Plant Biotechnology Journal (2022) 20, pp. 876–885 doi: 10.1111/pbi.

13766

Engineering of rice varieties with enhanced resistances to

both blast and bacterial blight diseases via CRISPR/Cas9
Yanbiao Zhou1,2,*,† , Shichong Xu1,3,†, Nan Jiang1, Xinhui Zhao1,4, Zhenan Bai1, Jinling Liu4, Wei Yao4,
Qianying Tang1, Gui Xiao5, Chao Lv1,3, Kai Wang1, Xiaochun Hu1, Junjie Tan6,* and Yuanzhu Yang1,3,4,5,6,*
1
Key Laboratory of Southern Rice Innovation & Improvement, Ministry of Agriculture and Rural Affairs/Hunan Engineering Laboratory of Disease and Pest Resistant
Rice Breeding, Yuan Longping High-Tech Agriculture Co., Ltd, Changsha 410001, Hunan, China
2
College of Life Sciences, South China Agricultural University, Guangzhou 510642, China
3
College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, China
4
College of Agronomy, Hunan Agricultural University, Changsha 410128, Hunan, China
5
State Key Laboratory of Hybrid Rice, Hunan Hybrid Rice Research Center, Changsha 410125, Hunan, China
6
State Key Laboratory of Crop Genetics and Germplasm Enhancement, Innovation Center for Genome Editing and Engineering, Jiangsu Collaborative Innovation
Center for Modern Crop Production, Nanjing Agricultural University, Nanjing 210095, China

Received 1 December 2020; Summary

revised 25 November 2021; Rice blast and bacterial blight represent two of major diseases having devastating impact on the
accepted 4 December 2021. yield of rice in most rice-growing countries. Developments of resistant cultivars are the most
*Correspondence
economic and effective strategy to control these diseases. Here, we used CRISPR/Cas9-mediated
(Tel +8613607442126; fax
gene editing to rapidly install mutations in three known broad-spectrum blast-resistant genes,
+86073187059179; e-mail:
Bsr-d1, Pi21 and ERF922, in an indica thermosensitive genic male sterile (TGMS) rice line
yzhuyah@[Link] (YY); Tel
+8615274918432; fax +86073187059179; Longke638S (LK638S). We obtained transgene-free homozygous single or triple mutants in T1
zhouyanbiao2005@[Link] (YZ); Tel generations. While all single and triple mutants showed increased resistance to rice blast
+8616651618276; fax +86073187059179; compared with wild type, the erf922 mutants displayed the strongest blast resistance similar with
tanjunjie@[Link] (JT)) triple mutants. Surprisingly, we found that Pi21 or ERF922 single mutants conferred enhanced
†
These authors contributed equally to this resistance to most of tested bacterial blight. Both resistances in mutants were attribute to the up-
work. regulation of SA- and JA-pathway associated genes. Moreover, phenotypic analysis of these
single mutants in paddy fields revealed that there were no trade-offs between resistances and
Keywords: rice, TGMS, CRISPR/Cas9, main agricultural traits. Together, our study provides a rapid and effective way to generate rice
rice blast, bacterial blight. varieties with resistance to both rice blast and bacterial blight.

activated upon perception of pathogen-associated molecular

Introduction
patterns (PAMPs) by cell surface pattern recognition receptors
Rice (Oryza sativa L.) is one of the most important staple foods (PRRs), leading to PAMP-triggered immunity (PTI). The first layer
feeding more than 50% population worldwide. However, its could be overcome by pathogens through secreting PTI-inhibited
production and quality are severely threatened by a variety of effectors into plant cells, which activate effector-triggered
pathogens. Among them, Magnaporthe oryzae (M. oryzae) and susceptibility (ETS). To respond this virulence strategy, the second
Xanthomonas oryzae pv. oryzae (Xoo) are two well-known layer was activated by expressing plant resistance (R) genes,
destructive pathogens causing rice blast and bacterial blight, which specifically recognize pathogen effectors to activate
respectively (Yin et al., 2018). The most economic and effective effector-triggered immunity (ETI) (Dangl et al., 2013). These
approach to control these diseases is to create the rice cultivars innate immune responses trigger many molecular events, includ-
with broad-spectrum resistance against both of them (Deng et al., ing induction of a large number of defense-responsive genes
2017). (Eulgem and Somssich 2007), production of defense signal
Hybrid rice with high yield plays a vital role in guarantee for molecules, reactive oxygen species (ROS) (Kaku et al., 2006)
staple food supply worldwide (Cheng et al., 2007). Compared and phytohormones (De Vleesschauwer et al., 2013). Salicylic acid
with conventional three-line hybrid rice, the two-line hybrid rice (SA) and jasmonic acid (JA) represent two most important
has attracted increasing attention as it is independent of restorer phytohormones in regulation of plant immune responses (Liu
genes and more efficient in production of hybrid seeds (Yuan, et al., 2016). Resistance to biotrophic pathogens is predominantly
1994). Thermo-sensitive genic male sterility (TGMS) and involved in SA-dependent pathway, whereas resistance to
photoperiod-sensitive genic male sterility (PGMS) lines are the necrotrophic pathogens and insects is usually associated with
central component for the two-line hybrid approach. LK638S is JA- and ET-driven defenses (De Vleesschauwer et al., 2013), e.g.,
an indica (Xian) TGMS line developed in the 2000s with low overexpression of OsWRKY13, a WRKY-type transcription factor,
critical sterility-inducing temperature (<23.5
C), high outcrossing enhanced rice resistance to rice blast and bacterial blight, which
rate, good combining ability, but susceptible to rice blast and was associated with the activation of SA synthesis- and responsive
bacterial blight. Thus, establishing its variants with multiple genes (Qiu et al., 2007); knock-outs of MPK15 enhanced disease
diseases resistance is of great importance. resistance in rice against blast and bacterial blight accompanied
Plants have evolved a two-layered innate immune system to by up-regulation of SA- and JA-pathway-associated genes (Hong
defend against pathogens (Zhao et al., 2018). The first layer is et al., 2019).

876 ª 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use,
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 Engineering of disease-resistant TGMS rice 877

Recently, more than 100 major rice blast R genes have been single guide RNA (sgRNA) scaffold under the rice small nuclear
identified and 37 of them have been cloned and characterized RNA promoters was constructed based on previously described
(Yin et al., 2021). Except for Pid2, pi21 and Ptr, most known R CRISPR/Cas9 vector (Yin et al., 2018). We took advantage of this
genes encode typical nucleotide-binding domain leucine-rich vector to target one or multiple sites via a single construct, to
repeat containing (NLR) domain, which directly or indirectly create bsr-d1- or pi21- or erf922-targeted single gene mutants
interact with fungal effectors to trigger ETI (Dangl et al., 2013; and triple bsr-d1/pi21/erf922 mutants (Figure 1d, e). These
Yin et al., 2021). The comprehensive understanding of immunity recombinant expression vectors were used to transform the
mechanisms of R genes can enhance the rice breeding with TGMS variety Longke638S (LK638S) via Agrobacterium-mediated
durable resistance. So far, an atypical R gene Pi21 and two transformation. Based on hygromycin-resistant selection, we
defense-related genes Bsr-d1 as well as ERF922 have attracted obtained 34 resistant lines for bsr-d1, 22 for pi21, 45 for
much more attention due to their broad-spectrum resistance. Bsr- erf922 as well as 35 for triple bsr-d1/pi21/erf922. The PCR-based
d1 encodes a C2H2-type transcription factor that directly binds to sanger sequencing was performed to produce about 82% (28/
the promoter of three peroxidase genes (Os01g73170, 34), 68% (15/22), 73% (33/45) and 71% (25/35) targeting
Os05g04470 and Os10g39170), thereby affecting H2O2 accu- efficiency for Bsr-d1, Pi21, ERF922 and triple Bsr-d1/Pi21/ERF922
mulation and broad-spectrum resistance to M. oryzae (Li et al., genes, respectively (Table 1). Seven, five, eight homozygous
2017; Tao et al., 2021; Zhu et al., 2020). In addition, Bsr-d1 was single mutants for Bsr-d1, Pi21, ERF922, respectively, as well as
shown to be transcriptionally regulated by MYSB1 through three homozygous triple mutants were identified in the T0
directly binding to its promoter and a single base change generations (Figure S1), but unfortunately no double-mutant
(SNP33-G) in the Bsr-d1 promoter leads to enhanced affinity plants were detected. In the T1 generation, for each gene, we
with MYSB1, which suppresses Bsr-d1 gene expression. Pi21 obtained two transgene-free mutants harbouring frameshift
encodes a proline-rich protein containing a putative heavy metal- mutations (Figure 1f, g and Figure S2), and the reverse transcrip-
binding domain and protein–protein interaction motifs (Fukuoka tion quantitative PCR (RT-qPCR) showed that the expression of
et al., 2009). Deletions of the proline-rich motifs cause a loss of Bsr-d1, Pi21, ERF922 were reduced in single or triple mutants
function. Knockout of Pi21 confers non-race specific and durable (Figure S3), which could be explained by cellular nonsense-
blast resistance (Tao et al., 2021). ERF922 encodes an APETELA2/ mediated mRNA decay (NMD) mechanism as described previously
ethylene response factor (AP2/ERF) type transcription factor and is (Lykke-Andersen and Jensen 2015). These results indicated that
strongly induced by M. oryzae (Liu et al., 2012). Knockdown or both protein and transcriptional abundance were affected in
knockout of ERF922 resulted in enhanced resistance against rice these single and triple mutants.
blast, indicating the negative regulation role of this gene in
disease resistance. To date, all the studies regarding Bsr-d1, Pi21
Rice blast resistance was enhanced in mutants
and ERF922 only separately explored their roles in resistance
against rice blast and were mainly carried out in the japonica rice To investigate their ability against rice blast, bsr-d1, pi21 and
background. The rice blast resistance comparison of these three erf922 mutants were evaluated for leaf blast resistance at
genes under the same rice variety as well as their roles in seedling stages in the blast nursery of Dawei Mountain. The
resistance to bacterial blight remains unknown. results showed that the LK638S leaves had much more lesions
CRISPR/Cas9 genome editing technology enables targeted that were significantly decreased in all single and triple mutant
mutagenesis of DNA (Baltes and Voytas,2015). Due to its lines (Figure 2a, b and Figure S4). To confirm these lesions were
simplicity, feasibility and versatility, it has been increasingly caused by M. oryzae, the amount of M. oryzae present in each
employed in a variety of organisms including crops that were inoculated leaf was measured by quantifying fungal-specific DNA.
edited for trait improvements. Moreover, because of its ability for DNA-based quantitative PCR (q-PCR) showed that the mutant
multiplexable genome targeting, it could be used to improve lines harboured less fungus than that in LK638S (Figure 2c).
multiple traits simultaneously (Oliva et al., 2019; Tang et al., Importantly, we observed that the erf922 mutants exhibited the
2017; Wang et al., 2016; Xu et al., 2019; Zhang et al., 2018; strongest resistance than the other two single mutants but no
Zhou et al., 2016). significant difference with triple mutants. To determine if ERF922
In this study, we have set an example of CRISPR/Cas9 was transcriptionally regulated by Bsr-d1 and Pi21, we carried out
applications in enhancing multiple disease resistance in a com- RT-qPCR to analyse the transcriptional abundance of ERF922 in
mercial indica TGMS line LK638S. We chose three broad- LK638S, bsr-d1 as well as pi21 (Figure S5). As the result showed,
spectrum blast-resistant genes (Bsr-d1, Pi21 and ERF922) and the expression levels of ERF922 exhibited no significant difference
created their single as well as triple mutants. We found that between LK638S and each mutant, consistent with the previous
ERF922 mutants showed the strongest blast resistance level report (Li et al., 2017). To test if enhanced blast resistance still
similar with triple mutants. Furthermore, we also explored their existed at reproductive stage, the blast evaluation on rice panicles
new roles in resistance to bacterial blight. Our study indicates was conducted, and the results showed that percentage of
CRISPR/Cas9 as a powerful tool in modern molecular breeding by diseased panicles in mutants were significantly lower than that in
targeted genes modifications that could significantly accelerate LK638S (Figure S6). These results demonstrated that the loss
the breeding of rice varieties with multiple disease resistance. function of Bsr-d1, Pi21 or ERF922 improved the blast resistance
at both seedling and reproductive stages. To further evaluate the
Results rice blast resistance of the mutants in greenhouse, two blast
isolates collected from different regions of China were used (110-
Targeted mutagenesis of Bsr-d1, Pi21 and ERF922 genes
2 and E2007046A2 from Hunan and Hubei, respectively). All
To generate Bsr-d1, Pi21 and ERF922 single mutants, three mutant lines exhibited increased resistance to rice blast, as
sgRNAs were designed (Figure 1a-c). An expression vector that reflected by the significantly decreased lesion length and fungal
contained the Cas9 cassette driven by the ubiquitin promoter and biomass in the mutant lines compared with the LK638S at

ª 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 20, 876–885
878 Yanbiao Zhou et al.

Figure 1 CRISPR/Cas9-induced mutations in the Bsr-d1, Pi21 and ERF922 genes. (a-c) Schematic of the Bsr-d1 (a), Pi21 (b) and ERF922 (c) gene structures
and target sites. Exons and introns are indicated with black rectangles and black lines, respectively. The spacer and PAM sequences were marked in red and
blue. (d, e) Schematic diagram of the T-DNA structures including the Cas9 and single sgRNA (d) or three sgRNAs (e) in genome editing construct. The
expression of Cas9 is driven by the maize ubiquitin promoter (OsUBQ); the expression of the sgRNA scaffold is driven by the rice OsU3 or OsU6a or OsU6b
small nuclear RNA promoter; the expression of hygromycin (HPT) is driven by CaMV35S promoter. NLS, nuclear localization signal; Tnos, the terminator; LB
and RB, left border and right border, respectively. (f, g) Homozygous mutations identified at the target sites of bsr-d1, pi21, erf922 mutant lines (f) and
triple bsr-d1/pi21/erf922 mutant lines (g) in the T1 generation. Amino acids were marked above the relative nucleotide triplets, and the first altered ones
from the frameshift were indicated in red, with the number representing the order in the proteins. The stars represent premature stop codons

Table 1 Targeting mutagenesis efficiency in T0 transgenic plants determining the expression level by RT-qPCR. The results showed
that these peroxidase genes in the bsr-d1 mutant lines were
T0 Mutant Homozygous Targeting reduced by 2- to 12-fold in comparison with wild-type LK638S,
Gene plants plants mutation plants efficiency (%) consistent with the previous study (Li et al., 2017) (Figure S7a-c).
The content of H2O2 in the bsr-d1 mutant lines was significantly
Bsr-d1 34 28 7 82
higher than that in LK638S (Figure S7d). These results indicated
Pi21 22 15 5 68
that loss function of Bsr-d1 reduced the expression of the
ERF922 45 33 8 73
peroxidase genes and increased the amount of H2O2, thereby
Bsr-d1/Pi21/ 35 25 3 71
leading to the resistance to a broad spectrum of rice blast.
ERF922
Activation of plant immune responses during pathogen attack
is accompanied by defense hormone pathways, including the SA
signalling pathway marker gene OsPR1a (Agrawal et al., 2000),
10 days’ post-inoculation (dpi) (Figure 3a, b). Notably, the lesion OsPR1b (Agrawal et al., 2001), OsWRKY45 (Tao et al., 2009), and
length and fungal biomass of erf922-1 and bsr-d1pi21erf922-1 jasmonate acid (JA) signalling pathway marker gene OsPR4
were significantly lower than those of the bsr-d1-9 and pi21-1 (Wang et al., 2011). Therefore, the expressions of these defense-
(Figure 3a, b). All together, these results suggested that knockout related genes were analysed in the mutant lines and LK638S at
of Bsr-d1, Pi21 or ERF922 enhanced rice blast resistance in the 0 h and 24 h after the leaves were inoculated with rice blast. The
background of LK638S, and among them, erf922 conferred the expression levels of these defense-related genes were markedly
strongest resistance. higher in mutant lines than those in LK638S at 24 h post-
inoculation (hpi) (Figure 4). These results indicated that knockouts
of Bsr-d1 or Pi21 or ERF922 activated the SA and JA signalling
The expression analysis of defense-related pathway genes by rice blast fungi, which may have contributed to
genes in mutants their improvement of rice blast resistance.
Rice Bsr-d1 suppresses the expression of peroxidase genes
through binding of the repressive MYB transcription factor Knockout of Pi21 or ERF922 conferred robust
(MYBS1) to the bsr-d1 promoter, resulting in broad-spectrum resistance to bacterial blight
resistance to rice blast (Li et al., 2017; Zhu et al., 2020). Thus,
three Bsr-d1 target peroxidase genes (LOC_Os01g73170, SA and JA are the most important plant hormones that play major
LOC_Os05g04470 and LOC_Os10g39170) were used for roles in regulation of plant defense responses against Xoo (Bari

ª 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 20, 876–885
 Engineering of disease-resistant TGMS rice 879

Figure 2 Enhanced blast resistance of the mutant lines in the blast nursery. (a) Rice mutant lines and wild-type LK638S were tested for resistance to M.
oryzae at the seedling stage. Scale bar, 1 cm. (b) The percentage of lesion areas (disease index) were measured via image analysis using ImageJ software
(n = 6 leaves). (c) Fungal growth was determined by the expression level of the Magnaporthe oryzae MoPot2 gene in the inoculated leaves by RT-qPCR,
and the levels were normalized to the expression level of the OsUbi gene (n = 3). Values and error bars represent the mean and standard deviation of three
independent biological replicates. ** indicates a significant difference (P < 0.01 from Student’s t-test)

and Jones 2009; De Vleesschauwer et al., 2013; Hou et al., 2019; single mutant bsr-d1-9, pi21-1 or erf922-1 (Figure 6). In addition,
Qiu et al., 2007). To investigate the bacterial blight resistance of there were no significant difference for the panicle length and
the mutant lines, we inoculated 6 Xoo strains (Table S2) to grain number between all mutant lines and LK638S. These results
mutant lines using the tip-cutting method (Ji et al., 2016). When suggested that knockouts of Pi21 or ERF922 exhibited increased
the mutant lines were inoculated with FuJ, PXO61 and PXO71, resistance against both rice blast and bacterial blight without
respectively, the pi21-1 and erf922-1 showed significantly shorter compromising major agricultural traits.
lesions length than LK638S (Figure 5a, b). The lengths of the
lesions on erf922-1 and pi21-1 caused by the PXO86 and PXO99
Discussion
were significantly shorter than those observed in LK638S,
respectively. Additionally, upon artificial inoculation of YN24, Currently, the objective for rice breeding is diversified, and
there were no significant difference of lesions length were breeders consider not only high yields but also rice quality and
detected between mutant lines and LK638S. These results disease resistance. Rice blast and bacterial blight are primary
demonstrated that knockout of Pi21 or ERF922 in LK638S destructive diseases that badly affect not only rice production but
background can improve resistance against bacterial blight also rice quality. In the current study, we took advantage of
disease. To further elucidate the regulation network, the CRISPR/Cas9 technology to edit three known broad-spectrum
transcriptional levels of SA and JA signalling pathway genes were blast resistant genes Bsr-d1, Pi21 as well as ERF922 in a very high
examined by RT-qPCR. The results showed that all the tested efficiency to generate each single and triple mutant in T0
genes were significantly up-regulated in pi21-1 and erf922-1 generations (Table 1). We further carried out inoculation exper-
when the mutant plants inoculated with Xoo, whereas these iments and proved that all the single and triple mutants conferred
genes remained unchanged in bsr-d1-9, except for OsWRKY45, enhanced resistance to rice blast (Figure 2 and 3). It is worth
which was induced in bsr-d1-9 (Figure S8). These results suggest noting that the erf922 mutants exhibited the strongest blast
that the knockout of Pi21 or ERF922 in LK638S-enhanced resistance among all single mutants similar with bsd-
resistance to bacterial blight disease, which may result from the d1pi21erf922 triple mutants indicating that ERF922 is preferred
activation of the SA and JA signalling pathway genes. to being employed to increase the blast resistance under the
background of LK638S. In addition, similar blast resistances in
erf922 and bsd-d1pi21erf922 triple mutants suggested possibility
Characterizations of main agronomic traits in
that ERF922 may act downstream of Bsd-d1 and Pi21. ERF922
mutants
may integrate Bsd-d1- and Pi21-mediated resistant signalling to
The above results showed that bsr-d1-9, pi21-1, erf922-1 and respond pathogen attack. Expression levels of ERF922 exhibited
bsr-d1pi21erf922-1 mutants were more resistant to rice blast no significant difference between WT and each single mutant
(Figure 2, 3), and the pi21-1 and erf922-1 mutants showed (Figure S5), suggesting the possibility that ERF922 was post-
increased resistance to bacterial blight (Figure 5). We further transcriptionally regulated. The detailed post-transcriptional
investigated whether increased resistances against rice blast and mechanism will be the subject of future work. Previously, all
bacterial blight could affect rice growth. At the mature stage, the studies regarding Bsr-d1, Pi21 and ERF922 only explored their
main agronomic traits including plant height, tillers per plant, roles in resistance against rice blast. It was unknown whether
panicle length and grain number per panicle were measured. these three genes played roles in resistance to bacterial blight. In
Compared with that in wild-type LK638S, the plant height and the present study, by testing six typical Xoo strains, we showed
tiller number in triple mutant bsr-d1pi21erf922-1 were signifi- that pi21-1 and erf922-1 mutants exhibited resistances to four
cantly lower, while there were not significantly different in the out of six Xoo strains with one Chinese isolates (FuJ) and three

ª 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 20, 876–885
880 Yanbiao Zhou et al.

Figure 3 Blast resistance evaluation by

punch inoculation. (a, b) The mutant lines
were inoculated with the M. oryzae isolate
110-2 (a) and E2007046A2 (b),
respectively. Two leaves for each of mutant
lines and LK638S are shown. Lesion length
and fungal growth were determined on
inoculated leaves at 10 dpi. Scale bar,
1 cm. Values and error bars represent the
mean and standard deviation of three
independent biological replicates. **
indicates a significant difference (P < 0.01
from Student’s t-test)

Philippine strains (PXO61, PXO71, PXO99 or PXO86) (Figure 5). 2019). TALEs activate the expression of host gene through binding
More Xoo strains will be tested in these mutants to investigate into specific promoters of host target genes (Wang et al., 2015).
their broad-spectrum resistance to the bacterial blight. A recent For example, the Os8N3 was activated by the TAL effector PthXo1
study showed that CRISPR/Cas9-mediated multiplex editing of from Xoo strain PXO99 by recognition of TAL effector binding
the Pi21, Bsr-d1 as well as a well-known Xoo-resistant gene Xa5 elements (EBEs) located at the promoter region of Os8N3 (Romer
generated pi21-bsr-d1-xa5 triple mutant which conferred resis- et al., 2010). Likewise, based on the enhanced resistances to Xoo
tant to both blast and bacterial blight (Tao et al., 2021). In our strains in pi21 and erf922 mutants, we speculated that TALEs
study, on one hand, we further compared the blast resistance secreted by these Xoo strains might target the promoter region of
level among Bsr-d1, Pi21 and ERF922 single mutants and Pi21 and ERF922 to activate Pi21 and ERF922 expressions and
identified the erf922 mutant with the strongest blast resistance. hence causing diseases, whereas the loss function of Pi21 or
On the other hand, our study described the new roles of Pi21 as ERF922 was conducive to the bacterial blight resistances.
well as ERF922 in the resistance to Xoo, which could well explain Plants response to pathogen attack by a combination of
why the pi21-bsr-d1-xa5 triple mutant exhibited much stronger constitutive and inducible defense responses. Many of these
resistance to Xoo than xa5 single mutant in the previous study responses are modulated through complex interconnecting signal
(Tao et al., 2021). transduction pathways, within which hormones fulfill central
It has been reported that many plant pathogenic Xanthomonas roles (De Vleesschauwer et al., 2013). Plant immunity is usually
spp. secreted a class of proteins designated transcription activator- associated with SA and JA hormone signalling pathways (Bari and
like (TAL) effectors that were injected into plant cells via the type III Jones 2009). For example, knockdown of OsBON1 conferred
secretion system (TTSS) to activate the expression of host suscep- enhanced resistance to rice fungal and bacterial pathogens,
tibility genes that result in diseases (Hu et al., 2014; Xu et al., which was accompanied by the activation of SA- and JA-

ª 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 20, 876–885
 Engineering of disease-resistant TGMS rice 881

Figure 4 Transcriptional abundances of

SA- and JA-regulated defense-related
genes in mutant lines. (a-d) Expression
levels of the genes involved in SA (a-c) and
JA (d) signal transduction in bsr-d1-9,
pi21-1, erf922-1, bsr-d1pi21erf922-1 and
wild-type LK638S were determined by RT-
qPCR. Values and error bars represent the
mean and standard deviation of three
independent biological replicates. **
indicates a significant difference (P < 0.01
from Student’s t-test)

responsive genes (Yin et al., 2018). Expressions of SA and JA blight by CRISPR/Cas9, which could significantly accelerate the
pathway-associated genes were significantly upregulated in the breeding of rice varieties with multiple disease resistance.
mpk15 mutant, resulting in enhanced disease resistance to rice
blast and bacterial blight (Hong et al., 2019). The SA signalling Experimental procedures
marker gene OsWRKY45 from indica rice varieties positively
Construct and rice transformation
regulates rice resistance to rice blast and bacterial blight (Tao
et al., 2009). The expression of OsWRKY45 was induced not only The target sites of Bsr-d1, Pi21 and ERF922 were selected by the
by M. grisea but also Xoo (Ryu et al., 2006; Tao et al., 2009). CRISPR-Plant Web server (Xie et al., 2014) and were constructed
OsWRKY45-overexpressing plants showed enhanced resistance into the CRISPR/Cas9 vector as described previously (Ma et al.,
to rice blast and bacterial blight. In this study, upon plants were 2015). Briefly, the designed targeting sequence was synthesized
infected with the rice blast, higher levels of SA signalling related and annealed to form the oligo adaptors. The oligo adaptors were
genes OsPR1a, OsPR1b, OsWRKY45 and JA signalling pathway inserted into the sgRNA expression cassette vectors at a Eco31I
gene OsPR4 were detected in mutants than that in wild-type site. The integrated sgRNA expression cassette was then amplified
plants (Figure 4). In addition, when the mutant plants were by PCR using universal primers, and the amplicons were cloned
inoculated with bacterial blight, the OsPR1a, OsPR1b, OsWRKY45 into the CRISPR/Cas9 plant expression vector. The CRISPR/Cas9
and OsPR4 were significantly up-regulated in pi21-1 and erf922-1 plasmids were introduced into Agrobacterium tumefaciens
(Figure S8). These results indicated that the enhancement of EHA105. Rice transformation of LK638S was performed as
resistance to rice blast and bacterial blight may be associated with described previously (Zhou et al., 2018). Specific primer pairs
the activation of SA and JA signalling genes in mutants. However, Cas9-F/Cas9-R and HPT-F/HPT-R were used to confirm T0
detailed molecular mechanisms of Pi21- and ERF922-mediated transgenic-positive plants. Bsr-d1-CX-F/Bsr-d1-CX-R, Pi21-CX-F/
immunity to bacterial blight still require further study. Of note, Pi21-CX-R and ERF922-CX-F/ERF922-CX-R were used to amplify
the inconsistence of gene expression in Figure 4 and Figure S8 at the genome regions containing each target site, and the resulting
0 h (e.g. expression of OsWRKY45 is slightly higher in each PCR products were followed by sequencing to detect mutations
mutant than LK638S in Figure 4 but similar in Figure S8) could be in T0 and T1 generations. Sequencing results were decoded by an
explained by the fact that the samples used for mRNA detection online tool DSDecodeM ([Link] dsdecode/) (Liu
in the two experiments were different in terms of developmental et al., 2015b). Transgene-free plants were identified using the
stage, growth environment as well as the sampling area. primer pairs Cas9-F/Cas9-R and HPT-F/HPT-R and determined by
Together, our work provides a convenient, rapid and effective both showing negative amplification. Sequences of the primers
way to obtain rice cultivars against the blast as well as bacterial are listed in Table S1.

ª 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 20, 876–885
882 Yanbiao Zhou et al.

Figure 5 Phenotypical characterization of bsr-d1-9, pi21-1, erf922-1 and wild-type LK638S against Xoo. (a) Phenotypes of disease reactions in the LK638S
and the mutant lines after inoculation with Xoo strain FuJ, PXO61, PXO71, PXO86, PXO99 and YN24. Scale bar, 2 cm. (b) Measurements of disease lesion
lengths (cm) in wild-type and mutant lines at 2 weeks after inoculation. Values and error bars represent the mean and standard deviation of six
independent biological replicates. ** indicates a significant difference (P < 0.01 from Student’s t-test)

mutant rice plants were sown in single-row plots, and three

Rice blast Inoculation extremely susceptible cultivars 9311, Xiangaizao7 and Xiang-
The rice blast nursery investigation was performed during April to wanxian11 were sown on the plot borders used as an inducer to
May in two consecutive years (2019-2020) in Dawei Mountain ensure uniform blast infection. The diseased straws were cut into
(Hunan Province, 28°490 N, 113°990 E). Fifty seeds of LK638S and small segments (2-3 cm) and sprinkled over the susceptible

ª 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 20, 876–885
 Engineering of disease-resistant TGMS rice 883

Figure 6 Comparison of major agronomic

traits between wild-type LK638S and bsr-
d1-9, pi21-1, erf922-1 as well as bsr-
d1pi21erf922-1 rice plants. (a) Phenotypes
of LK638S and each mutant plant at the
heading stage. Scale bar, 10 cm. (b-e) The
major traits including plant height (b),
tillering number per plant (c), panicle
length (d) and grain number per plant (e)
are displayed in histograms. Agronomic
traits were investigated in paddy filed
located at Guanshan village (28°19’32’’N,
112°40’38’’E), Changsha, in 2020. Data
are presented as mean  SD (n = 20). **
indicates a significant difference (P < 0.01
from Student’s t-test)

spreader cultivars at the two-leaf stage to induce natural of diseased panicle = % (the number of diseased panicles/ total
infection. Twenty-five days later, flag leaves were harvested to number of panicles).
analyse lesion area and relative fungal biomass. The relative
fungal biomass was calculated using the threshold cycle value (Ct)
Bacteria strains and inoculation
of M. oryzae Pot2 gene against the Ct of the rice genomic
ubiquitin (OsUbi) gene (Li et al., 2017). Sequences of the primers To examine resistance of bacterial blight, Xoo strains were grown
are listed in Table S1. in TSA medium (10 g/L tryptone, 10 g/L sucrose, 1 g/L glutamic
Punch inoculation was performed as previously described (Liu acid, 15 g/L agar and PH 6.8-7.0). Bacteria were suspended in
et al., 2015a) with slight modification. Detached leaf sheaths sterile water to an OD600 = 1.0 for inoculation on rice leaves. Rice
from the fourth leaf of five-leaf-stage seedlings were inoculated plants at the booting stage were inoculated with the leaf-clipping
with two M. oryzae isolates collected from different regions of method as previously described (Xu et al., 2019). Disease
China, with 110-2 strain from Hunan and E2007046A2 from phenotype was scored by measuring the lesion length at 2 weeks
Hubei. M. oryzae isolates were grown on complete agar medium after inoculation. The sources of the Xoo strains tested in this
for 15 d before producing spores. Spores were collected via study are listed in Table S2.
flooding of the fungal agar cultures with sterile water, and the
spore concentration in the suspension was adjusted to 5 9 105
RNA Isolation and RT-qPCR
conidia/mL before punch inoculation. Dip 5 lL spore suspension
for each drop using pipette tip at two spots on each leaf. Total RNA isolation and reverse transcription quantitative PCR
Inoculated detached leaves were placed in 0.1% 6- (RT-qPCR) were carried out as described previously (Zhou et al.,
benzylaminopurine (6-BA) in sterile water to keep moist. The 2015). To analyse the influence of M. oryzae infection on gene
lesion lengths of disease reactions were measured at 10 days’ expression, three-week-old rice plants growing in the chamber
post-inoculation (dpi). Relative fungal biomass was calculated were sprayed with 110-2 spores. The inoculated plants were kept
using the threshold cycle value (Ct) of M. oryzae Pot2 gene in the growth chamber for 24 h in the dark at 25°C with 90%
against the Ct of the rice genomic ubiquitin (OsUbi) gene (Li et al., relative humidity. The inoculation leaves were harvested to
2017). analyse the gene expression. To examine the influence of
Blast resistance at productive stage was also investigated in an bacterial blight infection on gene expression, flag leaves from
experimental paddy field in Dawei Mountain. Twenty-four plants 2-month-old rice plants in the paddy field were used. About 3 cm
were transplanted for each line in early June. After flowering, the leaf fragments next to Xoo strain FuJ infection sites were used for
blast symptoms in rice neck were evaluated. The total number of RNA isolation. The primers used for RT-qPCR are listed in
panicles and the number of diseased panicles (with at least one Table S1. The expression of rice Actin gene (LOC_Os03g50885)
black neck symptom) were counted for each plant. The panicle was used as an internal control. The relative expression levels
severity of a plot was defined as the mean of percentage of were measured as previously described (Livak and Schmittgen
diseased panicle from 20 plants (Sester et al., 2014). Percentage 2001).

ª 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd., 20, 876–885
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This work was supported by the National Natural Science resistance. Nat. Commun, 7, 1–9.
Foundation of China (31901516), the special funds for the Kaku, H., Nishizawa, Y., Ishii-Minami, N., Akimoto-Tomiyama, C., Dohmae, N.,
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Conflict of interest resistance. Cell, 170(1), 114–126.
The authors declare no conflicts of interest. Liu, D., Chen, X., Liu, J., Ye, J. and Guo, Z.(2012) The rice ERF transcription
factor OsERF922 negatively regulates resistance to Magnaporthe oryzae and
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Over-expression of a glutamate dehydrogenase gene, MgGDH, from

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