Phytopathology®
2021
111:1008-1016
https://doi.org/10.

1094/PHYTO-08-20-0360-R

Molecular and Physiological Plant Pathology

Editing miR482b and miR482c Simultaneously by CRISPR/Cas9 Enhanced

Tomato Resistance to Phytophthora infestans
Yuhui Hong,1 Jun Meng,2 Xiaoli He,1 Yuanyuan Zhang,1 Yarong Liu,1 Chengwei Zhang,3 Hongyan Qi,4,†
and Yushi Luan1,†
1
School of Bioengineering, Dalian University of Technology, Dalian 116024, China
2
School of Computer Science and Technology, Dalian University of Technology, Dalian 116024, China
3
Beijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding, Beijing Academy of Agriculture & Forestry Sciences, Beijing
100000, China
4
College of Horticulture, Shenyang Agricultural University/Key Laboratory of Protected Horticulture, Ministry of Education/Northern National &
Local Joint Engineering Research Center of Horticultural Facilities Design and Application Technology (Liaoning), Shenyang 110866, China
Accepted for publication 26 November 2020.

ABSTRACT

Late blight, caused by Phytophthora infestans, is severely damaging to expression of their common target nucleotide binding site-leucine-rich
the global tomato industry. Micro-RNAs (miRNAs) have been widely repeat genes and decreased levels of reactive oxygen species. Furthermore,
demonstrated to play vital roles in plant resistance by repressing their silencing miR482b and miR482c simultaneously was more resistant than
target genes. Recently, the clustered regularly interspaced short silencing miR482b alone in tomato. More importantly, we found that
palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) knocking out miR482b and miR482c can elicit expression perturbation of
method has been continuously improved and extensively applied to edit other miRNAs, suggesting cross-regulation between miRNAs. Our study
plant genomes. However, editing multiplex miRNAs by CRISPR/Cas9 demonstrated that editing miR482b and miR482c simultaneously with
in tomato has not been studied yet. We knocked out miR482b and CRISPR/Cas9 is an efficient strategy for generating pathogen-resistant
miR482c simultaneously in tomato through the multiplex CRISPR/Cas9 tomatoes, and cross-regulation between miRNAs may reveal the novel
system. Two transgenic plants with silenced miR482b and miR482c mechanism in tomato–P. infestans interactions.
simultaneously and one transgenic line with silenced miR482b alone were
obtained. Compared with wild-type plants, the disease symptoms of three Keywords: CRISPR/Cas9, disease resistance, late blight, miRNA, plant
transgenic plants upon infection were reduced, accompanied by increased immune responses, tomato

Tomato is not only a major crop in global agriculture but also an target gene expressions at the posttranscriptional or posttranslational
important model for investigating plant–pathogen interactions. Late level (Ha and Kim 2014; Song et al. 2019; Yu et al. 2017). The bio-
blight (LB) has triggered widespread damage in the global tomato synthesis of miRNAs in plants starts with the transcription of
industry since the mid-1840s (Fry 2016; Fry et al. 2015). The MIRNA genes into the primary miRNA transcripts, which sequen-
causal agent of LB, Phytophthora infestans, ranked first among 10 tially are cleaved by DCL1 and generate short hairpins (called pre-
selected plant pathogenic oomycete species (Kamoun et al. 2015). miRNA), finally resulting in an miRNA/miRNA* duplex (about
As a hemibiotroph, P. infestans initially infects living tissues and 21 bp) (Ha and Kim 2014; Song et al. 2019; Yu et al. 2017). During
extracts nutrients in its biotrophic stages, when minimal symptoms the function stage, miRNAs are loaded into RNA-induced silencing
are exhibited by the plant. Subsequently, P. infestans transforms to complexes to complement mRNAs partly or fully, leading to deg-
necrotrophic stages, feeding on dead plant tissue (Leesutthiphonchai radation or translational repression of target transcripts (Song et al.
et al. 2018). Currently, the primary strategy for suppressing tomato 2019). The biological roles of most plant miRNAs have not yet
late blight relies on frequent applications of fungicides, which are been discovered, so it remains challenging to accurately define their
costly and harmful to human health and the environment (Cohen functions. With the development of genome editing technology,
et al. 2018). Consequently, identifying and incorporating a new various reverse genetics approaches have been used to manipulate
source of genetic resistance to P. infestans has become a priority in MIRNA genes, such as artificial miRNA (amiRNA) (Eamens et al.
tomato breeding. 2011), short tandem target mimic (STTM) (Yan et al. 2012), and
Micro-RNAs (miRNAs) are endogenous noncoding RNAs of 20 miRNA sponge (Reichel et al. 2015; Zhou et al. 2020) techniques.
to 24 nt playing crucial roles in plant stress response by regulating
The amiRNA is an artificially modified miRNA that mimics the
structures of endogenous miRNA, which can effectively silence
†
Corresponding authors: Y. Luan; ysluan@dlut.edu.cn, endogenous miRNA by targeting its stem-loop precursor sequence
and H. Qi; qihongyan@syau.edu.cn (Eamens et al. 2011). The STTM technology bridges two target
mimics (TMs) with a specific 48-nt sequence. The three-base mis-
Funding: This study was financially supported by the National Natural Science match structure of these two TMs allows miRNA to bind to it but
Foundation of China grants 32072592, 31872116, and 61872055.
cannot be cleaved (Yan et al. 2012). Furthermore, miRNA sponges
*The e-Xtra logo stands for “electronic extra” and indicates that four supplemen- contain many repeats of miRNA binding sites, which can bind to
tary figures and two supplementary tables are published online. target miRNAs without being cleaved by them. The aforementioned
The author(s) declare no conflict of interest. methods have been used extensively to study the functions of
miRNAs in plant response to biotic stresses. For instance, suppres-
© 2021 The American Phytopathological Society sion of miR1916 activity by STTM and amiRNA technologies in

1008 PHYTOPATHOLOGY®
tomato improved plant resistance to Botrytis cinerea and P. infestans were prepared by rinsing the surface of the medium covered by
(Chen et al. 2019b). Circular RNA 477-3p and long noncoding mycelium with distilled water, then incubating the sporangiophore
RNA 39026 have been reported to be involved in tomato resistance at 4 C for 1 to 2 h. The concentration of spore suspensions was
by acting as miRNA sponges (Hong et al. 2020; Hou et al. 2020). adjusted to 106 zoospores/ml with a microscope and a hemocytom-
Apart from the methods mentioned previously, a newly devel- eter (Hong et al. 2019). For infection, six-leaf stage tomato plants
oped powerful approach, clustered regularly interspaced short palin- with consistent growth were sprayed with P. infestans spores sus-
dromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), has pension (106 zoospores/ml), then placed in a shed at 20 ± 1 C
been continuously improved and widely applied to edit genomes of under 100% relative humidity. Subsequently, leaves were sampled
many plants (Chen et al. 2019a). A typical CRISPR/Cas9 system at 0, 3, 6, 12, 24, 48, 72, and 96 h postinfection (hpi) with three bio-
includes a single guide RNA (sgRNA) and a Cas9 enzyme (Jinek logical replicates.
et al. 2012). The sgRNA directs Cas9 to cleave the target locus Prediction of miR482b and miR482c common target genes.
adjacent to the 50-NGG-30 protospacer adjacent motif and generate The precursor and mature sequences of miR482b and miR482c in
double-stranded DNA breaks, which are repaired mainly by nonho- tomato were acquired from miRBase (version 22) (Kozomara et al.
mologous end joining in higher eukaryotes (Chen et al. 2019a). The 2019). Common targets of mature miR482b and mature miR482c
nonhomologous end joining repair pathway can generate variable- were identified via two different algorithms, psRNATarget (Dai
size insertions or deletions (indels), ultimately leading to gene func- et al. 2018) and TAPIR (Bonnet et al. 2010). Tomato transcripts
tion disruption (Wang et al. 2013). Because of its convenience, (JGI Genome Project, iTAG version 2.3) were used as references
CRISPR/Cas9 technology has been used to study gene functions in for prediction in psRNATarget. The expectation value £1.5 was
tomato resistance to various biotic stresses. For example, powdery set as a limitation, and the other parameters were set as defaults.
mildew-resistant tomatoes were generated by knocking out the The score and free energy ratio set for TAPIR prediction were 4
MILDEW RESISTANT LOCUS O (Mlo) gene through the CRISPR- and 0.7, respectively. The target genes of miR482b and miR482c
Cas9 system (Nekrasov et al. 2017). Likewise, Atypical Receptor were separately predicted by TAPIR first, and the shared genes
Kinase 1 (TARK1) CRISPR mutants were resistant to Pseudomonas were reserved as set A. Similarly, the target genes of miR482b and
syringae pv. tomato strain DC3000 (Guzman et al. 2020). Addi- miR482c were separately predicted by psRNATarget, and the
tionally, CRISPR/Cas9 can simultaneously edit multiple genes. shared genes were reserved as set B. Subsequently, the intersec-
Through the multiplex CRISPR/Cas9 system, multisite genome tions of A and B were considered the common target genes of
knockout mutations were generated by editing of five key genes in miR482b and miR482c.
tomato c-aminobutyric acid shunts (Li et al. 2018b); desirable traits Vector construction. We found that disrupting the premiRNA
were introduced into four wild tomato accessions by simultaneous stem–loop structure by editing miRNA and miRNA* sequences is
editing of multiple domestication genes (Li et al. 2018c). Recently, likely to impede miRNA biogenesis and function (Bi et al. 2020).
mir knockout mutants have been widely generated to investigate Using the online tool chopchop (http://chopchop.cbu.uib.no/)
miRNA roles in plant biological processes. Loss of miR529c in (Montague et al. 2014), we designed the sgRNA1, sgRNA2, and
Marchantia polymorpha triggered ectopic development on thalli sgRNA3 to disrupt the stem–loop structure of premiR482b. The
(Tsuzuki et al. 2019); loss of miR528 impaired rice tolerance to salt sgRNA4, sgRNA5, and sgRNA6 were designed to disrupt the stem–
stress (Zhou et al. 2017). Apart from single miRNA knockout, mul- loop structure of premiR482c. The cassette of the Arabidopsis U6
tiple miRNA knockout has become a potential strategy to elucidate promoter–six tRNA–sgRNAs–Oryza sativa U3 terminator was
gene functions comprehensively. assembled into the modified binary vector pBI121 containing a plant
MIR482 is a conserved and extensive family playing pivotal roles codon-optimized Cas9 gene driven by 35S promoter via PCR, BsaI
in regulating plant defense mechanisms via targeting transcripts with digestion, and ligase. The primers used to construct the CRISPR/Cas9
nucleotide binding site-leucine-rich repeat (NBS-LRR) motifs (de vector (named CR-miR482b, c) are listed in Supplementary Table S1.
Vries et al. 2015). The NBS-LRR genes participate in plant immunity Transient expression of CR-miR482b, c in tomato. Transient
by mediating the recognition of pathogen effectors (Belkhadir et al. agroinfiltration assay was performed according to our earlier study
2004), and their functions in plant resistance have been broadly (Hong et al. 2019). The infiltration culture containing CR-miR482b, c
studied in cotton (Li et al. 2018a), soybean (Xun et al. 2019), tomato was introduced into tomato leaves by infiltration, and the infiltration
(Jiang et al. 2018a), and other species. We previously reported over- culture containing empty vector (EV) was used as a control. After
expressing lncRNA23468, which acts as an miR482b sponge in transient expression of CR-miR482b, c for 3 days at 20 C without
tomato, reinforced plant resistance to P. infestans (Jiang et al. 2019). light, the expression levels of miR482b and miR482c were investi-
Also, overexpression of miR482c impaired tomato resistance (Hong gated. Subsequently, each infiltrated leaf region was inoculated with
et al. 2019). However, earlier strategies were less convenient when 20 ml of P. infestans droplets. Three days after inoculation, the nec-
multiple miRNAs exerted functions simultaneously because they rotic lesions were observed and the diameters of the lesions were
silence only a single miRNA at a time. Moreover, there has been no statistically calculated with a sample size of 20.
research on editing multiple miRNAs in tomato. We hypothesized Stable transformation and genotyping of genome editing
that compared with silencing a single miRNA, silencing multiple events. The agrobacterium-mediated transformation method was
miRNAs at the same time will increase resistance in tomato. Our applied to generate transgenic tomato plants (Jiang et al. 2018b). The
results provide empirical guidelines on targeting two miRNAs simul- T0 seedlings were screened on kanamycin-resistant medium. Total
taneously with CRISPR/Cas9 in tomato, which contribute to the genomic DNA of each T0 seedling was isolated from 100 mg of fresh
understanding of the molecular mechanisms of tomato–P. infestans frozen leaves with a Plant Genomic DNA Kit (Tiangen, Beijing,
interactions and serve as an effective strategy to study plant disease China). PCR amplifications of the neomycin phosphoryl transferase
resistance in other species. II (nptII) gene (Supplementary Table S1) were performed by using
their DNA as templates to confirm the presence of the transgene. The
MATERIALS AND METHODS verified transgenic plants were grown in the tissue culture room for 4
weeks (16/8 h of light/dark, 25 ± 3 C) and then transferred to the soil
Plant material and pathogen inoculation. Solanum lycopersi- and grown under the same conditions for 2 weeks.
cum-susceptible cultivar Zaofen no. 2 was grown in a greenhouse The specific genomic regions of the premiR482b and premiR482c
under a photoperiod of 16 h light/8 h dark at 25 ± 3 C. P. infestans were amplified with the primers flanking the target sites (Supplemen-
strain P12103 was cultured in oat medium in a dark microbial incu- tary Table S1). The PCR products were subjected to the Hi-TOM
bator at 20 C for 20 days. The spore suspensions of P. infestans sequencing platform for tracking mutations induced by the CRISPR/

Vol. 111, No. 6, 2021 1009

Cas9 system (Liu et al. 2019). The secondary structures of pre- in technical triplicate for each sample, with three independent bio-
miR482b and premiR482c were analyzed by the online tool RNA- logical replicates. The Sl-actin gene was used as an internal control
fold (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi) (Mascia et al. 2010). The relative expression level of each gene was
(Hofacker 2003). calculated via 2–DDCt analysis (Livak and Schmittgen 2001). All the
Assay of resistance to P. infestans. Detached leaf inoculation primers used in qRT-PCR assay are listed in Supplementary Table S1.
and whole plant infection assays were performed to confirm disease Statistical analysis methods. The relative expressions (log2) of
resistance of wild-type (WT) and transgenic plants, as previously the genes in transgenic lines versus WT were displayed in bar
reported (Hong et al. 2019). In the detached leaf inoculation assay, graphs according to previous work (Canto-Pastor et al. 2019). Stat-
the diameter of the lesion area on each leaf was measured 5 days istical comparisons of the data were implemented with Duncan’s
after inoculation with a sample size of 20, then the leaves were sub- multiple range test. Significant differences (P < 0.05) were indi-
jected to trypan blue staining solution (2.5 mg/ml) for 12 h; finally, cated by different lowercase letters.
an ethanol solution was applied to decolor the leaves (Li et al.
2015). Also, the expression of P. infestans actin gene was detected
to indicate the growth status of P. infestans in lesion areas. In the RESULTS
whole plant infection assay, the zoospore suspension was sprayed
on the whole plant of WT and transgenic plants with a sample size Identification of miR482b and miR482c common targets
of 12, and the blight symptoms were observed. The disease severity and their expression characteristics upon infection. As
index (DSI) of each plant was evaluated and assigned a score predicted by TAPIR, 12 genes were targeted by miR482b and
10 genes were targeted by miR482c. Among them, seven genes
between 0 and 4, where 0 indicated asymptomatic, 1 indicated 1 to
(Solyc05g008070, Solyc12g017800, Solyc11g006530, Solyc12g016220,
5% leaf area infected, 2 indicated 5 to 25% of leaf area infected, 3
Solyc10g054940, Solyc10g055170, and Solyc10g057970) were shared.
indicated 26 to 50% of leaf area infected, and 4 indicated 51 to
As predicted by psRNATarget, six genes were targeted by miR482b
75% of leaf area infected. The disease severity rating (DSR) was
and 10 genes were targeted by miR482c. Among them, three genes
calculated with the following formula (Kim 2012):
(Solyc05g008070, Solyc12g017800, and Solyc11g006530) were
DSR (%) 5
Rðnumber of plants with symptoms  DSIÞ ×100. shared. Combined with these two prediction results, three NBS-LRR
4  number of plants genes, Solyc05g008070, Solyc11g006530, and Solyc12g017800,
were considered simultaneously targeted by miR482b and miR482c.
Histochemical staining and physiological assay. Diamino- The expression patterns of miR482c and Solyc11g006530 upon
benzidin and nitro blue tetrazolium staining assays were carried infection were detected previously (Hong et al. 2019). In the current
out to observe the H2O2 and O2
– accumulation in leaves of work, to explore the expression patterns of miR482b, Solyc05g008070,
whole plant infection assays, respectively. Peroxidase (POD) and and Solyc12g017800 on P. infestans, the relative expressions of
superoxide dismutase (SOD) are reactive oxygen species (ROS) these genes at 3, 6, 12, 24, 48, 72, and 96 hpi were compared with
scavengers that can catalyze the removal of H2O2 and O2
–, 0 hpi. In the biotrophic stage of P. infestans, both miR482b and
respectively, to maintain the intracellular ROS concentration miR482c were induced by infestation as early as 6 hpi (Fig. 1).
(Brigelius-Floh
e and Floh
e 2020; Torres 2010). Malondialdehyde Compared with miR482c, miR482b showed continuous induction
(MDA) content is an indicator of membrane system damage within 24 hpi. Moreover, the regulation of miR482b and miR482c
(Farmer and Mueller 2013). Phenylalanine ammonia-lyase (PAL) with their target genes was robust within 24 hpi. In the necro-
is a key enzyme in the phenylpropane metabolism pathway and trophic stage of P. infestans, there appeared to be a similar induc-
is related to the formation of disease-resistant secondary biomass tion rhythm for both miR482b and miR482c, because at 48 hpi,
(Wang et al. 2019). Therefore, the content of MDA and the the expression levels of both miRNAs dropped and stayed steady
enzyme activities of POD, SOD, and PAL were measured at 72 hpi (Fig. 1) (Hong et al. 2019). The expression of target
according to the previous method (Li et al. 2015). genes showed low coregulation with miR482b and miR482c at
Gene expression analysis by quantitative real-time PCR. this stage, indicating that they were probably regulated by other
Quantitative real-time PCR (qRT-PCR) assay was applied to deter- mechanisms.
mine the expression levels of pri-miR482b, pri-miR482c, miR482b, Structure of CRISPR/Cas9 vector and transient silencing
and miR482c, and common target genes of miR482b and miR482c. of miR482b, c in tomato. To disrupt the stem–loop structure of the
Additionally, the other five members in miR482 gene family miRNA precursor, sgRNAs were designed to target the mature and
(miR482a, miR482d-3p, miR482d-5p, miR482e-3p, and miR482e-5p) passenger strands (miRNA*) of miR482b and miR482c (Fig. 2A).
and six randomly selected miRNAs (miR156a, miR164a, miR476b, Afterward, the tRNA–sgRNA system was used in the engineered
miR5300, miR5302, and miR6027) were also subjected to qRT-PCR CRISPR/Cas9 binary vector (Fig. 2B). The successfully constructed
assay to detect the off-targeting effects. All reactions were carried out vector was named CR-miR482b, c.

Fig. 1. Expression characteristics of miR482b, solyc05g008070, and solyc12g017800 on Phytophthora infestans infection. Relative expression (log2) of
A, miR482b, B, solyc05g008070, and C, solyc12g017800 in infected plants at 3, 6, 12, 24, 48, 72, and 96 h postinfection (hpi) compared with 0 hpi (n 5 3).

1010 PHYTOPATHOLOGY®
 After transient expression of CR-miR482b, c for 3 days, the larger the absolute value of MFE, the more stable the structure is
expression of miR482b and miR482c decreased by 70 and 55%, (Ng and Mishra 2007). Compared with WT, the absolute MFE val-
respectively, in CR-miR482b, c plants compared with empty vector ues of premir482b and premir482c in transgenic lines were lower
plants (Fig. 2C). Moreover, transgenic tomatoes that transiently (Supplementary Fig. S2), indicating that the precursor structures in
expressed CR-miR482b, c displayed less severe disease symptoms the mutants were not as stable as in WT. The previous study showed
upon infection (Fig. 2D), with almost 50% reduction in diameter of that DCL1 is responsible for processing most miRNAs into 21-nt
lesions (DOL) (Fig. 2E). miRNA/miRNA* duplexes; the unstable precursor secondary struc-
CRISPR/Cas9 system-mediated mutagenesis of miR482b ture may interfere with the recognition activity of DCL1 (Bi et al.
and miR482c in tomatoes. Transgenic plants were generated by 2020). Partial inhibition of DCL1 activity may lead to the accumula-
the introduction of CR-miR482b, c plasmid into the tomato. Three tion of pri-miRNA in mutants. It was conceivable that the cleavage
transgenic T0 plants were selected and named L1, L2, and L3 by of normal premiR482b and premiR482c structures was impaired,
PCR application of nptII genes for further examination. The leading to higher pri-miRNA accumulation and lower mature
Hi-TOM platform was applied to identify transgenic plant mutation miRNA accumulation.
types. Transgenic line L1 carried both biallelic and heterozygous Off-target effects of miRNA-targeting sgRNAs. To investi-
mutations at target sites, L2 carried biallelic mutations, and L3 car- gate the possibility of off-targeting, we examined six sgRNAs used
ried heterozygous mutations (Fig. 3A to D). For miR482b, L1 con- in this study, and we checked L1, L2, and L3 transgenic tomato
tains both 4 base-pair (CATG) deletion and single “T” insertion, plants. We selected three high-probability off-target sites for each
L2 contained both 3 base-pair (ATG) deletion and single “T” inser- sgRNA assay, with the exception of sgRNA2, with one high-prob-
tion, and L3 contained 2 base-pair (AT) deletion (Fig. 3A). For ability off-target site. Surveying these potential off-targeting sites
miR482c, L1 contained 3 base-pair (GCG) deletion, L2 contained via Sanger sequencing of PCR products, we found no mutations
single “A” insertion and 3 base-pair (GCG) deletion, and L3 (Supplementary Table S2). Although we could not observe all
showed no mutations (Fig. 3C). potentially off-target sites extensively, the results suggested that
The transcript accumulations of pri-miR482b and pri-miR482c off-targeting effects from these sgRNAs are of less concern.
were greater in L1 and L2 than in WT (Fig. 3E and F). The relative Silencing of miR482b, c increased tomato resistance to P.
expression of miR482b declined in all three transgenic plants
infestans. The expression patterns of Solyc05g008070, Solyc11g006530,
(Fig. 3G), and the expression of miR482c decreased only in L1 and
and Solyc12g017800 in WT and positive transgenic plants were iden-
L2 (Fig. 3H). It was hypothesized that expression of the MIRNA
genes is not attenuated but rather the cleavage of the stem-loop tified by qRT-PCR. Compared with WT plants, the expression of
structure is reduced, resulting in less mature miRNAs and an accu- Solyc05g008070, Solyc11g006530, and Solyc12g017800 increased
mulation of primary miRNA transcripts. 3.8-fold, threefold, and twofold in L1, and the expression of
As mentioned previously, miRNA biogenesis proceeds through Solyc05g008070 and Solyc11g006530 increased 5.2-fold and
steps from pri-miRNA, premiRNA, and miRNA/miRNA* duplex to 7.8-fold in L2 and 1.5-fold and 1.3-fold in L3, respectively (Fig. 4A).
mature miRNA. The biogenesis of miRNA depends on the stem- The expression of Solyc12g017800 in L2 and L3 was lower (Fig.
loop structure of premiRNA. Because mutations in the mature 4A), probably because it was affected by other regulatory
miRNA sequences had been created, some sequence changes may mechanisms.
cause a slight disruption in the premiRNA stem–loop structure. In In the detached-leaves infection assay, after inoculation for 5
order to detect the biogenesis changes due to the altered sequences days, the dead cells on the inoculated leaves were observed by
within the mature miRNA sequences, the secondary structures of trypan staining assay. We found that the number of dead cells, indi-
premiR482b and premiR482c in WT and three transgenic lines were cated by darker blue marks on transgenic tomato leaves, was much
predicted. The results showed that, compared with WT, the stem– less than that on WT leaves (Fig. 4B). The DOLs of L1, L2, and
loop structure of premiR482b and premiR482c in transgenic plants L3 leaves were smaller than on WT leaves; L2 had the smallest
showed abnormal bulges (indicated by black boxes) and asymmetric DOL, followed by L1, and finally L3 (Fig. 4D). Furthermore, the
duplex structures (Supplementary Fig. S2). In addition, minimum expression levels of P. infestans actin gene in L1, L2, and L3 were
free energy (MFE) is an index of secondary structure stability. The lower than in WT (Fig. 4C).

Fig. 2. MiR482b, c gene editing via CRISPR/Cas9 and transient expression of CR-miR482b, c in tomato. A, Schematic illustration of multiple targeting sites
in miR482b and miR482c gene sequence. The single guide RNA (sgRNA) sequences are underlined, and the protospacer adjacent motif sequences are high-
lighted. B, Structure of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) vector built on the pBI121
backbone. After transient expression of CR-miR482b, c for 3 days, we compared C, expression of miR482b and miR482c in CRISPR versus wild-type plants
(n 5 3), D, necrotic lesions (scale bars 5 5 mm), and E, the diameter of lesions (DOL) on the detached leaves (n 5 20).

Vol. 111, No. 6, 2021 1011

Fig. 3. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)–mediated mutagenesis of miR482b and
miR482c in tomatoes: mutation sites in A, mature miR482b, B, miR482b*, C, mature miR482c, and D, miR482c* in three transgenic plants. The base inser-
tion is indicated by small triangles, and the base deletion is represented by a dash. The relative transcript accumulations of E, pri-miR482b, F, pri-miR482c,
G, mature miR482b, and H, mature miR482c in transgenic plants (n 5 3) are log2 transformed and displayed. WT, wild type.

Fig. 4. Transgenic plants with silenced miR482b and miR482c showed increased resistance to Phytophthora infestans. A, Relative expression (log2) of
Solyc05g008070, Solyc11g006530, and Solyc12g017800 in transgenic plants compared with wild-type (WT) plants (n 5 3). B, Necrotic lesions and trypan
blue staining on leaves of WT and transgenic plants (scale bars 5 5 mm) (n 5 20) in the detached leaves 5 days after inoculation with P. infestans. C, Relative
expression (log2) of P. infestans actin gene in transgenic plants compared with WT plants (n 5 3). D, Diameter of lesions (DOL) on detached leaves of WT
and transgenic plants (n 5 20). In the whole plant infection assay, 5 days after infection, E, disease symptoms (scale bars 5 5 mm) and F, disease severity
rating (DSR) of WT and transgenic plants (n 5 12).

1012 PHYTOPATHOLOGY®
 Similarly, 5 days after whole plant infection, all transgenic plants miR482c tended to be stable, and the inhibitory effect of miR482b
exhibited fewer disease symptoms than those in WT (Fig. 4E). and miR482c on the target genes decreased, leading to increased
Meanwhile, the DSRs of L1, L2, and L3 were 0.36-fold, 0.33-fold, expression of the target genes (Fig. 1) (Hong et al. 2019).
and 0.73-fold lower, respectively, than those of WT (Fig. 4F). These It has been reported that modest but impactful changes in
results indicated that the silencing of miR482b, c increased tomato miRNAs genes might have phenotypic consequences (Chuck et al.
defense. 2007). In our study, all three transgenic plants contained mutations
Altered ROS level and physiological indicators increased for miR482b (Fig. 3A). L1 and L2 contained mutations for
tomato resistance. To further explore the resistance mechanism miR482c, and L3 showed no mutation (Fig. 3C). The expressions of
mediated by gene editing of miR482b and miR482c, we analyzed miR482b and miR482c common target genes were higher in trans-
the ROS levels in WT and transgenic plants. Compared with WT genic plants (Fig. 4A). The disease symptoms and DSR of transgenic
plants, the transgenic plant leaves displayed fewer brown spots, plants were less than those of WT plants in the whole plant infection
indicating a decrement in H2O2 accumulation after diaminobenzidin assay (Fig. 4E and F). Furthermore, the disease resistance of L1 and
staining (Fig. 5A and B), and fewer blue spots indicated a reduction L2, with silenced miR482b and miR482c simultaneously, was super-
in O2
– accumulation after nitro blue tetrazolium staining (Fig. 5C ior to that of L3, with silenced miR482b alone, indicating miR482c
and D). Furthermore, the SOD and POD enzymes in the transgenic may play a negative role in resistance (Supplementary Fig. S1B).
plants exhibited higher activity, indicating increased ROS scaveng- This finding was a supplement to our previous work (Hong et al.
ing activity (Fig. 5E and F). Moreover, lower MDA contents 2019) and further proves the role of miR482c in disease resistance.
reflected alleviated damage of the membrane system in transgenic Moreover, compared with controls, in the transiently expressed
plants (Fig. 5G). Additionally, the PAL enzyme activities in trans- CR-miR482b, c plant, the expression of miR482b was 70% lower
genic lines were higher than in WT plants (Fig. 5H), suggesting (Fig. 2C), and the DOL was almost 50% lower (Fig. 2E). We previ-
higher accumulation of disease-resistant secondary biomass. These ously reported that overexpression of lncRNA23468, which acts as
results indicate that altered ROS level and physiological indicators an miR482b sponge, led to a 60% decrease in miR482b expression
contribute to increased resistance of transgenic tomato plants. (Supplementary Fig. S1A) and a 40% decrease in DOL (Supplemen-
tary Fig. S1B) (Jiang et al. 2019). It was inferred that CRISPR/Cas9
DISCUSSION technology is more efficient in silencing miR482b than the miRNA
sponge method.
In the biotrophic phase of P. infestans, resistance proteins were Under pathogen attack, the pathogen-associated molecular pat-
highly expressed, and percept pathogens subsequently activate tern-triggered immunity in plants was elicited and a number of
hypersensitive response/cell death, which in turn would restrain defense mechanisms were induced, including stomatal closure to
pathogen spread (Vleeshouwers et al. 2000). In our work, both limit the entry of bacteria (Bigeard et al. 2015; Melotto et al.
miR482b and miR482c were induced by infection as early as 2008); generation of phytoalexins, such as camalexin and patho-
6 hpi, a vital time point for infection success, because early hyper- gen resistance proteins (Ahuja et al. 2012; Bednarek 2012);
sensitive response significantly advances resistance to P. infestans programmed cell death at the site of infection to limit the progres-
(Vleeshouwers et al. 2000). Additionally, in this phase two NBS- sion of pathogens (Mur et al. 2008); and accumulation of ROS
LRR proteins, soly05g008070 and solyc12g017800, were highly (Torres 2010). In the dynamic balance between the formation and
expressed, and the regulation of miR482b and miR482c to target removal of active oxygen in the plant, POD and SOD enzymes
genes was robust (Fig. 1), which was consistent with a previous exerted important functions, and they were related to the synthesis
work (de Vries et al. 2018). During the necrotrophic phase of of lignin and antivirals, increasing the resistance of plants to vari-
P. infestans (after 72 hpi), the expressions of miR482b and ous pathogens (Brigelius-Floh
e and Floh
e 2020). In our study,

Fig. 5. Altered reactive oxygen species level and physiological indicators contributed to tomato resistance. A, Diaminobenzidin staining for H2O2 (scale
bars 5 5 mm) and B, relative accumulation of H2O2 of wild-type (WT) and transgenic plants 5 days after whole plant infection assay. C, Nitro blue tetrazo-
lium staining for O2
– (scale bars 5 5 mm) and D, relative accumulation of O2
– in WT and transgenic plants. E, peroxidase (POD) activity (U/g FW),
F, superoxide dismutase (SOD) activity (U/g FW), G, malondialdehyde (MDA) content (mmol/g FW), and H, phenylalanine ammonia-lyase (PAL) activity
(mmol/g FW) of WT and transgenic plants.

Vol. 111, No. 6, 2021 1013

after P. infestans infection, the transgenic plants all showed lower so when one miRNA in the cluster is knocked out, the secondary
ROS levels in comparison with WT plants (Fig. 5A to D and 6), structure of their common primary precursor is changed, and the bio-
which implies resistance. The change in other physiological indi- genesis of other miRNAs in the cluster is thus affected (Chang et al.
cators, such as MDA content and PAL enzyme activity, can also 2016). Therefore, when miR482b and miR482c were knocked out,
contribute to plant resistance. MDA is a final decomposition prod- expression levels of miR482d-3p and miR482d-5p in the cluster also
uct of lipid peroxidation, which reduces the fluidity of the cell decreased. In addition to the miR482 family, there are numerous
membrane and destroys membrane function (Farmer and Mueller miRNAs, including miR476b, miR5300, and miR6027, with the
2013); the PAL enzyme was involved in the formation of disease- potential to target NBS-LRRs (Luan et al. 2015). Interestingly, we
resistant secondary biomass including plant protection factors, lig- found that expression of miR476b, miR5300, and miR6027
nin, and phenolic compounds (Wang et al. 2019). Lower MDA decreased in the miR482b and miR482c knockout background (Sup-
content and higher PAL activity in transgenic plants compared plementary Fig. S4). We inferred that there was cross-regulation
with WT plants suggests that transgenic plants sustain less mem- between miRNAs at the genome level, which may be attenuated by
brane damage and produce more disease-resistant secondary bio- altered expression of NBS-LRRs and miR482s. Similarly, Zhou et al.
mass (Fig. 5G and H and 6). These results were consistent with (2017) identified a list of differentially expressed miRNAs in
previous findings that elevated ROS scavenging ability, lower OsMIR408 and OsMIR520 mutant backgrounds using miRNA
MDA content, and higher PAL activity increase tomato resistance sequencing data and qRT-PCR analysis We also selected three miR-
(Luan et al. 2018). NAs (miR156a, miR164a, and miR5302) that were not closely
miRNAs play critical roles in regulating expression of many per- related to disease resistance to verify their expression levels. The
ipheral genes involved in various biological processes. Expression expressions of miR156a, miR164a, and miR5302 were not affected
profiling in an miRNA knockout background should reveal the regu- by the miR482b and miR482c knockout background (Supplementary
latory functions of miRNAs. To explore this prospect, we first Fig. S4). These results indicate that knocking out miR482b and
detected the expression levels of the other five members of the miR482c elicited expression perturbation of other miRNAs. Because
miR482 gene family (miR482a, miR482d-3p, miR482d-5p, no mutations were found in our off-targeting analysis, it can be
miR482e-3p, and miR482e-5p) in our transgenic lines and WT. The inferred that there are miRNA–miRNA regulation networks, which is
results showed that the transcript accumulation of miR482d-3p and of great significance for elucidating the novel regulatory mechanism
miR482d-5p was lower in transgenic lines than in WT, whereas the of miRNAs in tomato–P. infestans interactions.
expression of miR482a, miR482e-3p, and miR482e-5p did not differ In summary, we edited two miRNAs simultaneously by
(Supplementary Fig. S3). According to the location indicated by CRISPR/Cas9 in tomato for the first time (Fig. 6), compared with
miRBase, miR482d, miR482b, and miR482c belong to a gene clus- the traditional method of silencing a single miRNA; this strategy
ter. Cluster miRNAs are generally cotranscribed at a genomic locus, saves time, labor, and material resources, and the procedure is more

Fig. 6. Working model of silencing miR482b and miR482c by CRISPR/Cas9 system in tomato resistance to Phytophthora infestans.

1014 PHYTOPATHOLOGY®
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