Currently, providing enough food for the world’s expanding population is the most serious challenge. Due to the increased mandate for crops, the population of the globe has reached around 7.7 billion and is predicted to reach 9.7 billion by 2050 (
Rajam, 2020). Enhancing food crop productivity and efficiency is essential to meeting global food demand
(Saurabh et al., 2014).
However, the unexpected occurrence of environmental pressures and global climate change, as well as the scarcity of agricultural land and the significant rise in biotic variables, exacerbate the issues and pose a challenge to crop productivity. Accordingly, it is necessary to produce crops that are resilient to various environmental pressures and climate changes
(Raza et al., 2019).
To increase output in tandem with the growing human population, it is imperative to produce superior varieties of climate change-resistant crops (
Begna, 2021).
To address concerns with food security resulting from the world’s population growth at a rapid pace, agricultural productivity needs to be raised
(Varshney et al., 2021). The process of creating new and improved crop varieties with better agronomic traits-such as abiotic stress tolerance, disease resistance, extended shelf lives and herbicide resistance-was made easier by genetic engineering
(Nerkar et al., 2022). For the previous thirty years, transgenic methods have been used to enhance crops. Nonetheless, at the matter of edible crop species, the society is troubled about the generic and occasionally unstable integration of transgenes into the host genome (
Stephens and Barakate, 2017).
The society strongly opposes genetically modified crops, which are typically thought of as “unnatural” since they incorporate genetic material from several hosts
(Schmidt et al., 2020). New avenues for crop improvement without introducing foreign DNA from other organisms have been made possible by the development of modern plant breeding technologies such as sequence-specific gene silencing, or RNAi
(Altpeter et al., 2016).
Since RNAi produces genetically modified (GM) plants free of the transgenic protein or proteins, it is seen to be preferable to more conventional transgenic methods (
Rajam, 2020).
According to
Dubrovina and Kiselev (2019) and
Dalakouras, Wassenegger et al., (2020), exogenously applied dsRNAs have been seen as an additional option to genetic transformation that could offer comparable advantages without endangering ecological stability and societal acceptance (
Dubrovina and Kiselev, 2019);
(Dalakouras et al., 2020).
In order to improve crops in a way that the public may find more acceptable, new approaches and safe techniques must be developed. Accordingly, scientists worldwide who specialize in various facets of molecular biology have been interested in RNA silencing or RNA interference (RNAi) technology (Anavarali, 2020).
According to
Saurabh, Vidyarthi et al., (2014), these modifications include improved nutrition, decreased levels of food allergens and toxic compounds, enhanced defense against biotic and abiotic stresses, morphological changes, male sterility crafting, enhanced secondary metabolite synthesis and seedless plant varieties.
A natural occurrence in most eukaryotes, including plants and animals, is RNA interference. Long double-strand breaks (dsRNAs) are primarily processed into tiny RNA molecules, which range in length from 21 to 24 nucleotides (nt). The target messenger RNA (mRNA) is subsequently recognized by these short RNAs through homology-based binding and effector proteins assist in causing its destruction.
(Chaudhury et al., 2021).
Although RNA is acknowledged to play major functions in biology, the widespread competences and intricacies of this nucleic acid have remained intangible and unknowable, RNAi has enabled researchers to gain an understanding of gene function, pest resistance and physiological developments in plants
(Koeppe et al., 2023).
Numerous crops have benefited greatly from the widespread application of the CRISPR/cas9 method, including sorghum
Cai et al., (2015);
Li et al., (2015), wheat (
Wang et al., 2016).
Liang et al., (2017) and maize
Svitashev et al., (2016);
Zhu et al., (2016), Many rice crop genes, including OsHAK1, OsERF922, OsPDS, TMS5 and Badh2, have been knocked out with the aid of CRISPR/Cas9 technology, yielding predicted phenotypic consequences (
Zhang et al., 2014:
Zhou et al., 2016:
Nieves-Cordones et al., 2017; Shao et al., 2017). A few studies on elite rice demonstrate direct CRISPR/Cas9 genome editing of cultivars. A CRISPR/Cas9-mediated pooled sgRNA assembly was refined in an upland cotton (
Gossypium hirsutum) study, offering a platform for sgRNA designing that targets numerous genes.
Objectives
To evaluate the impact of RNA enzymes and RNA protein complexes on large-scale uses of RNA interference for agricultural improvement.
Review
History of RNA interference
The concept of an RNA silencing narrative originated during a hunt for transgenic petunia flowers that were predicted to be more purple. Jorgensen’s lab intended to increase the activity of a gene for chalcone synthase (chsA), which is involved in the creation of anthocyanin pigments.
Surprisingly, several transgenic petunia plants with the chsA coding area under a 35S promoter lost both endogene and transgene chalcone synthase activity, resulting in variegated or white flowers
(Napoli et al., 1990).
The reduction of cytosolic chsA mRNA was not related with decreased transcription, as evidenced by transcription experiments in isolated nuclei (
Van Blokland et al., 1994). Jorgensen proposed the term “co-suppression” to explain the loss of mRNA from both the endo and transgene.
Enzyme and proteins involved in RNA interference
Dicer, Drosha , RISC (RNA-induced silencing complex), Argonaute
Dicer is an RNase III endonuclease that converts miRNA precursors into functional 21-23 nucleotide RNAs, which are then integrated into the RNA-induced silencing complex. Dicer inactivation causes the loss of mature miRNAs and the buildup of miRNA precursors, making it an effective method for analyzing miRNAs’ global roles.
Dicer-like proteins with roles and protein domains akin to those of animal and insect dicers are encoded in plant genomes. For instance, four dicer-like proteins, DCL1 through DCL4, are produced in the model organism
Arabidopsis thaliana. DCL1 has a role in the synthesis of sRNA from inverted repeats and miRNA. Cis-acting antisense transcripts are converted into siRNA by DCL2, which supports viral immunity and defense. DCL4 is engaged in trans-acting siRNA metabolism and transcript silencing at the post-transcriptional level, while DCL3 produces siRNA that helps with chromatin modification. Furthermore, DCLs 1 and 3 are necessary for
Arabidopsis to blossom. DCL deletion in
Arabidopsis does not result in significant developmental issues.
Since it cleaves these double-strand breaks (dsRNAs) into small interfering RNAs, or microRNAs, which are then incorporated into the RNA-induced silencing complex, or RISC complex, the Human Dicer and its mechanism are crucial.
(Macrae et al., 2006; Khraiwesh et al., 2010) Next, this complex binds mRNA, damaging the targeted gene to stop translation. Gene silencing cannot happen without Dicer. Thus, it is impossible to control DNA and RNA without Dicer. Dicer-like (DCL) proteins are found in plants. There are four DCLs in
Arabidopsis; the genomes of rice and maize have eight and five DCL genes, respectively
(Kapoor et al., 2008; Qian et al., 2011), with four of these genes matching those of
Arabidopsis.
Since the dicer mechanism is a common defensive mechanism of many organisms, DCLs are also produced by rice and grapes. Compared to Arabidopsis, rice’s five DCLs have acquired new roles and are more crucial to the plant’s growth and function. Furthermore, the expression patterns in the various plant cell types of rice vary, whereas the expression in
Arabidopsis is more uniform. Drought, salinity and cold are examples of biological stress factors that can impact rice DCL expression. As a result, these stressors can make a plant less resistant to viruses. In contrast to
Arabidopsis, developmental abnormalities in rice are caused by loss of function of DCL proteins
(Liu et al., 2009).
The DROSHA (formerly RNASEN) gene encodes the class 2 ribonuclease III enzyme known as Drosha. The primary nuclease responsible for initiating the processing of microRNA (miRNA) in the nucleus is RNase III Drosha. The short RNA molecules known as microRNAs are generated and they operate in concert with the RNA-induced silencing complex (RISC) to split complementary messenger RNA (mRNA) as part of the RNA interference pathway, thereby regulating a multitude of other genes. However the significance of Drosha is yet to be defined in plants.
A single strand of a single-stranded RNA (ssRNA) fragment, such as microRNA (miRNA) or double-stranded small interfering RNA (siRNA), is incorporated into the RISC, a multiprotein complex as in Fig 1, specifically a ribonucleoprotein
(Filipowicz et al., 2008). To enable RISC to identify the corresponding mRNA (mRNA) transcript, the single strand serves as a template. Once located, the mRNA is activated and cleaved by Argonaute, one of the RISC proteins. According to
Fire, Xu et al., (1998), it is a crucial step in both gene silencing and resistance against viral infections.
Plant miRNA biogenesis varies mostly from animal biogenesis in the nuclear processing and export stages. Rather than undergoing two distinct cleavages-once within and once outside the nucleus-the plant miRNA cleaves twice, with each cleavage being carried out by a Dicer homolog known as Dicer-like1 (DL1). Plant cells’ nuclei are the only places where DL1 is expressed, suggesting that both processes occur there. Hua-Enhancer1 (HEN1) is an RNA methyltransferase protein that methylates the 3' overhangs of plant miRNA:miRNA* duplexes prior to their transportation out of the nucleus. A protein known as Hasty (HST), an Exportin 5 homolog, subsequently moves the duplex from the nucleus into the cytoplasm, where it disassembles and the maturemiRNA is integrated into the RISC (
Lelandais-Brière et al., 2010).
As “critical machinery” of the RNA-induced silencing complex (RISC), the Argonaute protein family plays a crucial role in RNA silencing progressions and is consequently in charge of gene silencing processes. The name of this protein family is derived from a mutant phenotype resulting from mutation of AGO1 in
Arabidopsis thaliana, which was likened by
(Bohmert et al., 1998) to the appearance of the pelagic octopus
Argonauta argo. Argonaute proteins bind several kinds of non-coding RNAs, including as small interfering RNAs (siRNAs) and microRNAs (miRNAs). By complementarity in base pairing, the short RNAs guide the Argonaute proteins to their specific targets, resulting in translation inhibition or mRNA cleavage
(Bohmert et al., 1998).
After the target mRNA is used to create de novo double-stranded (ds) RNA duplexes in plants, an unidentified RNase-III-like enzyme generates new siRNAs. These siRNAs are then loaded onto Argonaute proteins with PIWI domains but without the catalytic amino acid residues, which may cause an additional degree of specific gene silencing.
Mechanism of RNA interference in plants
Short-interfering RNAs (siRNAs), microRNA mediated gene silencing and deletion of RNA
Gene silencing can happen at the post-transcriptional, transcriptional, or both levels. MicroRNAs and short-interfering RNAs (siRNAs) are the two main kinds of tiny RNAs that are needed for many sequence-specific silencing scenarios. Through directing mRNA degradation or translational repression, miRNAs aid in post-transcriptional gene silencing. Compared to animal siRNAs, endogenous siRNAs are more varied in plants and can control transcriptional gene silencing by inducing DNA methylation and histone modifications or post-transcriptional gene silencing by causing mRNA degradation.
Long dsRNA or short-hairpin RNA (shRNA) precursors that are homologous in sequence to the gene to be silenced can start the process of gene silencing by RNA interference. Dicer cleaves this double-stranded RNA (dsRNA), which is produced by transcribing inverted repeat DNA, convergent transcription, viral replication and ssRNA copying by plant-specific RNA-dependent RNA polymerase (RDR). This dicer splits the dsRNA into short, duplex siRNA segments of 21–25 bp, which are then methylated at the 3 ‘end by Hua Enhancer1 (HEN1). The siRNA guide strand is loaded onto RISC when methylated duplex siRNA is transferred to the cytoplasm. Based on siRNA-mRNA complementarity, Si RISC attaches itself to the target mRNA and proceeds to break it.
Translation is halted when RISC including Argonaute (AGO) and other effector proteins cleaves the target mRNA. Inhibiting protein synthesis leads to post-transcriptional gene silence and active RISC can frequently take part in mRNA degradation
(Pattanayak et al., 2013).
Plants use miRNAs for a wide range of purposes, including development, stress responses and nutritional balance.Gaining knowledge of miRNA-mediated gene regulation may help develop new approaches to enhancing plant characteristics like stress tolerance While a significant number of
Arabidopsis miRNAs have been conserved over a long evolutionary distance, new research using short RNA cloning techniques has made it evident that a much larger number of miRNAs are not
(Sunkar et al., 2005; Lu et al., 2005; Rajagopalan et al., 2006).
MiRNA homologs (miR396d,e, miR437 and miR444) found in rice small RNA libraries are conserved in barley, maize, wheat, sorghum and sugarcane, but neither in
Arabidopsis or
Populus, indicating they are unique to monocotyledonous plants
(Sunkar et al., 2005a).
Since miRNAs are transcribed from MIR genes, they have an endogenous origin (
Rajam, 2020). The Dicer-like-1 enzyme (DCL1) and its associated proteins, such as Hyponastic Leaves 1 (HYL1) and Serrate (SE), help excise the primary miRNA (pri-miRNA) transcripts that are created when RNA polymerase II transcribes the MIR genes
(Pareek et al., 2015; Tyagi et al., 2019 and Kaur
et al., 2023).
These enzymes work together to produce the dicing microprocessor unit, which is phosphorylated by phosphatases and activated by certain kinases.
After additional processing of premiRNA, the DCL1 enzyme creates an unstable miRNA/miRNA* duplex. This duplex is methylated by HUA Enhancer 1 (HEN1) and it is then transported to the cytoplasm by HASTY, an exportin-like protein
(Tyagi et al., 2019; Kaur
et al., 2023).
The two routes for the synthesis of miRNAs and siRNAs now converge and proceed along the same path. To facilitate the creation of the final assembly of the RNA-induced silencing complex (RISC) for the purpose of silencing the target gene or genes, the two strands of the mature RNAi precursor are split. The passenger strand, sense, is broken down by the sRNA degrading enzyme (SDN) during the incorporation of the guide strand, antisense, into specific effector proteins known as Argonaute (AGO) to produce activated RISC
(Kaur et al., 2022). After being loaded, the RISC locates its cognate mRNA and permits translational repression or target mRNA degradation to down-regulate the chosen gene (
Borges and Martienssen, 2015;
Tyagi et al., 2019). dsRNA is delivered using a variety of techniques. One of the most popular approaches is micro-injection; however, despite its merits, this strategy is not appropriate for controlling insect pests due to a number of highly technical and difficult to implement restrictions
(Walshe et al., 2009). Products based on RNA interference that can be sprayed on are being developed and introduced to the market. The transitory presence of dsRNA in plant tissues is caused by root irrigation, spray delivery and microinjections.
In order to accomplish this, dsRNA is homologous to the insect’s essential gene and subsequently introduced into the plant to create a transgenic plant. When the insect feeds on the transgenic plant, the insect absorbs the dsRNA and activates its RNAi machinery, which results in the down-regulation of the insect’s essential genes, either causing the insect pest to die off or to grow more slowly
(Wang et al., 2017).
Application of RNA interference for crop improvement measures
Enhanced nutritional value of crops
Crop nutritional value has been increased through the application of RNA silencing-based technologies. Utilizing RNAi constructs, important genes involved in plant metabolic pathways are downregulated. A vital protein called glutenin can be found in the majority of food crops, such as maize, wheat and rice. According to
Wang et al., (2017), it is primarily in charge of the functional aspects of dough that raise its viscoelasticity. The primary functions of gliadins in gluten and dough are extensibility and viscosity, whereas polymeric glutenins are responsible for elasticity.
Using RNA silencing technology, which results in a variety of crops dubbed LGC (low glutenin content), the amount of glutenin could be decreased. It has been shown that it is possible to systematically silence particular groups of gluten proteins by RNA interference (RNAi) when used to silence the expression of particular Y gliadins
(Marin et al., 2022).
RNA interference is used to increase the amylose content of wheat. Drum wheat SBEIIa genes are found on the long arm of homologous group 2 chromosomes and they are responsible for determining the amylose content of the grain. RNA interference was used to significantly increase the amylose content in transgenic lines of durum wheat that showed the SBEIIa gene was silenced
(Sestili et al., 2010).
One of the most important steps in the agriculture industry to make rice more marketable is the development of fragrant rice. 2-Acety-1-pyrroline (2AP) is an aroma that may impart scent to rice plants. However, the OSBADH2 gene’s (betaine aldehyde dehydrogenase) output naturally inhibits the creation of 2AP, preventing the production of fragrance in rice. The agrobacterium-mediated binary vector gene delivery method was used to introduce RNAi into the IR-64 rice variety. The transformed rice seed increased 2AP production by about 30-40%, which increased the rice’s fragarncy and improved its marketability
(Khandagale et al., 2020).
Carotenoids and flavonoids, which are very good for human health, are found in tomatoes. The endogenous photomorphogensis gene DET1 in tomatoes functions as a negative regulator of light signal transduction or as a regulatory gene involved in the suppression of several light signaling pathways. Improved flavonoids or carotenoids can be found in tomatoes thanks to the inhibition of the DET1 gene. Tomatoes with higher levels of flavonoids and carotenoids are due to fruit-specific promoters suppressing DET1 through RNA interference. DELT1 has been effectively eliminated
(Kamthan et al., 2015).
Lipid-soluble pigments called carotenoids are essential to many plant processes. Additionally, because they reduce the risk of certain diseases and act as a precursor for the synthesis of vitamin A, carotenoids are significant components of the human diet
(Wenefrida et al., 2009). The beta-carotene hydrogenase gene prevents the assembly of carotenoids in potatoes. Silencing the beta-carotene hydroxylase gene (BCH) is necessary to stop betacarotene from being converted to zeaxanthin. In this sense, RNAi constructs were introduced into potato lines using the Agrobacterium tumefaciens-mediated transformation method (
Van Eck et al., 2007).
A resurgent oil crop called Camelina sativa must have its saturated fatty acid content reduced in order to satisfy various application needs. According to a study by
Ozseyhan et al., (2018), downregulating the genes producing fatty acyl-ACP thioesterases (FATB) allowed for a decrease in saturated fatty acids. In contrast to the wild type, seeds expressing a particular form of amiFATB increased the amount of oleic acid while decreasing palmitic acid by 54% and stearic acid by 38%. An artificial microRNA that targets regions specific to the FATB gene in camelina can successfully delete the gene
(Ozseyhan et al., 2018).
While corn protein is an important source of nutrition for both people and animals, the majority of it (60%) is composed of zein, a storage protein that lacks important amino acids like lysine and tryptophan22. Through the use of conventional genetic methods, the opaque-2 gene and a corn line containing lysine and tryptophan mutants were isolated. This led to the development of quality protein maize (QPM). Zein genes and other endosperm genes are regulated by an endosperm transcriptional factor that is encoded by O2. Zein levels in kernels of the loss-of-function o2 mutant are significantly lower, but non-zein protein levels are rising concurrently.
It is thought that the lysine and tryptophan content of o2 kernels is enhanced by this protein replacement. Similar nutritional gains were seen in transgenic corn kernels using a fusion IR transgene that targets zein gene families. In theory, specific zein reduction through RNA silencing can lessen the pleiotropic effects brought on by the o2 mutation. Unlike the prior examples of increasing free lysine by changing the metabolic lysine, this strategy primarily increases lysine in the protein fraction. route (
Hasan and Rima, 2021).
Because it is an apotent ion chelator and accounts for 60-80% of the phosphorus reserve in soybeans, phytotic acid deficiency can result in malnourishment. It’s antinutrients, which are substances that attach to nutrients in meals and reduce the body’s ability to absorb them
(Kumar et al., 2019). found that the phytate content of RNAi, which was induced by repressing the expression of GmMlPS1 as the target gene, was reduced by 41%. In soybeans, it increases mineral bioavailability.
Enhanced shelf life
Utilizing RNA interference technology, the biotechnology business Calgene preserves the tomato’s original flavor and color while delaying the ripening process and preventing premature softness. This would enable it to mature on the vine in the right way while preserving its ability to travel long distances without softening. Scientists at Calgene used the modified bacterial parasite Agrobacterium tumefaciens to introduce genetic material into Flavr Savr plant cells. The bacterium “infects” plants with foreign genes as part of its life cycle.
The desired genes were inserted into the bacterial T-plasmid in place of the parasite’s genetic material. The FLAVR SAVR tomato was created by using RNA to modify the activity of polygalacturonase (PG) or pectin depolymerase in the ripened fruit of Lycopersicon esculentum. Mature organisms have large amounts of this enzyme; ethylene, the well-known ripening hormone, initiates, controls and coordinates the expression of multiple genes involved in the ripening process.
The shelf life of tomatoes, a tropical fruit vegetable, is short. Fruit ripening, or softening, can lead to increased fruit rotting during transportation. Tomatoes might deteriorate before they reach customers if they are plucked while ripe because of their limited shelf life. To address this, tomatoes that are being shipped are often picked “green,” or underripe and then treated with ethylene gas, a plant hormone, to cause them to mature just before they are delivered. This method’s drawback is that the tomato’s natural growing process is not completed, which lowers the tomato’s final flavor.
A spike in ethylene synthesis marks the start of ripening in fruits with high respiration rates, such as tomatoes. By inhibiting the ACC synthase (ACS) gene during ripening, RNA interference (RNAi) technology was utilized to postpone the tomato ripening process. The synthesis of ethylene in tomatoes was effectively suppressed by the chimeric RNA interference-ACC synthase product, which targets homologs of ACC synthase. According to Diretto,
Frusciante et al., (2020), fruits from these lines had superior juice quality and matured later, lasting a longer six weeks.
Thus, extending the life of these plants is crucial as an additional agronomic trait that can lessen fruit and vegetable deterioration and spoiling (
Behera and Singh, 2019). RNA interference (RNAi) method to extend tomato life. They induced the expression of the oxidase gene 1-aminocyclopropane-1carboxylate (ACC) in tomatoes by initiating a dsRNA unit. The manufacture of ethylene and the reduction of water loss in plants are facilitated by the ACC oxidase gene, which codes for a big protein in plant cells. To guarantee that tomatoes have a long shelf life,
Xiong et al., (2019) limit the rate of ethylene generation in ripening plants and fruits.
Other strategies involved reducing fruit softness by inhibiting the gene that codes for the cell wall-degrading protein. By breaking down the cell wall, N-glycans are thought to play a major role in the ripening process of tomatoes. β-d-N-acetyl hexosaminidase (β-Hex) and α-mannosidase (β-Man) are the two recognized enzymes that alter ripening-specific N-glycoproteins. Suppressing these enzymes extended the shelf life by approximately one month because of the slower rate of softening
(Meli et al., 2010).
The ripening gene in bananas requires the genetic components of the MaMADS box genes, MaMADS1 and MaMADS2. Bananas with transgenic plants exhibit delayed ripening and extended shelf life due to RNA interference blocking one or both of the genes
(Elitzur et al., 2016). Fruit ripening delays are associated with decreased synthesis of the ripening hormone ethylene and a delay in climacteric respiration; in the most severe repressed lines, ripening was best delayed and no ethylene was produced
(Elitzur et al., 2016).
In Potato genes TALEN (SDN1), PEG mediated systems whose genomes were edited to hydrolyzes the sucrose produced from starch breakdown into one molecule of glucose and one of fructose, thereby delaying ripening
Clasen et al., (2016) Again in Potato genes CRISPR/Cas9 (SDN1) PEG mediated systems StPPO2 genomes was edited to catalyze the oxidation of phenolic compounds into quinones (highly reactive form) thereby also delaying ripening
González et al., (2020). In the ornamental flower Petunia, genes CRISPR/Cas9 (SDN1) in systems A.T. of explants PhACO whose genomes were edited to catalyze aminocyclopropane-1 carboxylic acid to ethylene in ethylene biosynthesis pathway in order to improve shelf life
(Xu et al., 2020).
Seedless fruit development (Parthenocarpy)
The process of growing fruit from seedless crops using ovaries without pollination or fertilization is known as parthenocarpy, or seedless fruit improvement. An agronomic characteristic that is greatly desired is parthenocarpy seedless, especially in fruit crops that are safe to consume. It requires neither fertilization nor pollination, thus even in difficult environmental conditions, it yields high quantities. Despite the fact that fresh fruit may be produced at any time of year, consumers are always demanding the same.
Certain fruits yield hard seeds that are difficult to come out of dormancy, while other fruits yield seeds that test difficultly because the lack of seeds can also be an advantage for both industrial applications (frozen eggplants and tomato sauce) and direct clean consumption (grape, citrus and banana)
(Meli et al., 2010).
One of the most important primary technologies for resolving parthenocarpy vegetation problems is RNA interferers. Parthenocarpy in
Solanaceae plants is caused by loss of function of the Pad-1 aminotransferase gene, which is important in auxin homeostasis
(Matsuo et al., 2020). All nine phytohormone pathways are regulated centrally by transcriptional co-repressors (TPL).
The interaction between SlTPL1 and SlIAA9, in addition to its high expression levels during flower development, encouraged the investigation of its functional importance through RNA interference (RNAi) technology, which selectively degrades the mRNA that encodes a protein to prevent its translation. The consequence of downregulating SlTPL1 was facultative parthenocarpy. Under normal conditions, the fruits produced by plants of the SlTPL1-RNAi transgenic lines were identical and did not exhibit any pleiotropic effects.
On the other hand, when they were emasculated and exposed to heat stress, they produced fruits without seeds. Moreover, SlTPL1-RNAi flower buds had lower abscisic acid levels and increased cytokinin levels. An RNA sequence was done to find the genes that SlTPL1 regulates in ovaries before and after fruit set in order to understand how SlTPL1 controls facultative parthenocarpy. The findings demonstrated that all transcription factors, including MYB, CDF and ERF and genes involved in cytokinin metabolism were expressed at lower levels when SlTPL1 was down-regulated.
In contrast, genes involved in the formation of the cytoskeleton and cell wall were expressed when SlTPL1 was downregulated. SlTPL1 is a critical regulator of these processes and our studies offer new insights into the molecular mechanism of facultative tomato parthenocarpy
(He et al., 2021).
One important hormone that regulates and develops the fruit set is gibberellin (GA). According to
Wang, Wu and colleagues (2020), the overexpression of the PbGA20ox2 gene enhanced the GA biosynthetic pathway and solidified A4 production, hence stimulating fruit set and augmenting parthenocarpic fruit enhancement in pears. According to
Ding et al., (2019) and
Wang et al., (2020a), the PpIAA19 gene is specifically regulated in relation to the quantity of lateral roots, stem elongation, parthenocarpy and fruit structure of tomatoes and grapevines. In contrast to plants of the wild type, overexpression of the MaTPD1A gene in banana plants results in fruits devoid of seeds
(Hu et al., 2020).
The capacity of different cultivars of summer squash (
Cucurbita pepo L.) to produce parthenocarpic fruit was compared. Certain cultivars produce no parthenocarpic fruit at all, while others exhibit varying levels of fruit set in the absence of pollination. Seasons affected the degree of parthenocarpy, but overall, cultivars ranked similarly in terms of parthenocarpy. The dark cultivars exhibited the best parthenocarpic fruit set
(Patel et al., 2022).
The generation of genetically engineered Parthenocarpic eggplants and their corresponding controls were the subject of an experiment by
Acciarri et al., (2002). When transgenic Parthenocarpic hybrids were compared to controls, they discovered effective results. Transgenic parthenocarpic accumulated more fruit weight and more yield per plant.
Globally, hybrids of gynoecious and parthenocarpic cucumbers have transformed the greenhouse sector. However, each hybrid’s unique popularity is based on how well it adapts to certain growth conditions, including consistency in pistillate blooming (
More, 2002;
Patel et al., 2022).
Applying plant growth regulators to watermelon yielded favorable results. It has been observed that the use of growth regulators increases fruit yield, size and parthenocapic fruit development. Growth regulators have also been shown to increase the output of certain horticultural crops that are used in parthenocarpic development production
(Patel et al., 2022).
The spiny gourd, or Mordica dioica Roxb., is a member of the
Cucurbitaceous genus
Mordica. It is also referred to as a spine gourd or kakrol. It exhibits dioecious, annual, viny characteristics and spreads vegetatively by means of tuberous roots. It has a lot of protein, carbohydrates and carotene (
Rashid, 1993). It is possible to produce seedless fruit and avoid manual pollination using genetic (natural) parthenocarpy or chemically induced parthenocarpy
(Patel et al., 2022).
In an experiment using the pat and WT lines of tomatoes
andrea et al., (1998) found that while a small proportion of low-seeded fruits were also produced in the NTR environment, all WT fruits from the HTR and LTR regimens had more than five seeds. Parthenocarpy degree in pat plants is expressed as the percentage of mature fruits without seeds
(Patel et al., 2022).
Tolerance to biotic stress
Tillage, crop rotation, the use of synthetic and organic pesticides, field monitoring, etc. are some conventional crop protection strategies. Growers must be trained and have a suitable plant protection strategy in place before introducing efficient farming methods, though useful they have their limitations.
Proteins and enzymes, chemical substances and morphological and structural barriers are the defense methods against biotic stress by the plant. These provide products strength and stiffness, as well as protection, therefore granting tolerance or resistance to biotic stimuli naturally nevertheless invasion by disease and insect pests are the biotic stresses blamed for a large loss of crop yield. Because these are conventional methods, there are inherent restrictions to using pesticides for pest management. Applications of RNA interference for disease inhibition and insect-pest resistance crops have proven effective. This involves identifying a target pest’s critical gene and using host plant-mediated RNA interference, also known as host-induced gene silencing (HIGS), to silence it. Suppression of this essential gene product’s expression will result in insect death because it is essential to the growth and development of insects (
Rajam, 2020).
One of the most damaging diseases to rice and a major source of yield loss is leaf blight, which is caused by the bacterial pathogen
Xanthomonas oryzae pv.
oryzae. RNA interference (RNAi) can suppress the OsSSI2 gene (OsSSI2-kd) in rice, increasing resistance to the pathogen (
Jiang, Shimono et al., 2009; Tamang et al., 2021).
By altering stress-sensitive genes and hormone accumulation, overexpression of the rice chorismate mutase (OsCM) gene changed the aromatic amino acid pathway downstream, weakening the stress of bacterial leaf rot (BLB)
(Jan et al., 2020). A few of the well-known plants that shown that bacteria could be resisted by modifying RNAi genes are Arabidopsis
(Wang et al., 2020), citrus (
Yu and Killiny, 2020), tomatoes
(Bento et al., 2020) and soybeans
(Tian et al., 2020).
The transcription of vitellogenesis (Vg) within C. suppressalis was significantly reduced by the RNA interference-mediated suppression of CsKrh1, which is crucial for suppressing rice pests
(Tang et al., 2020). dsRNA exhibits great promise for controlling pests in the field by topical application or spraying; soybean aphids, Aphis glycines, have been shown to have a mortality rate of up to 81.67%
(Yan et al., 2020), while soybean viridula, Nezara viridula, have been shown to have a mortality rate of up to 90%
(Sharma et al., 2021).
Enhanced resistance to parasitic weeds
Weeds that harm plants are common in and around fields in a number of nations, leading to notable losses in crop production. There are several classic control techniques like removal of weeds by pulling out with the hand. Removal of weeds by using the trowel. Removal of weeds by some agricultural techniques like ploughing, burning
etc. Spraying weedicides. In regions where the target weed is imported and problematic but its natural enemies are lacking, the classical or inoculative technique entails importing and releasing one or more natural enemies that prey on the weed in its native habitat.Use of glyphosate, imidazolinones and sulfonylureas are most frequently used to control parasitic weeds on faba beans
(Joel et al., 2007). however they have some drawbacks. Therefore, in order to tackle parasitic weeds, biotechnology instruments must be developed.
A subsequent study showed how to use RNA interference technology to advance plant kinds that are resistant to weeds. Transgenic tomato plants containing the M6PR dsRNA expression cartridge were created by
(Ally et al., 2009). It was discovered that there was a significant increase in the percentage of dead tubercles in
Orobanche aegyptiaca tubercles, a significant reduction in mannitol levels and a 60-80% decrease in endogenous M6PR mRNA in the tubercles and underground shoots of the plant grown on transgenic tomato plants. Using hpRNA-mediated RNAi resistant to
Striga asiatica L., a parasitic weed-resistant variety of maize was created (
de Framond et al., 2007; Yoder et al., 2009).
Small RNAs (sRNAs) that can induce gene silencing in weeds (known as spray-induced gene silencing, or SIGS) would be sprayed on them. This would allow for the achievement of traditional chemical control targets, targets that are not currently more sensitive to herbicides and even new targets. Because stable molecules that are easy to enter plants, do not pose a risk to crops when they go off target and can be obtained in small amounts that ensure effective systemic silencing, have proven more difficult to obtain than those found in other crop protection fields like entomology and plant pathology, the development of SIGS in weed science has progressed more slowly. After certain obstacles are removed, SIGS technology may be sprayed directly onto crops to selectively eliminate weeds, just how herbicides are now used in the field. This will offer a fresh method for controlling weeds, managing herbicide resistance and possibly even exploring novel plant enzyme targets that have never been reached through chemical control
. (
Zabala-Pardo et al., 2022).
Enhancement of resistance for insects and nematodes
Billions of dollars are spent on insecticides and crop losses brought on by pest insects. Despite this, farmers constantly face the threat of pesticide resistance, which leads them to continuously search for new pest-control strategies
(Ferry et al., 2006; Gordon and Waterhouse, 2007).
Every year, the phytoparasitic nematodes reported agricultural losses estimated to be worth US$125 billion. Nematode-resistant plants showed evidence of dsRNA articulation in a host plant resistant to parasitism or housekeeping genes in the root knot nematode by (
Gheysen and Vanholme, 2007).
Traditionally nematode management usually involves hot water treatment. Before planting, seed materials such onion bulbs, banana corms, tubers and seedling roots can be soaked in hot water between 5 and 55°C for ten minutes. The nematode is also killed by radiation. Nematode control techniques can be divided into three main groups: cultural methods, chemical methods and biological methods. One effective method of managing nematodes is crop rotation. A crop that is extremely vulnerable to nematodes might be grown in a field alongside less sensitive crops through rotation.
For conventional insects control majority of targeted insect control techniques fall into one of the following main categories: chemical, mechanical, biological, host resistance, cultural, physical, or mechanical control.
The process of enhancing the uptake of dsRNA in a buffered solution by neurostimulants during in vitro soaking conditions led to the first demonstration of RNA interference (RNAi) of plant-parasitic nematode (PPN) genes in the cyst nematodes
Heterodera glycines and
Globodera pallida (Urwin et al., 2002).
A wide range of lengthy dsRNAs, notably in plants, have been identified as having the ability to influence gene expression in the species that ingest them. In Arabidopsis, for example, 200 base-paired short non-coding RNAs (sRNAs) that may be processed for eRNAi were identified. Eukaryotes have two fundamental methods for RNA-based gene silencing: transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS). TGS inhibits transcription by methylating the 52-untranslated region (52 UTR), preventing transcription factor binding, whereas PTGS methylates the coding area, tagging the transcript for destruction
(Choudry et al., 2024).
Huang et al., (2006) were the first to develop resistance to multiple worm species by targeting a parasitism-complex gene as opposed to a nematode housekeeping gene. New objectives for HD-RNAi are likely to be revealed by the recent sequencing of the genome of
Meloidogyne hapla (Oppermana et al., 2008).
The sugar beet cyst nematode (
Heterodera schachtii) has four nematode parasitism genes: 3B05, 4G06, 8H07 and 10A06. The researchers used host-induced RNA interference to manipulate these genes. The number of mature nematode females in several RNAi lines decreased by 23-64% despite the fact that no conclusive resistance was detected
(Sindhu et al., 2009).
Fusarium wilt is caused by the filamentous fungal pathogen
Fusarium oxysporum, which infects a number of crop species, including tomatoes. A recent study found that RNA interference (RNAi) effectively suppressed fusarium wilt by blocking the pathogen’s critical polyamine (PA) biosynthesis gene, ornithine decarboxylase (ODC). This is because the disease needs PAs, namely putrescine, spermidine and spermine, to develop properly
(Singh et al., 2020).
In plants, RNA silencing is also an essential component of the antiviral defense mechanism (
Pathak and Gogoi, 2016). Targeted viral mRNA is broken down in a way that is self-replicating (RNA-dependent RNA polymerase) and results in resistance to RNA viruses (
Auer and Frederick, 2009). The single-stranded RNA genomes of the majority of plant viruses replicate via dsRNA intermediates.
Due to the virus’s single-stranded RNA genome, the dsRNA is converted into virus-derived miRNAs, which cause any similar RNA to be degraded
(Dalakouras et al., 2020). The studies done on tobacco demonstrated that short interference RNA (SiRNA) or double-stranded RNA (dsRNA) molecules complementary to the viral coat protein could be used to accomplish host-delivered RNA interference (HD-RNAi)
(Tilahun et al., 2021). The primary RNA silencing technology-mediated antiviral strategy in plants depends on the cleavage of viral dsRNA by Dicer-like enzymes, which results in virus-derived small interfering RNAs (siRNAs)
(Meena et al., 2017).
In tropical and subtropical areas, whiteflies are a significant agricultural pest. They are highly polyphagous and severely harm a variety of crops. RNA interference (RNAi)-based plasmids with an interfering cassette that target a novel v-ATPase transcript in the white fly have been developed successfully. Whiteflies fed transgenic plants had lower levels of endogenous v-ATPase gene expression, according to quantitative reverse transcription PCR
(Ibrahim et al., 2017).
The Development of Arabidopsis Nematode Resistance through the Effector Gene Mi-msp2’s silencing mediated by HD-RNAi. Intriguing parasites known as root knot nematodes (RKNs) prey on the thin roots of plants. Oesophageal gland effector genes, such as Mi-msp2, are known to facilitate RKN infection. Based on domain analysis, the Mi-MSP2 protein contains the aShKT domain, which is likely involved in blocking K+ channels and may aid in evading the plant’s defense response. Plant resistance to nematodes increases when the expression of the Mi-MSP2 effector gene is blocked by RNA interference (RNAi)
(Joshi et al., 2019).
Elimination of unwanted toxic compounds
The process of decaffeinating coffee involves taking the caffeine out of it. Coffee, tea, Coca-Cola and other goods all contain caffeine, a naturally occurring chemical stimulant (
Berkowitz and Spector, 1971). Because caffeine has stimulatory effects that can cause palpitations, elevated blood pressure and insomnia in vulnerable persons, there is an increasing demand for decaffeinated coffee
(Ogita et al., 2003).
RNA interference (RNAi) suppresses the theobromine synthase expressions (CaMXMT1) gene, which is important for the manufacture of caffeine. Coffee can now be produced that is decaffeinated and has very little to no caffeine thanks to RNA silencing technology (
Pathak and Gogoi, 2016).
According to
Siritunga, Arias-Garzon et al., (2004), cassava has potentially hazardous amounts of cyanogenic glycosides (linamarin), which shield the plant against theft and herbivory. A cyanogenic glucoside called linamarin is present in the roots and leaves of cassava plants.
It breaks down into a hazardous chemical compound in the human gut when it comes into contact with enzymes and intestinal flora
(Mansoor et al., 2006). According to McMahon,
Sayre et al., (2022), cytochrome P450 enzymes CYP79D1 and CYP79D2 generate linamarin in leaves and then transfer it to roots. The linamarin level of transgenic plants’ roots was 99% lower when these enzymes were inhibited specifically in the leaves
(Meena et al., 2017).
The primary carcinogen found in grass pea dry seeds and seedlings is 3-N-oxalyl-L-2,3-diaminopanoic acid (beta-ODAP), a neurotoxic. According to
Lambein, Khan et al., (1993), ODAP is considered a possible explanation for the condition neurolathyrism, which causes weakness, rigidity in the muscles and paralysis of the lower extremities in both people and livestock.
The neurotoxic â-N-oxalyl-L-á, â-diaminopropionic acid (β-ODAP) found in its seedlings and seeds is the cause of this irreversible nerve illness in people and animals
(Kumar et al., 2011). Oxalyl-CoA synthetase and ODAP synthase are two-terminal enzymes that are used in a two-step procedure to produce ODAP. ODAP synthesis can be prevented by blocking the biosynthesis pathway by silencing any one of the two enzymes utilizing cosuppression or RNA interference technologies
(Xu et al., 2017).
Induction of male sterility
In a number of crops, male sterility has been studied as a specialized mechanism to control gene outflow; the desired transgene is converted into the male sterile (female) inbred to generate a hybrid and this containment strategy is widely discussed
(Moon et al., 2010). To stop such gene transfer between crops and weeds (transgenic containment) or, in the case that it does, stop the transgene from being established and proliferating within the population (transgene mitigation). Maintaining the transgene of choice within the crop is the aim of gene containment, which stops it from invading related weeds, crop varieties and wild species. Numerous transgenic molecular pathways have been proposed to incorporate genes, particularly transgenes, into the crop.
Male sterility is largely cytoplasmic and transmitted through the mitochondrial genome, or chondriome. Since nothing is known about chondriome engineering, this will be challenging to accomplish. When tested in the field, four male fertile individuals from a population of a thousand had the marker, demonstrating the ability of cytoplasmic male sterility to prevent sorghum transgene flow through pollen (using non-transgenic pollinators to decrease the risk of viable pollen flow)
(Pedersen et al., 2003; Gressel, 2012).
The most widely used method of male sterility in agriculture is still the natural cytoplasmic sterility system. Although CMS has been successfully applied to several crops, such as rice, sorghum, millet, onion, sugar beet and carrot, it is noted that there are still drawbacks to this genetic system.
Three categories exist for male sterility: (1) cytoplasmic, (2) genetic and (3) cytoplasmic-genetic.
A crucial feature in the development of hybrid seeds is male sterility. Male sterility is induced using RNA silencing techniques. Scientists have produced male sterile tobacco strains by decreasing the expression of TA29, a gene required for pollen production
(Tehseen et al., 2010). TA29 is responsible for the creation of pollen. For plants to become male-sterile again, RNA silencing is also essential
(Sinha et al., 2023).
RNAi technique to jointly control male fertility and rice plant height, resulting in sterile dwarf rice.
Agrobacterium-mediated transformation of rice was carried out using the RNAi construct pTCK-EGGE, which targets the OsGA20ox2 and OsEAT1 genes. Dwarf male sterile plants are transgenic T0 plants that exhibit full male sterile traits and significantly decreased plant height
(Ansari et al., 2017).
The CRISPR/Cas 9 technology was effectively used to modify the targeted genes, which were linked to male sterility in cotton. Using this method, 112 genes related to plant development were knocked out (
Ramadan et al., 2021;
Chen et al., 2021).
Tolerance to abiotic stress
Enhancement of drought stress tolerance
Sunkar and Zhu (2004) documented the function of microRNAs (miRNAs) in relation to abiotic stressors in Arabidopsis plants, including salinity, cold, drought and oxidative stress. These plants were exposed to a variety of abiotic stimuli and it was shown that abscisic acid (ABA), cold, dehydration and elevated salinity levels all significantly up-regulated miR393. Moreover, abiotic stress in Arabidopsis altered miR402, miR319c, miR397b and miR389a to varying degrees. Because RNAi technology has so many advantages, including sequence-based gene silencing and high specificity, it may be used as an adjunct to sophisticated molecular approaches. Many plant species have successfully incorporated desired features for abiotic stress tolerance through the use of RNA interference (RNAi)
(Singh et al., 2018).
RACK1 gene expression was inhibited by RNA interference (RNAi) in rice, one of the genetically modified plants. This finding suggests that RACK1 may play a role in drought stress situations affecting rice crops. Compared to non-transgenic rice plants, transgenic rice was found to have a greater tolerance level
(Cao et al., 2015).
Using a microarray platform, miRNA analysis and genome sequencing profiling were carried out in rice studied in drought at different growth stages. 16 miRNAs (miR1126, miR1050, miR1035, miR1030, miR896, miR529, miR408, miR156, miR171, miR170, miR168, miR159, miR396, miR319, miR172 and miRNA1088) were remarkably involved in down-regulation in response to drought stress, while 14 miRNAs (miR1125, miR159, miR903, miR169, miR171, miR896, miR395, miR854, miR475, miR845 and miRNA1026) were found to be up-regulated under drought stress. A few miRNA gene families, such as miR319, miR896 and miR171, were recognized as both up- and down-regulated groups
(Meena et al., 2017).
(Singroha et al., 2021) have examined the upregulation of miR474 during drought stress in the maize crop, which interacts with proline dehydrogenase. Using a plant miRNA microarray technology, researchers have recently investigated the patterns of drought tolerance expressed by wild emmer wheat miRNA in response to drought stress
(Kantar et al., 2010).
In tomato, overexpression of the miR169c gene reduced transpiration rate, stoma openings and leaf water loss, improving transgenic plants’ resistance to drought in comparison to wild-type controls
(Zhang et al., 2011a). In order to make rice more susceptible to drought stress, the OsTBP2.2 gene was knocked down
(Zhang et al., 2020).
Enhancement of tolerance to cold and heat stress
The two abiotic stressors that might affect plant growth are cold and heat. Variable weather causes the plants to become pale, reduces the output and quality of plant products and can also result in financial loss. Utilizing RNA silencing coding, transgenic plants can withstand this stress. It has been proposed that
Arabidopsis, rice and sugar cane exhibit altered expression of MiR319 in response to cold stress
(Lv et al., 2020).
After plants were cold-acclimated (12C), overexpression of the Osa miR319 gene resulted in improved tolerance to cold stress (4C)
(Yang et al., 2013). A transgenic rice plant that is resistant to cold was created by overexpressing OsPCF5 and OsTCP21
(Yang et al., 2013). A novel type of thermotolerant mechanism in plants was discovered by
Guan et al., (2013), specifically to save the reproductive organs.
It also involves down-regulating its target genes, CSD (copper/zinc superoxide dismutase), CSD1 and CSD2 and CCS (a gene that codes for copper chaperones for CSD1 and CSD2). This is accomplished by inducing miR398. According to
Guan et al., (2013), they found that csd1, csd2 and ccs mutant plants were significantly less damaged by heat stress and had a greater tolerance to it than wild-type plants. They also demonstrated an enhanced accumulation of heat stress transcription factors and heat shock proteins.
Yu et al., (2018) and
Suksamran et al., (2020) on corn.
MiRNA expression in response to cold stress has been examined in
Arabidopsis, Brachypodium and
Populus species
(Liu et al., 2008; Arenas-Huertero et al., 2009; Zhang et al., 2009). All of the aforementioned species showed up-regulated levels of miR169 and miR397, while
Brachypodium and
Arabidopsis showed upward-regulated levels of miR172. Furthermore, during cold stress, many miRNAs (miR408, miR393, miR165/166 and miR396) were elevated in Arabidopsis; however, other miRNAs (miR398, miR156/157, miR394, miR159/319 and miR164) showed either transient or mild regulation (
Liu et al., 2008;
Younis et al., 2014).
Using Solexa high-throughput sequencing, researchers were able to clone the miRNAs from the wheat leaves that had been subjected to heat stress, as evidenced by the variable expression of miRNA in the wheat response. 32 families of miRNA were found in wheat and nine of those miRNAs were thought to be heat-responsive. For example, in response to heat stress, miR172 was clearly downregulated, but miRNAs such as miR827, miR156, miR169, miR159, miR168, miR160, miR166 and miR393 were observed to be upregulated (
Xin et al., 2010;
Younis et al., 2014).
Enhancement of tolerance to salt stress
The effects of salt stress on crop yield and plant growth highlight the need of comprehending the mechanisms behind plant salt tolerance. An “unfolded protein response” (UPR) is triggered when the endoplasmic reticulum’s (ER) ability to fold proteins is disrupted, leading to a buildup of unfolded proteins and ER stress. It is yet unknown how salt stress causes UPR, despite data indicating that it does so in a variety of species, including plants. In many different types of organisms, zinc shortage also causes UPR.
Ionic imbalance and cellular toxicity are caused by increased concentrations of cations and anion, as well as by their accumulation and dispersion. Plants can use one of two strategies to deal with salt stress: they can either limit their input through their root systems or manage their distribution and storage.
Breeding techniques shed light on the tolerance to salt stress in various crops, including wheat and rice (
Muthu et al., 2020; Al-Ashkar
et al., 2019). Incorporating structural, functional and comparative genomics would strengthen conventional breeding initiatives. Crop plants have been genetically engineered to identify genes linked to salt tolerance and their introgression (
Roy et al., 2011). The most beneficial outcome of biotechnology is the use of molecular tools in breeding programs (
Singh et al., 2021:
Kordrostami et al., 2015), Still, there remains a huge gap between crop yields under stress and under ideal circumstances.
Depending on the stage of development and length of stress, different amounts of salt stress quantitatively inhibit the growth of numerous plants. Different strategies have been created by plants to tolerate salt stress. Using microRNAs (miRNAs), which can affect post-transcriptional gene regulation under a variety of environmental circumstances, including salt, is one of the primary tactics.
Plant endogenous miR genes encode the 20-22 nucleotide regulatory RNAs known as microRNAs (miRNAs) (
Wang et al., 2019). Dicer-like (DCL) proteins transcribe their primary transcripts into precursor RNAs with a partly double-stranded stem-loop structure, which mature miRNAs are made of. Primordial miRNAs (pri-miRNAs) are produced by RNA polymerase II (Pol II) from nuclear-encoded miR genes in the miRNA biogenesis pathway. This process produces precursor transcripts with a unique hairpin structure (
Tyagi et al., 2002;
Waqar et al., 2022).
With the aid of the proteins SERRATE (SE) and HYPONASTIC LEAVES 1 (HYL1), DCL1 transforms pri-miRNA into pre-miRNA [10,11]. De novo synthesis of the pre-miRNA hairpin precursor, which is controlled by DCL1, HYL1 and SE, results in the formation of miRNA duplexes. Hua enhancer 1 (HEN1) methylates HASTY (HST1), an exportin protein and sends it into the cytoplasm
(Naqvi et al., 2012; Wen-wen et al., 2014). The catalytic component of the RNA-induced silencing complex (RISC), the Argonaute (AGO) protein, binds one strand of the duplex (miRNA) in the cytoplasm and tells RISC to target transcripts based on sequence complementarity
(Chen et al., 2005). Through modifying post-transcriptional targets and causing epigenetic modifications such DNA and histone methylation, miRNAs affect the expression of genes
(Jung et al., 2009; Chang et al., 2020; Waqar et al., 2022).
There have been reports of many regulated miRNAs in plants under salinity stress. Salinity stress increased the expression of miR397, miR156, miR394, miR158, miR393, miR159, miR319, miR165, miR171, miR167, miR169, miR168 and miR398 in
Arabidopsis, but it decreased the accumulation of miR398. With the addition of NaCl
3, it was seen that the accumulation of miR159.2 and miRS1 in
P. vulgaris progressed.
While miR1450 and miR482.2 were favorably regulated throughout the salt stress period71, miR171l-n, miR1447, miR530a, miR1445 and miR1446a-e were negatively regulated in
P. trichocarpa. Recently, an investigation using microarrays was conducted to explain the salinity-tolerant miRNA profile in both a salt-sensitive and salinity-tolerant line of maize. The results showed that in saline-stressed maize roots, members of the miR162, miR474, miR395 and miR168 groups were up-regulated, while members of the miR396, miR167 and miR156 groups were down-regulated
(Ding et al., 2009).
Enhancement of grain yield
A pleiotropic feature, grain yield uses several genes that work in intricate signaling cascades. Numerous regulatory elements, including as microRNAs (miRNAs), which are short non-coding RNAs with 20-22 nucleotides (nt) that have become the major ribo-regulators of eukaryotic genes, regulate the yield-related genes. The secret to achieving high yields in rice has long been thought to be having an ideal plant architecture (IPA) and it has been determined that several miRNAs are crucial in coordinating essential regulatory processes that lead to the best plant morphological yield-related characteristics, such as fewer unproductive tillers, more panicle branches and heavier grains
(Kaur et al., 2020).
Achieving an ideal plant architecture (IPA) in rice has been considered the secret to high yield and it has been discovered that several miRNAs are crucial in coordinating essential regulatory processes that lead to the best plant morphological yield-related characteristics, such as fewer unproductive tillers, more panicle branches and heavier grain
(Kaur et al., 2020).
MiRNAs are important regulators of plant architecture that target several transcription factor genes, according to mounting evidence (
Tang and Chu, 2017). OsSPL14 is encoded by the rice IPA1 (Ideal Plant Architecture 1) quantitative trait locus, which is in vivo controlled by OsmiR156. A point mutation in OsSPL14 increases OsSPL14 transcription by interfering with the OsmiR156-directed cleavage of its mRNA. Improved plant architecture-related features follow, including fewer tillers, greater lodging resistance and higher grain output
(Jiao et al., 2010).
The taller rice variety QX1 has semi-dwarf plant life due to the inhibition of the GA 20-oxidase gene (OsGA20ox2), which is mediated by RNA. increases both the weight and number of seeds per panicle. The objective of Osa-miR156 in rice is to accelerate grain yield, decrease tiller number and increase rice grain yield via OsSPL14 (Souamosa promoter binding protein like14) (
Mamta and Rajam, 2018). Additionally, various deficiencies in yield parameters, such as grain counts and seed putting rate in rice, were caused by the overexpression of OsamiR1873, as reported by
Dekeba (2021). Increased grain weight and size as well as the promotion of stem development in rice have previously been linked to overexpression of the OsAGO17 gene
(Zhong et al., 2020).
As a result, miRNAs have a variety of functions in controlling plant design through their targeting of key transcription factors or signaling proteins, which makes them promising candidates for improving crop plant architecture.
Spray-induction using gene silencing
SIGS, or spray-induced gene silencing, is a newly developed crop pest management technique. It makes use of endogenous RNA interference machinery and exogenously administered double stranded RNA to precisely decrease the expression of pest target genes. Widespread obligatory biotrophic fungus known as powdery mildews attack ornamentals like roses as well as agricultural crops including wheat, barley, cucurbits and grapevine
(McRae et al., 2023).
Because RNAi has the potential to be developed into a workable and specific biopesticide for commercial use, there is a particular interest in using it as a foliar spray
(Koeppe et al., 2023). According to
Sang et al., (2020), this approach is known as Spray-Induced Gene Silencing (SIGS) and it enables non-transformative control of plant pests and diseases. This procedure involves spraying the afflicted plant with a foliar solution containing extended siRNAs and dsRNAs, which, when consumed by the intended insect or pathogen, will cause RNAi. With this method, harmful microorganisms can be precisely controlled without having the negative impacts that chemical-based pesticides have on the nearby ecology
(Puyam et al., 2020).
Widespread obligatory biotrophic fungus known as powdery mildews attack ornamentals like roses as well as agricultural crops including wheat, barley, cucurbits and grapevine. Using the
Golovinomyces orontii-Arabidopsis thaliana pathosystem and the known azole-fungicide target CYP51, SIGS techniques were created and optimized for powdery mildews in a study. Following more screening, conserved gene targets and processes linked to the proliferation of powdery mildew were found. These included the secreted effector EC2, the lipase a, lipase 1 and acetyl-CoA oxidase in lipid catabolism, the putative abscisic acid G-protein coupled receptor, 9-cis-epoxycarotenoid dioxygenase and xanthoxin dehydrogenase, all predicted to function in manipulation of the plant hormone abscisic acid. The most common disease affecting grapes is powdery mildew and reports of widespread powdery mildew resistance to fungicide application have been made
(McRae et al., 2023).
Late blight disease in potatoes and tomatoes is caused by
Phytophthora infestans; resistance cultivars and heavy fungicide spraying are the current methods of prevention. Spraying potato leaves with double-stranded RNAs (dsRNA) that target the vital
P. infestans genes. A study demonstrated that the green fluorescent protein (GFP)-expressing sporangia of
P. infestans may directly absorb in vitro generated dsRNAs homologous to GFP from their environment, including leaves, resulting in a decreased relative expression of GFP
(Kalyandurg et al., 2021).
By focusing on developmentally significant genes in
P. infestans like guanine-nucleotide binding protein â-subunit (PiGPB1), haustorial membrane protein (PiHmp1), cutinase (PiCut3) and endo-1,3(4)-â-glucanase (PiEndo3), further illustratrates potential of spray-induced gene silencing (SIGS) in managing potato late blight disease. Such findings show that SIGS may be used to reduce potato late blight; however, the choice of target genes will determine how much the disease is controlled
(Kalyandurg et al., 2021).
SIGS is an interesting promise for commercial plant disease control due to its flexibility, specificity, decreased dangers to the environment and human health and quick transition from the bench to the field.
