RNAi and Plant Parasitic Nematodes

Introduction

With over four thousand species, invasion of plant-parasitic nematodes takes an enormous effort to manage in farmlands across the world (Jones et al., 2013). Every year they reduce the global agricultural output by one hundred and seventy-three billion US dollars (Elling, 2013). Meloidogyne incognitaGöldi; (Tylenchida: Heteroderidae) is one of the major species causing global yield loss. Though numerous resilient seeds are marketed, they are effortlessly overcome by the plant parasitic nematode populations. Finding reliable and long-lasting methods to protect global crop production have become the need of the hour. In this regard, the prospect of RNAi mediated gene editing in plant-parasitic nematodes for developing new nematicides is promising.

Within a living cell, when mRNAs get interpreted the corresponding genes are expressed. RNA interference is the cleaving of mRNA transcripts before they are translated. Consequently, the cleaving prevents the corresponding gene expression. RNAi is used as natural protection from invading viruses and other foreign transcripts after they have infiltered the cell’s physical barriers. In (Fire et al., 1998) it was experimentally proved that the induction of a tailor made double-stranded RNA (dsRNA) of a particular sequence in the cytoplasm of the nematode can stimulate RNA interference. Later, a deeper understanding of the RNAi mechanism was made possible with the discovery of the Dicer. Dicer is a ribonuclease that cleaves the introduced dsRNA in the cytoplasm into small interfering RNA (siRNA). The newly formed siRNA is unstable, and it combines with an RNA-induced silencing complex(RISC) to start cleaving all the mRNA it comes into contact if the mRNA has the similar transcripts as the siRNA (Tijsterman et al., 2004).

The advent of induced RNAi has changed the world of gene editing and functional genomics. Since the discovery and standardization of the methodology, the application of induced RNAi has reached far and wide from finding the functions of a target gene and analyzing its characteristic phenotypes in plants and animals to gene therapy. In plant parasitic nematodes, RNAi is predominantly used to decrease the mRNA levels or suppress the expression of effector genes. Effectors genes are essential to the plant parasitic nematodes for entering the host plants. The plant parasitic nematodes dispense an array of effectors that influence intracellular functions into the plant cell. The plants respond with its natural defense mechanisms. Some effectors serve as the nodal chemicals for host defense suppression. Nematode effectors are also used for disrupting the defense signaling pathways within the plant from the roots and meristematic tissues (Haegeman et al.,2012).

This review paper on RNAi and plant parasitic nematodes comprises of three sections. In the first section, In vitroRNAi and In plantaRNAi based experiments for silencing of several nematode genes that modulate movement, reproduction, sex ratios, feeding, reduced population and poor development of egg masses are discussed at length. In the second section, Modern dsRNA delivery mechanisms are examined. Molecular mechanisms that are responsible for deviations such as off-target effects, RNAi efficiency, and barriers that prevent the effectiveness of dsRNA and siRNA delivery are reviewed in the third section of the paper. RNAi and plant-parasitic nematodes

a) In vitroRNAi and its efficiency

The delivery of the dsRNA into the nematode’s body in a controlled laboratory environment is called In vitroRNAi. Microinjection, electroporation, soaking in dsRNA are some commonly used means of dsRNA delivery in In vitroRNAi. In one very early works by (Urwin et al.,2002) silencing of three groups of target genes cysteine proteinases, msp gene, and H.glycines hgctl gene was studied. Two nematode species Globodera pallidaStone; (Tylenchida: Heteroderidae) and Heterodera glycinesIchinohe;(Tylenchida: Heteroderidae) were chosen as the target nematodes. The cysteine proteinases are the genes which when inhibited leads to deterioration of the health and metabolism of the plant parasitic nematodes. mspgene is major sperm protein gene which is only expressed in the males. TheHgctlwas chosen because even though the function of the gene is unknown, there is a possibility that this gene affects the natural development of the plant parasitic nematodes.

Choosing the right stage of the nematodes for RNAi is essential. J2 are candidate stages for several experiments on nematode species. Nematodes are obligate parasites in the host plants; they cannot survive outside for extended duration. Only the juvenile nematodes in their second stage can move before the nematodes enter the plant. One big hindrance to introducing the dsRNA by feeding is that the plant parasitic nematodes do not ingest food outside the host plants. In (Urwin et al.,2002) it was successfully demonstrated that RNA interference could be made possible by chemically forcing the ingestion of dsRNA with the help of octopamine. Octopamine helps in stimulating the pumping of the pharyngeal region, and fluoroscein isothiocyanate was used as a reporter for further analysis (Urwin et al.,2002).

In H.glycineswhile targeting the cysteine proteinases, the proportion of the population that was to develop into females declined from seventy-five percent to fifty percent. Similarly, in G. pallida, there was an observed reduction from seventy-seven percent to fifty-six percent while targeting the cysteine proteinases. (Urwin et al.,2002). The reasons for this abnormal phenotype is not clear. Also while targeting the mspgene in both plant-parasitic nematode species, it was observed that there was a decrease in transcript abundance in J2 mRNA for the mspgene. The inhibition of the mspgene expression was evident fifteen days after the induced RNAi via soaking the nematodes in dsRNA targeting themsp gene. The function of the H.glycines hgctl gene is unknown. However, RNA interference of this gene exhibited fascinating results. It was found that silencing of the hgctlgene resulted in the reduction of nematode population by forty-one percent after fourteen days. These conclusions from (Urwin et al.,2002) helped establish the protocol for induced RNAi via feeding the nematodes with dsRNA for functional genomics and nematicide development in plant-parasitic nematodes.

In experiments by (Kimber et al., 2007) flpgenes silencing on G.pallidawas examined. Nematode flpgenes Gp flp6, Gp flp12, Gp flp14, Gp flp1, and Gp flp18are found to be responsible for proper motion and migration. The flpgenes are expressed predominantly in the nerve cells as they modulate movement, reproduction, and feeding (Dalzell et al.,2009). Soaking was the method of delivery adopted. The silencing of the genes helped in achieving an inhibiting effect on locomotory behavior in J2 plant-parasitic nematodes. Though the effect was pronounced while knocking down some flpgenes over others, knocking down of eachflpgene demonstrated inhibition in locomotory behavior at a minimum of over seventy percent after the first twenty-four hours. Gp-flp-12silencing resulted in one hundred percent inhibition of the nematode population after 24 hours. On the second day, all the treatments demonstrated a successful inhibition at a rate of over ninety-five percent. Some abnormal phenotypes like shuddering and non-motility were also observed on the seventh day (Kimber et al., 2007). Soaking concentration of the dsRNA was considered an essential attribute for successful RNAi in the nematode. To verify the influence of the dsRNA concentration on the success of the RNAi, the nematodes were exposed to ten-fold serial dilutions of the dsRNA between 0.1 mg/ml and one pg/ml for twenty-four hours (Kimber et al., 2007). The optimal concentration was found to be between ten μg/ml and 0.1 μg/ml (Kimber et al., 2007).

The efficiency of the induced RNAi primarily depends on the efficiency of the delivery of the dsRNA. The dsRNA soaking method seems to work well in many critical species (Dalzell et al.,2009; Urwin et al.,2002) However, investigations have shown that the efficiency of RNAi is high when microinjection or electroporation is used as the means of delivery in pinewood nematodes (Parket al., 2008). In (Park et al., 2008) experiments were conducted to see which among the three major in vitroRNAi methods (soaking, electroporation, and microinjection) is the most efficient in inducing RNAi in B.xylophilus. Pinewood nematode genes Bx myo3corresponding to myosin heavy chain, Bx tmy1 gene corresponding to tropomyosin, Bx hsp1 gene corresponding to heat shock protein 70, and Bx cyt2:1gene corresponding to cytochrome C were selected as target genes for RNAi.

In(Park et al., 2008) fifty J2 nematodes were soaked in the dsRNA for twenty-four hours at a constant incubation temperature of 25°C. It was observed that an average of twenty five percent of the nematode population was reduced as a result of the RNAi by soaking in the dsRNA. In the following generation, the survival was reduced by twenty three percent. When RNAi was initiated by electroporation of the dsRNA in the J2 nematodes, the survival rate was reduced to thirty two percent. This result is higher than the soaking method, however, when using the lethality was not observed in the following generation. The survival rate was reduced by forty six percent in the F1 generation when the dsRNA is delivered to J2 nematodes via microinjection. This reduction is a much more impactful and efficient reduction in the nematode population. Hence, through this study, it is evident that microinjection is the most efficient method compared to other delivery mechanisms for silencing these five particular genes in the pinewood nematode (Park et al., 2008) — thus proving that the dsRNA delivery method needs to be chosen appropriately taking the nematode species, target genes and other requirements into consideration.

b) In plantaRNAi and its efficiency

In plantaRNAi is the delivery of the dsRNA via the host plant by incorporating the transcripts in the seeds of the plants and other primary transformants. Plant mediated RNAi was studied and experimentally proven in (Dinh, P et al.,2014). In this study, various experiments were conducted on two potato cultivars Russet Burbank and Desiree, and a breeding line PA99N82 to understand the potato plants’ resistance to nematode Meloidogyne chitwoodiFinley; (Tylenchida: Heteroderidae) where the effector gene, Mc16D10Lwas the gene silenced by plant mediated RNAi. The breeding line carries the nematode resistance gene R-Mc1(blb). Experimental evidence shows that thirty five days after M. chitwoodi inoculation when compared with control lines, the number of egg masses developed on the gene silenced lines declined significantly by sixty eight percent in both the potato cultivars. It was also analytically confirmed with certainty that the RNAi effect was inherited in future generations and there was a marked reduction in the plant parasitic nematode population (Dinh, P et al.,2014). The edited lines of the advanced breeding line with dsRNA that can silence the nematode’s effectors significantly diminished the growth and development of eggs by forty-four percent and egg masses produced by forty-seven percent. The in plantasilencing effect was also, observed to be inherited by the offspring of the nematodes, thus proving that the plant-mediated RNAi works across cultivars and regardless of variation in plant genotype for resistance against M. chitwoodi.

Radopholus similisCobb; (Tylenchida: Pratylenchidae) is an important horticultural pest that causes enormous loss of yield and is extremely challenging to control. In (Li et al., 2017) the control of burrowing nematodes in Nicotiana benthamianaDomin; (Solanales: Solanaceae) by in plantaRNAi is examined. The study also analyses the effect of in planta RNAi on the nematode control. The major difference between the burrowing nematode and other plant-parasitic nematodes is that both the young and adult stage burrowing nematodes can survive inside and outside the plant tissues by feeding on the cortex cells’ cytoplasm. As Cysteine proteinases have multiple roles in the normal functioning of the nematodes, cathepsin S also known as Rs-cps is chosen as the target gene for performing in planta RNAi (Li et al., 2017).

Three experiments were conducted in the study by (Li et al., 2017). A feeding bioassay was experimented to see the effectiveness of the in plantaRNAi. After inoculation, the nematodes are allowed to complete their lifecycle on the T2 generation plants which are used for successfully delivering the dsRNA (Li et al., 2017). The second experiment is for understanding the effect of the RNAi on reproduction. Thirty females were then isolated and cultured on carrot callus for fifty days at 25 °C to analyze the change in the reproduction rate of the burrowing nematodes. Similarly, one hundred nematodes were inoculated on carrot callus for twenty-five days at 25 °C for observing hatching rates. Each experiment was conducted two times, with five replications each. The feeding bioassay revealed that the transgenic plants have significantly higher resistance, and the transcription levels of the target gene were reduced by seventy-five percent (Li et al., 2017).

The reproductive capacity of the nematode was calculated by counting the population after fifty days. It was found that the nematode population was reduced to thirteen percent compared to the control groups. The hatching of the burrowing nematode eggs that were isolated from the transgenic plants were found to be reduced to just thirty-two percent (Li et al., 2017). Thus In plantaRNAi resulted in lowering of the transcripts as compared to knockout when RNAi is performed on the plant parasitic nematodes. This distinction could be because of two reasons. There is a possibility of lower accumulation of the double-stranded RNA in the transgenic plants. Another reason could be that host plant enzymes dice the dsRNA within the plant cells (Li et al., 2017).

Vector mediated RNAi

The Usage or assemblage of vectors for dsRNA delivery and viral vectors mediated RNAi are modern approaches that demonstrate promising results in laboratory trials. Two different experiments for the creation of a fungal transformant vector and a viral vector are discussed below. In (Wang et al., 2016) a pDH-RH was assembled as a vector for delivery of dsRNA. Bursaphelenchus xylophilusSteiner; (Aphelenchida: Parasitaphelenchidae) is an invasive parasite in forest ecosystems. In the experiments, the phenotypes of 4 dpygenes were examined. Silencing of the dpy genes can result in the production of shorter nematodes. The entire genome of B. xylophilushas been sequenced and published. This sequencing has resulted in several studies including genome-wide functional analyses of the pinewood nematode. The study also describes the feeding methodology for the pinewood nematode. The study describes the creation of a vector using the binary plasmid pDHt/SK, for silencing that comprises of a hairpin loop construction transcript with identical sequences to the genes of interest with the help of an Agrobacterium assisted fungal transformation. Strains of Fusarium oxysporumSchlecht; (Hypocreales: Nectriaceae) are used as the filamentous fungal transformants that can be fed to the nematodes for the dsRNA delivery (Wang et al.,2016). Decreased body length and size confirmed the successful silencing of dpygenes.

Viral vectors like tobacco rattle virus are employed to enable dsRNA generation at a fast phase. It can be beneficial in gene silencing within plant root tissues and meristems (Dubreuil et al., 2009). Viral vectors like TRV have a bipartite genome where proteins for polymerases, locomotion, and coat along with two other proteins are coded. These proteins are substituted by sections corresponding to the genes of interest. The Virus will serve as a means to release the dsRNA into the nematode feeding cellS. In (Dubreuil et al., 2009) N.benthamianawas the host plant of choice. M.incognita, one of the dominant species causing global yield loss in the tobacco is chosen as the target species for in viral vector-mediated resistance development. In this study, Virus is inoculated into the tobacco plants by co-agroinfiltration usingAgrobacterium tumefaciensSmith & Townsend; (Rhizobiales: Rhizobiaceae) strains (Dubreuil et al., 2009). Two genes Mi-tncand Mi-crtwere selected as the target genes. Mi-tncis responsible for proper egg, and development of the nematode juveniles and Mi-crtis responsible for an effector which is very important for parasitism. Two batches of six tobacco plants were inoculated with the viral vectors containing the dsRNA separately, and each plant is infested with five hundred nematodes in their J2 juvenile stages. This procedure is done three weeks after the agroinfiltration (Dubreuil et al., 2009). The root-knot nematodes in the transgenic plants were allowed to complete their lifecycle.

While investigating the effects of Mi-tncgene silencing it was observed that one-third of the plants exhibited more than fifty percent reduction in Mi-tnc transcripts, and another one-third of the plants exhibited eighty to ninety percent reduction when the transcript levels in the progeny are analyzed (Dubreuil et al., 2009. While analyzing the effects of Mi-crtgene silencing, it was observed that one-third of the plants exhibited a reduction in the transcript levels, where one plant exhibited more than fifty-five percent reduction in transcripts and another exhibited seventy-five percent reduction when the transcript levels in the progeny are analyzed (Dubreuil et al., 2009). Even though the vector-mediated dsRNA delivery method is experimentally proved to be effective, there remain many avenues for improvement like increased efficiency and better targeting.

Off-target effects and recent advancements in RNAi

In functional genomics, an abnormal phenotype formed as a result of gene silencing helps to understand the functions of the target gene. An aberrant phenotype can be formed due to the resemblance among dsRNA and non-target mRNA producing off-target effects. Off-target effects arise once there is ninety five percent resemblance between a target transcript and an mRNA in the cytoplasm (Rual et al.,2007).Also, the outcome of RNAi on the plant parasitic nematodes is determined by nematodes’ ability to offset the cost of the gene of interest that has been silenced. Standardization RNAi specificity and efficiency is the most challenging task as there is no one size fits all approach. The distinct sites of cleavage are the consequence of the endoribonuclease engaging a 3′ end-counting rule (Vermeulen et al.,2005). This cleaving is part of the RNA interference pathway.The Dicer’s functional domains are well studied; however, there is still a knowledge gap about the contributions that the double-stranded RNA makes to position the point of cleavage and its role on the efficiency of the division.Even though the silencing efficiency of siRNAs is predictable, the cleaving of chemically synthesized short hairpin RNA (shRNAs) by the Dicer is not simple, and it is challenging to achieve reliable results (Vermeulen et al.,2005).

The position of cleaving usually measures a distance of approximately twenty two nucleotides from the 3′ terminus to cut both strands of the RNA. This was questioned in (Rual et al.,2007) where it was found that the length varies from 30 to 50~nt. The structural specifications required for a particular Dicer to cleave a shRNA are largely unknown adding to the already existing constraints of RNAi. In (Vermeulen et al.,2005) the role of the dsRNA in determining Dicer cleavage patterns are analyzed. It is found that the dsRNA and the shRNA comprising of blunt and overhang termini are cleaved by the dicer at well-defined sites and the structure of the overhang along with its composition, length and sequence plays a vital role in determining the cleavage specificity and efficiency. Experiments analyzing the silencing of several genes that modulate movement, reproduction, sex ratios, feeding, reduced population and poor development of egg masses on different plant parasitic nematodes have shown that there is potential for controlling the nematode population using RNAi (Dinh et al.,2014; Urwin et al.,2002).

Recent advancement in understanding the RNAi molecular mechanisms in other model organisms has made the future of RNAi plant-parasitic nematode management on the farm bright. In (Reynolds et al.,2004) to distinguish the siRNA-specific characteristics that are anticipated to influence the efficiency of the RNAi, one hundred and eighty siRNAs targeting specific mRNAs of two target genes were systematically analyzed. It was found that the lack of inverted repeats is an essential characteristic linked to siRNA functionality (Reynolds et al.,2004). Low guanine-cytosine content is also found as an essential factor as it is already well known that according to the base pairing rule, guanine-cytosine content determines the stability of siRNA which leads to the possibility that it can affect cleavage of dsRNA substrates (Ho et al.,2007).

The straight forward and high effectiveness demonstrated when RNAi is performed in vitro is shadowed by some practical difficulties. Silencing of genes with siRNA is complicated by the presence of multi-scale barriers, such as rapid excretion of the siRNA, and low stability of the siRNA. The aggregation of the siRNA in tissues which are outside the regions where the target genes are expressed is also a significant barrier for in-vitro RNAi (Shim et al.,2010). The delivery of the siRNA can be improved by a large extent when the cellular uptake rate and intracellular release rate are increased. Two novel approaches to overcoming barriers are as follows. Identifying and incorporating targeted binding of siRNA to specific cells like targeting moieties and promoting intracellular movement by using cell piercing peptides, and polymers that respond to external stimuli (Shim et al.,2010).

Conclusion

The advent of RNAi has enabled post-transcriptional gene silencing. RNAi has transcended from a gene silencing tool to a powerful weapon for demystifying the function of individual genes. Within a few years of its discovery, the application of RNAi has reached far and wide. The scientific and economic importance of RNAi mediation in gene silencing is felt in all quarters of the world. RNAi enabled resistance in plant-parasitic nematodes is an exciting work in progress. Research in the recent past has opened up exciting opportunities by discovering important molecular traits that can be used as targets for controlling and managing several plant-parasitic nematode species. However, like all tools, the RNAi mechanism is not perfect. The efficiency of dsRNA delivery mechanisms and Dicer specificity can still be enhanced. The reduction of off-target effects and improving the overall efficiency of the RNAi are also key research questions in many contemporary research papers. Several barriers discussed in the paper still need to reproducible solutions.

Further understanding of the structural demands for Dicer specificity and the role of the dsRNA in the RNAi efficiency can help in providing the instruments for creating more precise and useful shRNA and thereby reducing off-target effects. The position of cleaving from the 3′ terminus to cut both strands of the RNA varies from 30 to 50~nt (Rual et al., 2007). More research in this aspect can help in improving the efficiency of the cleaving process. The approachability of the siRNA-binding site on the target gene is another significant barrier that needs further investigation for improving the efficiency of RNAi (Kurreck, 2006). Recent advancement in understanding the RNAi molecular mechanisms in other model organisms has made the future of RNAi plant-parasitic nematode management on the farm bright. With increasing focus on specificity improvement, efficiency improvement and rigorous field trials, RNAi enabled resistance development through extremely precise nematicides can very soon become the order of the day for controlling plant parasitic nematodes.

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