The type V -I CRISPR-Cas system is becoming increasingly more attractive for genome editing. However, natural nucleases of this system often exhibit low efficiency, limiting their application. Here, we used structure -guided rational design and protein engineering to optimize an uncharacterized Cas12i nuclease, Cas12i3. As a result, we developed Cas-SF01, a Cas12i3 variant that exhibits significantly improved gene editing activity in mammalian cells. Cas-SF01 shows comparable or superior editing performance compared to SpCas9 and other Cas12 nucleases. Compared to natural Cas12i3, Cas-SF01 has an expanded PAM range and effectively recognizes NTTN and noncanonical NATN and TTVN PAMs. In addition, we identified an amino acid substitution, D876R, that markedly reduced the off -target effect while maintaining high on -target activity, leading to the development of CasSF01HiFi (high-fidelity Cas-SF01). Finally, we show that Cas-SF01 has high gene editing activities in mice and plants. Our results suggest that CasSF01 can serve as a robust gene editing platform with high efficiency and specificity for genome editing applications in various organisms.

种植抗病品种一直是防控水稻白叶枯病(bacterial blight,BB)最有效的措施.近年来,白叶枯病在我国多地呈现"老病新发"态势.为查明近期白叶枯病成灾的原因,2019-2021年间,在南方8省(海南、云南、广西、广东、福建、湖南、浙江和江苏)病害重发生田块采集叶片,分离获得野生白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo).通过对主效毒性因子基因型tale(transcription activator-like effectors)进行Southern杂交检测,将新分离的97株Xoo菌株划分为10个基因型,其中基因型V是优势种群代表.选取各基因型的代表菌株,剪叶接种携带主要抗病基因(R gene)的水稻(Oryza sativa)品种,毒力测试结果显示,Xa3和Xa4等传统抗病基因对田间大部分菌株已经丧失抗性,Xa7和Xa23等优异抗病基因对白叶枯病仍具有广谱抗性.研究结果表明,近期"老病新发"的主要原因可能是水稻新品种选育过程中忽视了优异抗白叶枯病基因资源的引入,挖掘与利用优异抗病基因资源仍是防控白叶枯病最理想的途径.

Summary Plant leaf senescence, caused by multiple internal and environmental factors, has an important impact on agricultural production. The lectin receptor‐like kinase (LecRLK) family members participate in plant development and responses to biotic and abiotic stresses, but their roles in regulating leaf senescence remain elusive. Here, we identify and characterize a rice premature withered leaf 1 ( pwl1 ) mutant, which exhibits premature leaf senescence throughout the plant life cycle. The pwl1 mutant displayed withered and whitish leaf tips, decreased chlorophyll content, and accelerated chloroplast degradation. Map‐based cloning revealed an amino acid substitution (Gly412Arg) in LOC_Os03g62180 ( PWL1 ) was responsible for the phenotypes of pwl1 . The expression of PWL1 was detected in all tissues, but predominantly in tillering and mature leaves. PWL1 encodes a G‐type LecRLK with active kinase and autophosphorylation activities. PWL1 is localized to the plasma membrane and can self‐associate, mainly mediated by the plasminogen‐apple‐nematode (PAN) domain. Substitution of the PAN domain significantly diminished the self‐interaction of PWL1. Moreover, the pwl1 mutant showed enhanced reactive oxygen species (ROS) accumulation, cell death, and severe DNA fragmentation. RNA sequencing analysis revealed that PWL1 was involved in the regulation of multiple biological processes, like carbon metabolism, ribosome, and peroxisome pathways. Meanwhile, interfering of biological processes induced by the PWL1 mutation also enhanced heat sensitivity and resistance to bacterial blight and bacterial leaf streak with excessive accumulation of ROS and impaired chloroplast development in rice. Natural variation analysis indicated more variations in indica varieties, and the vast majority of japonica varieties harbour the PWL1 Hap1 allele. Together, our results suggest that PWL1, a member of LecRLKs, exerts multiple roles in regulating plant growth and development, heat‐tolerance, and resistance to bacterial pathogens.

Bacterial blight (BB) and bacterial leaf streak (BLS), caused by phytopathogenic bacteria Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), respectively, are the most serious bacterial diseases of rice, while blast, caused by Magnaporthe oryzae (M. oryzae), is the most devastating fungal disease in rice. Generating broad-spectrum resistance to these diseases is one of the key approaches for the sustainable production of rice. Executor (E) genes are a unique type of plant resistance (R) genes, which can specifically trap transcription activator-like effectors (TALEs) of pathogens and trigger an intense defense reaction characterized by a hypersensitive response in the host. This strong resistance is a result of programed cell death induced by the E gene expression that is only activated upon the binding of a TALE to the effector-binding element (EBE) located in the E gene promoter during the pathogen infection. Our previous studies revealed that the E gene Xa23 has the broadest and highest resistance to BB. To investigate whether the Xa23-mediated resistance is efficient against Xanthomonas oryzae pv. oryzicola (Xoc), the causal agent of BLS, we generated a new version of Xa23, designated as Xa23p1.0, to specifically trap the conserved TALEs from multiple Xoc strains. The results showed that the Xa23p1.0 confers broad resistance against both BB and BLS in rice. Moreover, our further experiment on the Xa23p1.0 transgenic plants firstly demonstrated that the E-gene-mediated defensive reaction is also effective against M. oryzae, the causal agent of the most devastating fungal disease in rice. Our current work provides a new strategy to exploit the full potential of the E-gene-mediated disease resistance in rice.

Grain quality has become one of the foci in rice breeding worldwide since it is crucial for farmers, millers and consumers. Previous studies have revealed that rice gain quality is mainly determined with several major characteristics including milling, physical appearance, eating and cooking, sensory and nutritional value. In genetics, rice grain quality is a complex trait controlled by polygene/QTL. Better understanding the mechanism underlying the grain quality is fundamental for developing new breeding strategies. Therefore, abundant studies had been conducted to discover the factors that control the quality traits, and led to dozens of QTL identified and several major genes cloned. This has enabled development of the functional markers to facilitate selection of the complex traits. But breeding for rice varieties with desired grain quality needs to pyramid multiple and beneficial loci or alleles, some of which are still untapped. In this chapter, we first summarize the literatures on rice grain quality traits and the relevant phenotyping approaches, and then review the progress in genetic dissection of these traits. This review also presented the breeding attempts using marker-assisted selection, genomics-assisted breeding approaches, as well as genome editing technologies, and finally provided innovative insights into the future of rice grain quality breeding by using the modern technology with integration of machine learning and genomic selection.

Transmembrane kinases (TMKs) play important roles in plant growth and signaling cascades of phytohormones. However, its function in the regulation of early leaf senescence (ELS) of plants remains unknown. Here, we report the molecular cloning and functional characterization of the WATER-SOAKED SPOT1 gene which encodes a protein belongs to the TMK family and controls chloroplast development and leaf senescence in rice (Oryza sativa L.). The water-soaked spot1 (oswss1) mutant displays water-soaked spots which subsequently developed into necrotic symptoms at the tillering stage. Moreover, oswss1 exhibits slightly rolled leaves with irregular epidermal cells, decreased chlorophyll contents, and defective stomata and chloroplasts as compared with the wild type. Map-based cloning revealed that OsWSS1 encodes transmembrane kinase TMK1. Genetic complementary experiments verified that a Leu396Pro amino acid substitution, residing in the highly conserved region of leucine-rich repeat (LRR) domain, was responsible for the phenotypes of oswss1. OsWSS1 was constitutively expressed in all tissues and its encoded protein is localized to the plasma membrane. Mutation of OsWSS1 led to hyper-accumulation of reactive oxygen species (ROS), more severe DNA fragmentation, and cell death than that of the wild-type control. In addition, we found that the expression of senescence-associated genes (SAGs) was significantly higher, while the expression of genes associated with chloroplast development and photosynthesis was significantly downregulated in oswss1 as compared with the wild type. Taken together, our results demonstrated that OsWSS1, a member of TMKs, plays a vital role in the regulation of ROS homeostasis, chloroplast development, and leaf senescence in rice.

Recent studies have shown that reprogramming of gene expression in a genome can induce the production of proteins enabling yield increase. The transcription activator-like effectors (TALEs) from several species of bacterial Xanthomonas have been extensively studied, and a series of research tools, such as genome editing tool TALENs and gene expression activators, have been developed based on the specific protein-nucleic acid recognition and binding mechanisms of TALEs. In this proof-of-principle study, we designed and constructed a designer TALE (dTALE), designated as dTALE-NOG1, to specifically target the promoter of OsNOG1 gene in rice, and demonstrated that this dTALE can be used as a new type of plant growth regulator for better crop growth and harvest. In doing so, the dTALE-NOG1 was transferred into the non-pathogenic Xanthomonas oryzae pv. oryzae (Xoo) strain PH to generate a genetically engineered bacteria (GEB) strain called PH-dtNOG1. Functional verification showed that dTALE-NOG1 could significantly induce the expression of OsNOG1. By spraying cell suspension of PH-dtNOG1 on the rice plants during the tillering stage, the transcription level of OsNOG1 was highly enhanced, the grain number of rice plants was increased by more than 11.40%, and the grain yield per plant increased by more than 11.08%, demonstrating that the dTALE-NOG1 was highly effective in enhancing rice yield. This work provided a new strategy for manipulating agronomical traits by reprogramming gene expression in a crop genome.

Executor (E) genes comprise a new type of plant resistance (R) genes, identified from host–Xanthomonas interactions. The Xanthomonas-secreted transcription activation-like effectors (TALEs) usually function as major virulence factors, which activate the expression of the so-called “susceptibility” (S) genes for disease development. This activation is achieved via the binding of the TALEs to the effector-binding element (EBE) in the S gene promoter. However, host plants have evolved EBEs in the promoters of some otherwise silent R genes, whose expression directly causes a host cell death that is characterized by a hypersensitive response (HR). Such R genes are called E genes because they trap the pathogen TALEs in order to activate expression, and the resulting HR prevents pathogen growth and disease development. Currently, deploying E gene resistance is becoming a major component in disease resistance breeding, especially for rice bacterial blight resistance. Currently, the biochemical mechanisms, or the working pathways of the E proteins, are still fuzzy. There is no significant nucleotide sequence homology among E genes, although E proteins share some structural motifs that are probably associated with the signal transduction in the effector-triggered immunity. Here, we summarize the current knowledge regarding TALE-type avirulence proteins, E gene activation, the E protein structural traits, and the classification of E genes, in order to sharpen our understanding of the plant E genes.

The two-line rice hybrid “Super 1000” (GX24S × R900) represents a major landmark achievement of breeding for super-hybrid rice in China. However, both male parent R900 and hybrid “Super 1000” have an obvious defect of high susceptibility to rice bacterial blight (BB) and blast. Thus, improving disease resistance and maintaining the original high-yield capacity are essential for the sustainable application of “Super 1000.” In this study, the application of closely linked single-nucleotide polymorphism (SNP) markers for foreground selection of dominant resistance gene loci together with genome-wide SNP markers for the background selection rapidly improved the disease resistance of R900 without disturbing its high-yield capacity. A series of improved R900 lines (iR900, in BC 2 Fn and BC 3 Fn generations) were developed to stack resistance genes ( Xa23 + Pi9, Xa23 + Pi1 + Pi2/9 ) by marker-assisted backcrossing and field selection for phenotypes, and further crossed with the female line GX24S to obtain improved hybrid variety Super 1000 (iS1000). The genetic backgrounds of iS1000 and “Super 1000” were profiled by using a 56 K SNP-Chip, and results showed that they shared 98.76% of similarity. Meanwhile, evaluation of the field disease resistance showed that the iR900 lines and iS1000 hybrids possess significantly enhanced resistance to both BB and rice blast. Resistance spectrum assays revealed that the iR900 lines and their derived hybrids exhibited high-level resistance to 28 Xoo strains tested, and enhanced resistance to leaf blast at the seedling stage when infected with 38 Magnaporthe oryzae isolates. Between 2019 and 2020, the multi-location field trials across the middle and lower reaches of the Yangtze River were launched and showed that the iS1000 slightly out-yielded than the original variety. In a large-scale demonstration site (6.73 ha, Yunnan, China), the iS1000 achieved 17.06 t/hm 2 of yield in 2019. Moreover, the high similarity was observed in main agronomic traits and grain quality when comparing the improved lines/hybrids to original ones (iR900 vs. R900, iS1000 vs. S1000). This work presented a typical genomics-assisted breeding strategy and practice, which involves in directional introgression and rapid stack of multiple disease resistance genes, endowing the super-high-yield hybrid rice variety with holistic disease resistance but without yield penalty.

Multiplex genome-editing (MGE) technologies are recently developed versatile bioengineering tools for modifying two or more specific DNA loci in a genome with high precision. These genome-editing tools have greatly increased the feasibility of introducing desired changes at multiple nucleotide levels into a target genome. In particular, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) [CRISPR/Cas] system-based MGE tools allow the simultaneous generation of direct mutations precisely at multiple loci in a gene or multiple genes. MGE is enhancing the field of plant molecular biology and providing capabilities for revolutionizing modern crop-breeding methods as it was virtually impossible to edit genomes so precisely at the single base-pair level with prior genome-editing tools, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Recently, researchers have not only started using MGE tools to advance genome-editing applications in certain plant science fields but also have attempted to decipher and answer basic questions related to plant biology. In this review, we discuss the current progress that has been made toward the development and utilization of MGE tools with an emphasis on the improvements in plant biology after the discovery of CRISPR/Cas9. Furthermore, the most recent advancements involving CRISPR/Cas applications for editing multiple loci or genes are described. Finally, insights into the strengths and importance of MGE technology in advancing crop-improvement programs are presented.

Splicing of precursor mRNAs (pre-mRNAs) is a critical biological process of gene expression. Normally, pre-mRNAs are processed by spliceosomes to produce mature mRNAs by removing introns at 5′-(donor) and 3′-(acceptor) splice sites based on the canonical GU-AG rule (Reddy et al., 2013). Mutation at either the intron donor or acceptor sites should cause mRNA mis-splicing. Recently, an exon skipping method has been developed to generate loss of gene function in mammalian cells using base editors to mutate nucleotides at the acceptor sites (Gapinske et al., 2018). Moreover, it has been approved that CRISPR/Cas9-mediated exon skipping in rabbits depends on non-sense-associated altered splicing induced by premature termination codon (PTC) mutation (Sui et al., 2018). In contrast, triggering exon skipping by genome editing in plants is still controversial as disruption of the intron donor or acceptor sites in plant genes did not trigger exon skipping but allowed either intron retention or aberrant splicing (Li et al., 2019), following the fact that intron retention is the most frequent mode of alternative splicing in plants (Ner-gaon et al., 2004). Fragrance is one of the most important rice quality traits (Abdelrahman and Zhao, 2020). The natural aroma substance 2-acetyl-1-pyrroline (2AP) is the major contributor to the aroma flavour of fragrant rice. The rice gene OsBADH2 encodes a betaine aldehyde dehydrogenase (BADH) that inhibits 2AP biosynthesis. Mutations in OsBADH2 could result in 2AP accumulation and rice fragrance (Chen et al., 2008). To endow the indica rice cultivar R317 with fragrance, we adopted CRISPR/Cas9 to mutate the OsBADH2 gene. A sgRNA targeting immediate upstream of the splice donor site (GT) of the intron 2 was designed (Figure 1a, b) and the vector pYLCRISPR-Cas9Pubi-H_BADH2 was introduced into R317 by Agrobacterium-mediated transformation. Two types of mutants (M190-5 and M190-13) were retrieved in the T0 generation plants (Figure 1c). M190-5 is a homozygous mutant with a single-nucleotide deletion (ΔG) at the exon–intron junction immediately upstream of the 5′-splice site of intron 2, while M190-13 is a bi-allele edited mutant with a single-nucleotide deletion (ΔG) and a trinucleotide deletion (ΔAAG). The homozygous mutation in M190-5 was faithfully transmitted to T1 generation, while the heterozygous mutations in M190-13 were transmitted to the T1 generation following the Mendelian law of segregation, resulting in three genotypes: ΔG/ΔG, ΔG/ΔAAG and ΔAAG/ΔAAG. Homozygous T1 plants were analysed for the presence of Cas9 and/or off-target mutations. From 88 T1 plants tested, 6 plants were free of Cas9 gene and with no off-target mutation, belonging to two genotypes (Rbadh2ΔG and Rbadh2ΔAAG; Figure 1c). The OsBADH2 gene encodes a functional protein with 503 amino acids (Chen et al., 2008). To check the alteration of the OsBADH2 mRNA in the edited plants, RT-PCR with a forward and a reverse primer in exons 1 and 6, respectively, was conducted to amplify the cDNAs from the homozygous mutants (Rbadh2ΔG and Rbadh2ΔAAG) and the wild type (WT). A 528bp-cDNA fragment was amplified from the mutant plants, while the WT generated a 671bp-fragment as expected (Figure 1d). Sanger sequencing revealed that the cDNA fragment from the WT is the expected size of exons of OsBADH2, indicating that the spliceosome recognizes the 5′-splice sites and cleaves the sites following normal splicing process. To our surprise, not only the introns but also the exon 2 were absent in the 528bp-cDNA from the edited plants (Figure 1e, f), indicating that the deletion of nucleotides (ΔG or ΔAAG) caused an exon skipping splicing pattern (Figure 1f). Apparently, the alteration (ΔG or ΔAAG) at the donor site of intron 2 prevented the spliceosome from normal splice site recognition. The Osbadh2 mRNA missing exon 2 from Rbadh2ΔG and Rbadh2ΔAAG proves that the exonic nucleotide (G) immediately upstream of the exon–intron junction is critical for the normal splicing process. Deletion of this exonic nucleotide caused the entire exon skipping. Recently, it has been shown that base editing-mediated disruption of either the native intron donor site or acceptor site in plant genes caused intron retention or mis-spliced segments, but no exon skipping mutation (Li et al., 2019). Our present study showed that exon skipping could happen to occur in plants by CRISPR/Cas9-mediated mutations at the exon end immediately upstream of the exon–intron junction. However, it is unknown that whether the deletion of exonic nucleotide upstream of the intron donor site can always lead to exon skipping in plants. It has been reported that base editing-mediated exon skipping depends on PTC mutations in rabbits (Sui et al., 2018). However, our results showed that exon skipping occurred due to the CRISPR/Cas9-mediated alterations at the splice site of a plant gene. Moreover, we found the exon 2 removal during the splicing process in the Rbadh2ΔG and Rbadh2ΔAAG mutants caused shifting of reading frame in the processed mRNA, resulting in a PTC in the exon 3 (Figure 1g). The existence of the PTC did not cause any further exon skipping in Rbadh2ΔG and Rbadh2ΔAAG, which was in line with the results obtained by Lee et al. (2020). The transcripts containing a PTC will be degraded by non-sense-mediated decay (Capito et al., 2018), as indicated by the reduced expression level of the mutated Osbadh2 compared with the WT (Figure 1d, h). Since loss of OsBADH2 function promotes accumulation of 2AP, we explored the consequence of Osbadh2 exon 2 skipping on the 2AP content in grains of the homozygous transgene-free plants of Rbadh2ΔG and Rbadh2ΔAAG using gas chromatography–mass spectrometry (GC-MS). The Chinese fragrant rice variety Daohuaxiang No.2 (DHX2), which harbours a fragrant allele with mutation at exon 7 of OsBADH2, was utilized as a positive control. The GC-MS internal standard for 2AP measurement was 2,4,6-trimethyl pyridine (TMP) (Laohakunjit and Kerdchoechuen, 2007), as it has similar chemical properties to 2AP. Results showed that the 2AP content in the mutants (Rbadh2ΔG and Rbadh2ΔAAG) is as high as that in the positive control (about 0.08 mg/kg), while the 2AP in the WT was null (Figure 1i). These results indicate that the Osbadh2 in the mutants is not functional to hinder the production of 2AP, resulting in the grain fragrance. We further investigated the effect of the OsBADH2 exon 2 skipping on the phenotypic characteristics of the mutants. Data showed that there were no significant differences between the edited plants and WT for vegetative and yield characteristics, indicating that the edited gene has no effect on the agronomic traits except the 2AP content or grain fragrance. In conclusion, this study provides the first evidence that CRISPR/Cas9-mediated deletion of the exonic nucleotide at the exon–intron junction of a plant gene could cause exon skipping during pre-mRNA splicing. Furthermore, the OsBADH2 exon 2 skipping caused shifting in the reading frame, resulting in a downstream PTC in exon 3. Moreover, our results highlighted that CRISPR/Cas9-mediated exon skipping could facilitate improvement of agronomically important trait of plants. The authors thank Professor Yaoguang Liu, College of Life Sciences of South China Agricultural University, for providing the CRISPR/Cas9 system. The authors declare that they have no competing interests. This research was supported by grants from the Major Science and Technology Project to Create New Crop Cultivars Using Gene Transfer Technology (2016ZX08001002), the Innovation Program of Chinese Academy of Agricultural Sciences (to K.Z.) and the Talented Young Scientist Program of China (to M.A.). K.Z., C.W. and F.W. designed experiments. Y.T., J.L., Z.J. and C.W. performed experiments. H.Q. provided the rice seeds. Y.T., M.A. and K.Z. analysed the data and wrote the manuscript. All authors read and approved the final manuscript.

Lesion mimic mutants (LMMs) are ideal materials for studying programmed cell death and defense response in plants. Here we report investigations on two LMMs ( msl-1 and msl-2 ) from the indica rice cultivar JG30 treated by ethyl methyl sulfone. Both of the mutants showed similar mosaic spot lesions at seedling stage, but they displayed different phenotypes along with development of the plants. At tillering stage, larger orange spots appeared on leaves of msl-2 , while only small reddish-brown spots exhibit on leaves of msl-1 . At heading stage, the msl-2 plants were completely dead, while the msl-1 plants were still alive even if showed apparent premature senility. For both the mutants, the mosaic spot lesion formation was induced by light; DAB and trypan blue staining showed a large amount of hydrogen peroxide accumulated at the lesion sites, accompanied by a large number of cell death. Consequently, reactive oxygen species were enriched in leaves of the mutants; SOD and CAT activities in the scavenging enzyme system were decreased compared with the wild type. In addition, degraded chloroplasts, decreased photosynthetic pigment content, down-regulated expression of genes associated with chloroplast synthesis/photosynthesis and up-regulated expression of genes related to senescence were detected in the mutants, but the abnormality of msl-2 was more serious than that of msl-1 in general. Genetic analysis and map-based cloning revealed that the lesion mimic and premature senescence traits of both the mutants were controlled by recessive mutated alleles of the SL (Sekiguchi lesion) gene, which encodes the CYP71P1 protein belonging to cytochrome P450 monooxygenase family. The difference of mutation sites and mutation types (SNP-caused single amino acid change and SNP-caused early termination of translation) led to the different phenotypes in severity between msl-1 and msl-2 . Taken together, this work revealed that the CYP71P1 is involved in regulation of both premature senescence and cell death in rice, and its different mutation sites and mutation types could cause different phenotypes in terms of severity.

The fast-and ever-growing human population and rapid climate change are threatening the global food security.As a staple food crop,rice(Oryza sativa)feeds nearly half of the world's population.However,bacterial blight(BB),the most devastating bacterial dis-ease of rice(Mew et al.,1993),caused by Xanthomonas oryzae pv.oryzae(Xoo),affects millions of hectares of cultivated rice plants annually,with an estimated crop loss of as high as 75％.Using host resistance bestowed by genetically inheritable resistance(R)genes has proved to be the best choice to achieve economical and sustainable management of this disease.

Rice (Oryza sativa) is one of the most important food crops as well as a model plant for basic research. Rice bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a devastating bacterial disease worldwide (Mew et al., 1993Mew T.W. Alvarez A.M. Leach J.E. Swing J. Focus on bacterial blight of rice.Plant Dis. 1993; 77: 8Crossref Google Scholar), affecting millions of hectares of rice annually, with an estimated crop loss of as high as 75%. Upon invasion of rice cells, Xoo secretes transcription activator-like effectors (TALEs) to activate the host Sugars Will Eventually be Exported Transporters (SWEET) genes to obtain nutrition for bacterial growth, because the induced SWEET transports more sugars to where the bacteria reside (Boch and Bonas, 2010Boch J. Bonas U. Xanthomonas AvrBs3 family-type III effectors: discovery and function.Annu. Rev. Phytopathol. 2010; 48: 419-436Crossref PubMed Scopus (644) Google Scholar). During the long-term plant–pathogen arms race, rice has evolved a resistance mechanism in which the effector binding element (EBE) located in the promoter region of an executor R gene traps the pathogen’s TALEs to activate its expression, and the expressed executor R protein kills the infected rice cells immediately, resulting in a short supply of nutrition to bacteria and BB resistance (Boch and Bonas, 2010Boch J. Bonas U. Xanthomonas AvrBs3 family-type III effectors: discovery and function.Annu. Rev. Phytopathol. 2010; 48: 419-436Crossref PubMed Scopus (644) Google Scholar). Transcription of executor R genes are usually tightly suppressed but can be activated by TALEs upon attachment of the pathogen (Zhang et al., 2015Zhang J. Yin Z. White F.F. TAL effectors and the executor R genes.Front. Plant Sci. 2015; 6: 641Crossref PubMed Scopus (80) Google Scholar). More than 20 SWEET genes exist in rice genome but only three, namely OsSWEET11, OsSWEET13, and OsSWEET14, are major targets of TALEs from Xoo. In parallel, only three to five members among a dozen of TALEs in a Xoo strain are major virulence effectors (Boch and Bonas, 2010Boch J. Bonas U. Xanthomonas AvrBs3 family-type III effectors: discovery and function.Annu. Rev. Phytopathol. 2010; 48: 419-436Crossref PubMed Scopus (644) Google Scholar). Based on a deep understanding of the mechanisms governing the interactions between rice SWEET genes and Xoo TALEs, broad-spectrum BB resistance has been engineered by CRISPR-Cas9-mediated genome editing, in which the EBEs in promoters of the three major target SWEET genes were destroyed to avoid recognition by the major virulent TALEs from Xoo (Oliva et al., 2019Oliva R. Ji C. Atienza-Grande G. Huguet-Tapia J.C. Perez-Quintero A. Li T. Eom J.S. Li C. Nguyen H. Liu Bo. et al.Broad-spectrum resistance to bacterial blight in rice using genome editing.Nat. Biotechnol. 2019; 37: 1344-1350Crossref PubMed Scopus (211) Google Scholar; Xu et al., 2019Xu Z. Xu X. Gong Q. Li Z. Li Y. Wang S. Yang Y. Ma W. Liu L. Zhu B. et al.Engineering broad-spectrum bacterial blight resistance by simultaneously disrupting variable TALE-binding elements of multiple susceptibility genes in rice.Mol. Plant. 2019; 12: 1434-1446Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Here, we report a new strategy to engineer broad-spectrum BB resistance through CRISPR-Cas9-mediated precise homology directed repair (HdR). Several successful cases about CRISPR-Cas-mediated HdR in plants have been reported, but most of them relied on chemical selection of the inserted cassette (Li et al., 2016Li J. Meng X.B. Zong Y. Chen K.L. Zhang H.W. Liu J.X. Li J.Y. Gao C.X. Gene replacements and insertions in rice by intron targeting using CRISPR-Cas9.Nat. Plants. 2016; 2: 16139Crossref PubMed Scopus (204) Google Scholar; Dong et al., 2020Dong O.X. Yu S. Jain R. Zhang N. Duong P.Q. Butler C. Li Y. Lipzen A. Martin J.A. Barry K.W. et al.Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9.Nat. Commun. 2020; 11: 1178https://doi.org/10.1038/s41467-020-14981-yCrossref PubMed Scopus (80) Google Scholar). Recently, a 5.2-kb carotenoid biosynthesis cassette has been successfully inserted into the genomic safe harbors in rice. The authors intended to insert the cassette by HdR-mediated precise gene replacement, but unexpectedly achieved the insertion via NHEJ (non-homologous end joining). Whether the presence of the homology arms in the donor plasmid facilitated the insertion of large DNA fragments by NHEJ is not clear yet (Dong et al., 2020Dong O.X. Yu S. Jain R. Zhang N. Duong P.Q. Butler C. Li Y. Lipzen A. Martin J.A. Barry K.W. et al.Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9.Nat. Commun. 2020; 11: 1178https://doi.org/10.1038/s41467-020-14981-yCrossref PubMed Scopus (80) Google Scholar). Our study was based on investigation of the interaction between rice R gene Xa23 and the cognate TALE AvrXa23 from Xoo. The executor R gene Xa23 confers the broadest resistance to rice BB (Wang et al., 2014Wang C.L. Qin T.F. Yu H.M. Zhang X.P. Che J.Y. Gao Y. Zheng C.K. Yang B. Zhao K.J. The broad bacterial blight resistance of rice line CBB23 is triggered by a novel TAL effector of Xanthomonas oryzae pv. oryzae.Mol. Plant Pathol. 2014; 15: 333-341Crossref PubMed Scopus (32) Google Scholar). We have previously revealed that Xa23 expression is completely suppressed under normal conditions, but can be activated immediately upon Xoo’s attack, resulting in a strong hypersensitive response at the infection site, thereby limiting the growth of the pathogen, resulting in a high-level BB resistance (Wang et al., 2015Wang C.L. Zhang X.P. Fan Y.L. Gao Y. Zhu Q.L. Zheng C.K. Qin T.F. Li Y.Q. Che J.Y. Zhang M.W. et al.XA23 is an executor R protein and confers broad-spectrum disease resistance in rice.Mol. Plant. 2015; 8: 290-302Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). The molecular mechanism underlying the Xa23-mediated broad-spectrum and high-level BB resistance is the interaction between Xa23 and the avirulence gene (avrXa23) from Xoo. Intriguingly, avrXa23, encoding the TALE AvrXa23, presents in virtually all the tested naturally occurring Xoo strains (Wang et al., 2014Wang C.L. Qin T.F. Yu H.M. Zhang X.P. Che J.Y. Gao Y. Zheng C.K. Yang B. Zhao K.J. The broad bacterial blight resistance of rice line CBB23 is triggered by a novel TAL effector of Xanthomonas oryzae pv. oryzae.Mol. Plant Pathol. 2014; 15: 333-341Crossref PubMed Scopus (32) Google Scholar). During Xoo’s infection of rice, the secreted AvrXa23 can be trapped by a 28-bp EBE (EBEAvrXa23) in the promoter of Xa23 and this interaction activates expression of Xa23, resulting in the strong BB resistance. Most strikingly, the 28-bp EBEAvrXa23 originated from wild rice species exists in only a few BB-resistant cultivars of rice, but the coding region for the XA23 protein is present in most of the BB-susceptible rice cultivars (Cui et al., 2017Cui H. Wang C.L. Qin T.F. Xu F.F. Tang Y.C. Gao Y. Zhao K.J. Promoter variants of Xa23 alleles affect bacterial blight resistance and evolutionary pattern.PLoS One. 2017; 12: e0185925Crossref PubMed Scopus (7) Google Scholar). In other words, although the alleles corresponding to Xa23 in the susceptible cultivars harbor the open reading frame of Xa23 (designated as xa23ORF), they cannot be expressed owing to lack of the EBEAvrXa23 in their promoter regions, and therefore it was called as susceptible xa23 allele (Cui et al., 2017Cui H. Wang C.L. Qin T.F. Xu F.F. Tang Y.C. Gao Y. Zhao K.J. Promoter variants of Xa23 alleles affect bacterial blight resistance and evolutionary pattern.PLoS One. 2017; 12: e0185925Crossref PubMed Scopus (7) Google Scholar). Based on the aforementioned discoveries, we conveived that a BB-susceptible rice cultivar can be turned into a BB-resistant variety by adding EBEAvrXa23 in the promoter region of the otherwise susceptible xa23 allele. As a proof-of-concept study, we adopted the CRISPR-Cas9-mediated precise HdR to insert EBEAvrXa23 into the promoter region of the xa23 allele in PXO99A-susceptible Nipponbare, aiming to turn it into a BB-resistant variety. In doing so, we selected target sites T1 and T2 (Figure 1A) with minimum potential of off-targets by using an online tool (http://skl.scau.edu.cn/targetdesign), and constructed the CRISPR-Cas9 targeting vector pCxa23 using a previously described method (Ma et al., 2015Ma X.L. Qunyu Zhang Q.Y. Zhu Q.L. Liu W. Chen Y. Qiu R. Wang B. Yang Z.F. Li H.Y. Lin Y.R. et al.A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants.Mol. Plant. 2015; 8: 1274-1284Abstract Full Text Full Text PDF PubMed Scopus (979) Google Scholar; Wang et al., 2016Wang F.J. Wang C.L. Liu P.Q. Lei C.L. Hao W. Gao Y. Liu Y.G. Zhao K.J. Enhanced rice blast resistance by CRISPR/Cas9-targeted mutagenesis of the ERF transcription factor gene OsERF922.PLoS One. 2016; 11: 1-18Google Scholar) as well as the donor vector pDxa23 harboring the EBEAvrXa23 fragment with a neighboring BamHI site (Figure 1A). The vectors pCxa23 and pDxa23 were mixed in 1:1 molar ratio and co-transformed into calli derived from Nipponbare embryos by particle bombardment (Supplemental Figure 1). As a result, 830 transgene-positive plantlets (T0 generation) were regenerated from 112 calli after hygromycin (50 μg/ml) selection and confirmation by PCR analysis for the Cas9 gene. Molecular identification of the target sites revealed that the activity efficiency of Cas9 is 90.2% (Supplemental Table 1). These T0 plants were also characterized by PCR coupled with restriction enzyme digestion assays (Li et al., 2016Li J. Meng X.B. Zong Y. Chen K.L. Zhang H.W. Liu J.X. Li J.Y. Gao C.X. Gene replacements and insertions in rice by intron targeting using CRISPR-Cas9.Nat. Plants. 2016; 2: 16139Crossref PubMed Scopus (204) Google Scholar) using the primer pair PF/PR (Figure 1A) and restriction enzyme BamHI. Results showed that PCR amplicons from 15 T0 plants could be digested by BamHI, among which 12 originated from one callus (w1694-2) and the other three plants originated from another callus (w1699-3, Supplemental Table 1). Further sequencing of the PCR amplicons revealed that the 12 plants from callus w1694-2 were identical in terms of genotype at the editing sites and were designated as type 1 plants (Figure 1B; Supplemental Figure 2; Supplemental Table 1). Similarly, the three plants from callus w1699-3 had another identical genotype at the editing sites and were designated as type 2 plants (Figure 1B; Supplemental Figure 2; Supplemental Table 1). Thus, the HdR-mediated EBEAvrXa23 insertion frequency is about 1.8% (2/112). Both type 1 and type 2 plants were bi-allelic mutants, with an accurate EBEAvrXa23 fragment at the editing site of one allele, while the editing site of another allele was repaired by NHEJ with deletions (ΔTTCACAT and ΔCCTT for type 1 plants; Δ77 bp for type 2 plants) (Figure 1B). Pathogen inoculation assay revealed that these heterozygous T0 plants were resistant to Xoo strain PXO99A, and thereby were self-pollinated to generate progenies for further study. In the T1 generation, we first conducted genotyping at the editing sites in the T1 plants. As expected, three new genotypes that resulted in chromosome combinations were identified and the corresponding individuals were classified into type 3, type 4, and type 5 plants (Figure 1B). Furthermore, the segregation ratio of different genotypes was 1:2:1 (e.g., type 3:type 1:type 4) in all the T1 families, consisting with the segregation ratio of a single Mendel factor (Figure 1C). To check the effect of the EBEAvrXa23 presence on BB resistance, leaves of the T1 plants along with the susceptible wild-type Nipponbare and resistant cultivar CBB23 (containing Xa23 as a positive control) were inoculated with highly pathogenic Xoo strain PXO99A at booting stage using a leaf-cutting method (Wang et al., 2014Wang C.L. Qin T.F. Yu H.M. Zhang X.P. Che J.Y. Gao Y. Zheng C.K. Yang B. Zhao K.J. The broad bacterial blight resistance of rice line CBB23 is triggered by a novel TAL effector of Xanthomonas oryzae pv. oryzae.Mol. Plant Pathol. 2014; 15: 333-341Crossref PubMed Scopus (32) Google Scholar). Two weeks after inoculation, the average lesion length (five leaves per plant) of type 1, type 2, and type 3 plants were about 0.5 cm, comparable with that of CBB23. In contrast, the average lesion length of type 4 and type 5 plants was about 10.0 cm, similar to the wild-type Nipponbare (Figure 1D and 1E). These results demonstrated that the EBEAvrXa23 inserted into Nipponbare genome activated expression of the otherwise silent xa23 allele and thereby prevented growth and spreading of the pathogen and resulted in BB resistance. The activation of xa23 expression was confirmed by qRT–PCR assays (Figure 1F). In addition, the 10 most likely off-target sites were examined by PCR and sequencing in the type 3 plants, and results proved that there was no off-target activity for both sgRNAs targeting the T1 and T2 sites (Supplemental Table 2). Furthermore, we selected the editing site homozygous and transgene/cas9-free T1 plants (type 3 plants) for subsequent experiments. For the T2 generation progenies, genotyping assays showed that the knocked-in EBEAvrXa23 was still faithfully transmitted. Meanwhile, the PX099A resistance of both the homozygous plants carrying the EBEAvrXa23 element was confirmed in the T2 progenies. These observations confirmed the stability of the replaced EBEAvrXa23 segment as well as its effect on BB resistance in different generations. To test the BB resistance spectrum of the transgene-free edited plants with EBEAvrXa23, a type 3 plant line in T2 generation was used for inoculation with 20 Xoo strains, including 10 Philippine strains (P1–P10), three Japanese strains (T1–T3), and seven Chinese strains (C1–C7) at booting stage using a leaf-cutting method (Wang et al., 2014Wang C.L. Qin T.F. Yu H.M. Zhang X.P. Che J.Y. Gao Y. Zheng C.K. Yang B. Zhao K.J. The broad bacterial blight resistance of rice line CBB23 is triggered by a novel TAL effector of Xanthomonas oryzae pv. oryzae.Mol. Plant Pathol. 2014; 15: 333-341Crossref PubMed Scopus (32) Google Scholar). Results showed that the homozygous type 3 plants displayed high resistance to all the tested Xoo strains, similar to that of the positive control CBB23 (Supplemental Table 3; Supplemental Figure 3). qRT–PCR assay was also carried out to demonstrate the inducible xa23 expression in the type 3 plants but not in the wild-type plants upon Xoo inoculation (Supplemental Figure 4). We further investigated the effect of the EBEAvrXa23 replacement on the overall phenotypic characteristics of three randomly selected lines derived from the type 3 edited plants. Data showed that there were no significant differences between the type 3 plants and the wild-type plants grown under normal conditions for vegetative and yield traits, indicating that the replacement with EBEAvrXa23 has no effect on plant characteristics, especially on yield-related traits (Figure 1G). In conclusion, this study successfully turned the BB-susceptible rice cultivar Nipponbare into BB-resistant lines by using CRISPR-Cas9-mediated homology-directed knockin of EBEAvrXa23 at the otherwise susceptible xa23 allele, establishing a new strategy for engineering broad-spectrum BB resistance of rice. This is a significant expansion to application of both the elite BB-resistance gene Xa23 and the newly emerged genome-editing technology in rice improvement, providing a paradigm for studying and usage of important genomic elements in plant. This work was supported by grants from the National Priority Program-Breeding New Rice Varieties for Southern and Southwest China Areas ( 2017YFD0100100 and 2017YFD0100202 ), the National Science Foundation of China ( U20A2035 ), the Beijing Municipal Natural Science Foundation ( 6202031 ), the Innovation Program of the Chinese Academy of Agricultural Sciences to K.Z. and C.W., and the Talented Young Scientist Program of China to M.A. and M.R.

The emerging pests and phytopathogens have reduced the crop yield and quality, which has threatened the global food security. Traditional breeding methods, molecular marker-based breeding approaches and use of genetically modified crops have played a crucial role in strengthening the food security worldwide. However, their usages in crop improvement have been highly limited due to multiple caveats. Genome editing tools like transcriptional activator-like effector nucleases and clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 (CRISPR/Cas9) have effectively overcome limitations of the conventional breeding methods and are being widely accepted for improvement of crops. Among the genome editing tools, the CRISPR/Cas9 system has emerged as the most powerful tool of genome editing because of its efficiency, amicability, flexibility, low cost and adaptability. Accumulated evidences indicate that genome editing has great potential in improving the disease resistance in crop plants. In this review, we offered a brief introduction to the mechanisms of different genome editing systems and then discussed recent developments in CRISPR/Cas9 system-based genome editing towards enhancement of rice disease resistance by different strategies. This review also discussed the possible applications of recently developed genome editing approaches like CRISPR/Cas12a (formerly known as Cpf1) and base editors for enhancement of rice disease resistance.