR (RT‐PCR) (Galipienso et al ., ), using primers flanking the insertion site of the FT gene in the vector (Agüero et al ., ). Some genotypes flowered in successive flushes along the year, whereas others only flowered in the spring flush, suggesting that factors other than the FT protein may be necessary to induce flowering.…”

Section: Resultsmentioning

confidence: 97%

“…These results suggest that the FT protein delivered by the viral vector could terminate the vegetative growth by inducing the transition from vegetative to reproductive meristematic tissues in the shoot apex. The ability of CLBV to invade meristematic regions in citrus (Agüero et al ., ) may favour delivery of the FT protein in this region. Previous studies on expression of flowering‐related genes in the shoot apex of mature citrus trees found increased CiFT mRNA levels upon transition from the vegetative to the reproductive growth state (Muñoz‐Fambuena et al ., ; Nishikawa et al ., ).…”

Section: Discussionmentioning

confidence: 99%

“…However, clbvINpr‐AtFT was able to induce flowering during the long period studied (almost 5 years). PCR analysis of individual AtFT clones showed that only 8.5% of the clones analysed yielded a DNA band of a size smaller than that expected for the complete gene, confirming that CLBV‐based vectors are highly stable (Agüero et al ., , ). Although some of the clones sequenced showed nucleotide substitutions leading to amino acid changes, none of these changes affected the amino acids essential for the FT function (Hanzawa et al ., ; Ho and Weigel, ).…”

Section: Discussionmentioning

confidence: 99%

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Precocious flowering of juvenile citrus induced by a viral vector based on Citrus leaf blotch virus: a new tool for genetics and breeding

Velázquez

Agüero

Vives

et al. 2016

Plant Biotechnology Journal

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SummaryThe long juvenile period of citrus trees (often more than 6 years) has hindered genetic improvement by traditional breeding methods and genetic studies. In this work, we have developed a biotechnology tool to promote transition from the vegetative to the reproductive phase in juvenile citrus plants by expression of the Arabidopsis thaliana or citrus FLOWERING LOCUS T (FT) genes using a Citrus leaf blotch virus‐based vector (clbvINpr‐AtFT and clbvINpr‐CiFT, respectively). Citrus plants of different genotypes graft inoculated with either of these vectors started flowering within 4–6 months, with no alteration of the plant architecture, leaf, flower or fruit morphology in comparison with noninoculated adult plants. The vector did not integrate in or recombine with the plant genome nor was it pollen or vector transmissible, albeit seed transmission at low rate was detected. The clbvINpr‐AtFT is very stable, and flowering was observed over a period of at least 5 years. Precocious flowering of juvenile citrus plants after vector infection provides a helpful and safe tool to dramatically speed up genetic studies and breeding programmes.

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“…Previously, we have used Citrus leaf blotch virus (CLBV) for either silencing or protein expression in citrus (Agüero et al ., ; Velázquez et al ., ). The CLBV gRNA has 8747 nts organized in three ORFs and 5′‐ and 3′‐UTRs of 73 and 541 nts, respectively.…”

Section: Introductionmentioning

confidence: 99%

The resistance of sour orange to Citrus tristeza virus is mediated by both the salicylic acid and RNA silencing defence pathways

Gómez-Muñoz

Velázquez

Vives

et al. 2016

Molecular Plant Pathology

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Citrus tristeza virus (CTV) induces in the field the decline and death of citrus varieties grafted on sour orange (SO) rootstock, which has forced the use of alternative decline-tolerant rootstocks in affected countries, despite the highly desirable agronomic features of the SO rootstock. Declining citrus plants display phloem necrosis below the bud union. In addition, SO is minimally susceptible to CTV compared with other citrus varieties, suggesting partial resistance of SO to CTV. Here, by silencing different citrus genes with a Citrus leaf blotch virus-based vector, we have examined the implication of the RNA silencing and salicylic acid (SA) defence pathways in the resistance of SO to CTV. Silencing of the genes RDR1, NPR1 and DCL2/DCL4, associated with these defence pathways, enhanced virus spread and accumulation in SO plants in comparison with non-silenced controls, whereas silencing of the genes NPR3/NPR4, associated with the hypersensitive response, produced a slight decrease in CTV accumulation and reduced stunting of SO grafted on CTV-infected rough lemon plants. We also found that the CTV RNA silencing suppressors p20 and p23 also suppress the SA signalling defence, with the suppressor activity being higher in the most virulent isolates.

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Effectiveness of gene silencing induced by viral vectors based on Citrus leaf blotch virus is different in Nicotiana benthamiana and citrus plants

Cited by 46 publications

References 51 publications

<i>Peanut stunt virus</i>-induced gene silencing in white lupin (<i>Lupinus albus</i>)

<i>Peanut stunt virus</i>-induced gene silencing in white lupin (<i>Lupinus albus</i>)

Precocious flowering of juvenile citrus induced by a viral vector based on Citrus leaf blotch virus: a new tool for genetics and breeding

The resistance of sour orange to Citrus tristeza virus is mediated by both the salicylic acid and RNA silencing defence pathways

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