SlymiR482e‐3p mediates tomato wilt disease by modulating ethylene response pathway

Fusarium oxysporum f. sp. Lycopersici, a necrotrophic pathogen, is a causal agent of tomato wilt disease. Plants have two major sophisticated innate immune systems, Pathogen‐Associated Molecular Pattern (PAMP)‐triggered immunity (PTI) and Effector‐Triggered Immunity (ETI), to perceive and resist pathogen offences (Jones and Dangl, 2006). MicroRNAs (miRNAs) contribute to PTI and ETI by fine‐tuning plant hormones and/or silencing the genes involved in pathogen virulence by regulating the expression of target genes, thereby acting as crucial regulators of the plant immune system (Fei et al., 2016). Many plants produce microRNAs belonging to the miRNA482/2118 superfamily. These miRNAs target R‐genes of the class NBS‐LRR (nucleotide‐binding site‐leucine rich repeat) through recognizing the P‐loop motif in the NBS‐LRR mRNA. Our previous studies showed that SlymiR482e‐3p, a members of the miR482/2118 superfamily in tomato, negatively regulated the resistance to Fusarium oxysporum f. sp. lycopersici (race 2) (Fol) by targeting several NBS‐LRR genes (Ouyang et al., 2014). However, the exact mechanism underlying the basic function of SlymiR482e‐3p during the response to Fol attack needs further exploration. In this study, two near‐isogenic tomato cultivars, Moneymaker (susceptible, i‐2/i‐2) and Motelle (resistant, I‐2/I‐2) to Fol infection, were recruited (Ouyang et al., 2014).

To characterize the functions of SlymiR482e‐3p in response to tomato wilt disease, we generated a CRISPR/Cas9‐related knock‐out mutant lacking the SlymiR482e‐3p gene in the susceptible cultivar Moneymaker (Deng et al., 2018). Three regenerated plants, termed as SlymiR482e‐3p‐KO‐Line 3, 7 and 11, carried 2‐, 9‐ and 6‐nucleatide deletion in front of the mature miRNA region respectively, were identified (Figure 1a). Compared with the control, the expression levels of SlymiR482e‐3p was dramatically reduced by more than 90% in individual transgenic plants (Figure 1b). SlymiR482e‐3p has been proved as a negative regulator for several targeted NBS‐LRR genes, including Soly08g075630 and Soly08g076000 in tomato (Ouyang et al., 2014). As expected, basal expression levels of both Soly08g075630 and Soly08g076000 were increased in all transgenic Moneymaker plants (Figure 1b). Furthermore, no visible difference in major agronomic traits, including leaves, flowers and fruits, were observed in transgenic plants compared with the control (Figure 1c).

Figure 1.

SlymiR482e‐3p mediates tomato wilt disease by modulating ethylene response pathway. (a) Clustalx nucleic acid sequence alignments of SlymiR482e‐3p‐KO plants. The sequence of mature miRNA is highlighted with grey. (b) Expression of known targets of SlymiR482e‐3p in KO plants. (c) Agricultural phenotypic traits of SlymiR482e‐3p‐KO plants. (d) Knock‐out of SlymiR482e‐3p enhances the resistance to Fol in Moneymaker. Two‐week‐old seedlings of the indicated control or transgenic plants were treated with water or Fol and photographed 2 weeks later. Red arrows indicating vascular tissue. (e) Level of target mRNAs. qRT‐PCR was used to determine relative levels of SlSGT1 in Nicotiana benthamiana leaves expressing target mRNA and empty vector, target mRNA and the appropriate miRNA, target mRNA and a control miRNA (SlymiR166). Values were normalized to N. benthamiana actin. Asterisks indicated significant differences (* P < 0.05, ** P < 0.01). (f) SlSGT1 protein level was detected by Western blot using anti‐GFP antibody. (g) The cleavage site in the SlSGT1 mRNA was determined using 5′ RLM‐RACE. The arrow indicates the 5′ terminus of miRNA‐guided cleavage products and the frequency of clones (13/14) was shown. (h) Accumulation of SlSGT1 during a time course in different cultivars and transgenic tomato plants infected with Fol. (i) SlymiR482e‐3p regulates ethylene signalling by suppressing the expression of SlERFs. Total RNA was isolated from tomato seedling root at 24 hpi and subjected to qRT‐PCR to evaluate expression with gene‐specific primers. (j) Effect of exogenous ethylene on Fol defence in wild and SlymiR482e‐3p‐KO plants. Two‐week‐old tomato seedlings were inoculated with Fol for 30 min followed by the first spraying with 1‐Aminocyclopropane‐1‐carboxylic acid (ACC, 100 μm). All plants were treated three times with an interval of 24 h (Left). Accumulation of Fol in tomato stems as visualized by staining with lactic acid phenol Medan dye (Middle). Red arrows indicate vascular tissue boundary of stems with extensive staining. Recovery of Fol from tomato stem slices incubated on PDA medium (Right). (k) Model for SlymiR482e‐3p‐mediated ethylene signalling in promoting resistance to Fol. During fungal pathogen Fol invasion, down‐regulated endogenous SlymiR482e‐3p releases SlSGT1 accumulation, triggering CDPK‐depending programmed cell death (PCD). Consequently, SlERFs, components of the ethylene signalling transduction pathway, are repressed to enhance the resistance to tomato wilt disease caused by Fol.

To further evaluate the function of SlymiR482e‐3p in tomato wilt disease susceptibility, we inoculated the SlymiR482e‐3p‐KO transgenic plants as well as resistant Motelle and susceptible Moneymaker controls with Fol. As gauged, SlymiR482e‐3p‐KO plants exhibited enhanced resistance to Fol relative to the Moneymaker control while displayed an appearance similar to the treated Motelle plants (Figure 1d). This result further confirms that SlymiR482e‐3p functions as a negative regulator of resistance to tomato wilt disease.

We utilized the psRNATarget algorithm (Dai et al., 2018) to predict potential targets of SlymiR482e‐3p. Intriguingly, Solyc11g010660, a homolog of SGT1 (suppressor of the G2 allele of skp1), was predicted as a target of SlymiR482e‐3p and termed as SlSGT1. SGT1 was first reported as a component of the SCF E3 ubiquitin ligase complex in yeast (Kitagawa et al., 1999) and interacted with RAR1 to trigger disease resistance in plants (Azevedo et al., 2002). It has been documented that SGT1 homologs in plants are triggered by various plant defence response pathways, including ethylene‐mediated cross‐talk between calcium‐dependent protein kinases (CDPK) and mitogen‐activated protein kinase (MAPK) signalling (Ludwig et al., 2005; Peart et al., 2002). To determine whether SlymiR482e‐3p regulate the SlSGT1 expression, we conducted an Agrobacterium‐mediated transient co‐expression experiment in N. benthamiana, as previously implemented in our laboratory (Ouyang et al., 2014). qRT‐PCR data showed that the SlSGT1 transcripts were greatly decreased in the presence of SlymiR482e‐3p (Figure 1e). Consistently, GFP fluorescence and Western blot assays using an anti‐GFP antibody further demonstrated that SlSGT1 protein levels were significantly down‐regulated in the presence of SlymiR482e‐3p (Figure 1f). To identify the cleavage site in the SlSGT1 mRNA targeted by SlymiR482e‐3p, we performed a 5′‐RNA ligase‐mediated rapid amplification of cDNA ends (5′ RLM‐ RACE) analysis. The result showed the cleavage site occurred at the 999th nt of the SlSGT1 mRNA in 13 out of 14 clones (Figure 1g). AdSGT1 transcripts were strong up‐regulated by ethephon resulting in enhanced disease resistance in tobacco and peanut (Kumar and Kirti, 2015). In this study, SlSGT1 was dramatically induced during Fol infection in the susceptible cultivar Moneymaker, meanwhile, the basal level of SlSGT1 was elevated in the SlylmiR482e‐3p‐KO mutant possibly resulting in the resistance to Fol (Figure 1h). Considering all of the above results, we concluded that SlymiR482e‐3p regulates SlSGT1 expression by chopping the intact mRNA.

To further understand the role of SlymiR482e‐3p in mediating resistance to Fol in tomato, we constructed and sequenced six RNA‐seq libraries, including Moneymaker treated with water (MM_H₂O) or Fol (MM_Fol), as well as SlymiR482e‐3p‐KO lines 3 and 7 treated with water or Fol (KO‐Line3_H₂O, KO‐Line3_Fol, KO‐Line7_H₂O, and KO‐Line7_Fol) (The raw sequence data are available in the Genome Sequence Archive in BIG Data Centre, under accession numbers CRA002427). Intriguingly, we determined that genes in several phytohormone signalling pathways, particularly the ethylene (ET) signal transduction pathway, may participate in the response to Fol infection in tomato. To evaluate further a possible role for SlymiR482e‐3p in regulating ethylene signalling, we monitored expression of key genes in the pathway in tomato plants after inoculation with Fol spores or water over a 24 h period. The basal expression levels (water control) of SlERF1, SlERF3, SlERF4, SlERF5, SlERF9, and SlERF11 were depressed in all SlymiR482e‐3p‐KO plants relative to Moneymaker. However, all these genes except SlERF3 were induced after Fol infection in both Moneymaker and SlymiR482e‐3p‐KO plants (Figure 1i). These results prompted us to speculate that the ethylene signal transduction pathway might be important during the response to Fol infection. We next asked whether application of a precursor of ET biosynthesis, 1‐Aminocyclopropane‐1‐carboxylic acid (ACC), would exacerbate wilt disease symptoms. For these experiments, WT and transgenic plants were treated with Fol followed by spraying 100 μm ACC (optimal concentration was determined through our preliminary experiments). After ACC treatment, all tomato plants displayed aggravated wilt disease symptoms and faster disease progression compared to treatment with Fol alone (Figure 1j). Particularly, ACC overrode the resistance to Fol infection in Motelle against Fol (Figure 1j).

In summary, we present evidence that supports a key role of SlylmiR482e‐3p‐mediated ethylene signalling in promoting resistance to a fungal necrotroph Fol. We propose that during fungal pathogen Fol invasion, endogenous SlylmiR482e‐3p promotes SlSGT1 accumulation, thereby triggering CDPK‐depending PCD in tomato. Consequently, SlERFs, components of the ethylene signalling pathway are regulated to enhance resistance to tomato wilt disease (Figure 1k). Our research provides a basis to elucidate the complex SlylmiR482e‐3p‐mediated resistance to Fol in tomato, which will be beneficial for the design of strategies to improve tomato wilt disease resistance.

Conflicts of interest

The authors declare no conflict of interest.

Author contributions

SQO designed the experiments. SQO contributed to data analysis and interpretation and wrote the paper. KAB contributed to design this project and revised this manuscript. YG and SJL performed the experiments in cooperation with SWZ, TF, ZYZ, SJL and HYM. All authors read and approved the final manuscript.

Gao, Y. , Li, S.‐J. , Zhang, S.‐W. , Feng, T. , Zhang, Z.‐Y. , Luo, S.‐J. , Mao, H.‐Y. , Borkovich, K. A. and Ouyang, S.‐Q. (2021) SlymiR482e‐3p mediates tomato wilt disease by modulating ethylene response pathway. Plant Biotechnol. J., 10.1111/pbi.13439