Overexpression of Sly-miR398b Compromises Disease Resistance against Botrytis cinerea through Regulating ROS Homeostasis and JA-Related Defense Genes in Tomato

MicroRNAs (miRNAs) have been shown to be critical components in plant immunity. MicroRNA398 (miR398) is a highly conserved miRNA in all land plants and plays crucial roles in diverse biotic stress responses. However, the role of miR398 has not yet been characterized in tomato resistance against Botrytis cinerea. In this report, the transcript levels of sly-miR398b were strongly decreased in B. cinerea-infected leaves and the overexpression of sly-miR398b resulted in enhanced susceptibility. The attenuated expression of cytosol Cu/Zn-SOD (CSD1), chloroplast Cu/Zn-SOD (CSD2), and guaiacol peroxidase (GPOD), as well as the decreased activities of superoxide dismutase (SOD) and GPOD, collectively led to increased hydrogen peroxide (H2O2) accumulation in sly-miR398b overexpressing plants. Furthermore, sly-miR398b was induced by methyl jasmonate (MeJA) treatment. The overexpression of sly-miR398b suppressed the expression of TomLoxD, LapA, and PR-STH2 in response to B. cinerea and MeJA treatment. Our data demonstrate that sly-miR398b overexpression negatively regulates the resistance to B. cinerea in tomato by inducing the accumulation of reactive oxygen species (ROS) and downregulating the expression of MeJA-responsive defense genes.


Introduction
Tomato (Solanum lycopersicum) is the most important vegetable crop worldwide (Food and Agriculture Organization of the United Nations; http://faostat.fao.org/site/339/ default.aspx (accessed on 20 January 2022)). With the improvement in people's living standards and the increasing demand for fruits and vegetables, the annual supply of fruits and vegetables is becoming more and more important. Due to low temperatures, high humidity, low light, and other reasons, overwinter-grown tomatoes and greenhouse-grown tomatoes are prone to Botrytis cinerea infection. Botrytis species are responsible for gray mold rot and cause devastating diseases and significant crop losses in over 1000 plant species, including tomato, grape, and strawberry [1]. A large number of studies have shown that most cultivated tomato varieties are susceptible to B. cinerea [2], causing about 20-40% losses in tomato crops, which could rise to 50-70% during the disease epidemic periods [3].
Plant pathogens could be classified into necrotrophs, hemi-biotrophs, and biotrophs based on their lifestyles. In response to different invasion modes of pathogens, plants have evolved complex and fine defense response pathways controlled by plant hormones such as jasmonic acid (JA) and salicylic acid (SA). It is generally believed that plants use the SAmediated signaling pathway to defend against biotrophic and hemibiotrophic pathogens, and use the JA-mediated signaling pathway to defend against necrotrophic pathogens such pathosystems. In addition, given the role of the JA pathway in this interaction, a possible molecular connection between miR398 and JA signaling also seems worthy of investigation.
The aim of this work was to characterize the function of sly-miR398b in tomato defense against B. cinerea. We first analyzed the expression in response to infection and then investigated the effects of sly-miR398b overexpression on resistance. In addition, the activities and gene expression of defense enzymes, as well as the transcripts of JAresponsive genes, were also measured. Our results in this report extend the knowledge of the role of miR398 in tomato-B. cinerea interactions.

Effects of B. cinerea Infection on the Expression of Pri-miR398b and Sly-miR398b
To elucidate the role of sly-miR398b in plant resistance, we first assessed MIR398b expression profiles by quantifying pri-miR398b in tomato leaves upon B. cinerea infection via a quantitative reverse transcription PCR (qRT-PCR) assay. We also quantified the effects of mature sly-miR398b in tomato leaves upon B. cinerea infection via a stem-loop qRT-PCR assay. As the results showed that the amounts of pri-miR398b and sly-miR398b in tomato leaves decreased during infection ( Figure 1A,B), we thus postulated that sly-miR398b might participate in regulating tomato immunity against B. cinerea.
can possibly be due to connections with different pathogen lifestyles. Whether miR398 participates in regulating tomato defense against B. cinerea and its role in tomato-B. cinerea interaction remains unknown, and needs to be verified experimentally without simply relying on results obtained in different pathosystems. In addition, given the role of the JA pathway in this interaction, a possible molecular connection between miR398 and JA signaling also seems worthy of investigation.
The aim of this work was to characterize the function of sly-miR398b in tomato defense against B. cinerea. We first analyzed the expression in response to infection and then investigated the effects of sly-miR398b overexpression on resistance. In addition, the activities and gene expression of defense enzymes, as well as the transcripts of JA-responsive genes, were also measured. Our results in this report extend the knowledge of the role of miR398 in tomato-B. cinerea interactions.

Effects of B. cinerea Infection on the Expression of Pri-miR398b and Sly-miR398b
To elucidate the role of sly-miR398b in plant resistance, we first assessed MIR398b expression profiles by quantifying pri-miR398b in tomato leaves upon B. cinerea infection via a quantitative reverse transcription PCR (qRT-PCR) assay. We also quantified the effects of mature sly-miR398b in tomato leaves upon B. cinerea infection via a stem-loop qRT-PCR assay. As the results showed that the amounts of pri-miR398b and sly-miR398b in tomato leaves decreased during infection ( Figure 1A,B), we thus postulated that sly-miR398b might participate in regulating tomato immunity against B. cinerea. Figure 1. Effects of B. cinerea infection on pri-miR398b (A) and sly-miR398b levels (B). Four-weekold tomato plants were spotted with a 5-µL spore suspension (10 6 spores/mL) of B. cinerea or with 1% Sabouraud Maltose Broth (mock). Leaf samples were collected 48 h after inoculation. For (A,B), relative levels were calculated by comparing with the corresponding values of uninfected leaves (mock). SlACTIN was used as a reference gene for pri-miR398b in qRT-PCR. Tomato U6 was used as a reference gene for sly-miR398b in a stem-loop qRT-PCR assay. Values presented are the means ± SD from three biological replicates. Asterisks indicate significant differences detected using Student's t test (*, p < 0.05) when compared with uninfected leaves (mock).

Overexpression of Sly-miR398b Enhances Tomato Susceptibility to B. cinerea
To investigate whether sly-miR398b would affect the immune function of tomato regarding B. cinerea, we obtained sly-miR398b-overexpression (sly-MIR398b#OE) transgenic plants and pBI121#OE control plants in the cultivar Micro-Tom [38]. The transcript level of pri-miR398b and sly-miR398b in sly-MIR398b#OE was noticeably higher than that in the pBI121#OE control plants (Figure 2A,B). The detached leaves from four-week-old tomato plants were incubated with B. cinerea spore suspensions for 48 h. The results indicated that B. cinerea infection leads to significantly larger necrotic lesions in sly-MIR398b#OE plants than in the pBI121#OE control plants ( Figure 2C,D). Together, these results substantiate sly-miR398b overexpression negatively regulating tomato resistance against B. cinerea infection. Figure 1. Effects of B. cinerea infection on pri-miR398b (A) and sly-miR398b levels (B). Four-week-old tomato plants were spotted with a 5-µL spore suspension (10 6 spores/mL) of B. cinerea or with 1% Sabouraud Maltose Broth (mock). Leaf samples were collected 48 h after inoculation. For (A,B), relative levels were calculated by comparing with the corresponding values of uninfected leaves (mock). SlACTIN was used as a reference gene for pri-miR398b in qRT-PCR. Tomato U6 was used as a reference gene for sly-miR398b in a stem-loop qRT-PCR assay. Values presented are the means ± SD from three biological replicates. Asterisks indicate significant differences detected using Student's t test (*, p < 0.05) when compared with uninfected leaves (mock).

Overexpression of Sly-miR398b Enhances Tomato Susceptibility to B. cinerea
To investigate whether sly-miR398b would affect the immune function of tomato regarding B. cinerea, we obtained sly-miR398b-overexpression (sly-MIR398b#OE) transgenic plants and pBI121#OE control plants in the cultivar Micro-Tom [38]. The transcript level of pri-miR398b and sly-miR398b in sly-MIR398b#OE was noticeably higher than that in the pBI121#OE control plants (Figure 2A  Total RNAs were extracted from 18-day-old seedlings. SlACTIN was used as reference gene for pri-miR398b. Tomato U6 was used as reference gene for sly-miR398b. Values presented are the means ± SD from three biological replicates. ** above the columns indicate significant differences at p < 0.01 level. (C,D) Response of PBI121#OE and sly-MIR398b#OE plants to B. cinerea infection. Inoculation was carried out by dropping spore suspensions (10 6 spores/mL) on detached leaves of fourweek-old plants. Photographs (C) were taken and the lesion areas (D) were analyzed 48 h after inoculation. Error bars represent the SE from six replicates (n = 6). Asterisks indicate a significant difference from the wild type according to Student's t test at * p < 0.05.

Overexpression of Sly-miR398b Results in the Accumulation of H2O2
Previous studies have demonstrated that miR398b mediates plant immunity by regulating ROS homeostasis [39,40]. To analyze the effects of sly-miR398b overexpression on ROS homeostasis, the concentration of O2 •− and H2O2 in B. cinerea -inoculated tomato leaves were compared between pBI121#OE control plants and sly-MIR398b#OE plants. As the results show in Figure 3A, no difference was observed in both the untreated and treated leaves of pBI121#OE control plants and sly-MIR398b#OE plants in terms of O2 •− . However, incubation with B. cinerea upregulated the level of H2O2 ( Figure 3B). The H2O2 content in the sly-MIR398b#OE plants was significantly higher than that in the pBI121#OE control plants at 48 h after B. cinerea infection, while there was no difference detected between the uninfected leaves of either genotypes ( Figure 3B). Total RNAs were extracted from 18-day-old seedlings. SlACTIN was used as reference gene for pri-miR398b. Tomato U6 was used as reference gene for sly-miR398b. Values presented are the means ± SD from three biological replicates. ** above the columns indicate significant differences at p < 0.01 level. (C,D) Response of PBI121#OE and sly-MIR398b#OE plants to B. cinerea infection. Inoculation was carried out by dropping spore suspensions (10 6 spores/mL) on detached leaves of four-week-old plants. Photographs (C) were taken and the lesion areas (D) were analyzed 48 h after inoculation. Error bars represent the SE from six replicates (n = 6). Asterisks indicate a significant difference from the wild type according to Student's t test at * p < 0.05.

Overexpression of Sly-miR398b Results in the Accumulation of H 2 O 2
Previous studies have demonstrated that miR398b mediates plant immunity by regulating ROS homeostasis [39,40]. To analyze the effects of sly-miR398b overexpression on ROS homeostasis, the concentration of O 2 •− and H 2 O 2 in B. cinerea -inoculated tomato leaves were compared between pBI121#OE control plants and sly-MIR398b#OE plants. As the results show in Figure 3A

Overexpression of Sly-miR398b Decreases the Activities of Antioxidant Enzymes and the Relative Expression of Antioxidant Genes
To further investigate the effects of sly-miR398b overexpression on ROS homeostasis in tomato plants, activities of SOD, CAT, GPOD, and APX in pBI121#OE control plants and sly-MIR398b#OE plants were examined. The activities of SOD, CAT, GPOD, and APX in sly-MIR398b#OE plants were decreased compared to pBI121#OE control plants, with values 28.21%, 24.66%, 26.88%, and 13.61% lower than those in pBI121#OE control plants 48 hpi (hours post inoculation), respectively ( Figure 4). However, SOD and GPOD activities in sly-MIR398b#OE plants were significantly lower than those in pBI121#OE control plants at 48 hpi ( Figure 4C).

Overexpression of Sly-miR398b Decreases the Activities of Antioxidant Enzymes and the Relative Expression of Antioxidant Genes
To further investigate the effects of sly-miR398b overexpression on ROS homeostasis in tomato plants, activities of SOD, CAT, GPOD, and APX in pBI121#OE control plants and sly-MIR398b#OE plants were examined. The activities of SOD, CAT, GPOD, and APX in sly-MIR398b#OE plants were decreased compared to pBI121#OE control plants, with values 28.21%, 24.66%, 26.88%, and 13.61% lower than those in pBI121#OE control plants 48 hpi (hours post inoculation), respectively ( Figure 4). However, SOD and GPOD activities in sly-MIR398b#OE plants were significantly lower than those in pBI121#OE control plants at 48 hpi ( Figure 4C).

Overexpression of Sly-miR398b Decreases the Activities of Antioxidant Enzymes and the Relative Expression of Antioxidant Genes
To   In uninfected tomato leaves, the relative transcript levels of antioxidant genes including CSD1, CSD2, CAT, GPOD, and APX were not affected in sly-MIR398b#OE plants ( Figure 5). Moreover, compared to pBI121#OE control plants, the transcript levels of CAT and APX in sly-MIR398b#OE plants showed no difference 48 hpi, but both CAT and APX transcripts were decreased significantly by infection ( Figure 5C,E). However, the transcript levels of CSD1, CSD2, and GPOD were increased significantly by infection, and they were significantly lower in sly-MIR398b#OE plants than those in pBI121#OE control plants 48 h after B. cinerea infection ( Figure 5A,B,D). In uninfected tomato leaves, the relative transcript levels of antioxidant genes including CSD1, CSD2, CAT, GPOD, and APX were not affected in sly-MIR398b#OE plants (Figure 5). Moreover, compared to pBI121#OE control plants, the transcript levels of CAT and APX in sly-MIR398b#OE plants showed no difference 48 hpi, but both CAT and APX transcripts were decreased significantly by infection ( Figure 5C,E). However, the transcript levels of CSD1, CSD2, and GPOD were increased significantly by infection, and they were significantly lower in sly-MIR398b#OE plants than those in pBI121#OE control plants 48 h after B. cinerea infection ( Figure 5A,B,D). , and SlSAND were used as reference genes. Each value represents a mean ± SD of three biological replicates. Different letters above the bars indicate significant differences (p < 0.05, one-way ANOVA) according to the least significant difference (LSD) test.

Effects of Sly-MIR398b Overexpression on JA-Related Defense Genes
Jasmonic acid (JA) signaling is believed to have a pivotal role in plant defense against necrotrophic pathogens [4,6]. Thus, we explored whether sly-miR398b affected tomato resistance to B. cinerea by influencing JA signaling. First, the accumulation pattern of sly-miR398b in response to MeJA was assessed. Stem loop RT-qPCR assays showed that the abundance of sly-miR398b was upregulated 3 and 12 h after treatment with MeJA ( Figure  6A). Second, MeJA-induced transcript levels of TomLoxD, LapA, and PR-STH2 were decreased in sly-MIR398b#OE plants compared with pBI121#OE control plants ( Figure 6B-D).
To further investigate the regulatory mechanism of sly-miR398b-mediated susceptibility to B. cinerea, the transcripts of JA-inducible defense genes were quantified 48 h after B. cinerea infection. Results showed that, compared to pBI121#OE control plants, the abundance of TomLoxD, LapA, and PR-STH2 transcripts in sly-MIR398b#OE plants were significantly decreased 48 hpi ( Figure 6E-G). These results indicated that the overexpression of sly-MIR398b hampered the expression of JA-responsive defense genes in response to B. cinerea infection. , and SlSAND were used as reference genes. Each value represents a mean ± SD of three biological replicates. Different letters above the bars indicate significant differences (p < 0.05, one-way ANOVA) according to the least significant difference (LSD) test.

Effects of Sly-MIR398b Overexpression on JA-Related Defense Genes
Jasmonic acid (JA) signaling is believed to have a pivotal role in plant defense against necrotrophic pathogens [4,6]. Thus, we explored whether sly-miR398b affected tomato resistance to B. cinerea by influencing JA signaling. First, the accumulation pattern of sly-miR398b in response to MeJA was assessed. Stem loop RT-qPCR assays showed that the abundance of sly-miR398b was upregulated 3 and 12 h after treatment with MeJA ( Figure 6A). Second, MeJA-induced transcript levels of TomLoxD, LapA, and PR-STH2 were decreased in sly-MIR398b#OE plants compared with pBI121#OE control plants ( Figure 6B-D).
To further investigate the regulatory mechanism of sly-miR398b-mediated susceptibility to B. cinerea, the transcripts of JA-inducible defense genes were quantified 48 h after B. cinerea infection. Results showed that, compared to pBI121#OE control plants, the abundance of TomLoxD, LapA, and PR-STH2 transcripts in sly-MIR398b#OE plants were significantly decreased 48 hpi ( Figure 6E-G). These results indicated that the overexpression of sly-MIR398b hampered the expression of JA-responsive defense genes in response to B. cinerea infection.

Discussion
miRNAs have been found to play important roles in response to various biotic and abiotic stresses in plants, including biotic stresses (bacteria, fungi, viruses, insects, nematodes, etc.) and abiotic stresses (salinization, drought, low/high temperature, nutrient deficiencies, etc.) [41]. Uncovering the mechanisms mediated by miRNAs in these stress responses will help us to utilize miRNA-mediated defense mechanisms and promote resistance breeding in tomato. miR398 is a highly conserved miRNA and has been demonstrated to play important roles in both plant development and stress responses [39]. As computationally predicted by the base-pairing principle in Arabidopsis and rice, firstly, the target genes of miR398 were CSD1 and CSD2, which are responsible for scavenging ROS [31][32][33]42]. Interestingly, For MeJA treatment, 18-day-old seedlings of the indicated genotypes with two fully expanded leaves were exposed to MeJA vapor for the indicated times (hours) before extracting total RNAs for RT-qPCR assays. (E-G) RT-qPCR assays of B. cinerea-induced relative transcript amounts of TomLoxD (E), LaPA (F), and PR-STH2 (G) in PBI121#OE and sly-MIR398b#OE plants. Leaves from PBI121#OE and sly-MIR398b#OE plants were harvested 48 h after inoculation with a 5-µL spore suspension (10 6 spores/mL) of B. cinerea or with 1% Sabouraud Maltose Broth (mock). For (B-G), SlACTIN was used as a reference gene. Each value represents a mean ± SD of three biological replicates. For (A), asterisks indicate significant differences from PBI121#OE plants according to Student's t test at ** p < 0.01 and *** p < 0.001. For (B-G), different letters above the bars indicate significant differences (p < 0.05, one-way ANOVA) according to the least significant difference (LSD) test.

Discussion
miRNAs have been found to play important roles in response to various biotic and abiotic stresses in plants, including biotic stresses (bacteria, fungi, viruses, insects, nematodes, etc.) and abiotic stresses (salinization, drought, low/high temperature, nutrient deficiencies, etc.) [41]. Uncovering the mechanisms mediated by miRNAs in these stress responses will help us to utilize miRNA-mediated defense mechanisms and promote resistance breeding in tomato. miR398 is a highly conserved miRNA and has been demonstrated to play important roles in both plant development and stress responses [39]. As computationally predicted by the base-pairing principle in Arabidopsis and rice, firstly, the target genes of miR398 were CSD1 and CSD2, which are responsible for scavenging ROS [31][32][33]42]. Interestingly, miR398 was further shown to regulate CSD2 at the protein level through translational inhibition [22,23]. A growing body of research suggests that the miR398-CSD regulation module is implicated in biotic stress responses [39]. Here, sly-miR398b overexpression was used to investigate how sly-miR398b influences tomato resistance against B. cinerea. Results showed that overexpression significantly enhances susceptibility. The abundance of sly-mi398b decreased significantly 48 h after B. cinerea infection. Consistent with this, CSD1 and CSD2 transcripts were upregulated 48 hpi. These results suggest that the negative correlation between sly-miR398b, CSD1 and CSD2 affects tomato-B. cinerea interactions. Indeed, several studies have revealed that miR398 and its targets CSD1/2 play crucial roles in disease resistance responses. For instance, in the Arabidopsis-P. syringae pv. tomato DC3000 interaction system, miR398b negatively regulates Arabidopsis defense, with the downregulation of miR398b and upregulation of CSD1 upon infection [28,32]. In the common bean-Sclerotinia sclerotiorum interaction system, the downregulation of miR398b and an increase in CSD1 transcripts have been observed upon infection. Moreover, the overexpression of miR398 promotes infection [43]. Recently, miR398b has been demonstrated to negatively regulate cotton immune responses to Verticillium dahliae via downregulating GhCSD1, GhCSD2, and GhCCS [44]. In contrast to the negative roles of miR398 in plant resistance in these studies, as well as in our work, many pieces of evidence have proposed an opposite role for miR398; for example, in rice against M. oryzae [35,37]. As for viruses, miR398 facilitates BMV accumulation in N. benthamiana [33], and it was speculated that miR398 enhances N. benthamiana resistance against the beet necrotic yellow vein virus in the same species [45]. Taken together, in response to various pathogens, miR398 has diverse regulatory mechanisms in plant immune responses. In plant-pathogen interactions, plants have evolved efficient mechanisms to combat pathogen attack. During pathogen infection, oxidative burst, hypersensitive response (HR), and ROS-induced cell death are considered to be major contributors to disease resistance [46]. Hypersensitive cell death is thought to inhibit infection by biotrophic pathogens; however, it facilitates the growth of necrotrophic pathogens, such as B. cinerea [46]. And indeed, B. cinerea infection results in ROS accumulation and triggers an HR in the affected tissue for its own benefit [47][48][49][50]. In the present study, hypersensitive cell death and increased H 2 O 2 content were found in tomato leaves inoculated with B. cinerea 48 h prior. Compared to PBI121#OE, sly-MIR398b#OE leaves exhibited a higher H 2 O 2 accumulation with larger lesion sizes, suggesting that higher H 2 O 2 levels in sly-MIR398b#OE plants increase the susceptibility to B. cinerea compared to PBI121#OE control plants.
The phytohormone JA has long been known to positively regulate plant defenses against B. cinerea [6]. Mutants in the JA signaling pathway that display compromised resistance against B. cinerea are, for example, the JA receptor mutants coi1-1 in Arabidopsis and jai1 in tomato, which increased susceptibility to B. cinerea [5,6]. TomLoxD encodes a 13-lipoxygenase, which catalyzes a key step in JA biosynthesis [51,52]. The TomLoxD mutation in tomato increases susceptibility to B. cinerea infection, whereas TomLoxD overexpression has the opposite effect [7]. Additionally, TomLoxD was characterized as an early JA-responsive gene [5]. In tomato, LapA regulates defenses and wound signaling acting downstream of JA biosynthesis and perception [10]. This exopeptidase is induced by wounding and JA treatment and is known to mediate protein turnover during defense gene activation [53]. PR-STH2 is a pathogen-responsive marker gene [5,54]. A previous study indicated that JA signaling is required for B. cinerea to induce the activation of PR-STH2 in tomato [5]. Consistent with this, the transcript levels of TomLoxD, LapA, and PR-STH2 were significantly upregulated in response to MeJA treatment and B. cinerea infection. Since the MeJA-and B. cinerea-induced expressions of these genes was significantly decreased in sly-MIR398b#OE plants compared with controls, we propose that the JA signaling pathway is involved in the sly-miR398b-mediated resistance against B. cinerea in tomato, and the decreased JA-responsive defense genes in sly-MIR398b#OE plants might contribute to their increased susceptibility to B. cinerea. JA modulates both plant growth and defense, and mediates the trade-offs between them [55,56]. Given the metabolic cost of sustained defense responses to plant growth, turning off JA signaling is of equal importance as turning it on. sly-miR398b might act as a built-in negative feedback regulation mechanism to avoid excessive JA-induced defense responses at the expense of plant growth. Of course, JA quantification in our experimental system might help to understand whether this subset of miR398 effects is due to altered JA synthesis and/or sensitivity. In addition to sly-MIR398b (located on chromosome 05), tomato contains two other MIR398 members, sly-MIR398a (located on chromosome 11) and sly-MIR398c (located on chromosome 12). They encode three different mature sly-miR398, sly-miR398b (5 -uuguguucucaggucaccccu-3 ), sly-miR398a (5 -uauguucucaggucgccccug-3 ), and sly-miR398c (5 -uguguucucagguuaccccu-3 ) [57,58]. The roles of sly-miR398a and sly-miR398c in tomato responses to B. cinerea infection also need to be tested in the future.
Overall, the present study demonstrated that the overexpression of sly-miR398b negatively regulates the resistance to B. cinerea in tomato. These results reveal a new role of sly-miR398b in regulating tomato responses to B. cinerea by modulating ROS homeostasis and JA-responsive defense genes (Figure 7), and sly-miR398b might be a potential gene that can be applied for tomato resistance against B. cinerea. Further work in uncovering upstream regulators will help to elucidate the molecular mechanisms of sly-miR398b in suppressing tomato defense against B. cinerea, and the knockout mutant of sly-miR398b will also be required to fully unravel the role of sly-miR398b. of sustained defense responses to plant growth, turning off JA signaling is of equal importance as turning it on. sly-miR398b might act as a built-in negative feedback regulation mechanism to avoid excessive JA-induced defense responses at the expense of plant growth. Of course, JA quantification in our experimental system might help to understand whether this subset of miR398 effects is due to altered JA synthesis and/or sensitivity. In addition to sly-MIR398b (located on chromosome 05), tomato contains two other MIR398 members, sly-MIR398a (located on chromosome 11) and sly-MIR398c (located on chromosome 12). They encode three different mature sly-miR398, sly-miR398b (5′-uuguguucucaggucaccccu-3′), sly-miR398a (5′-uauguucucaggucgccccug-3′), and sly-miR398c (5′uguguucucagguuaccccu-3′) [57,58]. The roles of sly-miR398a and sly-miR398c in tomato responses to B. cinerea infection also need to be tested in the future.
Overall, the present study demonstrated that the overexpression of sly-miR398b negatively regulates the resistance to B. cinerea in tomato. These results reveal a new role of sly-miR398b in regulating tomato responses to B. cinerea by modulating ROS homeostasis and JA-responsive defense genes (Figure 7), and sly-miR398b might be a potential gene that can be applied for tomato resistance against B. cinerea. Further work in uncovering upstream regulators will help to elucidate the molecular mechanisms of sly-miR398b in suppressing tomato defense against B. cinerea, and the knockout mutant of sly-miR398b will also be required to fully unravel the role of sly-miR398b.

Plant Materials and Growth Conditions
Tomato (Solanum lycopersicum cv 'Micro-Tom') was used as the WT tomato plants. pBI121#OE (pBI121 overexpressed control plants) and sly-MIR398b#OE (sly-miR398b overexpressing lines) plants were generated in 'Micro-Tom' background. Specifically, sly-MIR398b precursor (410 bp) was cloned from tomato and then inserted into vector pBI121 (14,758 BP, a binary Agrobacterium vector with a GUS reporter gene for plant transformation) downstream of CaMV 35S promoter. The exact sequence length of sly-MIR398b precursor and the primer pair used for amplifying sly-MIR398b precursor sequence were

Plant Materials and Growth Conditions
Tomato (Solanum lycopersicum cv 'Micro-Tom') was used as the WT tomato plants. pBI121#OE (pBI121 overexpressed control plants) and sly-MIR398b#OE (sly-miR398b overexpressing lines) plants were generated in 'Micro-Tom' background. Specifically, sly-MIR398b precursor (410 bp) was cloned from tomato and then inserted into vector pBI121 (14,758 BP, a binary Agrobacterium vector with a GUS reporter gene for plant transformation) downstream of CaMV 35S promoter. The exact sequence length of sly-MIR398b precursor and the primer pair used for amplifying sly-MIR398b precursor sequence were included in the previous paper [38]. The resultant vector was then introduced into Agrobacterium tumefaciens strain GV3101 for tomato transformation. Homozygous transgenic plants were generated and identified as described previously [38]. Two sly-miR398b overexpressing lines (MIR398b#OE1 and MIR398b#OE8) were obtained [38]. The amounts of sly-miR398b increased sharply to a similar extent in the two miR398b overexpressing lines (MIR398b#OE1 and MIR398b#OE8) compared with those in control line, and they showed similar phenotypes with regard to the effects of salt stress on plant growth, oxidative damage, antioxidant response, and photosynthesis performance [38]. Therefore, in the present project, we selected one (MIR398b#OE1) of them (MIR398b#OE1 and MIR398b#OE8) for further study. Tomato seedlings were grown in a growth chamber with temperature of 25 • C/20 • C day/night and photoperiod of 16/8 h light/dark (with a white light intensity of 200 µmol m −2 s −1 ).

B. cinerea Inoculation Assays
B. cinerea isolate B05.10 was cultured on potato dextrose agar for 14 d at 20 • C under a 12 h photoperiod prior to spore collection. Spore suspensions were prepared by harvesting the spores in 1% Sabouraud Maltose Broth, filtering them through four layers of medical gauze to remove hyphae, and adjusting the concentration to 1 × 10 6 spores/mL. B. cinerea inoculation of tomato plants was performed as previously described [59], with minor modifications. To quantitatively measure lesion sizes, detached leaves from four-week-old tomato plants were placed in Petri dishes containing 0.8% agar medium (agar dissolved in sterile water), with the petiole embedded in the medium. Each leaflet was spotted with a single 5 µL droplet of B. cinerea spore suspension at a concentration of 1 × 10 6 spores/mL. The trays were covered with lids and then kept in the growth chamber. Photographs were taken after 48 h, and the lesion sizes were recorded and calculated with Image J software [60]. For RT-qPCR experiments and antioxidant enzyme activity analysis, inoculations were performed in planta. Leaves of four-week-old plants were spotted with a 5 µL B. cinerea spore suspension (10 6 spores/mL). To obtain saturating humidity conditions, the plants were covered with a clear plastic moisturizing lid and then incubated in the growth chamber. Spotted leaves were harvested 48 h later. In addition, leaves spotted with 1% Sabouraud Maltose Broth were considered uninfected leaves (mock). The leaf samples were frozen in liquid nitrogen and stored at −80 • C. To facilitate B. cinerea infection and tissue colonization, the light intensity in the growth chamber was set to 50 µmol m −2 s −1 for both in vitro and in planta B. cinerea inoculation assays.

RNA Extraction and Quantitative RT-PCR (qRT-PCR) Analysis
Total RNA was isolated from 0.1 g tomato leaves using TRIzol™ reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). For qRT-PCR, first strand cDNA was reverse transcribed from one microgram of DNA-free RNA with a PrimerScript II 1st Strand cDNA Synthesis Kit (TaKaRa). Oligo-dT primer and a miR398-specific primer were used to prepare the first cDNA strand of mRNA and miR398, respectively. The qRT-PCR was carried out with the TB Green Premix Ex Taq (TaKaRa) using UVP ChemStudio (Analytics Jena) following the protocol of 95 • C for 30 s, and 40 cycles at 95 • C for 5 s and 60 • C for 30 s. Levels of miR398b were normalized to the U6 gene using the ∆Ct method. Transcript abundance of all other loci was normalized to either one (SlACTIN: Solyc11g005330), or three reference genes (SlACTIN, SlEF1α: Solyc06g009970, SlSAND: Solyc03g115810) with the ∆Ct method. Normalization factors were calculated as the geometric mean of their transcript levels when three reference genes were used [61]. Primers used referred to the previous paper [5,7,61] or were designed in this work with Primer-BLAST tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast (accessed on 15 May 2022)), and they are listed in Supplementary Table S1. Each reaction was performed with three biological replicates, and each biological replicate contained three technical replicates. Each biological replicate consisted of the pooled leaves of four plants from one tray (different genotypes were grown together in a tray). Biological replicates were grown in different trays with different locations in the growth chambers and treated separately.

In Situ Staining and Measurement of O 2 •− and H 2 O 2
For detection of O 2 •− and H 2 O 2 , inoculations were performed in planta as described in 4.2. Uninfected and B. cinerea-infected tomato leaves were collected 48 h after inoculation and stained with nitroblue tetrazolium (NBT) and 3,3-diaminobenzidine (DAB), respectively. Leaves were immersed in NBT solution (1 mg/mL, pH 7.4) or DAB solution (1 mg/mL, pH 3.8) followed by vacuum infiltration until they were completely infiltrated, and were then incubated for 3 h in the dark at room temperature. The NBT-and DABtreated leaves were placed in 95% ethanol and kept in 65 • C water bath for 30 min to elute the chlorophyll. Subsequently, the accumulation of O 2 •− and H 2 O 2 in the leaves was captured with a digital camera.

Determination of Antioxidant Enzyme Activity
Uninfected and B. cinerea-infected tomato leaves were collected 48 h after inoculation to measure antioxidant enzyme activity. The activities of SOD, CAT, GPOD, and APX were determined using the methods described in a previous study [35], with some modifications. Briefly, 0.3 g of frozen leaves was ground with liquid nitrogen into powder. The frozen power was suspended in 2 mL of 50 mM phosphate-buffered saline (PBS, pH 7.8) for 5 min. The mixtures were centrifuged at 12,000× g for 20 min at 4 • C, and then supernatants were collected. The activity of SOD was calculated from its ability to inhibit the photochemical reduction in NBT. One unit of SOD activity was defined as the photochemical reduction that caused a 50% inhibition of NBT. The enzyme activity of CAT was defined by measuring the decrease in absorbance at 240 nm due to the decomposition of H 2 O 2 . The enzyme activity of GPOD was defined by calculating the increase in absorbance at 470 nm due to the polymerization of guaiacol to tetraguaiacol. The enzyme activity of APX was defined by calculating the decrease in absorbance at 290 nm due to ascorbate oxidation.

MeJA Treatment
Firstly, 18-day-old seedlings were kept in an airtight container, and then six cotton wicks that each contained 150 µL of 50 mM MeJA were placed evenly within the container. Tomato seedlings exposed to MeJA vapor were harvested at indicated time points and used for extracting total RNA [62].