Sclerotinia sclerotiorum is a cosmopolitan pathogen capable of infecting over 600 plant species, including economically important crops such as canola, sunflower, soybean, and several bean species [1]. White mold infections, e.g. Sclerotinia stem rot in soybean and sunflower, cause devastating diseases responsible for significant crop losses worldwide [2]. There is a scarcity of effective genetic resistance derived from plants to manage losses caused by S. sclerotiorum due to the polygenic trait of resistance genes. Although widely used to treat fungal diseases, chemical fungicides are not very effective at preventing and controlling white mold infections and require constant monitor the emergence of fungicide resistance and often lack of specificity and may harm beneficial insects and microbes.
Recent developments in the management of pathogenic fungi explore the use of the conserved and essential RNA interference (RNAi) process, which is a component of eukaryotic gene regulation. [3, 4]. RNAi is activated by double stranded RNA (dsRNA) which is processed by the dicer enzyme into typically 21–24 nucleotide small interfering RNA. These small interfering RNAs are next loaded onto an RNA-induced silencing complex (RISC). The sense strand of the small interfering RNA (siRNA) duplex is degraded, and the antisense strand binds to a target mRNA sequence through base complementation. Upon binding to a target mRNA sequence, the small RNA guides the RNase protein, argonaute, as part of the RISC complex to cleave mRNA and halt the translation of the transcript [5]. The argonaute PIWI/RNaseH domain functions in the dsRNA guided hydrolysis of mRNA which gives argonaute endonuclease activity. Hence, two main RNAi strategies have been developed to protect crop plants from pests and diseases. One strategy known as host-induced gene silencing (HIGS) involves the expression of dsRNA or antisense RNA in the plant to silence the genes of pests or pathogens [6]. The second is spray-induced gene silencing (SIGS), which is the process of externally applying dsRNA to plant tissues with the goal of silencing the genes of pests and pathogens [7, 8]. SIGS is considered environmentally friendly and does not involve relatively lengthy transgenic processes as opposed to HIGS.
Identifying suitable target species and genes for SIGS and the knockout of target genes of interest can ensure the success of SIGS development for disease prevention. Specifically, fungal RNAi genes contributing to anti-viral function are promising SIGS targets, when confirmed by knockout mutants, but also provide evidence for the efficacy of SIGS treatment. This is due to how viruses activate small RNA pathways in fungi by accumulating significant amounts of dsRNA known as defective interfering RNAs (reviewed in [9]) and how RNAi functions as the primary antiviral mechanism in fungi. Some fungi without active RNAi viral defense may not be adequate targets for SIGS. RNAi genes affecting the virulence of fungal pathogens made apparent under virus infection have so far been identified in Cryphonectria parasitica, Colletotrichum higginsianum, Magnaporthe oryzae, Fusarium graminearum, Sclerotinia sclerotiorum, and Neurospora crassa (reviewed in [10]). Important evidence for active RNAi includes virus-derived small RNAs with a distinct peak in abundance at a particular size and a significant uracil bias at the 5' position, demonstrating argonaute processing. For example, S. sclerotiorum generates a 22-nt peak with a 5’ uracil bias under two different virus infections, and secondary phased siRNAs derived from long noncoding RNAs were detected indicating amplification for long-range RNAi [11, 12].
In both mammalian and plant model organisms, it has been reported that argonaute homeostasis involves the coordinated action of the miRNA and siRNA pathways, and the multi-layered control of argonaute can determine important cell fate decisions [13–15]. In S. sclerotiorum, the effects of key RNA silencing pathway genes on growth, antiviral activities, and small RNA processing were determined. Mutants of dicers (Dcl1, Dcl2) and argonautes (Ago2, Ago4) were generated and transfected by mycoviruses [12, 16]. Specifically, the knockout mutant of Ago2, but not of Ago4, lacks anti-viral RNAi. Furthermore, detected by degradome and small RNA-Seq identified by cluster analysis, one particular miRNA was predicted to direct cleavage of the S. sclerotiorum Ago2 (SsAgo2), SS1G_00334, as a self-regulating mechanism [11].
The success of SIGS also depends on the ability of a fungal pathogen’s dsRNA uptake mechanism and possibly unidentified dsRNA receptors (reviewed in [17]), which may explain different uptake efficiency observed by a study comparing different filamentous pathogens [18]. Furthermore, due to its propensity to degrade quickly when exposed to outside environmental conditions, the stability of dsRNA is a main concern for SIGS application. Several strategies to improve RNA stability have been implemented. One commonly used strategy is to load the RNAs in nanoparticles (NPs) either synthetic inorganic materials such as layered double hydroxide (LDH) nanosheets [19] or organic materials such as carbon dots [20]. Artificial nano-vesicles also have been used to prolong SIGS effects for crop protection [21]. LDH is especially interesting because of its unique features such as biodegradability, capturing organic and inorganic anions and anionic exchange capacity [22, 23]. A study conducted by Worrall et al. [24] using LDH loaded with dsRNA targeting viral coding regions of bean common mosaic virus (BVMC) was sprayed to BCMV-inoculated plants and found to inhibit the development of viral symptoms for up to 20 days. According to Mitter et al. [25], spray-delivered CMV2b-dsRNA-Cy3 was washed off by irrigation and sudden rain, but CMV2b-dsRNA-Cys3-LDH with NPs was still adhered to the leaves and offered better crop protection against the pathogen. Similar observation were reported where the dsRNA-LDH complex showed stronger and more lasting effects in controlling Rhizoctonia solani infection by targeting pathogenicity-related genes by SIGS than using dsRNA alone [26]. Overall, the NPs loaded dsRNA have been found effective against numerous phytopathogenic fungi e.g., Rhizoctonia solani, Fusarium oxysporum, Sclerotinia sclerotiorum, Sclerotium rolfsii, Colletotrichum spp., etc. (reviewed in [27]).
In this study, we hypothesize that perturbing SsAgo2 homeostasis is a promising SIGS approach for reducing white mold infection. Here we report to have identified the regions suitable for SIGS within SsAgo2 as target segments, and demonstrated the use of MgFe-LDH nanosheets as a carrier for dsRNA produced by both in vitro and in vivo methods, with the later approach aiming to reduce the cost of producing dsRNA as sprays.