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
the global tomato industry. Micro-RNAs (miRNAs) have been widely
demonstrated to play vital roles in plant resistance by repressing their
target genes. Recently, the clustered regularly interspaced short
palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)
method has been continuously improved and extensively applied to edit
plant genomes. However, editing multiplex miRNAs by CRISPR/Cas9
in tomato has not been studied yet. We knocked out miR482b and
miR482c simultaneously in tomato through the multiplex CRISPR/Cas9
system. Two transgenic plants with silenced miR482b and miR482c
simultaneously and one transgenic line with silenced miR482b alone were
obtained. Compared with wild-type plants, the disease symptoms of three
transgenic plants upon infection were reduced, accompanied by increased
Tomato is not only a major crop in global agriculture but also an
important model for investigating plant–pathogen interactions. Late
blight (LB) has triggered widespread damage in the global tomato
industry since the mid-1840s (Fry 2016; Fry et al. 2015). The
causal agent of LB, Phytophthora infestans, ranked first among 10
selected plant pathogenic oomycete species (Kamoun et al. 2015).
As a hemibiotroph, P. infestans initially infects living tissues and
extracts nutrients in its biotrophic stages, when minimal symptoms
are exhibited by the plant. Subsequently, P. infestans transforms to
necrotrophic stages, feeding on dead plant tissue (Leesutthiphonchai
et al. 2018). Currently, the primary strategy for suppressing tomato
late blight relies on frequent applications of fungicides, which are
costly and harmful to human health and the environment (Cohen
et al. 2018). Consequently, identifying and incorporating a new
source of genetic resistance to P. infestans has become a priority in
tomato breeding.
Micro-RNAs (miRNAs) are endogenous noncoding RNAs of 20
to 24 nt playing crucial roles in plant stress response by regulating
†
Corresponding authors: Y. Luan; ysluan@dlut.edu.cn,
and H. Qi; qihongyan@syau.edu.cn
Funding: This study was financially supported by the National Natural Science
Foundation of China grants 32072592, 31872116, and 61872055.
*The e-Xtra logo stands for “electronic extra” and indicates that four supplemen-
tary figures and two supplementary tables are published online.
The author(s) declare no conflict of interest.
© 2021 The American Phytopathological Society
1008 PHYTOPATHOLOGY®
expression of their common target nucleotide binding site-leucine-rich
repeat genes and decreased levels of reactive oxygen species. Furthermore,
silencing miR482b and miR482c simultaneously was more resistant than
silencing miR482b alone in tomato. More importantly, we found that
knocking out miR482b and miR482c can elicit expression perturbation of
other miRNAs, suggesting cross-regulation between miRNAs. Our study
demonstrated that editing miR482b and miR482c simultaneously with
CRISPR/Cas9 is an efficient strategy for generating pathogen-resistant
tomatoes, and cross-regulation between miRNAs may reveal the novel
mechanism in tomato–P. infestans interactions.
Keywords: CRISPR/Cas9, disease resistance, late blight, miRNA, plant
immune responses, tomato
target gene expressions at the posttranscriptional or posttranslational
level (Ha and Kim 2014; Song et al. 2019; Yu et al. 2017). The bio-
synthesis of miRNAs in plants starts with the transcription of
MIRNA genes into the primary miRNA transcripts, which sequen-
tially are cleaved by DCL1 and generate short hairpins (called pre-
miRNA), finally resulting in an miRNA/miRNA* duplex (about
21 bp) (Ha and Kim 2014; Song et al. 2019; Yu et al. 2017). During
the function stage, miRNAs are loaded into RNA-induced silencing
complexes to complement mRNAs partly or fully, leading to deg-
radation or translational repression of target transcripts (Song et al.
2019). The biological roles of most plant miRNAs have not yet
been discovered, so it remains challenging to accurately define their
functions. With the development of genome editing technology,
various reverse genetics approaches have been used to manipulate
MIRNA genes, such as artificial miRNA (amiRNA) (Eamens et al.
2011), short tandem target mimic (STTM) (Yan et al. 2012), and
miRNA sponge (Reichel et al. 2015; Zhou et al. 2020) techniques.
The amiRNA is an artificially modified miRNA that mimics the
structures of endogenous miRNA, which can effectively silence
endogenous miRNA by targeting its stem-loop precursor sequence
(Eamens et al. 2011). The STTM technology bridges two target
mimics (TMs) with a specific 48-nt sequence. The three-base mis-
match structure of these two TMs allows miRNA to bind to it but
cannot be cleaved (Yan et al. 2012). Furthermore, miRNA sponges
contain many repeats of miRNA binding sites, which can bind to
target miRNAs without being cleaved by them. The aforementioned
methods have been used extensively to study the functions of
miRNAs in plant response to biotic stresses. For instance, suppres-
sion of miR1916 activity by STTM and amiRNA technologies in
tomato improved plant resistance to Botrytis cinerea and P. infestans
(Chen et al. 2019b). Circular RNA 477-3p and long noncoding
RNA 39026 have been reported to be involved in tomato resistance
by acting as miRNA sponges (Hong et al. 2020; Hou et al. 2020).
Apart from the methods mentioned previously, a newly devel-
oped powerful approach, clustered regularly interspaced short palin-
dromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), has
been continuously improved and widely applied to edit genomes of
many plants (Chen et al. 2019a). A typical CRISPR/Cas9 system
includes a single guide RNA (sgRNA) and a Cas9 enzyme (Jinek
et al. 2012). The sgRNA directs Cas9 to cleave the target locus
adjacent to the 50-NGG-30 protospacer adjacent motif and generate
double-stranded DNA breaks, which are repaired mainly by nonho-
mologous end joining in higher eukaryotes (Chen et al. 2019a). The
nonhomologous end joining repair pathway can generate variable-
size insertions or deletions (indels), ultimately leading to gene func-
tion disruption (Wang et al. 2013). Because of its convenience,
CRISPR/Cas9 technology has been used to study gene functions in
tomato resistance to various biotic stresses. For example, powdery
mildew-resistant tomatoes were generated by knocking out the
MILDEW RESISTANT LOCUS O (Mlo) gene through the CRISPR-
Cas9 system (Nekrasov et al. 2017). Likewise, Atypical Receptor
Kinase 1 (TARK1) CRISPR mutants were resistant to Pseudomonas
syringae pv. tomato strain DC3000 (Guzman et al. 2020). Addi-
tionally, CRISPR/Cas9 can simultaneously edit multiple genes.
Through the multiplex CRISPR/Cas9 system, multisite genome
knockout mutations were generated by editing of five key genes in
tomato c-aminobutyric acid shunts (Li et al. 2018b); desirable traits
were introduced into four wild tomato accessions by simultaneous
editing of multiple domestication genes (Li et al. 2018c). Recently,
mir knockout mutants have been widely generated to investigate
miRNA roles in plant biological processes. Loss of miR529c in
Marchantia polymorpha triggered ectopic development on thalli
(Tsuzuki et al. 2019); loss of miR528 impaired rice tolerance to salt
stress (Zhou et al. 2017). Apart from single miRNA knockout, mul-
tiple miRNA knockout has become a potential strategy to elucidate
gene functions comprehensively.
MIR482 is a conserved and extensive family playing pivotal roles
in regulating plant defense mechanisms via targeting transcripts with
nucleotide binding site-leucine-rich repeat (NBS-LRR) motifs (de
Vries et al. 2015). The NBS-LRR genes participate in plant immunity
by mediating the recognition of pathogen effectors (Belkhadir et al.
2004), and their functions in plant resistance have been broadly
studied in cotton (Li et al. 2018a), soybean (Xun et al. 2019), tomato
(Jiang et al. 2018a), and other species. We previously reported over-
expressing lncRNA23468, which acts as an miR482b sponge in
tomato, reinforced plant resistance to P. infestans (Jiang et al. 2019).
Also, overexpression of miR482c impaired tomato resistance (Hong
et al. 2019). However, earlier strategies were less convenient when
multiple miRNAs exerted functions simultaneously because they
silence only a single miRNA at a time. Moreover, there has been no
research on editing multiple miRNAs in tomato. We hypothesized
that compared with silencing a single miRNA, silencing multiple
miRNAs at the same time will increase resistance in tomato. Our
results provide empirical guidelines on targeting two miRNAs simul-
taneously with CRISPR/Cas9 in tomato, which contribute to the
understanding of the molecular mechanisms of tomato–P. infestans
interactions and serve as an effective strategy to study plant disease
resistance in other species.
MATERIALS AND METHODS
Plant material and pathogen inoculation. Solanum lycopersi-
cum-susceptible cultivar Zaofen no. 2 was grown in a greenhouse
under a photoperiod of 16 h light/8 h dark at 25 ± 3 C. P. infestans
strain P12103 was cultured in oat medium in a dark microbial incu-
bator at 20 C for 20 days. The spore suspensions of P. infestans
were prepared by rinsing the surface of the medium covered by
mycelium with distilled water, then incubating the sporangiophore
at 4 C for 1 to 2 h. The concentration of spore suspensions was
adjusted to 106 zoospores/ml with a microscope and a hemocytom-
eter (Hong et al. 2019). For infection, six-leaf stage tomato plants
with consistent growth were sprayed with P. infestans spores sus-
pension (106 zoospores/ml), then placed in a shed at 20 ± 1 C
under 100% relative humidity. Subsequently, leaves were sampled
at 0, 3, 6, 12, 24, 48, 72, and 96 h postinfection (hpi) with three bio-
logical replicates.
Prediction of miR482b and miR482c common target genes.
The precursor and mature sequences of miR482b and miR482c in
tomato were acquired from miRBase (version 22) (Kozomara et al.
2019). Common targets of mature miR482b and mature miR482c
were identified via two different algorithms, psRNATarget (Dai
et al. 2018) and TAPIR (Bonnet et al. 2010). Tomato transcripts
(JGI Genome Project, iTAG version 2.3) were used as references
for prediction in psRNATarget. The expectation value £1.5 was
set as a limitation, and the other parameters were set as defaults.
The score and free energy ratio set for TAPIR prediction were 4
and 0.7, respectively. The target genes of miR482b and miR482c
were separately predicted by TAPIR first, and the shared genes
were reserved as set A. Similarly, the target genes of miR482b and
miR482c were separately predicted by psRNATarget, and the
shared genes were reserved as set B. Subsequently, the intersec-
tions of A and B were considered the common target genes of
miR482b and miR482c.
Vector construction. We found that disrupting the premiRNA
stem–loop structure by editing miRNA and miRNA* sequences is
likely to impede miRNA biogenesis and function (Bi et al. 2020).
Using the online tool chopchop (http://chopchop.cbu.uib.no/)
(Montague et al. 2014), we designed the sgRNA1, sgRNA2, and
sgRNA3 to disrupt the stem–loop structure of premiR482b. The
sgRNA4, sgRNA5, and sgRNA6 were designed to disrupt the stem–
loop structure of premiR482c. The cassette of the Arabidopsis U6
promoter–six tRNA–sgRNAs–Oryza sativa U3 terminator was
assembled into the modified binary vector pBI121 containing a plant
codon-optimized Cas9 gene driven by 35S promoter via PCR, BsaI
digestion, and ligase. The primers used to construct the CRISPR/Cas9
vector (named CR-miR482b, c) are listed in Supplementary Table S1.
Transient expression of CR-miR482b, c in tomato. Transient
agroinfiltration assay was performed according to our earlier study
(Hong et al. 2019). The infiltration culture containing CR-miR482b, c
was introduced into tomato leaves by infiltration, and the infiltration
culture containing empty vector (EV) was used as a control. After
transient expression of CR-miR482b, c for 3 days at 20 C without
light, the expression levels of miR482b and miR482c were investi-
gated. Subsequently, each infiltrated leaf region was inoculated with
20 ml of P. infestans droplets. Three days after inoculation, the nec-
rotic lesions were observed and the diameters of the lesions were
statistically calculated with a sample size of 20.
Stable transformation and genotyping of genome editing
events. The agrobacterium-mediated transformation method was
applied to generate transgenic tomato plants (Jiang et al. 2018b). The
T0 seedlings were screened on kanamycin-resistant medium. Total
genomic DNA of each T0 seedling was isolated from 100 mg of fresh
frozen leaves with a Plant Genomic DNA Kit (Tiangen, Beijing,
China). PCR amplifications of the neomycin phosphoryl transferase
II (nptII) gene (Supplementary Table S1) were performed by using
their DNA as templates to confirm the presence of the transgene. The
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
and grown under the same conditions for 2 weeks.
The specific genomic regions of the premiR482b and premiR482c
were amplified with the primers flanking the target sites (Supplemen-
tary Table S1). The PCR products were subjected to the Hi-TOM
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-
miR482b and premiR482c were analyzed by the online tool RNA-
fold (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi)
(Hofacker 2003).
Assay of resistance to P. infestans. Detached leaf inoculation
and whole plant infection assays were performed to confirm disease
resistance of wild-type (WT) and transgenic plants, as previously
reported (Hong et al. 2019). In the detached leaf inoculation assay,
the diameter of the lesion area on each leaf was measured 5 days
after inoculation with a sample size of 20, then the leaves were sub-
jected to trypan blue staining solution (2.5 mg/ml) for 12 h; finally,
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
whole plant infection assay, the zoospore suspension was sprayed
on the whole plant of WT and transgenic plants with a sample size
of 12, and the blight symptoms were observed. The disease severity
index (DSI) of each plant was evaluated and assigned a score
between 0 and 4, where 0 indicated asymptomatic, 1 indicated 1 to
5% leaf area infected, 2 indicated 5 to 25% of leaf area infected, 3
indicated 26 to 50% of leaf area infected, and 4 indicated 51 to
75% of leaf area infected. The disease severity rating (DSR) was
calculated with the following formula (Kim 2012):
DSR (%) 5
Rðnumber of plants with symptoms  DSIÞ ×100.
4  number of plants
Histochemical staining and physiological assay. Diamino-
benzidin and nitro blue tetrazolium staining assays were carried
out to observe the H2O2 and O2
– accumulation in leaves of
whole plant infection assays, respectively. Peroxidase (POD) and
superoxide dismutase (SOD) are reactive oxygen species (ROS)
scavengers that can catalyze the removal of H2O2 and O2
–,
respectively, to maintain the intracellular ROS concentration
(Brigelius-Floh
e and Floh
e 2020; Torres 2010). Malondialdehyde
(MDA) content is an indicator of membrane system damage
(Farmer and Mueller 2013). Phenylalanine ammonia-lyase (PAL)
is a key enzyme in the phenylpropane metabolism pathway and
is related to the formation of disease-resistant secondary biomass
(Wang et al. 2019). Therefore, the content of MDA and the
enzyme activities of POD, SOD, and PAL were measured
according to the previous method (Li et al. 2015).
Gene expression analysis by quantitative real-time PCR.
Quantitative real-time PCR (qRT-PCR) assay was applied to deter-
mine the expression levels of pri-miR482b, pri-miR482c, miR482b,
and miR482c, and common target genes of miR482b and miR482c.
Additionally, the other five members in miR482 gene family
(miR482a, miR482d-3p, miR482d-5p, miR482e-3p, and miR482e-5p)
and six randomly selected miRNAs (miR156a, miR164a, miR476b,
miR5300, miR5302, and miR6027) were also subjected to qRT-PCR
assay to detect the off-targeting effects. All reactions were carried out
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).
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in technical triplicate for each sample, with three independent bio-
logical replicates. The Sl-actin gene was used as an internal control
(Mascia et al. 2010). The relative expression level of each gene was
calculated via 2–DDCt analysis (Livak and Schmittgen 2001). All the
primers used in qRT-PCR assay are listed in Supplementary Table S1.
Statistical analysis methods. The relative expressions (log2) of
the genes in transgenic lines versus WT were displayed in bar
graphs according to previous work (Canto-Pastor et al. 2019). Stat-
istical comparisons of the data were implemented with Duncan’s
multiple range test. Significant differences (P < 0.05) were indi-
cated by different lowercase letters.
RESULTS
Identification of miR482b and miR482c common targets
and their expression characteristics upon infection. As
predicted by TAPIR, 12 genes were targeted by miR482b and
10 genes were targeted by miR482c. Among them, seven genes
(Solyc05g008070, Solyc12g017800, Solyc11g006530, Solyc12g016220,
Solyc10g054940, Solyc10g055170, and Solyc10g057970) were shared.
As predicted by psRNATarget, six genes were targeted by miR482b
and 10 genes were targeted by miR482c. Among them, three genes
(Solyc05g008070, Solyc12g017800, and Solyc11g006530) were
shared. Combined with these two prediction results, three NBS-LRR
genes, Solyc05g008070, Solyc11g006530, and Solyc12g017800,
were considered simultaneously targeted by miR482b and miR482c.
The expression patterns of miR482c and Solyc11g006530 upon
infection were detected previously (Hong et al. 2019). In the current
work, to explore the expression patterns of miR482b, Solyc05g008070,
and Solyc12g017800 on P. infestans, the relative expressions of
these genes at 3, 6, 12, 24, 48, 72, and 96 hpi were compared with
0 hpi. In the biotrophic stage of P. infestans, both miR482b and
miR482c were induced by infestation as early as 6 hpi (Fig. 1).
Compared with miR482c, miR482b showed continuous induction
within 24 hpi. Moreover, the regulation of miR482b and miR482c
with their target genes was robust within 24 hpi. In the necro-
trophic stage of P. infestans, there appeared to be a similar induc-
tion rhythm for both miR482b and miR482c, because at 48 hpi,
the expression levels of both miRNAs dropped and stayed steady
at 72 hpi (Fig. 1) (Hong et al. 2019). The expression of target
genes showed low coregulation with miR482b and miR482c at
this stage, indicating that they were probably regulated by other
mechanisms.
Structure of CRISPR/Cas9 vector and transient silencing
of miR482b, c in tomato. To disrupt the stem–loop structure of the
miRNA precursor, sgRNAs were designed to target the mature and
passenger strands (miRNA*) of miR482b and miR482c (Fig. 2A).
Afterward, the tRNA–sgRNA system was used in the engineered
CRISPR/Cas9 binary vector (Fig. 2B). The successfully constructed
vector was named CR-miR482b, c.
 After transient expression of CR-miR482b, c for 3 days, the
expression of miR482b and miR482c decreased by 70 and 55%,
respectively, in CR-miR482b, c plants compared with empty vector
plants (Fig. 2C). Moreover, transgenic tomatoes that transiently
expressed CR-miR482b, c displayed less severe disease symptoms
upon infection (Fig. 2D), with almost 50% reduction in diameter of
lesions (DOL) (Fig. 2E).
CRISPR/Cas9 system-mediated mutagenesis of miR482b
and miR482c in tomatoes. Transgenic plants were generated by
the introduction of CR-miR482b, c plasmid into the tomato. Three
transgenic T0 plants were selected and named L1, L2, and L3 by
PCR application of nptII genes for further examination. The
Hi-TOM platform was applied to identify transgenic plant mutation
types. Transgenic line L1 carried both biallelic and heterozygous
mutations at target sites, L2 carried biallelic mutations, and L3 car-
ried heterozygous mutations (Fig. 3A to D). For miR482b, L1 con-
tains both 4 base-pair (CATG) deletion and single “T” insertion,
L2 contained both 3 base-pair (ATG) deletion and single “T” inser-
tion, and L3 contained 2 base-pair (AT) deletion (Fig. 3A). For
miR482c, L1 contained 3 base-pair (GCG) deletion, L2 contained
single “A” insertion and 3 base-pair (GCG) deletion, and L3
showed no mutations (Fig. 3C).
The transcript accumulations of pri-miR482b and pri-miR482c
were greater in L1 and L2 than in WT (Fig. 3E and F). The relative
expression of miR482b declined in all three transgenic plants
(Fig. 3G), and the expression of miR482c decreased only in L1 and
L2 (Fig. 3H). It was hypothesized that expression of the MIRNA
genes is not attenuated but rather the cleavage of the stem-loop
structure is reduced, resulting in less mature miRNAs and an accu-
mulation of primary miRNA transcripts.
As mentioned previously, miRNA biogenesis proceeds through
steps from pri-miRNA, premiRNA, and miRNA/miRNA* duplex to
mature miRNA. The biogenesis of miRNA depends on the stem-
loop structure of premiRNA. Because mutations in the mature
miRNA sequences had been created, some sequence changes may
cause a slight disruption in the premiRNA stem–loop structure. In
order to detect the biogenesis changes due to the altered sequences
within the mature miRNA sequences, the secondary structures of
premiR482b and premiR482c in WT and three transgenic lines were
predicted. The results showed that, compared with WT, the stem–
loop structure of premiR482b and premiR482c in transgenic plants
showed abnormal bulges (indicated by black boxes) and asymmetric
duplex structures (Supplementary Fig. S2). In addition, minimum
free energy (MFE) is an index of secondary structure stability. The
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).
larger the absolute value of MFE, the more stable the structure is
(Ng and Mishra 2007). Compared with WT, the absolute MFE val-
ues of premir482b and premir482c in transgenic lines were lower
(Supplementary Fig. S2), indicating that the precursor structures in
the mutants were not as stable as in WT. The previous study showed
that DCL1 is responsible for processing most miRNAs into 21-nt
miRNA/miRNA* duplexes; the unstable precursor secondary struc-
ture may interfere with the recognition activity of DCL1 (Bi et al.
2020). Partial inhibition of DCL1 activity may lead to the accumula-
tion of pri-miRNA in mutants. It was conceivable that the cleavage
of normal premiR482b and premiR482c structures was impaired,
leading to higher pri-miRNA accumulation and lower mature
miRNA accumulation.
Off-target effects of miRNA-targeting sgRNAs. To investi-
gate the possibility of off-targeting, we examined six sgRNAs used
in this study, and we checked L1, L2, and L3 transgenic tomato
plants. We selected three high-probability off-target sites for each
sgRNA assay, with the exception of sgRNA2, with one high-prob-
ability off-target site. Surveying these potential off-targeting sites
via Sanger sequencing of PCR products, we found no mutations
(Supplementary Table S2). Although we could not observe all
potentially off-target sites extensively, the results suggested that
off-targeting effects from these sgRNAs are of less concern.
Silencing of miR482b, c increased tomato resistance to P.
infestans. The expression patterns of Solyc05g008070, Solyc11g006530,
and Solyc12g017800 in WT and positive transgenic plants were iden-
tified by qRT-PCR. Compared with WT plants, the expression of
Solyc05g008070, Solyc11g006530, and Solyc12g017800 increased
3.8-fold, threefold, and twofold in L1, and the expression of
Solyc05g008070 and Solyc11g006530 increased 5.2-fold and
7.8-fold in L2 and 1.5-fold and 1.3-fold in L3, respectively (Fig. 4A).
The expression of Solyc12g017800 in L2 and L3 was lower (Fig.
4A), probably because it was affected by other regulatory
mechanisms.
In the detached-leaves infection assay, after inoculation for 5
days, the dead cells on the inoculated leaves were observed by
trypan staining assay. We found that the number of dead cells, indi-
cated by darker blue marks on transgenic tomato leaves, was much
less than that on WT leaves (Fig. 4B). The DOLs of L1, L2, and
L3 leaves were smaller than on WT leaves; L2 had the smallest
DOL, followed by L1, and finally L3 (Fig. 4D). Furthermore, the
expression levels of P. infestans actin gene in L1, L2, and L3 were
lower than in WT (Fig. 4C).
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).

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 Similarly, 5 days after whole plant infection, all transgenic plants
exhibited fewer disease symptoms than those in WT (Fig. 4E).
Meanwhile, the DSRs of L1, L2, and L3 were 0.36-fold, 0.33-fold,
and 0.73-fold lower, respectively, than those of WT (Fig. 4F). These
results indicated that the silencing of miR482b, c increased tomato
defense.
Altered ROS level and physiological indicators increased
tomato resistance. To further explore the resistance mechanism
mediated by gene editing of miR482b and miR482c, we analyzed
the ROS levels in WT and transgenic plants. Compared with WT
plants, the transgenic plant leaves displayed fewer brown spots,
indicating a decrement in H2O2 accumulation after diaminobenzidin
staining (Fig. 5A and B), and fewer blue spots indicated a reduction
in O2
– accumulation after nitro blue tetrazolium staining (Fig. 5C
and D). Furthermore, the SOD and POD enzymes in the transgenic
plants exhibited higher activity, indicating increased ROS scaveng-
ing activity (Fig. 5E and F). Moreover, lower MDA contents
reflected alleviated damage of the membrane system in transgenic
plants (Fig. 5G). Additionally, the PAL enzyme activities in trans-
genic lines were higher than in WT plants (Fig. 5H), suggesting
higher accumulation of disease-resistant secondary biomass. These
results indicate that altered ROS level and physiological indicators
contribute to increased resistance of transgenic tomato plants.
DISCUSSION
In the biotrophic phase of P. infestans, resistance proteins were
highly expressed, and percept pathogens subsequently activate
hypersensitive response/cell death, which in turn would restrain
pathogen spread (Vleeshouwers et al. 2000). In our work, both
miR482b and miR482c were induced by infection as early as
6 hpi, a vital time point for infection success, because early hyper-
sensitive response significantly advances resistance to P. infestans
(Vleeshouwers et al. 2000). Additionally, in this phase two NBS-
LRR proteins, soly05g008070 and solyc12g017800, were highly
expressed, and the regulation of miR482b and miR482c to target
genes was robust (Fig. 1), which was consistent with a previous
work (de Vries et al. 2018). During the necrotrophic phase of
P. infestans (after 72 hpi), the expressions of miR482b and
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.
miR482c tended to be stable, and the inhibitory effect of miR482b
and miR482c on the target genes decreased, leading to increased
expression of the target genes (Fig. 1) (Hong et al. 2019).
It has been reported that modest but impactful changes in
miRNAs genes might have phenotypic consequences (Chuck et al.
2007). In our study, all three transgenic plants contained mutations
for miR482b (Fig. 3A). L1 and L2 contained mutations for
miR482c, and L3 showed no mutation (Fig. 3C). The expressions of
miR482b and miR482c common target genes were higher in trans-
genic plants (Fig. 4A). The disease symptoms and DSR of transgenic
plants were less than those of WT plants in the whole plant infection
assay (Fig. 4E and F). Furthermore, the disease resistance of L1 and
L2, with silenced miR482b and miR482c simultaneously, was super-
ior to that of L3, with silenced miR482b alone, indicating miR482c
may play a negative role in resistance (Supplementary Fig. S1B).
This finding was a supplement to our previous work (Hong et al.
2019) and further proves the role of miR482c in disease resistance.
Moreover, compared with controls, in the transiently expressed
CR-miR482b, c plant, the expression of miR482b was 70% lower
(Fig. 2C), and the DOL was almost 50% lower (Fig. 2E). We previ-
ously reported that overexpression of lncRNA23468, which acts as
an miR482b sponge, led to a 60% decrease in miR482b expression
(Supplementary Fig. S1A) and a 40% decrease in DOL (Supplemen-
tary Fig. S1B) (Jiang et al. 2019). It was inferred that CRISPR/Cas9
technology is more efficient in silencing miR482b than the miRNA
sponge method.
Under pathogen attack, the pathogen-associated molecular pat-
tern-triggered immunity in plants was elicited and a number of
defense mechanisms were induced, including stomatal closure to
limit the entry of bacteria (Bigeard et al. 2015; Melotto et al.
2008); generation of phytoalexins, such as camalexin and patho-
gen resistance proteins (Ahuja et al. 2012; Bednarek 2012);
programmed cell death at the site of infection to limit the progres-
sion of pathogens (Mur et al. 2008); and accumulation of ROS
(Torres 2010). In the dynamic balance between the formation and
removal of active oxygen in the plant, POD and SOD enzymes
exerted important functions, and they were related to the synthesis
of lignin and antivirals, increasing the resistance of plants to vari-
ous pathogens (Brigelius-Floh
e and Floh
e 2020). In our study,
Vol. 111, No. 6, 2021 1013
after P. infestans infection, the transgenic plants all showed lower
ROS levels in comparison with WT plants (Fig. 5A to D and 6),
which implies resistance. The change in other physiological indi-
cators, such as MDA content and PAL enzyme activity, can also
contribute to plant resistance. MDA is a final decomposition prod-
uct of lipid peroxidation, which reduces the fluidity of the cell
membrane and destroys membrane function (Farmer and Mueller
2013); the PAL enzyme was involved in the formation of disease-
resistant secondary biomass including plant protection factors, lig-
nin, and phenolic compounds (Wang et al. 2019). Lower MDA
content and higher PAL activity in transgenic plants compared
with WT plants suggests that transgenic plants sustain less mem-
brane damage and produce more disease-resistant secondary bio-
mass (Fig. 5G and H and 6). These results were consistent with
previous findings that elevated ROS scavenging ability, lower
MDA content, and higher PAL activity increase tomato resistance
(Luan et al. 2018).
miRNAs play critical roles in regulating expression of many per-
ipheral genes involved in various biological processes. Expression
profiling in an miRNA knockout background should reveal the regu-
latory functions of miRNAs. To explore this prospect, we first
detected the expression levels of the other five members of the
miR482 gene family (miR482a, miR482d-3p, miR482d-5p,
miR482e-3p, and miR482e-5p) in our transgenic lines and WT. The
results showed that the transcript accumulation of miR482d-3p and
miR482d-5p was lower in transgenic lines than in WT, whereas the
expression of miR482a, miR482e-3p, and miR482e-5p did not differ
(Supplementary Fig. S3). According to the location indicated by
miRBase, miR482d, miR482b, and miR482c belong to a gene clus-
ter. Cluster miRNAs are generally cotranscribed at a genomic locus,
Fig. 6. Working model of silencing miR482b and miR482c by CRISPR/Cas9 system in tomato resistance to Phytophthora infestans.
1014 PHYTOPATHOLOGY®
so when one miRNA in the cluster is knocked out, the secondary
structure of their common primary precursor is changed, and the bio-
genesis of other miRNAs in the cluster is thus affected (Chang et al.
2016). Therefore, when miR482b and miR482c were knocked out,
expression levels of miR482d-3p and miR482d-5p in the cluster also
decreased. In addition to the miR482 family, there are numerous
miRNAs, including miR476b, miR5300, and miR6027, with the
potential to target NBS-LRRs (Luan et al. 2015). Interestingly, we
found that expression of miR476b, miR5300, and miR6027
decreased in the miR482b and miR482c knockout background (Sup-
plementary Fig. S4). We inferred that there was cross-regulation
between miRNAs at the genome level, which may be attenuated by
altered expression of NBS-LRRs and miR482s. Similarly, Zhou et al.
(2017) identified a list of differentially expressed miRNAs in
OsMIR408 and OsMIR520 mutant backgrounds using miRNA
sequencing data and qRT-PCR analysis We also selected three miR-
NAs (miR156a, miR164a, and miR5302) that were not closely
related to disease resistance to verify their expression levels. The
expressions of miR156a, miR164a, and miR5302 were not affected
by the miR482b and miR482c knockout background (Supplementary
Fig. S4). These results indicate that knocking out miR482b and
miR482c elicited expression perturbation of other miRNAs. Because
no mutations were found in our off-targeting analysis, it can be
inferred that there are miRNA–miRNA regulation networks, which is
of great significance for elucidating the novel regulatory mechanism
of miRNAs in tomato–P. infestans interactions.
In summary, we edited two miRNAs simultaneously by
CRISPR/Cas9 in tomato for the first time (Fig. 6), compared with
the traditional method of silencing a single miRNA; this strategy
saves time, labor, and material resources, and the procedure is more
convenient. We found that knocking out miR482b and miR482c
simultaneously increased tomato resistance more than knocking out
miR482b alone, indicating that both miR482b and miR482c act as
negative regulators, and the effects can be superimposed. Addition-
ally, knocking out miR482b with CRISPR/Cas9 was more effective
than the previously used miRNA sponge method. More import-
antly, we found that knocking out miR482b and miR482c can
reduce the expression of miR482d and other NBS-LRR targeting
miRNAs, which has no effect on other members, suggesting cross-
regulation between miRNAs. This cross-regulation may reveal the
novel mechanism of tomato–P. infestans interaction. Taken
together, the application of CRISPR/cas9 in multiplex miRNAs
editing in our work is of great significance and will strengthen our
capability to study the underlying functions of miRNA in plant
resistance.
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