Genome-Wide Expression Profiling of Small RNAs in Indian Strain of Rhizoctonia solani AG1-1A Reveals Differential Regulation of milRNAs during Pathogenesis and Crosstalk of Gene Regulation

Rhizoctonia solani AG1-1A is a necrotrophic fungus that causes sheath blight disease in rice. The reliable resistant source against this phytopathogenic fungus is not available in the gene pool of rice. Better understanding of pathogen genomics and gene regulatory networks are critical to devise alternate strategies for developing resistance against this noxious pathogen. In this study, miRNA-like RNAs (milRNAs) of an Indian strain of R. solani were identified by deep sequencing of small RNAs. We identified 128 known and 22 novel milRNAs from 20,963,123 sequence reads. These milRNAs showed 1725 target genes in the fungal genome which include genes associated with growth, development, pathogenesis and virulence of R. solani. Notably, these fungal milRNAs showed their target genes in host (rice) genome also which were later verified by qRT-PCR. The host target genes are associated with auxin metabolism, hypersensitive response, defense genes, and genes related to growth and development of rice. Osa-vacuolar-sorting receptor precursor: Rhi-milR-13, Osa-KANADI1:Rhi-milR-124, Osa-isoflavone reductase: Rhi-milR-135, Osa-nuclear transcription factor Y:Rhi-milR-131, Osa-NB-ARC domain containing protein: Rhi-milR-18, and Osa-OsFBX438: Rhi-milR-142 are notable potential regulons of host target genes: fungal milRNAs that need to be investigated for better understanding of the crosstalk of RNAi pathways between R. solani and rice. The detailed expression analysis of 17 milRNAs by qRT-PCR was analysed during infection at different time points of inoculation, at different growth stages of the host, in four different genotypes of the host, and also in four different strains of fungi which revealed differential regulation of milRNAs associated with pathogenesis and virulence. This study highlights several important findings on fungal milRNAs which need to be further studied and characterized to decipher the gene expression and regulation of this economically important phytopathogen.


Introduction
Rhizoctonia solani AG1-1AKühn (Teleomorph: Thanatephorus cucumeris (A.B. Frank) Donk.) is a basidiomycete semi-saprotrophic soil-borne fungal pathogen that causes sheath blight disease in rice. In Eastern Asia, R. solani causes a loss of 6 million tons of rice grains per year [1]. India suffers 10% yield loss in rice due to sheath blight disease every year [2]. The pathogen is present in the form of sclerotia or mycelium, and severely affects high yielding, semi-dwarf, and nitrogen-responsive rice cultivars. Initial symptoms are noticed on leaf sheaths near water level. Warm and humid weather, dense planting, and high nitrogen inputs are the favourable conditions for its aggravation. Until now, rice germplasm immune to R. solani has not been known. Among several other factors, poor understanding of pathogen biology is one of the key factors that hamper the development of resistant cultivars either through breeding or transgenic approaches. Recently, efforts have been made to understand the regulation of the genome and transcriptome of R. solani during pathogenesis [3][4][5].
Clear understanding of pathogen genomics and gene regulatory networks is extremely important for drawing effective strategies for resistance development against diseases. One such example is the rice-Xanthomonas oryzae pv. oryzae (Xoo) pathosystem where deeper understanding of bacterial TALE (transcription activator-like effector) genes and their interaction with rice SWEET genes has helped developing durable and broad spectrum bacterial blight-resistant rice genotypes through marker-assisted breeding and genome editing [6][7][8][9]. Genomics has been extremely useful to facilitate host-induced gene silencing (HIGS) technology for the control of Fusarium diseases through targeted disruption of key genes [10]. Recently, Dong and Ronald (2019) [11] presented comprehensive information on pathogen genes used for disease resistance development through genetic engineering, RNAi, and genome editing. In addition to genetic resistance, fungal genomics is crucial for fungicide development by identifying potential fungicide targets, their validation and mode of action, as demonstrated in several pathosystems [12]. For more than a decade, small RNAs have taken central stage in the area of genomics due to their versatile functions in gene regulation and metabolic pathways [13,14].
In the model fungi Neurospora crassa, four different types of miRNA-like RNAs (mil-RNAs) processed through a dicer-independent pathway were identified. The proteins responsible for the production of these milRNAs are different from the plants and animals to a large extent [24]. Considering the essential functions of miRNAs in gene regulation and metabolic processes, there is a necessity to study the fungal genome encoded milRNAs which would give different insights and roles of microRNAs in pathogenesis and disease development. To date, very few milRNAs have been reported in plant pathogenic fungi, which could be due to the low accumulation of milRNAs or the small number of cells at certain infection stages, and also due to lesser attention of researchers. Recently, Meng et al. (2021) [25] identified virulence-associated milRNAs from R. solani AG1-IA infecting maize crop. With this perspective, our current study focussed on discovering the milRNAs in R. solani causing sheath blight disease in rice. Genome-wide small RNAs isolated from a pure culture of the Indian strain of R. solani AG1-1Awere sequenced to identify the known and novel milRNAs. Later, the targets of these milRNAs were predicted in R. solani and its host (rice) genome. Expression of 17 fungal milRNAs was analyzed in infected tissue of rice at different time points of inoculation, in different genotypes and growth stages of rice, and also in different strains of fungi. Expression analysis of target genes present in fungal and rice genome suggested that milRNAs are critical regulators of host-pathogen interaction.

Fungal and Plant Material
In this study, four strains of R. solani AG1-IA were used. These strains were Wgl-2, Chn-1, Imph-2 and Lud-1 [26]. Four rice genotypes as a fungal host were used that include susceptible (TN1 and BPT5204) and resistant (Tetep and Pankaj) cultivars.

RNA Isolation and Sequencing
The highly virulent Indian strain of R. solani AG1-IA Wgl-2 was grown in growth medium having pectin as a carbon source. The fungal sclerotium was placed in liquid medium (0.7 g K 2 HPO 4 , 0.5 g KH 2 PO 4 , 0.5 g MgSO 4 , 0.01 g FeSO 4 , 0.001 g ZnSO 4 , and 10g pectin in 1 L distilled and autoclaved water) at 28 • C for 48-72 h as a still culture. The mycelium from three biological replicates was harvested and pooled to extract the RNA using TRIzol reagent (Invitrogen, Waltham, MA, USA). The quality of RNA was checked by Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA), Bioanalyzer (Agilent 2100), and agarose gel electrophoresis to check the integrity, concentration, and contamination. After quality control (QC), the sequencing library was constructed by TruSeq Small RNA Library Preparation Kit (Illumina, CA, USA). The cDNA library was prepared by sequencing adaptor ligation, reverse transcription, PCR enrichment, purification and size selection. The QC of library was analyzed by Qubit 2.0 (preliminary library concentration), Agilent 2100 (insert size), and qPCR (effective concentration of library). The QC passed libraries were fed into HiSeq 2500 sequencer for sequencing.

milRNAs Identification in R. solani
The sequence data were analyzed to identify the milRNAs in the R. solani genome. Clean reads without adaptorcontamination were mapped to reference genome available in the RSIADB database [27]. Further non-coding sRNAs like rRNA, snRNA, snoRNA, and tRNAs were removed by using the Rfamdatabase (https://rfam.xfam.org/, accessed on 1 July 2017). The mRNA sequences or coding transcripts were filtered using RSIADB database. Later, miRNA prediction was undertaken using miREAP and miRDeep2 software for identification of both known and novel milRNAs. The secondary structures of these potential novel milRNA precursors were predicted by the RNAfoldWebServer program (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi, accessed on 1 July 2017). Putatively novel milRNAs were identified based on the following criteria: (1) mature milRNA sequence should be in either of the arm of hairpin structure, (2) number of mismatches 4 or less, (3) no loop or break in milRNA sequences, (4) asymmetric bulges should be minimal in size (one or two bases) and frequency (typically one or less), especially within the milRNA/milRNA* duplex [3,20,28].

The Target Gene Prediction in R. solani and Rice Genome
The target genes of milRNAs in the R. solani genome were predicted by using Miranda software [21] and the RSIADB database. The target genes of fungal encoded milRNAs in rice genome were predicted by using the psRNA Target database [29]. The default criteria of the target prediction tool were used for identification of milRNA target genes.

Expression Analysis of Fungal milRNAs and Their Target Genes
Forty-five day old plants of rice cultivars TN1, Pankaj, BPT 5204, and Tetep were inoculated by R. solani AG1IA Wgl-2 strain. In parallel, TN1 was inoculated by three other strains, i.e., Chn-1, Imph-2 and Lud-1. For inoculation, the fungal sclerotium was placed in the sheath tissue of rice using a transparent cello tape. The sheath tissue was harvested from different rice genotypes (TN1, Pankaj, BPT 5204, and Tetep) infected by the Wgl-2 strain. Also, infected sheath tissue was harvested from TN1 at different time points of infection (18 h, 24 h, 48 h, 72 h, 96 h, and 5 days after inoculation) as well as from 45-(vegetative) and 80-(reproductive) day-old growth stages infected by the Wgl-2 strain. To analyze the strain specific milRNAs regulation, tissue was harvested from TN1 infected by three other strains (Chn-1, Imph-2, and Lud-1). Small RNAs and mRNAs were isolated from fungal infected rice tissue and pure culture of fungi (grown in potato dextrose agar medium) using the mirVana miRNA isolation kit (Ambion, TX, USA) according to the manufacturer's instructions. cDNA synthesis of normalized sRNAs and mRNAs was performed using the miScript II RT kit (Qiagen, Hilden, Germany) and Improm-II reverse transcription system (Promega, Madison, WI, USA), respectively. The respective cDNA was used as template for milRNA and target gene quantification using miScript SYBR Green PCR kit (Qiagen, Germany) and SYBR Premix Ex-Taq (Takara, Shiga, Japan), respectively. Three biological replicates and two technical replicates were used for all the qRT-PCR experiments. Following the manufacturer instructions, all the kit reagents along with the milRNA and gene-specific primers (Table S1) were used for setting up the qRT-PCR reaction in a CFX96 Real-Time System (BIO-RAD, Hercules, CA, USA). The temperature profile was followed as described in previous report [30]. To ensure specificity of amplified product, melt curve analysis and agarose gel electrophoresis were used. To quantify the relative expression of milRNAs and target genes, the comparative threshold cycle (C T ) method was followed. The relative expression was calculated from 2 −∆∆Ct . The standard error was calculated as reported previously [31].

Sequence Statistics
The milRNAs encoded by the R. solani AG1 IA genome were identified through Illumina deep sequencing of small RNAs. All the sequence reads were aligned to the reference genome of R. solani available in the RSIADB database [27] and the mapped clean reads were further used to identify the known and novel milRNAs. After deep sequencing of R. solani small RNAs 20,963,123 raw reads and 18,084,903 clean reads were obtained. The clean reads were used to filter 1,231,411 reads of 18-24 nts size of which 185,760 were unique reads. 2574 reads of R. solani aligned with hairpin sequences of known milRNAs [21], while 619,582 reads were utilized for prediction of novel milRNAs.

Identification of Target Genes in R. solani Genome
We identified 1725 target genes of 128 known and 22 novel milRNAs in the R. solani genome (Table S3). The known milRNAs targeting the genes encoding carbohydrate active enzymes, secretory proteins, and other critical proteins associated with fungal biology are listed (Tables 2 and S4). Also, the target genes of novel milRNAs were predicted (Table 3). Notably, Scaffold47_36931 showed the highest number (12) of target genes. Rhi-milR-141, Rhi-milR-120, Rhi-milR-41, and Rhi-milR-91 showed pectin esterase and pectate lyase domain-containing protein genes as their targets.

qRT-PCR Expression Analysis of milRNAs
Seventeen milRNAs (known and novel) were selected based on read count, target genes in R. solani, and also based on the target genes in rice. The relative expression of fungal encoded milRNAs during host (rice) infection was analyzed at different time points of infection, different age and genetic makeup of host tissue, and also in different strains of fungi. The number of expressed milRNAs varied in different experiments, therefore, only those milRNA were included which showed expression in at least one sample.

In Different Growth Stages of Host Plant
The expression of fungal encoded milRNAs was influenced by the growth stage of its host. All the milRNAs except Rhi-milR-16 showed down-regulation at both the growth stages during infection when compared with the expression level of milRNAs in fungi grown in PDA medium. Among the two growth stages, scaffold7_15416, Rhi-milR-160, and Rhi-milR-169 showed more expression at reproductive stage (80 days old) of host than vegetative (45 days old). Rhi-milR-43 showed down-regulation in a 45-dayold host and the expression levels further declined in an 80-day-old host. Interestingly, Rhi-milR-16 did not show expression in a 45-day-old host but was up-regulated in an 80-day-old host; whereas Rhi-milR-146 did not show expression in 80 days old host but was down-regulated in a 45-day-old host. Scaffold1_2221 and Rhi-milR-135 also showed stage specific expression, i.e., down-regulation in 45 and 80-day-old hosts, respectively. Scaffold7_15416, scaffold130_44481, scaffold47_37559, scaffold47_37005, Rhi-milR-43, Rhi-milR-35, Rhi-milR-160, and Rhi-milR-169 showed down-regulation in both 45-dayold and 80-day-old hosts ( Figure 3).

In Different Rice Genotypes
Expression of fungal-encoded milRNAs was analysed during the infection of four different genotypes of rice, i.e., Tetep, BPT5204, Pankaj, and TN1. Forty-five day-old plants of these genotypes were inoculated with Wgl-2 strain of R. solani and samples were collected after 5 days of inoculation. All the milRNAs except Rhi-milR-160 showed downregulation during infection of four rice genotypes when compared with the expression level of milRNAs in fungi grown in PDA medium. Seven milRNAs: scaffold7_15416, scaffold130_44481, scaffold47_37559, scaffold47_37005, Rhi-milR-35, Rhi-milR-160, and Rhi-milR-169 showed expression in all the genotypes. These milRNAs were down-regulated during infection of rice genotypes. Among these, expression of scaffold130_44481 was more in tolerant (Tetep and Pankaj) than the susceptible (TN1 and BPT5204) rice genotypes. Expression of scaffold47_37559 was greater in Pankaj than the other three genotypes while, Rhi-milR-169 expression was lesser in Pankaj than the other three genotypes. Rhi-milR-160 showed up-regulation in Tetep and BPT5204 but down-regulation in Pankaj and TN1. Rhi-milR-120 and Rhi-milR-135 showed exclusive expression in Pankaj while Rhi-milR-146 showed exclusive expression in TN1. Scaffold1_2221 expressed in all genotypes except BPT5204 ( Figure 4).

Expression Analysis of Fungal and Plant Target Genes
Expression of seven plant target genes and two fungal target genes was analysed in R. solani-infected rice (TN1) tissue. Along with the target genes, the expression of the respective regulatory milRNA was also analysed. Among the plant target genes, the regulons Osa-KANADI1:Rhi-milR-124, Osa-vacuolar-sorting receptor precursor:Rhi-milR-13, Osa-plastocyanin-like domain containing protein:Rhi-milR-22, and Osa-serine/threonineprotein kinase receptor precursor: Rhi-milR-51 showed anticipated reciprocal trend of expression. In these cases, the fungal encoded milRNAs and their respective plant target genes showed opposite trend of expression during the disease development (Figure 6a). Similarly, fungal genes AGA1IA_02889 and AGA1IA_07743 and their regulatory milRNAs Rhi-milR-141 and Rhi-milR-120 showed a contrasting expression pattern in infected tissue of rice (Figure 6b).

Discussion
MicroRNAs are regulatory genes that play critical roles in cellular processes of almost all eukaryotes [14]. It was believed that fungi do not possess microRNAs until the discovery of miRNA-like small RNAs (milRNAs) produced through at least four different biogenesis pathways in model fungi N. crassa [6,32]. Compared to plants and animals, there are very limited reports on milRNAs of phytopathogenic fungi which are primary agents causing diseases and huge loss to agriculture crops. In the absence of resistance in the rice germplasm against R. solani, it is extremely important to decipher the gene regulatory networks and biology of pathogens to devise effective strategies for controlling the menace of sheath blight disease in rice [31,[33][34][35]. We analyzed the milRNAs of sheath blight disease causing pathogen R. solani AG1 IA by RNAseq of small RNAs. A total of 128 known and 22 novel milRNAs were identified. Most of the milRNAs showed low abundance which is accordance with earlier study of fungal milRNAs [36]. Notably, known milRNAs-Rhi-milR-16, Rhi-milR-22, Rhi-milR-35, Rhi-milR-57, Rhi-milR-91, and Rhi-milR-112 and novel milRNAs-scaffold13_22000, scaffold47_37005, scaffold47_37523, scaffold47_37559, scaffold47_37341, scaffold47_37407, and scaffold47_36418 showed more abundance than other microRNAs suggesting their significant role in fungal gene regulation. The novel milRNA scaffold13_22000 showed >6000 read count which emphasizes that several novel players identified in this study might regulate fungal genome. Earlier studies suggested that such novel milRNAs may have crucial roles in pathogenesis and virulence of R. solani [21,25]. 1725 target genes of 128 known and 22 novel milRNAs were identified that are involved in various metabolic and developmental processes of fungi indicating that milRNAs are major regulators of the R. solani genome. The role of milRNAs in fungal growth and development has been recently described in Sclerotinia sclerotiorum [37]. A number of milRNAs were identified in the soil-borne fungal pathogen Verticillium dahlia causing wilt diseases in several agriculture crops. One of the milRNAs (VdmilRNA1) was demonstrated to mediate epigenetic repression of a virulence gene (VdHy1) in pathogenic fungi [38]. Furthermore, the role of milRNAs (CmmilR4 and CmilR16) in fruiting body development was demonstrated in Cordyceps militaris [39]. The milRNAs-Rhi-milR-141, Rhi-milR-120, Rhi-milR-41, and Rhi-milR-91 target pectinesterase and pectate lyase domaincontaining protein genes signifying their crucial role in plant cell wall degradation and pathogenesis. milRNAs of R. solani targeting carbohydrate active enzymes and other fungal genes including those involved in virulence and pathogen-host interaction were reported recently [25]. The genes encoding plant cell wall degrading enzymes of R. solani play significant role in disease initiation and establishment [4]. Vm-milR16 of Valsamali regulates the expression of pectinase genes during V. Mali-host interaction [40].
Interestingly, R. solani milRNAs showed their targets in the host (rice) genome also. A similar observation was made on R. solani targeting maize genes [25]. Recently, cross kingdom regulation of genes by miRNAs during host-pathogen interaction has been demonstrated in several pathosystems [41]. Bidirectional cross-kingdom transport of microRNAs or small RNAs through naked form, combined with RNA-binding proteins or enclosed by vesicles between fungal pathogens and its host plants has been reported [42]. In an interesting observation, Cui et al. (2019) [43] demonstrated that bba-milR1 encoded by pathogenic fungus Beauveria bassiana hijacks the RNA-interference machinery of a host (mosquito) to attenuate host immunity and facilitate infection. The fungus Sclerotinia sclerotiorum was reported to produce at least 374 distinct highly abundant sRNAs during infection of Arabidopsis thaliana and Phaseolus vulgaris. Targets of these small RNAs were significantly down-regulated during infection in A. thaliana [44].
Rhi-milR-124 targets Osa-KANADI1. In Arabidopsis, KANADI1 acts as a transcriptional repressor of genes involved in auxin biosynthesis, auxin transport, and auxin response [45]. Fungal pathogens control the auxin levels and auxin signalling pathways that significantly influence the defense network in plants [46]. In the wheat-Zymoseptoria tritici pathosystem, Ma et al. [47] showed that as an immune response, the fungal infection induced wheat small RNAs that regulate auxin-related genes. Rhi-milR-13 regulates Osa-vacuolar-sorting receptor (VSR) of rice which is responsible for targeting defenserelated soluble proteins to the vacuole. Plant vacuoles play an important role in defense by releasing hydrolytic enzymes and antimicrobial compounds upon pathogen infection that leads to programmed cell death due to hypersensitive response [48]. Suppression of rice VSR by R. solani milRNA might suppress the plant defense by inhibiting the hypersensitive response. Notably, the target probability in the case of Osa-vacuolar-sorting receptor precursorRhi-milR-13 is very high with expectedvalue of 1.0. Therefore, further probing of this milRNA-target gene regulation network can add significant information on host-pathogen molecular cross-talk. Rhi-milR-131 targets nuclear transcription factor Y (NFY) subunit of rice which is also regulated by the host miRNA, i.e., Osa-miR169. The over-expression of Osa-miR169 in rice led to repression of NFY resulting in hypersusceptibility of transgenic rice against blast fungus Magnaportheoryzae [49]. Repression of NFY by Rhi-milR-131 may weaken the defense response of rice against R. solani. Other host target genes of fungal milRNAs included isoflavone reductase, OsFBX438-F-box domain containing protein, NB-ARC domain containing protein, serine/threonine-protein kinase receptor, and rabGAP/TBC domain-containing protein which are involved in plant defense against pathogens [50][51][52][53][54]. R. solani-encoded milRNAs target maize defensegenesduring infection and negatively regulate resistance of maize [25]. Notably, the regulons such as Osa-KANADI1:Rhi-milR-124, Osa-vacuolar-sorting receptor precursor:Rhi-milR-13, Osa-plastocyanin-like domain containing protein:Rhi-milR-22, and Osa-serine/threonineprotein kinase receptor precursor: Rhi-milR-51 were verified by analysing their expression through qRT-PCR during host-fungal interaction and disease development. Recent reports demonstrate a role of cross-kingdom RNAi in regulation of genes associated with pathogen virulence and host resistance. For example, small RNAs of phytopathogenic fungi Botrytis cinerea trigger the silencing of immunity-related genes of its hosts A. Thaliana and Solanum lycopersicum [55]. A similar observation has also recently been reported inthe case of Puccinia triticina and P. striiformis [56,57]. In addition to plant defense genes, fungal milRNAs showed their target as genes associated with growth and the development of the host.
This study provides a first comprehensive analysis of milRNAs expression during pathogen infection as the expression of 17 milRNAs was analysed at different time points of infection, at different growth stages of the host, in different genotypes (with different genetic make-up) of host, and also in different strains of fungi. The down-regulation of all milRNAs except Rhi-milR-160 was noticed at all the time points of infection suggesting the induction of their target genes during the disease initiation and establishment. The differential regulation of milRNAs at different time points of infection suggests that fungal genes are regulated by milRNAs during disease initiation and progression. Scaffold130_44481, scaffold47_37005, Rhi-milR-35, and Rhi-milR-169 showed expression throughout all time points of infection indicating their central role in infection. Expression of Rhi-milR-22, Rhi-milR-43, Rhi-milR-120, and Rhi-milR-146 was observed specifically at 72 h, 5d, 48 h, and 5d respectively, suggesting that fungi recruit specific milRNAs as the infection progresses. During the infection and disease progression, R. solani requires mycotoxin production and penetration of mycelium inside the host cells. Studies in Aspergillus flavus and Fusarium oxysporum f. sp. Niveum highlighted the significant roles of milRNAs in the mycelium growth and mycotoxin biosynthesis [22,58]. Lin et al. (2016) [21] analysed the differential expression pattern of R. solani milRNAs during infection and suggested their roles in disease initiation and establishment [58]. Differential regulation of milRNAs during sclerotial development in Sclerotinia sclerotiorum was observed by analyzing small RNAs at different time points [59].
At the time of infection, expression of scaffold7_15416, Rhi-milR-160, and Rhi-milR-169 was more at the reproductive stage than vegetative stage of rice while Rhi-milR-16 did not show expression in vegetative stage but was up-regulated in the reproductive stage. By contrast, Rhi-milR-146 was down-regulated in the vegetative stage but no expression was observed in the reproductive stage. These results suggest that growth stage of host plant influences the milRNAs expression of fungi. Scaffold1_2221 was down-regulated in the vegetative stage while Rhi-milR-135 was down-regulated in reproductive stage, indicating their specific role during pathogenesis of different-aged hosts. Scaffold7_15416, scaffold130_44481, scaffold47_37559, scaffold47_37005, Rhi-milR-43, Rhi-milR-35, Rhi-milR-160, and Rhi-milR-169 were down-regulated at both the growth stages signifying their essential roles during infection.
Expression of seven-milRNAs-scaffold7_15416, scaffold130_44481, scaffold47_37559, scaffold47_37005, Rhi-milR-35, Rhi-milR-160, and Rhi-milR-169 was down-regulated while infecting the four rice genotypes suggesting their key roles in regulating the genes during infection. Expression of scaffold130_44481 and scaffold47_37559 was greater in tolerant genotypes than susceptible rice genotypes while Rhi-milR-120 and Rhi-milR-135 showed exclusive expression in Pankaj indicating that regulation by milRNAs is significantly influenced by host genetic architecture. Expression of other milRNAs also varied in different genotypes of its host.
Fungal genome-encoded microRNA-like RNAs are relatively less explored genetic elements that play a major role in regulation and expression of genes associated with growth, development, pathogenesis, and virulence. We made a comprehensive effort by combining RNAseq and qRT-PCR assays to decipher the milRNAs of an Indian strain of R. solani AG1-1A causing sheath blight disease of rice. Several milRNA candidates targeting key genes of R. solani were identified which need to be further characterized to understand their specific biological roles. The novel milRNAs are significant additions to understanding of the complexity of gene regulation of R. solani. The novel milRNA scaffold13_22000 showing very high expression needs to be characterized for its role in pathogen-host interaction and fungal metabolism. Further probing the regulation of rice genes by fungal milRNAs will be a milestone in understanding the molecular basis of host-pathogen interaction. Some of the gene regulatory networks such as Osa-KANADI1:Rhi-milR-124, Osa-vacuolar-sorting receptor precursor:Rhi-milR-13, and Osa-nuclear transcription factor Y:Rhi-milR-131 can be studied on priority. Also, the better understanding regulation of rice defense genes by R. solani milRNAs may help in executing more effective strategies against this deadly plant pathogen. Detailed expression analysis of milRNAs at different time points of infection, at different growth stages of hosts, in different genotypes of hosts, and in different strains of fungi provide ample information to plan future studies for understanding the gene regulation mechanism of R. solani. Our results indicate that milRNAs may contribute to almost all the metabolic processes of the R. solani pathogen.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/jof7070561/s1, Table S1: List of primers used in this study for qRT-PCR assays. Table S2: List of known milRNAs identified in this study. Table S3: List of target genes of known and novel milRNAs. Table S4: Gene ontology analysis of selected target genes. Data Availability Statement: All data generated or analysed during this study are included in this published article (and its Supplementary Information files). The sequence data is available on request.