IDENTIFICATION AND VALIDATION OF MIRNAS AND THEIR TARGETS THAT REGULATE THE RESISTANCE GENES AGAINST FUSARIUM WILT IN TOMATO

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INTRODUCTION
MicroRNAs (miRNAs) are non-coding single-stranded RNA molecules with a length of 20-24 nucleotides (nt) (Llave et al., 2002;Reinhart et al., 2000;Park et al., 2002;Pan et al., 2017;Huang et al., 2019). miRNAs are important in a variety of biological processes in plants, including floral morphogenesis, hormone balance, reproductive performance, and regulating growth and plant responses to abiotic (e.g., drought, salinity, and temperature) and biotic (e.g., pathogens such as fungi, bacteria, and viruses). Furthermore, in of presence of nutritional deficiency, miRNAs organize the metabolic balance of sulphur, copper, and phosphorus in the plants (Sunkar et al., 2007;Padmanabhan et al., 2009;Rubio-Somoza et al., 2009;Chen and Cao, 2015;Xu et al., 2021). During the pathogen invasion, the two-lined immune system is activated in plants to protect them from pathogens. The first line is called pathogen-associated molecular patterns (PAMPs), which discovers conserved molecular characteristics of pathogens, to trigger immunity (pathogen-associated molecular patterntriggered immunity [PTI]) that prevents infection of the host with pathogens (Zipfel and Felix, 2005;Boller and Felix, 2009). Successful microbes transfer effectors inside the host to inhibit PTI and establish the pathogen (Dou and Zhou, 2012). In turn, the second line of plant immunity, termed effector-triggered immunity (ETI), is initiated upon the recognition of effectors by the cognate intracellular immune receptors, such as nucleotidebinding site-leucine-rich repeat (NBS-LRR)-type proteins (Gao et al., 2021). ETI is commonly associated with a hypersensitive reaction that results in programmed cell death at the infection site to prevent microbe spread (Jones and Dangl, 2006). PTI and ETI provide pre-and post-invasive resistance to the host in plant-fungus interactions, respectively. Therefore, small RNAs are needed in both PTI and ETI signaling and are represented as key fine-tuning regulators (Katiyar-Agarwal and Jin, 2010;Huang et al., 2016;Zhang et al., 2016). The tomato (Solanum lycopersicum) belongs to the Solanaceae family. It is the main vegetable crop in Egypt and worldwide. However, tomatoes are greatly affected by various biotic stress factors which result in crop loss. Fusarium oxysporum f. sp. lycopersici a soil-borne plant pathogen, is an ascomycetous hemibiotrophic fungus that causes root infection by colonizing in the xylem vessels leading to wilt. Several miRNAs have been shown to respond to F. oxysporum f. sp. lycopersici infection in tomato (Jin and Wu, 2015;Srinivas et al., 2019). Information about miRNA function in the plant immune system against F. oxysporum f. sp. lycopersici is limited. Therefore, this study was carried out to identify and validate F. oxysporum f. sp. lycopersici-responsive miRNAs from the roots of tomato plants.
In this study, we studied the role of miRNAs in the defensive response against a fungal pathogen, Fusarium oxysporum f. sp. lycopersici, which causes wilt disease in tomatoes. Furthermore, the expression patterns of two novel miRNAs (miR30 and miR33) and their targets were validated by qRT-PCR. Moreover, two new miRNAs were further sequenced.

MATERIALS AND METHODS Fungal Strain, Plant Material and Culture Conditions
F. oxysporum f. sp. lycopersici wild-type strain 4471 and the seeds of tomato cv. Pusa Early Dwarf were obtained from Indian Agriculture Research Institute (IARI-New Delhi). Fusarium strain was maintained on potato dextrose agar (PDA) at 28°C in the dark (Tetorya and Rajam, 2021).

Inoculation of Tomato Plants
A tomato cultivar Pusa Early Dwarf (PED) was used for plant infection with the root pathogen F. oxysporum f. sp. lycopersici strain 4471. Two-month-old tomato seedlings were grown at 282°C with a 16/8-h photoperiod. Roots of tomato plants were removed from the soil and incubated for 30 min in a solution of F. oxysporum f. sp lycopersici conidia at a concentration of 1x10 6 /ml (Singh et al., 2020). Mock-inoculated tomato plants (control tomato plants) were treated with water. Tomato plants were then replanted in soil and kept in a growth room at 25°C/24 h with constant light. The plants were then removed from the soil, and the roots were rinsed and excised with a sterile blade. The roots were frozen in liquid nitrogen and stored at -80°C.

Prediction of miRNA and Target Genes
Prediction of the miRNA target gene was conducted by following the guidelines described by Allen, (2005) (http://ted.bti.cornell.edu/cgibin/TFGD/sRNA/miRNA.cgi). To identify the conserved miRNAs in tomato plants, sRNA sequences obtained by deep sequencing were compared with known mature plant miRNAs in miRBase software (Griffiths-Jones et al., 2007). After homology searches and further sequence analysis, a total of 103 conserved miRNAs from 24 different miRNA families were discovered, and all of which can be referred to in the important database (http://ted.bti.cornell.edu/cgibin/TFGD/sRNA/miRNA.cgi).

Designing oligonucleotide primers for miRNAs and target genes
The primers used in this work were designed by the OligoAnalyzer tool and are listed in Table 1. Isolation of Total RNA Total RNAs were isolated from uninfected control and F. oxysporium f. sp. lycopersici infected S. lycopersicum roots cv. Pusa Early Dwarf by TRIzol reagent according to the manufacturer's recommended protocol (Sigma, USA), followed by RNase-free DNase treatment. The quality and quantity of total RNAs were computed using a Nano-Drop ND-1000 spectrophotometer (Nano-Drop Technologies Inc., Wilmington, DE).

Stem-loop reverse transcription PCR (RT-PCR)
The total RNA (1 µg/sample) and stem-loop RT primers were mixed in 10 µl reaction at room temperature (RT) and incubated at 65°C for 10 min, followed by a quick transfer to ice. This step was performed to relieve all secondary structures in the mixture and promote better annealing of the primers during cDNA synthesis. The DNaseI-treated total RNA was then reverse-transcribed using the RT-PCR kit (Thermo Scientific, USA). The cDNA was then used for the amplification of miRNAs using the following reaction mixture of 14.3 µl sterile dsH2O, 2 µl 10X PCR buffer HF (Promega Corp.), 1.5 µl dNTPs (10 mM), the two primer combinations (0.5 µl each) (0.5 µM), 0.2 µl Taq DNA polymerase (Thermo Scientific, UK), and 1 µl of cDNA template (~400 ng) and PCR cycles of 16°C for 30 min; 60 cycles of 30°C for 30 s, 42°C for 30 s, 50°C for 15 s and 85°C for 5 min. For PCR amplification of the target genes, the cycle was 25°C/5 min; 42°C/60 min and 70°C/5 min.

Quantitative RT-PCR (qRT-PCR)
MicroRNAs were detected with an all-in-one TM miRNA qRT-PCR detection kit according to the manufacturer's instructions (Thermo Scientific, USA). The qRT-PCR was carried out using a SYBR Green I Master Mix from Roche on the Light Cycler 480 II Real-Time PCR (Roche). The reaction mix was prepared in a final volume of 10 μl containing 1X Master Mix, 1 pmol of each of the forward and reverse primers. Specifically designed forward primers for each individual miRNA and reverse primers were used for qRT-PCR reactions (Fiedler et al., 2010;Yanik et al., 2013). The sequences of the primers used in the qRT-PCR are mentioned in Table 1. The qRT-PCR conditions for miRNAs were set up as follows: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, 60°C for 1 min, 95°C for 15 s and 60°C for 15 s. Target genes were analyzed using the following conditions, 50°C for 2 min, 95°C for 10 min; 40 cycles of 95°C for 15 s and 55°C for 1 min. The experiments were performed in four replicates and technical repeats.

Agarose Gel Electrophoresis
The amplified PCR products for miRNAs and target genes were electrophoresed on 5% and 2% agarose respectively, containing ethidium bromide (0.5 μg/ml) in 1X TBE buffer at 75 volts and visualized using a UV transilluminator. The size of each amplicon was determined with reference to a GeneRuler Ultra Low Range DNA Ladder (Thermo Scientific, USA).

Validation of miRNAs
Four miRNAs (miR30, miR33, miR46, and miR49) were validated by miRBase v. 20 (http://www.mirbase.org) to provide integrated interfaces to comprehensive microRNA sequence data, annotation, and predicted gene targets. Cloning of the miRNA30 and miRNA33 Genes Two fragments of the expected size, 55 bp for two new miRNAs (miR30 and miR33) were cut from the agarose gels and further purified using a Gene Jet Gel DNA purification kit (Thermo Scientific, USA). The concentrations of the purified products were measured by a NanoDrop ND-1000 spectrophotometer. The purified DNA fragments were ligated into pGEM®-T Easy vector (Promega, Mannheim, Germany). Ligated plasmids were transformed into E. coli XL-1 Blue competent cells. Isolation of plasmid DNA from the putative colonies obtained was done by the alkaline lysis method according to Sambrook et al. (1989). Plasmid DNA was digested by EcoRI restriction enzyme at 37ºC overnight to confirm the presence of positive intact clones.

Sequencing
The positive clones of two new miRNAs (miR30 and miR33) were further sequenced by Applied Biosystems (Inst model/Name 3100/3130XL-1468-009, India) using gene-specific primers. The sequence was aligned with corresponding sequences from the database using miRBase software.

The Secondary Structure of miRNAs
The secondary structures of miR30 and miR33 sequences obtained after cloning were predicted by Vienna RNA package program V.1.7. Institute for Theoretical Chemistry, University of Vienna (http://rna.tbi.univie.ac.at//cgi-bin/RNA), which uses a free energy minimization algorithm (Mathews and Turner, 2006).

Statistical Analysis
The qRT-PCR results are expressed as a standard deviation. Statistical analysis was carried out to determine P values by one-tailed paired Student's t-test analysis using Microsoft Excel 2019. P values  0.05 were considered to be statistically significant.

Expression Profiling of F. oxysporum f. sp. lycopersici-Responsive miRNAs and Target Genes in Tomato
We have investigated the expression of miRNAs in tomato roots (cv. PED -moderately susceptible) during infection with the wilt fungus F. oxysporum f. sp. lycopersici, compared with the control (treated with water) (Figure 1). Our goal was to identify differentially expressed miRNAs in tomato upon infection with the pathogen. The literature survey and in silico analysis provided with four miRNAs in tomato, two novel (miR30 and miR33) and two known (miR46 and miR49) and their target genes. Therefore, the expression pattern of four miRNAs (miR30, miR33, miR46, and miR49) and two target genes (p4 and β-1,3-glucanase) was analyzed in tomato plants infected with F. oxysporum f. sp. lycopersici and control by qRT-PCR 30 dpi (days post-inoculation). The expression levels were dramatically changed during the infection (Figure 2). The transcript level of miR30 was not significantly altered in tomato plants infected with the pathogen (fold= 3.61), compared with the control. However, miR46 was down-regulated with a fold decrease of 33.33, upon fungal infection, compared with the control. On the contrary, miR33 and miR49 were upregulated, with a fold increase in the expression of 0.4 and 0.15, respectively. The target gene's relative expression compared with control displayed down-regulation with a fold decrease of 2.43 and 9.09 for p4 and β-1,3 glucanase, respectively. The target genes showed an inverse correlation in expression with miR30 and miR46 ( Figure  2). This expression pattern suggests that the target mRNAs are susceptible factors during plant defense in tomato (cv. PED) upon infection (Figure 2). Figure 2. Relative expression of four miRNAs (a) and two target genes (b) in tomato cultivar PED using qRT-PCR, compared with the control. Expression was normalized to that of Actin. All values represent as standard deviation of results obtained with four replicates in each group (n=4).*P value  0.05, **P value  0.01, ***P value  0.001, ns= not significant. (p4 and β-1,3-

glucanase) and miRNA
The amplicon size of target gene p4 was 106 bp ( Figure 3A), while the fragment sizes of target gene β-1,3-glucanase were 107 and 115 bp, were obtained by using two different gene-specific primers ( Figures 3B  and 3C). The four miRNAs (miR30, miR33, miR46, and miR49) were amplified with primers designed to anneal to undergo the stem-loop RT-PCR. Four miRNAs were amplified with the expected product size ~55 bp with no additional non-specific amplicons (Figure 4).

Cloning of miR30 and miR33
Since miR30 and miR33 were novel miRNAs, the sequence validation was carried out by cloning the miRNAs. Five colonies were screened for the desired insert. All colonies having miR30 and miR33 gave one specific band with a molecular size of ~55 bp (Figures S1 and S2). A positive colony was then utilized for restriction digestion using EcoRI, which showed the expected size of 55 bp release of miR30 and miR33 ( Figures S3 and S4).

Multiple Sequence Alignments
The sequence analysis using BLAST showed % similarity with the sequence reported in the database. The nucleotide sequences of miR30 and miR33 were 20 and 21 nt, respectively ( Figure S5). Unknown readings were analyzed with miRBase (Release 21.0) to find miRNA homologs. Homology analysis showed that miR30 and miR33 had 100% homology to mature conserved miRNA166 found in various plant species such as Arabidopsis lyrata, Hordeum vulgare, Citrus sinensis, Phaseolus vulgaris, Glycine max, Nicotiana tabacum, Helianthus paradoxus, Cucumis melo, Linumusit atissimum, Solanum tuberosum, and Prunus persica. The Primary Sequence and Secondary Structure of miRNAs The primary structures of two novel miRNAs (miR30 and miR33) were determined using the sequences ( Figure S5), while the secondary structures were predicted by the Vienna RNA package program V.1.7 ( Figure 5). Two miRNAs formed an intra-molecular base pairing among their bases, which included interior loops, hairpin loops, and external loops ( Figure 5 and Table 2). The bracket notation for miR30 and miR33 secondary structures was represented in the space-efficient bracket notation ( Figure S5). The symbols « ( and ) » correspond to the 5′ and 3̀′ bases in the base-pair, respectively. However, « . » represents an unpaired base. On the other hand, it was observed that miR30 (20 bp) has a mutation in the stem-loop region. This mutation was found between G and U, while miR33 (21 bp) has not displayed any mutations ( Figure 5).

Prediction of miRNA Secondary Structure using Minimal Free Energy (MFE)
The secondary structures of miR30 and miR33 were predicted from their primary sequences by summing the energy contribution of all base pairs, hairpin loops, external loops, and interior loops at 37C. Therefore, the MFE algorithm, which estimates experimental thermodynamic values was used to calculate the miRNA secondary structures. Table 2 illustrates ∆G  37 (Gibbs free energy) of external loops, interior loops, and hairpins depending on the predicted free-energy values (kcal/mol at 37C). The total free energy of miR30 and miR33 was found to be -1.2 and -0.4 kcal/mol respectively, using Vienna RNA package V.1.7. (Table 2).

DISCUSSION
Both plants and animals have miRNAs, which are a class of small RNAs. The miRNAs are single-stranded RNA molecules, non-coding, and composed of ~20-24 nucleotides in length. miRNAs are generated from stemloop structures harbored in primary miRNA transcripts (Bartel, 2004). miRNA links to its mRNA target primarily through a sequence complementary to the 3′untranslated region (3′UTR), triggering the case of either translational suppression or degradation of the mRNA transcript. In this study, the role of miRNAs in the defense response of tomato plants against fungus F. oxysporum f. sp. lycopersici was studied. The expression of four miRNAs, two unknown (miR30 and miR33) and two known miRNAs (miR46 and miR49) related to disease development and the two target genes p4 and β-1,3glucanse in tomato were quantified by qRT-PCR. Furthermore, qRT-PCR results showed variations in their expression, wherein miR46 was down-regulated and miR33 and miR49 were up-regulated compared with the control. However, miR30 was not significantly changed in tomato plants infected with F. oxysporum f. sp lycopersici, compared with the control. Moreover, the expression of two target mRNAs (p4) and (β-1,3glucanase) displayed a negative relationship in the expression with their corresponding miRNAs (miR46) (miR33 and miR49), respectively. On the contrary, there were no significant differences in the level of expression among the other genes and mock. Therefore, miR33 and miR49 may have influenced the expression of their target genes β-1,3-glucanase in the presence of fungal infection, the latter belongs to pathogenesis-related proteins (PR), which causes inhibition of root pathogens. Furthermore, β-1,3-glucanase is an important plant defense enzyme involved in F. oxysporum f. sp. lycopersici fungus cell wall destruction (Kavroulakis et al., 2005). Also, our results showed that tomato variety PE is moderately susceptible to Fusarium wilt because its resistance is not well-expressed enough to be attributed to miRNAs. These results were in agreement with Chopada Chopada et al. (2014) who mentioned that tomato variety PED is moderately susceptible to Fusarium wilt infection with a percent wilt incidence of 66.67%. Ouyang et al. (2014); Ji et al. (2021) investigated the production of miRNAs after infection with the wilt fungus F. oxysporum f. sp lycopersici in two tomato cultivars, Motelle (resistant) and Moneymaker (susceptible). The results of the study showed that target mRNA was up-regulated in a resistant cultivar Motelle as a defense response against fungus, but not Moneymarker after infection. In addition, the miRNAs were down-regulated in the resistant cultivar Motelle after infection, but the study did not identify any miRNAs that were decreased or increased after infection in the susceptible cultivar Moneymarker. Our findings showed that plant miRNAs are important in the defense response against fungal infections and provide a platform for differentially expressed miRNAs in tomato after infection with fungus. In the present work, the full length of two novel miRNAs (miR30 and miR33), from tomato infected with F. oxysporum f. sp. lycopersici was 20 and 21 nt, respectively. These findings were in agreement with those obtained by Chen et al. (2009) Jones-Rhoades (2012); Sun (2012) and Cardoso et al. (2018) who reported that miRNAs in plant species are conserved as well as nonconserved (unique) and belong to miRNA families. For example, miR156 is recognized as a conserved class that is known in several plant species. Zhang et al. (2008) identified miRNAs from many plant species against tomato nucleotide sequences and predicted 13 miRNA candidates grouped into nine miRNA families (miR159, miR157, miR167, miR162, miR172, miR395, miR171, miR399, and miR319) from over 57,8000 tomato sequences. Furthermore, mature miRNAs as well as miR171, miR162, and miR319 precursors have been cloned (Itaya et al., 2008). Yin et al. (2008) identified 21 conserved miRNAs from 14 miRNA classes (miR159, miR160, miR156/157, miR162, miR168, miR169, miR399, miR172, miR167, miR403, miR869.1, miR1030, miR437, and miR830), seven of which were known in the Expressed Sequence Tag (EST) database and 14 in the Genome Survey Sequences (GSS) database, while some of them were not found in the S. lycopersicum (Zuo et al., 2011). The entire class of miRNAs has been identified in cultivated tomato through whole-genome sequencing. However, a few numbers of miRNAs have been found to be involved in tomato-specific processes, like fruit ripening (Sato et al., 2012;Wang et al., 2011). In the current study, the total free energy, or Gibbs free energy (ΔG) of miR30 and miR33 was -1.2 and -0.4 kcal/mol respectively, predicated by the Vienna RNA package program V.1.7. These results are in accordance with Trotta (2014) who mentioned that minimum free energy (MFE) has been applied for different aims. For instance, normalization was applied to progress the secondary structure prediction by removing fragments with normalized equilibrium free energies less than a threshold value. Normalized MFE was also applied to compare evolutionary relationships between miRNA genes and their functions (Zhu et al., 2012), and its advantage in identifying new non-coding RNAs was compared with other criteria (Eva and Vincent, 2005). Free energy can be applied as a measure of biological system stability. If the binding of a miRNA: target mRNA interaction is predicated to be stable, it is considered more probable to be a true target of the miRNA (Yue et al., 2009). Normalized MFE assisted in indicating thermodynamic variables between nuclear-encoded miRNAs located in the cytosol and mitochondria (Bandiera et al., 2011). The MFE was used in the search to predict actual miRNAs precursors (Loong and Mishra, 2007), to improve RNA folding prediction algorithms, and for comparing the thermodynamic stability (Ni et al., 2010).

CONCLUSIONS
In summary, a total of four different miRNAs: two novel miRNAs (miR30 and miR33) and two known ones (miR46 and miR49) were identified from tomato cv. PED infected with F. oxysporum f. sp. lycopersici. After infection with the pathogen, qRT-PCR analysis revealed that the expression of four miRNAs and two target genes (p4 and 1,3-glucanase) were differentially expressed. We also observed that (p4) and (β-1,3-glucanase) displayed an inverse relationship in expression with their corresponding miRNAs (miR46) (miR33 and miR49) respectively. As a result, tomato cultivar PED appeared to be moderately susceptible to fungus because its resistance was not expressed sufficiently to be attributed to miRNAs. The result of this work could improve our understanding of the role of miRNAs in resistance to fusarium wilt disease in tomato.     Figure S5. Nucleotide sequencings of primarily and secondary structure (by dot brackt notation) of miRNA30 (20 nt) and miRNA33 (21 nt). « (and) » corresponding to the ' 5 and 3̀ bases in the base-pair, respectively, « . » represents an unpaired base.
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