A practical approach to the understanding and teaching of RNA silencing in plants

Bazzini, Ariel; Mongelli, Vanesa; Hopp, H. Esteban; Vas, Mariana del; Asurmendi, Sebastián

doi:10.2225/vol10-issue2-fulltext-11

Cited by 8 publications

(7 citation statements)

References 36 publications

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“…Post-transcriptional gene silencing (PTGS) often occurs in plants to reduce the amount of protein expression. PTGS is a natural defense mechanism relying on RNA interference for plants against viruses and pathogenes 18 . Production of excessive RNA transcripts over a threshold level, such as overexpressing a transgene under CaMV 35S promoter, causes transgene silencing through activation of PTGS 19 – 21 .…”

Section: Discussionmentioning

confidence: 99%

Improvement of the transient expression system for production of recombinant proteins in plants

Yamamoto

Hoshikawa

Ezura

et al. 2018

Sci Rep

143

144

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An efficient and high yielding expression system is required to produce recombinant proteins. Furthermore, the transient expression system can be used to identify the localization of proteins in plant cells. In this study, we demonstrated that combination of a geminiviral replication and a double terminator dramatically enhanced the transient protein expression level in plants. The GFP protein was expressed transiently in lettuce, Nicotiana benthamiana, tomatoes, eggplants, hot peppers, melons, and orchids with agroinfiltration. Compared to a single terminator, a double terminator enhanced the expression level. A heat shock protein terminator combined with an extensin terminator resulted in the highest protein expression. Transiently expressed GFP was confirmed by immunoblot analysis with anti-GFP antibodies. Quantitative analysis revealed that the geminiviral vector with a double terminator resulted in the expression of at least 3.7 mg/g fresh weight of GFP in Nicotiana benthamiana, approximately 2-fold that of the geminiviral vector with a single terminator. These results indicated that combination of the geminiviral replication and a double terminator is a useful tool for transient expression of the gene of interest in plant cells.

show abstract

Section: Discussionmentioning

confidence: 99%

Improvement of the transient expression system for production of recombinant proteins in plants

Yamamoto

Hoshikawa

Ezura

et al. 2018

Sci Rep

143

144

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show abstract

“…Transient expression was carried out by agroinfiltration in N. benthamiana as described previously ( Bazzini et al , 2007 ; Love et al , 2012 ). Vectors for expressing WT or mutant P6 were constructed using the Gateway cloning system (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

Identification of the domains of cauliflower mosaic virus protein P6 responsible for suppression of RNA silencing and salicylic acid signalling

Laird

McInally

Carr

et al. 2013

Journal of General Virology

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Cauliflower mosaic virus (CaMV) encodes a 520 aa polypeptide, P6, which participates in several essential activities in the virus life cycle including suppressing RNA silencing and salicylic acid-responsive defence signalling. We infected Arabidopsis with CaMV mutants containing short in-frame deletions within the P6 ORF. A deletion in the distal end of domain D-I (the N-terminal 112 aa) of P6 did not affect virus replication but compromised symptom development and curtailed the ability to restore GFP fluorescence in a GFP-silenced transgenic Arabidopsis line. A deletion in the minimum transactivator domain was defective in virus replication but retained the capacity to suppress RNA silencing locally. Symptom expression in CaMV-infected plants is apparently linked to the ability to suppress RNA silencing. When transiently co-expressed with tomato bushy stunt virus P19, an elicitor of programmed cell death in Nicotiana tabacum, WT P6 suppressed the hypersensitive response, but three mutants, two with deletions within the distal end of domain D-I and one involving the N-terminal nuclear export signal (NES), were unable to do so. Deleting the N-terminal 20 aa also abolished the suppression of pathogen-associated molecular pattern-dependent PR1a expression following agroinfiltration. However, the two other deletions in domain D-I retained this activity, evidence that the mechanisms underlying these functions are not identical. The D-I domain of P6 when expressed alone failed to suppress either cell death or PR1a expression and is therefore necessary but not sufficient for all three defence suppression activities. Consequently, concerns about the biosafety of genetically modified crops carrying truncated ORFVI sequences appear unfounded.

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“…Local and systemic PTGS experiments were performed as described in Bazzini et al (43). TMV MP and CP…”

Section: Methods Viral Infectionsmentioning

confidence: 99%

Infection and coaccumulation of tobacco mosaic virus proteins alter microRNA levels, correlating with symptom and plant development

Bazzini

Hopp

Beachy

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

212

159

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A practical approach to the understanding and teaching of RNA silencing in plants

Cited by 8 publications

References 36 publications

Improvement of the transient expression system for production of recombinant proteins in plants

Improvement of the transient expression system for production of recombinant proteins in plants

Identification of the domains of cauliflower mosaic virus protein P6 responsible for suppression of RNA silencing and salicylic acid signalling

Infection and coaccumulation of tobacco mosaic virus proteins alter microRNA levels, correlating with symptom and plant development

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