Recent investigations documented that plants can uptake and process externally applied double-stranded RNAs (dsRNAs), hairpin RNAs (hpRNAs), and small interfering RNAs (siRNAs) designed to silence ...important genes of plant pathogenic viruses, fungi, or insects. The exogenously applied RNAs spread locally and systemically, move into the pathogens, and induce RNA interference-mediated plant pathogen resistance. Recent findings also provided examples of plant transgene and endogene post-transcriptional down-regulation by complementary dsRNAs or siRNAs applied onto the plant surfaces. Understanding the plant perception and processing of exogenous RNAs could result in the development of novel biotechnological approaches for crop protection. This review summarizes and discusses the emerging studies reporting on exogenous RNA applications for down-regulation of essential fungal and insect genes, targeting of plant viruses, or suppression of plant transgenes and endogenes for increased resistance and changed phenotypes. We also analyze the current understanding of dsRNA uptake mechanisms and dsRNA stability in plant environments.
RNA interference (RNAi) is a natural post-transcriptional regulatory mechanism that can be artificially induced by exogenous application of double-stranded RNAs (dsRNAs) to the plant surfaces. Recent ...studies show that it is possible to silence plant genes and change plant properties using plant RNA spraying and other approaches for dsRNA delivery. In this study, we investigated the effect of exogenous gene-specific dsRNAs on the silencing of four tomato genes encoding MYB-family transcription repressors of anthocyanin biosynthesis in the leaves of tomato
L. We found that the exogenous application of dsRNAs encoding for the
,
,
, and
genes downregulated mRNA levels of these endogenous repressors of anthocyanin production, upregulated the expression of anthocyanin biosynthesis-related genes, and enhanced anthocyanin content in the leaves of
. The data demonstrated that exogenous gene-specific dsRNAs can induce post-transcriptional gene silencing in tomato leaves by direct foliar application of dsRNAs. This approach may be used for plant secondary metabolism induction and as a silencing tool for gene function studies without the need to produce genetically modified plants.
Recent investigations show that exogenously applied small interfering RNAs (siRNA) and long double-stranded RNA (dsRNA) precursors can be taken up and translocated in plants to induce RNA ...interference (RNAi) in the plant or in its fungal pathogen. The question of whether genes in the plant genome can undergo suppression as a result of exogenous RNA application on plant surface is almost unexplored. This study analyzed whether it is possible to influence transcript levels of transgenes, as more prone sequences to silencing, in
genome by direct exogenous application of target long dsRNAs. The data revealed that
synthesized dsRNAs designed to target the gene coding regions of enhanced green fluorescent protein (
) or neomycin phosphotransferase II (
) suppressed their transcript levels in Arabidopsis. The fact that, simple exogenous application of polynucleotides can affect mRNA levels of plant transgenes, opens new opportunities for the development of new scientific techniques and crop improvement strategies.
Calmodulin-like proteins (CMLs) represent a large family of plant calcium sensor proteins involved in the regulation of plant responses to environmental cues and developmental processes. In the ...present work, we identified four alternatively spliced mRNA forms of the grapevine
gene that encoded proteins with distinct N-terminal regions. We studied the transcript abundance of
,
, and
in wild-growing grapevine
Rupr. in response to desiccation, heat, cold, high salinity, and high mannitol stress using quantitative real-time RT-PCR. The levels of all four splice variants of
were highly induced in response to cold stress. In addition,
and
forms were highly modulated by all other abiotic stress treatments. Constitutive expression of
and
improved biomass accumulation of
callus cell cultures under prolonged low temperature stress. Heterologous expression of the grapevine
and
splice variants in
improved survival rates of the transgenic plants after freezing. The
overexpression enhanced activation of the cold stress-responsive marker genes
and
, while
overexpression-
,
,
, and
genes after freezing stress in the transgenic
. The results indicate that the grapevine
gene acts as a positive regulator in the plant response to cold stress. The detected variety of
transcripts and their distinct transcriptional responses suggested that this expansion of mRNA variants could contribute to the diversity of grapevine adaptive reactions.
Exogenous application of double-stranded RNAs (dsRNAs) and small-interfering RNAs (siRNAs) to plant surfaces has emerged as a promising method for regulation of essential genes in plant pathogens and ...for plant disease protection. Yet, regulation of plant endogenous genes via external RNA treatments has not been sufficiently investigated. In this study, we targeted the genes of chalcone synthase (CHS), the key enzyme in the flavonoid/anthocyanin biosynthesis pathway, and two transcriptional factors, MYBL2 and ANAC032, negatively regulating anthocyanin biosynthesis in Arabidopsis. Direct foliar application of AtCHS-specific dsRNAs and siRNAs resulted in an efficient downregulation of the AtCHS gene and suppressed anthocyanin accumulation in A. thaliana under anthocyanin biosynthesis-modulating conditions. Targeting the AtMYBL2 and AtANAC032 genes by foliar dsRNA treatments markedly reduced their mRNA levels and led to a pronounced upregulation of the AtCHS gene. The content of anthocyanins was increased after treatment with AtMYBL2-dsRNA. Laser scanning microscopy showed a passage of Cy3-labeled AtCHS-dsRNA into the A. thaliana leaf vessels, leaf parenchyma cells, and stomata, indicating the dsRNA uptake and spreading into leaf tissues and plant individual cells. Together, these data show that exogenous dsRNAs were capable of downregulating Arabidopsis genes and induced relevant biochemical changes, which may have applications in plant biotechnology and gene functional studies.
Recent investigations have shown the possibility of artificial induction of RNA interference (RNAi) via plant foliar treatments with naked double-stranded RNA (dsRNA) to silence essential genes in ...plant fungal pathogens or to target viral RNAs. Furthermore, several studies have documented the downregulation of plant endogenous genes via external application of naked gene-specific dsRNAs and siRNAs to the plant surfaces. However, there are limited studies on the dsRNA processing and gene silencing mechanisms after external dsRNA application. Such studies would assist in the development of innovative tools for crop improvement and plant functional studies. In this study, we used exogenous gene-specific dsRNA to downregulate the gene of chalcone synthase (CHS), the key enzyme in the flavonoid/anthocyanin biosynthesis pathway, in Arabidopsis. The nonspecific
-dsRNA encoding the nonrelated neomycin phosphotransferase II bacterial gene was used to treat plants in order to verify that any observed effects and processing of
mRNA were sequence specific. Using high-throughput small RNA (sRNA) sequencing, we obtained six sRNA-seq libraries for plants treated with water,
-dsRNA, or
dsRNA. After plant foliar treatments, we detected the emergence of a large number of
- and
-encoding sRNAs, while there were no such sRNAs after control water treatment. Thus, the exogenous
-dsRNAs were processed into siRNAs and induced RNAi-mediated
gene silencing. The analysis showed that gene-specific sRNAs mapped to the
and
genes unevenly with peak read counts at particular positions, involving primarily the sense strand, and documented a gradual decrease in read counts from 17-nt to 30-nt sRNAs. Results of the present study highlight a significant potential of exogenous dsRNAs as a promising strategy to induce RNAi-based downregulation of plant gene targets for plant management and gene functional studies.
Calmodulin-like proteins (CMLs) are an important family of plant calcium sensor proteins that sense and decode changes in the intracellular calcium concentration in response to environmental and ...developmental stimuli. Nonetheless, the specific functions of individual CML family members remain largely unknown. This study aims to explore the role of the Vitis amurensis VaCML92 gene in the development of its high stress resistance and the production of stilbenes. The expression of VaCML92 was sharply induced in V. amurensis cuttings after cold stress. The VaCML92 gene was cloned and its role in the abiotic stress responses and stilbene production in grapevine was further investigated. The VaCML92-overexpressing callus cell cultures of V. amurensis and soil-grown plants of Arabidopsis thaliana exhibited enhanced tolerance to cold stress and, to a lesser extent, to the drought, while their tolerance to heat stress and high salinity was not affected. In addition, the overexpression of VaCML92 increased stilbene production in the V. amurensis cell cultures by 7.8–8.7-fold. Taken together, the data indicate that the VaCML92 gene is involved as a strong positive regulator in the rapid response to cold stress, the induction of cold stress resistance and in stilbene production in wild grapevine.
Plant stilbenes have attracted special attention as they possess valuable health benefits and improve plant resistance to environmental stresses. Stilbenes are synthesized via the phenylpropanoid ...pathway, where stilbene synthase (STS, EC 2.3.1.95) directly catalyzes the formation of t‐resveratrol (monomeric stilbene). This review discusses the features of using STS genes in genetic engineering and plant biotechnology with the purpose to increase plant resistance to environmental stresses and to modify secondary metabolite production.
This paper discusses the features of using genes encoding stilbene synthases (STSs), catalyzing the formation of monomeric stilbene trans‐resveratrol, to increase plant resistance to environmental stresses and to modify production of plant secondary metabolites in transgenic plants and plant cell cultures.
Abiotic stresses, such as drought, salinity, cold and heat, are major environmental factors that limit crop productivity. Vitis amurensis Rupr. is a wild grapevine species displaying a high level of ...abiotic and biotic stress resistance. Protein kinases, including Ca2+-dependent protein kinases (CDPKs), are known to mediate plant acclimation to various environmental changes. However, the functions of most grape CDPKs have not been clarified. A recent CDPK gene expression analysis revealed that 10 CDPK genes of V. amurensis were up-regulated under different abiotic stress treatments. The expression of the VaCPK20 gene was significantly up-regulated under low and high temperature stress in V. amurensis. In the current study, the effects of overexpressing the VaCPK20 gene in callus cell lines of V. amurensis and transgenic plants of A. thaliana on their responses to abiotic stresses were investigated. Transgenic Arabidopsis overexpressing the VaCPK20 gene showed higher tolerance to freezing and drought stresses, and transgenic grape cell cultures overexpressing the VaCPK20 gene showed higher resistance to cold stress in comparison with the controls transformed by the “empty” vector. Heat and salt stress resistance of the transgenic V. amurensis calli and A. thaliana was comparable to that of the wild type and vector controls. Overexpression of the VaCPK20 gene increased the expression of stress-responsive genes, such as COR47, NHX1, KIN1, or ABF3, in the transgenic Arabidopsis plants under non-stress conditions, after freezing, and under drought stress. The results imply that VaCPK20 may act as a regulatory factor involved in cold and drought stress response pathways.
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