We have previously written about the possibility of using RNAi-based technologies to provide plants more sustainable and greater protection against viruses. RNAi, or RNA interference, is the protective process used in many eukaryotic cells against viruses which uses double stranded RNA (“dsRNA”) sequences complementary to that of a pathogen to silence the translation of that foreign RNA into proteins. It was recognised in a recent review article as one of the genetic technologies that could be used to provide sustainable crop protection in the future.
An article in January’s Nature Plants (sorry, the full-text article is behind a paywall) looked for a way to give RNAi the ability to withstand field conditions when topically applied to crop surfaces.
BioClay as a Delivery Mechanism
The researchers investigated the possibility of connecting the dsRNA to clay nanosheets (“LDH”) to form a substance, which the researchers called “BioClay”, that can be applied to crops and provide longer lasting protection than applying naked dsRNA.
BioClay nanosheets were created with an average diameter of 45nm. Loaded onto the nanosheets were dsRNA sequences complementary to segments of the pepper mild mottle virus (“PPMoV”) or the cucumber mosaic virus (“CMV”).
To check for successful loading, the dsRNA-LDH substances were subjected to electrophoresis. The fact that the dsRNA-LDH complexes didn’t migrate from the well at all was taken as evidence that the dsRNA had been successfully loaded onto the LDH. Sequences up to 1.8kbp were shown to be attachable to the LDH to form BioClay.
Transmission Electron Microscopy used to view the BioClay formed showed that the dsRNA chain is either adsorbed on the LDH surface or thread within a number of LDH particles.
The mechanism of delivering dsRNA to the plant relies on the LDH degrading into a residue when exposed to CO2 and moisture. This process and the ability for BioClay to delivery dsRNA to the plant surface was tested by suspending the BioClay on the leaves of tobacco plants and incubating under atmospheric-like conditions for 7 days. The residue left after 7 days showed decreases in aluminium and magnesium, the conclusion being drawn that the LDH had degraded. The process was also tested by incubating test plants with CMV-loaded BioClay and collecting the residue after a week, finding that the amount of loaded BioClay had been reduced, indicating that the BioClay is releasing the loaded dsRNA.
How topically applied dsRNA provides protection to the subject plant is still a matter for further research. To test whether the dsRNA was being taken up by the plant after being released from the degraded BioClay nanosheets, the researchers attached a Cy3 fluorophore to LDH alone, to a dsRNA alone and to a dsRNA-LDH compound and tested all three by applying them Arabidopsis thaliana. 48 hours after application, the leaves were examined with confocal microscopy to determine whether any fluorophores and therefore, presumably, either the LDH, dsRNA or dsRNA-LDH complexes, had been taken up by the plant. The researchers observed the fluorophore within xylem of the leaves treated with Cy3 attached to dsRNA and dsRNA-LDH complexes, but was not internalised in treatments that did not contain dsRNA. Further, in the dsRNA-Cy3-LDH treatment showed flurophore uptake in the spongy mesophyll.
Not only was the fluorophore shown to enter the plant when attached to dsRNA, but was also seen to be transported to new apical meristem leaves that had not been directly treated.
Further testing of the uptake of dsRNA was undertaken on transgenic Arabidopsis that contained a β-glucuronidase reporter, the aim being to test whether a dsRNA directed towards the reporter gene interfered with its expression. Interference was measured with a fluorometric assay and plants treated with dsRNA-GUS complexes (with or without LDH) showed decreased β-glucuronidase activity, indicating that RNA interference was being induced by the treatments.
Did the BioClay Persist Longer?
The first few tests showed the dsRNA was being taken up by the plants and causing RNAi, but does the use of the LDH nanosheets to deliver the dsRNA result in greater protection?
The researchers tested the usefulness of the LDH nanosheets in a number of ways. First, they again labeled the dsRNA complexes with Cy3 and applied them to Arabidopsis leaves. After leaving them on the leaves for 24 hours half of the leaves in each treatment group were rinsed and the fluorescence levels measured. Complexes that contained LDH displayed residual flourescence while non-LDH treatments had little-to-no fluorescence after rinsing.
The LDH complexes were next tested with an RNase to test the ability of the different complexes to withstand degradation. dsRNA and dsRNA-LDH were treated with RNase. After treating, the dsRNA was released from the LDH in that treatment group and the two sets of dsRNA subjected to northern blot analysis. It was shown that the dsRNA originally attached to LDH had been degraded to a lesser extent than the naked dsRNA.
Figure 3 from article – Figures a – d show the microscopy images of the 4 treatment types to detect remains of treatments after washing. Figure e is the northern blot result showing the levels of degradation of the dsRNA by RNase when attached to LDH or naked. Figure f compares the dsRNA present on leaves at different time points after being sprayed with either the naked dsRNA or the dsRNA-LDH complexes.
Similarly, when the dsRNA and dsRNA-LDH were applied to leaves and their continuing presence on the leaves detected after application, the non-LDH attached dsRNA was barely detected after 20 days while the LDH connect dsRNA was detected 30 days after the treatment.
Similar to the findings about the translocation of the dsRNA into untreated leaves, the researchers used northern blot analysis on purposefully untreated leaves to test for the presence of the dsRNA 20 days after the spray was applied, finding that where the dsRNA was attached to LDH, the dsRNA was still detectable.
But Does it Afford Protection Against Viruses?
Showing that the BioClay can caused directed RNAi in plants and persist longer on plants is all well and good, but it must also provide the plants with additional protection against viruses.
Using nectrotic lesions caused by CMV as a marker for virus resistance, the study showed a significant reduction in the number of lesions in leaves treated with dsRNA and BioClay. A similar test used a PMMoV challenge to test the number of lesions formed. The leaves were challenged with the virus 20 days after being treated with dsRNA complexes and the researchers found that only the BioClay complex provided significant protection at this time point, demonstrating a longer period of protection.
Similarly, when a double-antibody ELIZA was used to test for the presence of CMV in leaves 20-days post challenge, the percentage of leaves positive for CMV was significantly less in leaves treated with BioClay compared to those treated with LDH alone and the dsRNA alone.
Figure 4 from article. Fig 4a and 4b visualise the lesion number of lesions per leaf resulting from being challenged at different times after being sprayed with the various treatments. The most significant result was the significant reduction in lesion numbers in BioClay treated leaves when challenged 20 days after the treatment when compared to the number of lesions formed after the other treatments.
Protection afforded to the non-treated leaves was tested by taking leaves that emerged 20 days after treatment and using the same double-antibody ELIZA to detect the level of infection. The researchers found a reduced level of infection in the new leaves when the plant had been treated with BioClay.
Finally, the researchers used RNA-seq testing on leaves challenged with CMV, some of which had been treated with BioClay or dsRNA, seeking out viral RNA. Leaves treated with the dsRNA or BioClay showed virus specific RNA was at least 10 times less abundant than in non-treated leaves.
The researchers have demonstrated through a series of steps that LDH nanosheets have the ability to deliver dsRNA to plants, be subsequently taken up by the plant and seemingly distributed throughout the plant, to provide useful protection against viruses. Most importantly, the LDH nanosheets were demonstrated to provide better protection to the dsRNA from being washed off the plant or from being degraded.
Field trials are the next obvious steps for a technology that seemingly has the ability to provide significant protection in a sustainable manner. The ability of the BioClay to withstand field stress, UV radiation for example, would further cement this technology as one that may alter agricultural practices and improve food security. Whether the RNAi protection can be extended to other pests is even more exciting.
A great piece of research which gives hope that this biological phenomenon can be used to assist crop protection and food production.