In this study, an Indian strain of M. oryzae, i.e., B157 (international race IC9) isolated from Hyderabad, and its cytoplasmic GFP-expressing transformant (M. oryzaeGFP) were grown on yeast extract glucose (YEG) media (Saha et al., 2020). The blast-susceptible indica rice cultivar- C0-43, was grown at 27°C under a 16:8 light/dark photoperiod. The binary vector pCAMBIA1302 was streaked onto Luria Bertani (Himedia, Mumbai, India) agar plates supplemented with 50 mg/L Kanamycin (Himedia, Mumbai, India) and incubated overnight at 37°C. Fungal growth assays were performed on a complete medium (CM) supplemented with or without dsRNA, at 28°C for 7 days. For conidiation, the fungi were grown in dark on YEG plates for 8–10 days. The fungal biomass was then scraped on 10 days after inoculation and homogenized into uniform slurry by vortexing. Spores were separated from the mycelial debris by filtering the slurry through sterile MiraCloth (Calbiochem, Darmstadt, Germany), and then centrifuged at 7,000 rpm for 7 min. The conidia were visualized and counted under a microscope using a hemocytometer (Neubauer, Marienfeld, Germany). The length and breadth of conidia were measured keeping a 20-μm scale reference using an built-in microscope camera. Microscopic examination of at least 50 conidia per replicate was done in at least three independent experiments.

The fungal culture was grown in YEG broth by the inoculation of solid culture agar blocks. Fungal balls were filtered out of the 4-day-old culture, and mycelia were harvested, dried, and weighed before genomic DNA isolation was carried out (Dellaporta et al., 1983). Plasmid DNA was isolated using alkaline lysis method (Sambrook et al., 1989) from the fully grown culture. Each 20 μl PCR reaction was set up for 100 ng pure genomic DNA or plasmid DNA containing forward and reverse primers, deoxynucleotide triphosphates (dNTPs), 10 × reaction buffer, and 1.5 units of Taq DNA Polymerase (NEB, Ipswich, Massachusetts, United States). The PCR was carried out in a thermal cycler (Genetix, New Delhi, India), with standard PCR conditions, whereby the annealing temperatures were maintained at 52 (for GFP) and 54°C (for MoDES1) for 30 s per cycle. Resultant PCR products were used for in vitro transcription, as will be described later. The primers used are listed in Supplementary Table 2.

The sequence information of GFP [obtained from pCAMBIA-1302 (GenBank: AF234298.1)] and MoDES1 (MGG_04163) was obtained from NCBI.¹ Target regions were selected from their exonic regions, as indicated in Supplementary Figure 1. A target sequence homology of more than 19-mer with any other gene can render non-specificity to the precursor dsRNA. Hence, the target sequences were checked for homology across the whole genome assembly of the M. oryzae, O. sativa subsp. indica, O. sativa subsp. japonica, and O. sativa subsp. javanica groups using NCBI BLASTn. Homology was similarly checked between MoDES1 target sequence and its counterparts coding for its homologs known from other closely related Ascomycetous fungi (Chi et al., 2009). Only members that showed a significant percentage sequence similarity of greater than 40% were considered for BLAST analysis with the MoDES1 dsRNA sequence. After ensuring that there was no significant similarity among the interrogated sequences, the target regions (Supplementary Figure 1) were PCR-amplified from the M. oryzaeGFP genomic DNA using sense and anti-sense primers linked with a T7 promoter sequence at their 5′ ends (Supplementary Table 2). While the sense forward primer (FP) and the anti-sense reverse primer (RP) had a T7 promoter sequence attached to them, the sense RP and anti-sense FP did not have it. The PCR conditions have been discussed above, in the “genomic DNA isolation and PCR” section of “Materials and methods.” The amplicons were checked for band specificity, purified using Purelink PCR Purification Kit (Invitrogen, Waltham, MA, United States), and a further 1 μg each of individual amplicons were used as templates for T7 DNA-dependent RNA polymerase-based in vitro transcription. For this, TranscriptAid T7 High-Yield Transcription Kit (Invitrogen, Waltham, MA, United States) was used following the instructions of the manufacturer. The sense and anti-sense transcripts for each target sequence were incubated with RNase-free DNaseI (NEB, Ipswich, Massachusetts, United States) for 1 h at 37°C to remove residual template DNA. DNaseI digestion was stopped with0.5 M EDTA at 65°C for 10 min, followed by the phenol:chloroform-based extraction and ethanol-based precipitation of RNA transcripts. The single-stranded RNAs (ssRNAs), being self-complementary, were annealed in equimolar concentrations to generate respective dsGFP and dsDES1 by initially incubating at 75°C for 10 min, and gradually cooling them down at room temperature. The dsRNAs were further run on 2% agarose gel with their corresponding ssRNA transcripts for integrity analysis and successful annealing. To prevent the formation of secondary structures, the ssRNAs were mixed with an RNA-loading dye, and denatured and chilled before loading into the wells. Next, dsGFP and dsDES1 were purified using.a volume of 3 M sodium acetate (pH 5.2) and three volumes of chilled absolute ethanol. After overnight incubation at –20°C, the dsRNA was centrifuged in a Sigma 2–16 KL cooling-centrifuge at 13,000 rpm for 30 min. The precipitated pellets were washed twice with 70% ethanol, air-dried, and quantified with a UV spectrophotometer, and A_260/280 ratio was measured (Supplementary Figure 2). A schematic representation of the synthesis of dsRNAs and in vitro and in vivo proof-of-concept experiments is provided in Figure 1.

The in vitro treatment involved the co-incubation of wild type M. oryzae along with the purified dsRNA in both solid and liquid media. The fungus was grown on CM plates supplemented with 50 nM of dsDES1 by inoculating hyphal block and incubating it for 9 days, Spores were isolated from the culture. Then, the spore suspension was diluted and spreaded on dsDES1-supplemented CM plates, following the procedure as published (Guo et al., 2019). An individual colony was further sub-cultured onto a 50 nM dsDES1-amended CM plate and allowed to grow at 28°C. This dsRNA-fed strain was used for phenotypic assays and transcript analyses of MoDES1, to study the effects of dsDES1 on the wild type upon its uptake. The GFP fluorescence of M. oryzaeGFP was visualized by growing it both on sterile slides layered with YEG agar media, and on 24-well culture plates containing YEG broth. YEG agar was aliquoted, 2 ml each in a sterile centrifuge tube, cooled down, and supplemented with 5 nM dsGFP. The dsRNA-mixed YEG medium was layered onto sterile slides and allowed to solidify. A 10 μl drop of a 1 × 10⁵ spores/ml suspension was smeared along the length of the slide and incubated in the dark at 28°C for 2 days. In the liquid medium, a 10-μl drop of a 1 × 10⁵ spores/ml suspension was added to each well containing 1 ml of YEG broth and 1 ml of 5 nM dsGFP [dissolved in diethyl pyrocarbonate (DEPC)-water]. The culture plate was allowed to rotate at 45 rpm for mycelia formation and dsRNA-uptake. Conditions for the microscopic visualization of GFP fluorescence will be described later in the “Histochemical staining and microscopy” section. In order to mimic the oxidative stress in the environment, 1 mM H₂O₂ was added to the culture media, 30 min before RNA extraction from the dsRNA-fed and untreated axenic cultures. This was performed to elicit the transcription of MoDES1, which expresses in response to oxidative stress. The RNA was then used for the quantification of MoDES1 transcripts in the treated and untreated fungi.

An infection assay was performed on onion-peel, detached rice leaves and intact and abraded rice leaves, and 20 μl of a spore solution containing an equal number of spores (1 × 10⁵ spores/ml) was drop-inoculated on the sterile hydrophobic surface of the onion epidermis. Epidermal peels were placed on clean glass slides, kept inside moist petriplates, and incubated for 24 h at 28°C. Appressorium count was observed using a hemocytometer, and appressorium penetration was assessed following a published procedure (Chida and Sisler, 1987). The ease of cell-to-cell movement of invasive hyphae was determined by counting the infection units on onion epidermal cells and scoring them based on the penetration and branching pattern of invasive hyphae (IH) during in planta biotrophic growth. The percentage fraction of infection units showing 0, 1, and > 2-cell penetration was categorized considering 50 infection units in each case, with the experiment having three replicates; 10 μl drops of the 1 × 10⁵ spores/ml suspension [dissolved in 0.02% V/V Tween 20 (Merck, Bangalore, India) and 0.25% W/V gelatin] were spot-inoculated on the adaxial surface of detached leaves (Jia et al., 2003) and rice sheath (Kankanala et al., 2007) of 21 day-old plants. Mean lesion length and area were measured using the Image J software, whereas infection severity in terms of lesion densities per 5 cm² leaf area (Liu et al., 2010) was evaluated by spray-inoculating 3-week-old plants (Akagi et al., 2015). Among other whole-plant infections, the leaves were either abraded and punch-inoculated (Park et al., 2016), or spot-inoculated. In both cases, 50 μl spore drops were inoculated on 4-week-old plant leaves, and drops were secured to leaf surface with adhesive tapes. The plants were maintained in a growth chamber with 27°C temperature and 90% relative humidity settings.

Hyphal blocks of uniform size, from the 7-day old fungal culture were sub-cultured onto CM plates amended with 0, 3, and 7 mM H₂O₂, and incubated for 10 days at 28°C. ROS sensitivity was determined from the mean diameter of the growing culture at 5 and 10 dpi, and in all cases three biological replicates were maintained. Extracellular laccase activity was measured in solid CM media, supplemented with a 0.2 mM 2,2′-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) substrate, while peroxidase activity was measured from 200 μg/ml Congo red (CR)-amended CM (Guo et al., 2019). The mean diameter of degradation halos was measured on 5 dpi and in triplicates. The laccase and peroxidase activity of the fungal culture filtrates was measured from the absorbance at 420 nm using 10 mM ABTS and 3 mM H₂O₂ (Chi et al., 2009). For the protoplast release assay, which is an indicator of cell wall integrity, mycelia were grown in CM, and protoplasts were isolated from 3-day-old culture (Chen et al., 2017). Observations were conducted after 90 min of lysis with a lysing enzyme, and the experiments were repeated thrice.

For initial optimization, the detached leaves were sprayed locally with 50, 150, and 300 nM of dsGFP 24 h prior to M. oryzaeGFP inoculation. Later, for checking of systemic nature, 300 nM of dsGFP was sprayed in local and distal regions of sandpaper-abraded leaves, as described (Koch et al., 2016). The first and second (lower) leaves sprayed directly with dsRNA were considered as local regions, while the third and fourth leaves (upper) were considered as distal unsprayed parts. In case of infection on the punch-abraded leaves, 20 μl drops of 300 nM dsDES1 were spotted on abraded areas of the leaves and were secured with adhesive tapes. The same abraded regions, 24 h later, were inoculated with 20-μl drops of the spore suspension via syringes, without disturbing the tapes. Similarly, dsRNA treatments were performed for the intact leaves in whole plants, but without abrasion. For the whole plant leaf infection assays in the experimental sets, sandpaper-abraded leaves were sprayed with purified 300 nM dsDES1, 0, 12, and 24 h before fungal spray inoculation. While in set I he dsRNA and spore suspension were mixed together and sprayed simultaneously (0 h), in set II and III, dsRNA was sprayed 12 and 24 h, respectively, prior to infection.

Fungal infection units were observed by staining the infected onion epidermal cells with a ready-to-use lactophenol blue (Sigma Aldrich, MI, United States) solution. The epidermal peels infected with conidia were stained with one drop of lactophenol blue for 15 min, destained with lactophenol until the excess stain got removed, and finally mounted with 60% glycerol before visualization. The infected rice sheaths were excised, and the epidermal layers of mid-vein were lactophenol-fixed and stained with 0.01% Aniline Blue (Chi et al., 2009) for the observation of callose plugs and secondary wall deposition in and the around primary infected cells. In planta ROS generation was visualized by staining the infected sheath epidermal cells at room temperature with a 1 mg/ml 3,3′-diaminobenzidine (DAB) (BioSB, CA, United States) staining solution prepared as per the instructions of the manufacturer. The sheaths were destained with a clearing solution (ethanol:acetic acid = 94:4 V/V) for 1 h and visualized under a differential interference contrast (DIC) filter. The infected cells and their neighboring cells were color-coded based on staining intensity, representing the level of H₂O₂ generation. The cell death assay involved overnight staining of the leaf sheaths with 0.01% trypan blue (HiMedia) at room temperature, followed by destaining in chloral hydrate for 24 h. The stained epidermal tissues were visualized in a bright field. All DIC and fluorescence images were taken using an Axio mRm camera coupled with a Zeiss Axio Imager A1 fluorescence microscope (Carl Zeiss, Göttingen, Germany). Fungal GFP fluorescence in the leaf lesions was observed under a fluorescein isothiocyanate (FITC) filter, and all the images were processed using the Zen Light Blue software. The mean intensity of GFP expressed by M. oryzaeGFP in the dsRNA- sprayed and unsprayed sets were documented considering leaves from at least three plants from each set in three independent experiments.

Total RNA was isolated from both the dsRNA-fed fungal strain and DEPC water-treated wild type fungus [4 days post treatment (dpt)], and dsRNA-sprayed and unsprayed infected rice leaf-lesions 3, 4, 5, and 7 dpt. The fungus and infected leaf biomass were crushed using liquid nitrogen and taken forward for RNA extraction using RNeasy Plant Mini Kit (Qiagen, Germany), as recommended by the manufacturers. The RNA was treated with RNase-free DNaseI to remove traces of genomic DNA, assessed for quality and integrity, and further quantified by spectroscopy; 100 ng of RNA from local, distal-sprayed and unsprayed leaves was resolved on a 6% polyacrylamide gel (PAGE), and in vitro-transcribed dsGFP was kept as positive control. After the electro-transfer of the RNA onto a Ambion BrightStar positively charged nylon membrane, the membrane was hybridized with 150 ng of a non-radioactively labeled GFP probe (303 bp gel-purified PCR product). The primers used for PCR amplification of the GFP probe are listed in Supplementary Table 2. The labeling and detection of signals were performed using direct labeling reagents and CDP-Star (Amersham; GE Biosciences, United Kingdom) as per given instructions.

First-strand cDNA synthesis was performed by reverse transcribing a 1 μg template RNA with RevertAid H Minus M-MuLV Reverse Transcriptase (NEB, Ipswich, Massachusetts, United States). Reactions, 50 μl, were set up in a thermal cycler programmed at 42°C for 1 h, followed by termination of the reverse transcription at 70°C for 5 min. For transcript quantification by qRT-PCR, 2 μl of cDNA was taken as template in Power SYBR Green PCR Master Mix (Applied Biosystems, United Kingdom) consisting of gene-specific forward and reverse RT primers (Supplementary Table 2). Each reaction was carried out in triplicates, and the C_t values were normalized against the internal housekeeping control MoACTIN of the wild-type B157 strain. In the case of RT-PCR, 50 μl reactions were set up, each containing forward and reverse RT primers, deoxynucleotide triphosphates (dNTPs), 10 × reaction buffer, and 2.5 units of Taq DNA Polymerase (NEB, Ipswich, MA, United States). For molecular the analysis of fungal growth, the cDNA synthesized from the RNA extracted from infected leaf lesions were first diluted to different concentrations and calibrated using the rice housekeeping OsACTIN. Then, the amounts of cDNA corresponding to equal band intensity were used for the RT-PCR analysis of both the rice 25S rRNA and the fungal 28S rRNA. This was used to ensure that comparison of fungal growth was done in equal amounts of infected leaf tissue, from the experimental and control sets of rice plants. Standard PCR settings were maintained.

Each experiment was repeated at least three times independently. The statistical analyses were performed with the GraphPad Prism 8 (GraphPad, San Diego, CA, United States) software. Student’s t-test and pairwise comparison were performed by Tukey’s test with Bonferroni correction for the determination of significant outcomes. The alpha level was set at 0.05 in all cases.

All the relevant experiments from this study that involved the use of the phytopathogenic strain of M. oryzae, foliar spray of dsRNA, and infection assays of the rice plants were conducted following the biosafety norms as per Institutional Biosafety Committee (IBSC). To prevent environmental contamination through the dissemination of any of the above-mentioned agents, the experimental materials were handled under confined environment and laboratory conditions, restricted to growth chambers and the green house. Experimental wastes and unused materials were autoclaved and discarded in the end.

Double-stranded RNA-mediated gene silencing is a target mRNA sequence-specific phenomenon. Hence, the MoDES1 and GFP dsRNA sequences chosen as target were analyzed for selective specificity toward only the target gene and target organism. BLASTn analysis was done for the 303 bp and 300 bp target regions (Supplementary Figure 1) against M. oryzae and O. sativa genomic sequences. The GFP sequence showed significant similarity with neither the fungus nor the plant host. It only showed a 100% similarity with the GFP sequence from pCAMBIA1302. Similarly, the MoDES1 target did not show any significant similarity with the indica, japonica, and javanica groups of rice. The BLAST analysis of MoDES1 target across M. oryzae genome showed 100% sequence identity with only MGG_04163, which was the intended gene target. Three other M. oryzae strains, MZ5-1-6, LpKY97, and B71, showed 99.64% similarity with the query but had primary hosts other than rice (Supplementary Table 1). However, the chosen sequence for dsRNA generation would not lead to unspecific silencing in the B157 and 70–15 strains that are known to infect rice primarily. Ascomycetous fungi, such as Chaetomium globosum, Podospora anserina, Neurospora crassa, Fusarium sp., and Botrytis cinerea, have DES1 homologs and share a sequence similarity of greater than 44% with MoDES1 (Chi et al., 2009). Sequences for the homologs were obtained from the Locus information and compared with the MoDES1 target, and no significant similarity was observed (Supplementary Table 1). To this end, the target regions chosen from MoDES1 and GFP were found to be specific only toward the intended GFP gene and MGG_04163, with no predictable off-targets in rice and M. oryzae used in the experiments.

A proof-of-concept experiment was performed with an M. oryzae-GFP reporter system for the demonstration of SIGS in rice. Similar to the previous reports, out of the many parameters that determine the efficacy of RNAi phenotype, the optimum dosage of dsRNA or asiRNA, abundance of the target gene, size of the target dsRNA, and sequence complementarity of the designed dsRNA and its delivery method are the most crucial (Bennett et al., 2020). The 303 bp and 300 bp target regions (Supplementary Figure 1) chosen from GFP and MoDES1 were used, respectively, as templates for dsGFP and dsDES1 generation. In each case, an average concentration of 30 μg/μl of purified dsRNA was obtained from one set of transcription and annealing reaction (Supplementary Figure 2). It was demonstrated that 50 nM of asiRNAs, when supplemented in media, could induce silencing of the target M. oryzae gene (Guo et al., 2019). Hence the efficiency of the chosen target dsGFP was assessed by in vitro treatment of M. oryzaeGFP spores with 50 nM of dsGFP. The microscopic evaluation of the mycelia (Figure 2A) revealed a 76% reduction in the fluorescence intensity of the dsRNA-treated mycelia compared with the DEPC water-treated controls 3 dpt. The specificity of the observed GFP-silencing phenotype was further confirmed using a positive control, where dsDES1 was used for the in vitro treatment of M. oryzaeGFP. There was no observable reduction in fluorescence in this set, thereby attributing the formerly observed silencing effect to sequence-specific GFP knockdown via dsGFP. Since the effective dosage of dsRNA can often vary across organisms and delivery methods, optimization was performed by spraying 50, 150, and 300 nM of in vitro synthesized GFP-specific dsRNA (dsGFP) onto rice leaves, followed by M. oryzae GFP inoculation after 24 h. The silencing effect, in all the cases, was observed by measuring the mean intensity of GFP fluorescence in the inoculated GFP-transformant to optimize maximum silencing effect. It was observed that although the lower concentrations were able to induce silencing, the effect did not last beyond 36–48 h post treatment (data not shown). On the contrary, the effect of 300 nM dsGFP was perceived even after 4 days of application. During a 0–5 days time frame, the minimum intensity of GFP was observed 96 h post dsRNA treatment (hpt) or 72 h post inoculation (hpi) (Figure 2B), after which the effect of gene silencing started to reduce. Since 300 nM of the selected dsGFP was able to not only induce but sustain silencing for a longer period of time on the treated rice leaves, and considering that the abundance of our target pathogenicity gene MoDES1 is unknown, the 300 nM dsRNA concentration was used for spraying and infection-related experiments to ensure effective RNAi.

Ribonucleic acid molecules, being relatively more unstable than DNA or other biomolecules, have a limited longevity on plant surface. It has been shown that leaf cuticle can act as a barrier for uptake, hence, high-pressure spraying or abrading can ensure robust silencing (Dalakouras et al., 2016). Thus, we further wanted to check if removing physical barriers by sandpaper-mediated abrasion (Bennett et al., 2020), followed by spraying, would affect the longevity and efficiency of gene knockdown. Based on our previous observations on the detached leaf (Figure 2B), the 96 hpt time point was fixed for assessment and quantification of the silencing effect of dsGFP sprayed on both intact and wounded rice leaves. The microscopic observations revealed that the reduction in fluorescence beyond 4 dpt was more pronounced and sustained in the wounded dsGFP-sprayed sets (Figure 2C). Figure 2D further revealed a higher silencing efficiency in the wounded, dsRNA-sprayed leaves. The quantitative analyses showed that the relative abundance of the transcripts was relatively lower in the intact leaves, with 48% silencing, in contrast to the 67% knockdown observed in the case of the wounded leaves.

The efficiency of SIGS largely relies on its systemic nature, i.e., the ability of RNA signals to travel from the sprayed region (local) to the unsprayed (distal) parts of the plant. To determine if the dsRNA can traverse to unsprayed regions, the detached leaves of 2-week-old rice seedlings were sprayed locally and distally with 300 nM of dsGFP or Tris-EDTA buffer (mock) and given a lag period of 24 h to facilitate the uptake of dsRNA by the leaf tissue. The leaves were then inoculated with 5 × 10⁵ spores/ml of M. oryzaeGFP. The microscope images showed relatively more intense fluorescence in the mock-treated infected leaves (Figure 3A) compared with the directly and distally dsGFP-treated leaves. Based on this finding and some previous reports on Fusarium graminearum (Koch et al., 2016) indicating silencing in distal parts of the leaves, whole plant infection experiments were conducted to explore the effectiveness of SIGS in M. oryzae. DsRNA specific to GFP were sprayed onto the wounded leaves to ensure effective silencing. The RNA extracted 4 dpt from the direct and distally sprayed leaves was assessed for the presence of spray-supplied 300 bp dsGFP by northern blotting. The results suggested the presence of dsGFP in both locally and distally treated regions. While in the directly sprayed parts dsRNA was detectable up to 7 dpt, in the unsprayed distal regions, dsRNA was detected up to 4 dpt. The accumulation pattern of dsGFP in the local and distal leaves of the dsRNA-sprayed plants was aligned with the time dependent reduction of silencing phenotype in the sprayed leaves, as shown in Figure 3A.

MoDES1 is a novel gene encoding a hypothetical protein that was found to be involved in suppression of PTI in rice via host-derived ROS-scavenging activity. In a previous report, it has been functionally characterized and found to have homologs in other members of ascomycetes (Chi et al., 2009). Targeted gene silencing needs to be highly specific; hence, the selected target region of 300 bp is aligned with the genomic sequences of both rice and fungal members that possess a DES1 (Supplementary Table 1) homolog. The BLASTn and BLASTp results showed no significant homology, with no prediction of off-target effects. The in vitro-synthesized dsRNA was fed to the wild-type fungus, which after two generations of dsRNA treatment, showed a slight reduction in growth diameter, as compared with the untreated fungus (Figures 4A,E). The axenic cultures of the phenotypically distinguishable dsRNA-fed strain demonstrated an approximately 86% reduction in the MoDES1 transcripts (Figure 4B). Although conidiation rate was significantly higher, the treated strain produced a majority of short and broad deformed conidia (Figures 4C,E). Besides, an impediment was noted in appressorial penetration with respect to the untreated strain, although the rate of appressorium production was comparable in both strains (Figures 4D,E). In order to check ROS-scavenging activity, the treated and untreated strains were grown in media supplemented with ≥2 mM H₂O₂. The dsRNA-fed strain showed a marked reduction in culture diameter in all the concentrations 5 and 10 dpi (Figure 4A and Supplementary Figure 3), depicting the apparent reduction in ROS scavenging potential. As per previous studies conducted on the MoDES1 mutant, ROS sensitivity was due to impaired extra-cellular enzyme activity. Hence, enzymatic assays for laccases and peroxidases that are known to neutralize some free radicals were conducted. The dsRNA-fed strain that previously showed significant silencing of MoDES1 was observed to demonstrate a pronounced reduction in the aforementioned enzymatic activities in both the solid and liquid media (Figures 5A,B). This corresponded with the reduced ROS-scavenging activity that has been seen previously. The cell-wall integrity test, in terms of protoplast release assay, did not show any significant defects in cell wall integrity as a result of MoDES1 silencing (Supplementary Figure 4).

Based on previous studies, M. oryzae has been reported to use DES1 as a suppressor of basal defense for survival within rice. Also, it has an active role in the extension of the IH inside host cell and its colonization, which is essential for pathogenicity. As a proof-of-concept for the in vivo whole plant spray experiments, the in vitro impacts of dsDES1 on the M. oryzae wild-type strain were analyzed. Microscopy results of the onion peel assay showed that after 24 h, germination and IH development was defective in the dsDES1-fed strain as compared with the wild type. Most of the IH were thick, stout, and short, and confined to the primary host cell (Figures 5C,D), indicating impairment in the colonization of epidermal cells similar to the DES1 mutant (Chi et al., 2009). Our in vitro dsRNA feeding experiment indicated that the strain of M. oryzae that was fed with dsDES1 for two consecutive generations showed significant downregulation of MoDES1. Therefore, we further wanted to check if its virulence got affected because of dsRNA feeding. The detached leaves of 2-week-old rice seedlings were spot-inoculated with both the untreated and dsRNA-fed strains, and were observed for symptoms 5 dpi. While average lesion length in the case of the wild type was 12 mm, it was only 4.6 mm in the case of the leaves inoculated with the dsRNA-fed strain. Therefore, on an account of the silencing of MoDES1, there was almost a 62% reduction in lesion length (Figures 5E,F). As suggested by our previous results, this reduction in virulence could be attributed to the defects in IH and host colonization.

The dsRNA sprayed on the plant can be uptaken either directly by the fungus on the surface or via the plant cell post penetration. Since our results indicated that the silencing effect shows greater longevity in the wounded plants, it was assessed further if the fungus got silenced better via the plant cell. Four sets were kept, whereby no lag period was given in set I, and both the dsRNA and fungal spores were mixed together and sprayed. In set II, dsDES1 was sprayed on rice leaves 12 h prior to spray inoculation with the fungal spores; in set III, dsRNA was sprayed 24 h before the fungal inoculation to facilitate dsRNA uptake via the plant cells. Set IV was the control group where no dsDES1 was sprayed on the leaves before fungal infection. It was observed that in the plants where the dsRNA was sprayed 24 h before wild-type spray inoculation (set III), disease severity was around 60% less as compared with the set IV that was inoculated with wild type spores without any dsRNA treatment (set IV). On the contrary, when the wild-type spores were mixed together with dsRNA before spray-inoculation (set I), there was only a 25% reduction in symptoms, which was not significantly less than the untreated control (IV). The symptoms were milder in set II (where dsRNA and spores were sprayed 12 h apart) than in set I (Figure 6A). As studied by DAB staining of sheaths, the generation of in planta ROS indicated that the maximum number of heavily stained cells were observed in the plants that were sprayed with dsRNA first and then inoculated with fungal spores. In the plants where dsRNA and spores were mixed together and sprayed, the maximum number of cells showed medium staining or no staining, with lesser number of heavily stained cells (Figure 6B). This indicated lower levels of ROS-mediated resistance in the later. While in the first case, a lag period was given to ensure that the dsRNA would be uptaken by the host cell first, in the second case, the fungal spores were directly mixed with the dsRNA to ensure it takes up the dsRNA first. As our results indicated, the inhibitory effect of dsDES1 was more pronounced, leading to higher disease resistance, when the dsRNA was sprayed prior to fungal infection, allowing for a lag period of at least 12 h. Taken together with the finding from wounded leaves, it can be assumed that processing of sprayed dsRNA by the plant might have an important role in amplifying the sRNA signals.

After assessing the functionality of SIGS both in vitro and in vivo, we wanted to explore the efficiency of spray-induced downregulation of MoDES1 in developing resistance against fungal blast disease in whole plants. For this, Leaves of the susceptible CO-43 rice variety were sprayed with dsDES1 and inoculated with a 1 × 10⁵ spores/ml solution 24 h later (Figure 6C). The total RNA isolated 3 dpi from the dsRNA-sprayed leaves was analyzed for MoDES1 transcripts, and it was found to have only 35% of relative abundance of what was observed in unsprayed plants. The dsDES1-sprayed leaves that were spray-inoculated showed a relatively lesser number of lesions/cm² of infected leaves as compared with the control Tris-EDTA-treated (mock), spore-inoculated leaves. Similar results were obtained when the dsRNA-pre-treated leaves were punch- inoculated, whereby the area of lesions got drastically reduced (Figures 6D,E), with a notably reduced load of fungal biomass (Figure 7A). The fungal biomass was semi-quantitatively measured from the fungal specific 28S rRNA, with respect to the rice 25S rRNA, in both the sprayed and unsprayed infected leaves. When analyzed from the same amount of infected leaf tissue, the relative expression of 28S rRNA, both 6 and 8 dpi, was found to be lower in the sprayed set (Figure 7B). Since MoDES1 is known to suppress PTI, we wanted to check if the resistance developed via sprayed dsDES1 was due to innate host defense responses. The histochemical DAB and aniline blue staining of infected sheath sections showed that the host-derived ROS generation (Figure 7C), lignin deposition, and callose plug formation (Figures 7D,E) were more in the case of the dsRNA-sprayed sheaths. The microscopic analysis of dsRNA-sprayed infected sheaths showed that cell death response was at par with the resistance phenotype, as depicted by the trypan blue staining (Figure 7F). This corroborated with previous observations, as cell death is often a manifestation of mild HR, which is part of PTI in its mild form. The reduction in disease severity in the dsDES1 spray-treated set could, hence, be attributed to the reduced fungal load observed in the infected leaves.

The phytohormone salicylic acid-mediated pathway in the hemibiotrophic rice-blast pathosystem is central to the defense armor of the plant. Apart from early PTI responses, such as ROS-burst and callose deposition, the induction of defense-related PR genes on account of increased endogenous SA levels is a crucial indicator of a robust SAR response. OsPR1a is one such marker gene that has generally been used to assess defense responses in rice. Hence, its expression levels were checked in the dsRNA-treated and untreated infected (punch-inoculated) plants 2, 3, and 4 dpi. The semi-quantitative qPCR (Figure 8A) showed that 3 dpi, the dsDES1-treated leaves showed a greater accumulation of OsPR1a transcripts. The quantitative real-time PCR exhibited that, in comparison with the internal housekeeping OsACTIN, 2 dpi, the untreated plants showed a 2.5-fold change in the OsPR1a transcript, while there was a 64.8-fold change in the treated plants. While 3 dpi, the OsPR1a mRNA had twofold upregulation in the dsDES1-treated plants, 4 dpi, the relative expression of PR1a was found to be almost threefold higher in the sprayed set with respect to their corresponding untreated sets (Figure 8B). These observations were in support of the argument that the mild necrotic responses in the sprayed plants were a result of SAR, which was a manifestation of the sustained upregulation of PR gene.