Virus-induced silencing as a method for studying gene functions in higher plants

Zhirnov, I. V.; Trifonova, E. A.; Кочетов, А. В.; Shumny, V. K.

doi:10.1134/s1022795415050099

Cited by 4 publications

(5 citation statements)

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“…Examples include infiltration of lower leaves with a needleless syringe [ 26 ], pouring inoculum from the apical region and soaking wounded stems and leaves randomly pierced with a sterilized needle [ 16 ], infiltration of the abaxial side of both cotyledons [ 9 , 15 ], vacuum and co-cultivation agroinfiltration of germinated seeds [ 17 ], carpopodia of young fruit attached to the plant after pollination [ 5 ], soil adjacent to the plant roots (this method is called “agrodrenching”) [ 10 ] and infiltration at three different stages of seedling development [ 14 ]. It typically requires ~ 15–60 days to obtain transformants with these methods [ 1 , 17 ]. With the INABS method, the time period from inoculation to obtaining the target plants was reduced greatly (~ 10–12 days).…”

Section: Discussionmentioning

confidence: 99%

“…Virus-induced gene silencing (VIGS) is one of the most convenient and powerful methods of reverse genetics [ 1 ], and it is increasingly widely used to study plant gene functions [ 2 , 3 ]. Several viruses have been developed for use in VIGS [ 2 , 4 ].…”

Section: Introductionmentioning

confidence: 99%

“…[ 10 – 12 ], Arabidopsis thaliana [ 13 ], Papaver somniferum [ 14 ], Gossypium hirsutum [ 15 ], Amaranthus tricolor [ 16 ], Triticum aestivum , and Zea mays L. [ 17 ]. The molecular mechanisms of VIGS have been well studied, and the development and improvement of VIGS is presently focused on the creation of new viral constructs for different plant species, the search for new reporter genes to control VIGS efficiency, and the development of new, efficient infection methods [ 1 ].…”

Section: Introductionmentioning

confidence: 99%

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A rapid, simple, and highly efficient method for VIGS and in vitro-inoculation of plant virus by INABS applied to crops that develop axillary buds and can survive from cuttings

Liu

et al. 2021

BMC Plant Biol

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Background Virus-induced gene silencing (VIGS) is one of the most convenient and powerful methods of reverse genetics. In vitro-inoculation of plant virus is an important method for studying the interactions between viruses and plants. Agrobacterium-based infiltration has been widely adopted as a tool for VIGS and in vitro-inoculation of plant virus. Most agrobacterium-based infiltration methods applied to VIGS and virus inoculation have the characteristics of low transformation efficiencies, long plant growth time, large amounts of plant tissue, large test spaces, and complex preparation procedures. Therefore, a rapid, simple, economical, and highly efficient VIGS and virus inoculation method is in need. Previous studies have shown that the selection of suitable plant tissues and inoculation sites is the key to successful infection. Results In this study, Tobacco rattle virus (TRV) mediated VIGS and Tomato yellow leaf curl virus (TYLCV) for virus inoculation were developed in tomato plants based on the agrobacterium tumefaciens-based infiltration by injection of the no-apical-bud stem section (INABS). The no-apical-bud stem section had a “Y- type” asymmetric structure and contained an axillary bud that was about 1–3 cm in length. This protocol provides high transformation (56.7%) and inoculation efficiency (68.3%), which generates VIGS transformants or diseased plants in a very short period (8 dpi). Moreover, it greatly reduces the required experimental space. This method will facilitate functional genomic studies and large-scale disease resistance screening. Conclusions Overall, a rapid, simple, and highly efficient method for VIGS and virus inoculation by INABS was developed in tomato. It was reasonable to believe that it can be used as a reference for the other virus inoculation methods and for the application of VIGS to other crops (such as sweet potato, potato, cassava and tobacco) that develop axillary buds and can survive from cuttings.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A rapid, simple, and highly efficient method for VIGS and in vitro-inoculation of plant virus by INABS applied to crops that develop axillary buds and can survive from cuttings

Liu

et al. 2021

BMC Plant Biol

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“…The only identical stretch longer than 19 bp in the entire transcriptome was a 22-nucleotide fragment from the xylem Cys proteinase1-like transcript (XCP1). This single short stretch is less likely to cause significant silencing in off-target effects; furthermore, the xylem XCP1 is not involved in the herbivore defense response (Thomas et al, 2001;Gulati et al, 2013;Zhirnov et al, 2015). Still, to completely rule out any off-target effects, we evaluated the transcript levels of XCP1: no differences in levels were recorded between the wild-type and irAGO8 genotypes (Supplemental Fig.…”

Section: Generation and Characterization Of Stable Transgenic Lines Smentioning

confidence: 99%

Argonaute 8 (AGO8) Mediates the Elicitation of Direct Defenses against Herbivory

et al. 2017

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In , specific RNA-directed RNA polymerase (RdR1) and the Dicer-like (DCL3 and DCL4) proteins are recruited during herbivore attack to mediate the regulation of defense responses. However, the identity and role(s) of Argonautes (AGOs) involved in herbivory remain unknown. Of the 11 AGOs in the genome, we silenced the expression of 10. Plants silenced in expression grew normally but were highly susceptible to herbivore attack. Larvae of grew faster when consuming inverted-repeat stable transformants (ir) plants but did not differ from the wild type when consuming plants silenced in (, , and), , ( and ),, or expression. ir plants were significantly compromised in herbivore-induced levels of defense metabolites such as nicotine, phenolamides, and diterpenoid glycosides. Time-course analyses revealed extensively altered microRNA profiles and the reduced accumulation of transcripts and of the associated genes of the phenolamide and phenylpropanoid pathways as well as the nicotine biosynthetic pathway. A possible AGO8-modulated microRNA-messenger RNA target network was inferred. Furthermore, comparative analysis of domains revealed the diversity of AGO conformations, particularly in the small RNA-binding pocket, which may influence substrate recognition/binding and functional specificity. We infer that AGO8 plays a central role in the induction of direct defenses by modulating several regulatory nodes in the defense signaling network during herbivore response. Thus, our study identifies the effector AGO of the herbivore-induced small RNA machinery, which in now comprises RdR1, DCL3/4, and AGO8.

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“…To understand and control the undesirable self-incompatibility in P. fendleri we adapted virus-induced gene silencing (VIGS) knockdown methods to P. fendleri to evaluate self-incompatibility genes. VIGS is a simple, fast, and powerful reverse genetic tool widely used to study plant gene functions (Burch-Smith et al, 2004; Robertson, 2004; Zhirnov et al, 2015). We demonstrate that knockdown of SRK alone is sufficient to induce self-compatibility and produce seed pods without cross pollination.…”

Section: Introductionmentioning

confidence: 99%

Self-incompatibility based functional genomics for rapid phenotypic characterization of seed metabolism genes

Azeez,

Bates

2024

Preprint

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Reverse-genetic characterization of plant gene function through technologies such as CRISPR/Cas, RNAi, or gene overexpression requires the ability to efficiently transform the plant species of interest. However, efficient transformation systems are not available for most plant species. Physaria fendleri is an oilseed plant valued for its unusual hydroxylated fatty acids (HFA, e.g. lesquerolic acid) that accumulates up to 60% of seed oil and is a non-toxic alternative to castor (Ricinus communis) seeds as a source for HFA for the chemical industry. Domestication and improvement of P. fendleri seed oil requires characterization of genes involved in developing seed metabolism. Tissue culture-based transformation of P. fendleri is laborious, low-efficiency, and time-consuming (T1 ~18 months). Additionally, P. fendleri is self-incompatible requiring laborious hand pollination for propagation and seed collection from transgenic lines. We developed a rapid virus-induced gene silencing (VIGS) method to characterize genes within developing seeds. Identification of the self-incompatibility mechanisms in P. fendleri allowed the use of self-compatibility as a novel visual selectable marker by co-targeting the gene of interest (GOI) with the self-incompatibility gene S-locus receptor kinase (SRK). Seeds develop without cross-pollination from silenced SRK and each of those seeds contain the GOI silenced, allowing rapid phenotypic characterization of the seeds in the first generation. Through this methodology we confirmed the in vivo function of two key genes (FAH12, FAE1) involved in lesquerolic acid production. Thus, this self-compatibility based functional genomics approach is a rapid methodology for in vivo reverse-genetic gene characterization in self-incompatible plants.

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Virus-induced silencing as a method for studying gene functions in higher plants

Cited by 4 publications

References 100 publications

A rapid, simple, and highly efficient method for VIGS and in vitro-inoculation of plant virus by INABS applied to crops that develop axillary buds and can survive from cuttings

A rapid, simple, and highly efficient method for VIGS and in vitro-inoculation of plant virus by INABS applied to crops that develop axillary buds and can survive from cuttings

Argonaute 8 (AGO8) Mediates the Elicitation of Direct Defenses against Herbivory

Self-incompatibility based functional genomics for rapid phenotypic characterization of seed metabolism genes

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