Keywords

1 Introduction

Rice is one of the leading primary staple foods for the increasing world population, particularly in Asia. To meet the increasing global food demand, we will have to produce up to 40% more rice by 2030 (Khush 2005). We have to achieve the goal on a reduced sowing space because of urbanization and increasing environmental pollution. Improvement of yield per plant is not the only way to achieve this goal; reduction of losses by biotic and abiotic stress is also a potent solution. According to Food and Agriculture Organization estimates, diseases, insects, and weeds cause the maximum amount of annual yield losses in cereal crops (Khush 2005). In particular, fungal diseases can cause yield losses between 1% and 10%, regionally (Savary et al. 2000). Strong efforts have been invested across the world for improving disease resistance. Most of the efforts are capitalizing on the vast amount of information generated from studying different aspects of plant diseases.

Since the initial definition of the plant resistance (R) genes by Flor (1942), several R genes are known. The majority of the known R genes composed of proteins carrying nucleotide-binding sites and leucine-rich repeat motifs (NBS-LRR) (Jones and Dangl 2006). Most R genes recognize pathogen effectors, although there are some exceptions (Lee et al. 2009). Some of these effectors thus correspond to the initial definition by Flor of the avirulence gene. Depending on the presence/absence of the R gene and of the matching avirulence product, the interaction will be incompatible or compatible. Many R genes have been identified in rice and most code for NBS-LRR genes (Ballini et al. 2008). After recognition mediates by the R sequence, signal transduction occurs and requires regulators such as MAP kinases (Mishra et al. 2006). Finally, transcription factors like WKRYs modulate a transcriptional reprogramming within the cell (Eulgem 2005), leading to the activation of defense responses. These in term induce the production of secondary metabolites (Peters 2006), pathogenesis-related (PR) proteins (van Loon et al. 2006), strengthening of cell wall (Hückelhoven 2007), and programmed cell death leading to a hypersensitive response (HR) within the cell (Greenberg and Yao 2004).

Resistant cultivars and application of chemical pesticides have been widely used for disease control in practice. However, the useful life span of many resistant cultivars is only a few years, due to the breakdown of the resistance in the face of high variability of the pathogen population. Use of pesticides is costly as well as environmentally undesirable. Thus, novel ways offering protection for an extended time and over a broad geographical area are required. Such strategies will be particularly important in cases where the source of resistance is not available.

The most vital advancement within the space of vertical development for resistance is that the use of the techniques of recombinant DNA technology to develop transgenic plants immune to disease. Moreover, genome editing by programmable sequence-specific nucleases (SSN) like the zinc-finger nucleases (ZFNs) (Bibikova et al. 2003), transcription activator-like effector nucleases (TALENs) (Moscou and Bogdanove 2009), and Cas proteins (Jinek et al. 2012) has the potential to play a significant role in developing disease-resistant plants. Since ZFNs and TALENs are costly and not easy and straightforward to use, these two technologies have not become the method of choice. On the contrary, the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) system simplifies the operation of genome editing and provides a convenient and powerful tool for genome editing. The CRISPR/Cas methods have gained rapid popularity, and it is being used in rice functional genomics and disease resistance breeding (Molla and Yang 2019; Shao et al. 2017; Shen et al. 2017).

2 Genetic Engineering of Rice for Biotic Stress Resistance

Among all the diseases recorded so far, the blast (Magnaporthe grisea), bacterial leaf blight (Xanthomonas oryzae pv. oryzae), and sheath blight (Rhizoctonia solani) are the most serious constraints of rice production. Several methods are established for developing and raising rice resistance against the disease caused but fungus and bacteria through transgenic approaches. In this section, we describe different R genes identified from rice plants and other defense genes utilized for improving rice disease resistance.

2.1 Rice Disease Resistance (R) Genes

Biotechnological tools have been playing an instrumental in identifying rice disease resistance genes. Till now, more than 100 major blast resistance (R) genes have been identified, and 35 genes have been cloned successfully (Wang et al. 2017). Table 1 summarizes the cloned blast resistance genes. Similarly, for bacterial blight, a total of 42 resistance (R) genes identified and 9 have been molecularly cloned (Vikal and Bhatia 2017). Please see Table 2 for all bacterial blight resistance genes identified. Unlike blast and bacterial blight diseases, no resistance gene has been identified for rice sheath blight (Molla et al. 2019a, b).

Table 1 Summary of the cloned blast resistance genes
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Table 2 Summary of bacterial blight resistant genes in rice
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2.2 Other Defense Genes from Rice and Non-Rice Sources Utilized for Improving Disease Resistance

Genes from plants apart from rice have been extensively tested in rice. Since no resistant rice germplasm is known and resistance genes have not been identified for sheath blight disease, genes that do not fall in R gene category have been utilized for enhancing ShB resistance (Molla et al. 2019b). However, more than 50 genes regulating disease resistance have now been discovered from different plant species (Hammond-Kosack and Parker 2003). Some of these genes may not work properly in rice for some biological reasons. Transferring gene from one species to another may lead to detrimental effects. One of the most notable is the central regulatory gene NPR1 (Cao et al. 1998). Phenotypic cost has been observed when the Arabidopsis NPR1 gene was transferred to rice (Fitzgerald et al. 2004). The rice plants overexpressing AtNPR1 displayed an environmentally regulated and heritable lesion mimic phenotype. Moreover, a recent report on OsWRKY45 demonstrates that overexpression in japonica rice confers increased susceptibility to bacterial blight, whereas overexpressing in indica rice variety confers increased resistance to bacterial blight. These findings revealed that one should be careful before transferring a gene from one background to another, even within the Oryza sativa species.

2.3 Pathogenesis-Related (PR) Proteins

Pathogenesis-related (PR) proteins are a unique category of novel proteins synthesized and accumulated in infected plant tissues. Two well-known PR proteins are hydrolytic enzymes, chitinase, and ẞ-1,3-glucanase. Hydrolysis of cell wall generates chitin oligomer which is known to induce host defense mechanism. Genes encoding chitinase or ẞ-1,3-glucanase from plants and microbes have been extensively studied and used in the generation of transgenic rice resistant against fungal pathogens (Punja 2006). Transgenic plants overexpressing either a rice chitinase or a rice thaumatin-like protein showed enhanced resistance against R. solani (Datta et al. 1999, 2000, 2001). Green tissue-specific expression of rice oxalate oxidase 4 (PR-9 family of proteins) gene in transgenic rice showed improved resistance against sheath blight pathogen Rhizoctonia solani (Molla et al. 2013). Hydrolytic enzymes from microbial origin have also been demonstrated to be effective in engineering rice disease resistance against fungal pathogens. Bacterial chitinase ChiC from Streptomyces griseus showed clear inhibition on fungal hyphae under in vitro condition (Itoh et al. 2003). Majority of transgenic rice plants expressing ChiC had higher resistance against M. grisea than non-transformed control plants (Itoh et al. 2003). Three important genes, namely, ech42, nag70, and gluc78 which encode hydrolytic enzymes from Trichoderma atroviride, were introduced in rice either singly or in combination. Transgenic plants overexpressing Gluc78 showed enhanced resistance against M. grisea, while overexpression of endochitinase gene ech42 in transgenic rice showed significant resistance against R. solani, resulting in 62% resistance against sheath blight disease (Liu et al. 2004). There was a clear co-relation between ech42 expression and chitinase activity with disease resistance (Liu et al. 2004).

2.4 Antimicrobial Proteins

Antimicrobial peptides (AMP) are amphipathic small molecules with conserved α-helix and anti-parallel β-plated sheet and discrete patches of hydrophobic residues resulting in a structure capable of forming ion channels through the membrane. Majority of antimicrobial peptides contain cysteine residues which are joined to form disulfide bonds, leading to a compact structure. Different types of AMP have been identified from plant as well as microbes and exploited in molecular improvement of rice resistance against fungal and bacterial pathogens. Various types of antimicrobial peptides have been identified in plants, including thionins (Bohlmann and Broekaert 1994), maize zeamatin (Malehorn et al. 1994), coffee circulin (Tam et al. 1999), and wheat puroindoline (Krishnamurthy et al. 2001). Plant defensins are small peptides (45–54 amino acids) that share common characters among plants, insects, and mammals. Dm-AMP1 from Dahlia merckii, a defensin, was introduced into rice. Transgenic rice plants expressing Dm-AMP1showed significantly enhanced resistance against M. oryzae and R. solani but not accompanied by an activation of PR gene (Jha et al. 2009). In another study, overexpression of wasabi defensin or Mirabilis jalapa antimicrobial protein Mj-AMP2 gene in transgenic rice exhibited significant resistance against rice blast fungus (Kanzaki et al. 2002). There was 50% reduction in lesions size of the transgenic plants as compared to non-transformed control (Kanzaki et al. 2002). These reports highlight that expression of defensin in transgenic rice has the potential to provide broad-spectrum disease resistance against fungal pathogens. An antifungal protein (AFP) from Aspergillus giganteus showed in vitro antifungal activity against diverse economically important fungal pathogens including M. grisea (Hagen et al. 2007). The AFP protein from transgenic plants showed inhibitory activity on the in vitro growth of M. grisea and therefore enhanced resistance against blast disease (Coca et al. 2004). Transgenic rice plants constitutively expressing AFP protein exhibited inheritance of the transgene in subsequent generation without any phenotypic cost (Coca et al. 2004). Puroindolines, another small protein, reported to have in vitro antimicrobial activity. Transgenic rice plants with constitutively expressing wheat puroindoline genes PinA and/or PinB were generated. Puroindolines from leaf extracts of the transgenic rice plants reduced the in vitro growth of M. grisea and R. solani. Transgenic rice expressing PinA and/or PinB exhibited significantly increased resistance to M. grisea and R. solani (Krishnamurthy et al. 2001). Cecropins, a family of antimicrobial peptides, constitute a key component of insect immune response. The transgenic rice plants overexpressing cecropin A accumulated active cecropin A protein and showed resistance to rice blast disease (Coca et al. 2006). Similarly, transgenic rice plants overexpressing cecropin B gene revealed a significant reduction in lesion development of bacterial blight (Sharma et al. 2000). Oat thionin, when introduced into rice, showed potential to control bacterial leaf blight, caused by Burkholderia plantarii (Iwai et al. 2002). Plant defensin genes from B. oleracea and B. campestris conferred enhanced resistance in transgenic rice to blast and bacterial leaf blight (Kawata et al. 2003). Generally; it has been seen that constitutively expressed antimicrobial proteins in transgenic rice provide partial or moderate but not absolute resistance against disease-causing pathogens.

2.5 Defense Signaling Genes and Broad-Spectrum Disease Resistance

Broad-spectrum resistance is defined at two different levels, i.e., firstly, resistance to different isolates of the same pathogen localized at different regions of the world, and secondly, resistance to two or more unrelated pathogenic strains. Some of the known rice R genes have been found to confer broad-spectrum disease resistance against different races of a pathogen and thus have the potential to be used in breeding program or transferred into suitable elite rice varieties through genetic engineering. One of the novel strategies for broad-spectrum plant disease resistance has been to exploit the defense signaling network that modulates the innate plant defense mechanisms against pathogen (Jones and Dangl 2006). Functional genes or proteins belong to both plant and non-plant origins that positively regulate the broad-spectrum systemic acquired resistance against viruses, bacteria, and fungi will act as a useful source for genetic engineering. Recent studies have elucidated that salicylic acid (SA)- and ethylene (ET)/jasmonic acid (JA)-mediated signaling pathways, which act as prime candidate for activation of defense responses against biotrophic and necrotrophic pathogens, respectively, play important roles in rice disease resistance (Glazebrook 2005). Distinct mechanisms might be required for activation of defense responses in rice against different pathogens (Ahn et al. 2005). NPR1 is a master regulator in the SA-mediated signaling pathway in Arabidopsis thaliana. Transgenic rice plants expressing AtNPR1 exhibited enhanced disease resistance against M. grisea and X. oryzae by modulating the expression of SA-responsive endogenous PR genes (Chern et al. 2001; Fitzgerald et al. 2004; Quilis et al. 2008). Tissue-specific expression of AtNPR1 gene in transgenic rice showed enhanced and significant resistance to the sheath blight pathogen Rhizoctonia solani without any detrimental effect on rice phenotype (Molla et al. 2016). OsNPR1 is a rice orthologue of Arabidopsis NPR1. Five NPR1-like genes present in rice genome, and three among them, namely, OsNPR1, OsNPR2, and OsNPR3 were induced upon infection by X. oryzae pv. oryzae and M. grisea. Constitutive overexpression of OsNPR1 in rice conferred disease resistance against bacterial blight but also showed enhanced herbivore susceptibility (Chern et al. 2005). OsNPR1 might be a potential candidate gene that mediates crosstalk between the SA and JA signaling pathways and provides an approach for engineering rice plants against several diseases (Yuan et al. 2007). Genetic manipulation of JA biosynthesis pathway had shown to improve rice disease resistance against microorganisms. Previous study has shown that transgenic rice plants overexpressing a pathogen-inducible allene oxide synthase (OsAOS2) gene, which encodes a key enzyme in the JA biosynthetic pathway, upregulated expression of several PR genes and provide significant resistance against M. Grisea (Mei et al. 2006). Another study demonstrated that modification of JA-related fatty acid metabolism by suppressing beta-3 fatty acid desaturases, allene oxide cyclase, and 12-oxo-phytodienoic acid reductase exhibited increased disease resistance in transgenic rice against M. grisea (Yara et al. 2007, 2008).

2.6 Reactive Oxygen Species

Oxidative burst is a general phenomenon, mediated by hydrogen peroxide (H2O2), which has been recognized as a key component of the plant defense after infection. Glucose oxidase (GOX), an enzyme predominantly occurring in some microorganisms, brings about the oxidation of beta-D-glucose, generating H2O2, and gluconic acid. Transgenic rice plants transformed with Aspergillus niger GOX gene exhibited elevated levels of cellular H2O2, which in turn lead to cell death and activation of several defense responsive genes. The overexpression of GOX in transgenic rice plants exhibited enhanced resistance against both M. grisea and X. oryzae pv. oryzae (Kachroo et al. 2003). Similarly, enhanced H2O2 generation in infected rice plants with overexpressed oxalate oxidase gene showed improved resistance to sheath blight pathogen (Molla et al. 2013).

2.7 Microbe-Derived Elicitor Genes

Microbe-derived elicitor molecules are well-known plant defense activators. Broad-spectrum disease resistance could be achieved by expressing microbial genes coding for elicitors. Several proteinaceous elicitors from microbial origin have been shown to elicit systemic acquired resistance in plants by the activation of SA- and ET/JA-mediated defense signaling pathways. The bacterial harpin and flagellin have been extensively studied for generating broad-spectrum disease resistance in rice through genetic engineering. Recently, a harpin-encoding gene hrf1, derived from X. oryzae pv. oryzae, has been transferred into rice, and the generated transgenic rice lines showed high level of resistance to major races of M. grisea. Defense responses including elevated expression of several PR genes, increased content of silicon in leaves of overexpressing transgenic plants, and significant inhibition of mycelial growth on leaves of the transgenic rice plants were observed in hrf1 transgenic plants (Shao et al. 2008). This study revealed that harpins from phytopathogenic bacteria may offer new possibilities for generating broad-spectrum disease resistance in rice. In a similar note, the flagellin gene from Acidovorax avenae, a phytopathogenic bacterium, was introduced into rice to produce flagellin. The resultant transgenic plants exhibited increased expression of defense genes, elevated H2O2 production, and programmed cell death, signifying that the flagellin triggers innate plant immune responses. Flagellin transgenic rice plants exhibited enhanced resistance against M. grisea, accounting that the flagellin might provide a novel strategy for developing genetically engineered disease-resistant rice (Takakura et al. 2008).

2.8 Gene Pyramiding in Rice for Biotic Stress Tolerance

The newly released varieties lost their resistance quickly due to the high level of genetic instability in pathogen population. One way to combat this problem is to develop transgenic rice varieties with (i) a combination of genes encoding disease-resistant proteins which showed synergistic interaction between themselves to realize effective resistance against a particular or group of disease or (ii) pyramiding of genes associated with different diseases for broad-spectrum disease resistance. A previous report showed that pyramiding of three genes, namely, Xa21, chitinase, and Bt-fusion gene in IR72 rice variety through crossing of two independent homozygous transgenic rice lines, provide significant resistance against X. oryzae pv. oryzae, R. solani, and yellow stem borer (Datta et al. 2002). Using both marker-assisted breeding and genetic transformation yielded superior rice lines resistant against blast and leaf blight through pyramiding of Pi1, Piz5, and Xa21 (Narayanan et al. 2004). Genetic transformation of rice with a maize ribosome-inactivating protein and a rice chitinase gene exhibited enhanced resistance against three fungal pathogens, such as R. solani, Bipolaris oryzae, and M. grisea (Kim et al. 2003). Constitutive co-expression of rice chitinase and thaumatin-like protein in indica rice cultivar resulted in significant enhanced level of resistance against R. solani (Kalpana et al. 2006). Similarly, transgenic rice plants pyramided with chi11, tlp, and Xa21 exhibited an enhanced resistance against both sheath blight and bacterial blight diseases (Maruthasalam et al. 2007). Tissue specific co-expression of rice oxalate oxidase and chitinase genes in transgenic BR-29 rice lines conferred significantly enhanced resistance against R. solani (Karmakar et al. 2016). In another report, it has been shown that the dual gene expression cassette harboring Arabidopsis NPR1 (AtNPR1) and rice chitinase genes provide a superior level of resistance against sheath blight pathogen R. solani than the level of resistance from the individual gene cassette (Karmakar et al. 2017). Combinatorial expression of chitinase and 1,3-glucanase genes in indica rice showed enhanced resistance against sheath blight pathogen, R. solani (Sridevi et al. 2008). Transgenic rice lines expressing four antifungal genes, i.e., RCH10, RAC22, Glu, and B-RIP showed a heightened state of resistance to M. grisea, rice false smut (Ustilaginoidea virens), and rice kernel smut disease (Tilletia barclayana) (Zhu et al. 2007). Therefore, an ingeniously planned genetic engineering strategy involving a balanced expression of different transgenes with a potential different mode of action would ensure broad-spectrum and durable tolerance against diverse group of pathogens.

3 Genome Editing System

Genome editing systems with engineered nuclease (GEEN) allow cleavage and rejoining of DNA molecules in specified target sites to successfully modify the genetic loci. Special enzymes such as restriction endonucleases (RE) and ligase can be used for cleaving and rejoining of DNA molecules in small genomes like bacterial and virus. However, using only these two enzymes such as restriction endonucleases and ligases, it is very difficult to manipulate large and complex genomes of higher organisms, including plants. Target specificity of RE is enough for short DNA sequences such as bacterial and viral genomes, it is not sufficient to work with large genomes such as plant.

Invention of engineered nucleases for genome editing revolutionized biological study. There are three well-known nucleases such as zinc finger nuclease (ZFN), transcription activator-like effector nucleases (TALEN), and CRISPR/Cas9 available as genome editing tools. ZFN and TALEN depend on protein-DNA interaction, whereas CRISPR/Cas9 relies on RNA-DNA interaction through Watson-Crick base pairing. These engineered nucleases bind to targeted loci of the genome and make a highly specific double-strand break (DSB). Upon recognition of the DSB, the error-prone cellular repair machinery inserts or deletes few nucleotides at the DSB. Due to this indel (insertion/deletion) formation, the targeted gene suffers from frameshift mutation and that ultimately causes knockout of the gene. Similarly, utilizing cellular homology-directed repair (HDR) system, precise editing could be achieved with additional supply of donor template with homologous arms. Since working with CRISPR/Cas9 is the simplest among the three tools, it gains rapid popularity within a very short period of time. All the abovementioned three tools are discussed below briefly.

3.1 Tools Available for Editing Rice Genome

3.1.1 Zinc Finger Nucleases (ZFNs)

ZFNs (zinc finger nucleases) are the first-generation genome editing tools, which are chimerically engineered nucleases, and developed after the discovery of the working principles based on functional Cys2-His2 zinc finger (ZF) domain (Kim et al. 1996). Each Cys2-His2 ZF domain consists of about 30 amino acid residues, which are capable of binding to target DNA by inserting a α-helix of the protein into the major groove of the DNA-double helix (Pavletich and Pabo 1991). Each zinc finger (ZF) protein has the ability to recognize three tandem nucleotides in the target DNA. ZFN monomer consists of about two different functional domains: an artificial zinc finger (ZF) Cys2-His2 domain at the N-terminal portion and a FokI DNA cleavage domain at the C-terminal region (Fig. 1). Dimerization of FokI domain is critical factor for ZFN enzymatic activity (Kim et al. 1996). The modular recognition of zinc finger domains represents consecutive three bp targets enabled the realization that each of the individual zinc finger domains could be interchangeable and manipulation of the domains would lead to unique binding specificities to the proteins, enabling targeting of specific unique sequences in the genome.

Fig. 1
figure 1

Basic structure and design of a zinc finger nuclease (ZFN). ZFNs are created by joining a DNA-binding region to the catalytic domain of the nonspecific Fok1 endonuclease. Each zinc finger, illustrated by an individual circle, recognizes 3–4 nucleotides, and, by assembling three or four suitable zinc finger motifs, a sequence-specific DNA-binding domain can be created. Fok1 nuclease activity requires dimerization, and so the customized ZFNs function in pairs. As shown, the zinc finger-binding domain brings two Fok1 units together in the right orientation over the target sequence; this induces Fok1 dimerization and target sequence cleavage

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The application of ZFNs involves assembly, optimization, and modular design of zinc fingers against specific target DNA sequences. Over the past few years, zinc finger domains have been generated to recognize a large number of triplet nucleotides, which provide the accurate selection and linking of zinc fingers with a particular sequence that would permit recognition of the target sequence. Many successful studies on genome editing in plants have been reported using zinc finger nucleases (ZFNs). Utilization of ZFNs to induce a double-strand break in the soluble starch synthase gene (SSIVa) in rice leads to the regulation of the SSIVa expression. ZFN-mediated targeted gene disruption in the coding sequence of the SSIVa rice gene is an effort to elucidate the functional role of the gene (Jung et al. 2018).

3.1.2 Transcription Activator-Like Effector Nucleases (TALENs)

The efficient manipulation of target genomic DNA led to the identification of unique transcription activator-like effector (TALE) proteins that recognize and activate specific plant promoters through a set of tandem repeats which form basis for the creation of a new genome editing tool consisting of chimeric nucleases, called TALE nucleases (TALENs) (Jankele and Svoboda 2014). DNA-binding ability of these proteins was first discovered in the year 2007; after a year later, two scientific groups have decoded the recognition code of target DNA sequence by TALE proteins (Boch et al. 2009).

TALE monomers consist of a central repeat domain (CRD) that provides DNA binding and host specificity. The central repeat domain (CRD) consists of 34 amino acid tandem repeats. Two of the amino acids at positions 12 and 13 of the repeat are highly variable and are responsible for the recognition of specific nucleotide (Fig. 2). These two positions are known as repeat variable diresidue (RVD) (Moscou and Bogdanove 2009). The DNA binding specificity of RVD domain has been repurposed for designing specific DNA binding artificial TALE proteins. The fusion of Fok1 nuclease domain with TALE DNA binding domain has been demonstrated to successfully create a new class of target-specific nucleases (Christian et al. 2010).

Fig. 2
figure 2

A scheme for introducing a double-strand breaks using chimeric TALEN proteins. One monomer of the DNA-binding protein domain recognizes one nucleotide of a target DNA sequence. Two amino acid residues in the monomer are responsible for binding. Recognition sites are located on the opposite DNA strands at a distance sufficient for dimerization of the FokI catalytic domains. Dimerized FokI introduces a double-strand break into DNA

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With the use of TALENs, it will be possible to introduce double-strand breaks in any location of the genome as long as that location harbors the recognition sequence corresponding to the DNA-binding domains of TALENs.

The pathogen Xanthomonas oryzae pv. oryzae (Xoo) produces and translocates its virulence proteins with the TAL effectors into the host cells through a type-III secretion system. After internalization, TAL effectors are localized into the nuclei of the host cells and bind to the promoters of susceptibility (S) genes. After that, TAL effectors activate the S-gene expression that in turn leads to more susceptibility of host plants to bacterial infection. SWEET11, SWEET13, and SWEET14 are known rice susceptibility genes (Yang et al. 2006). SWEET14 gene has been disrupted using TALEN to develop bacterial blight resistant rice plants (Li et al. 2012). Similarly, Cai et al. showed that TALEN-mediated editing of rice gene Os09g29100 enhances resistance to the bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Cai et al. 2017).

3.1.3 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)

A novel genome editing system that has been discovered recently and became so demanding and popular is the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein system, popularly known as CRISPR/Cas system. The technology is derived from CRISPR/Cas type II immune system found in the bacterium Streptococcus pyogenes. It is comprised of CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and Cas9 protein. crRNA-tracrRNA hybrid guides the Cas9 nuclease to bind to a homologous nucleic acid and make a specific double-strand break. Jinek et al. (2012) first demonstrated successfully this system to make targeted DSB in DNA. The study also showed that a single chimeric RNA (comprised of crRNA and tracrRNA) known as single guide RNA (sgRNA) could direct the Cas9 to any DNA sequences of interest if they have a NGG sequence nearby. This 5′-NGG-3′ is known as protospacer adjacent motif (PAM). The 5′ 20 bp sequence in the sgRNA sequence is known as protospacer sequence which can be designed as per the requirement of a specific experiment. Hence, the design of a CRISPR/Cas experiment is easy and straightforward.

Since the initial study by Jinek et al. (2012), CRISPR/Cas9 system has extensively been used in various fields of applied biology, biotechnology, and genome engineering, due to its simplicity, efficiency, and wide applicability. Besides the conventional CRISPR/Cas9-mediated knockout techniques (Fig. 3), various CRISPR-derived technologies have been generated. CRISPR interference (CRISPRi) and CRISPR activator (CRISPRa) have been generated for gene repression and activation, respectively (Qi et al. 2013; Gilbert et al. 2013). Recently, CRISPR/Cas-mediated base editing systems have been developed to install precise point mutation in the genome (reviewed by Molla and Yang 2019). Base editing system has been used successfully to precisely install A to G conversion in the rice genome (Molla et al. 2020).

Fig. 3
figure 3

Schematic depiction of CRISPR/Cas9 genome editing mechanism. sgRNA guides Cas9 to bind and cut specific genomic locus. Once a double-strand break (induced by Cas9) is detected, cellular repair machinery repairs it through either non-homologous end joining (NHEJ) or homology directed repair (HDR) pathways. Error-prone NHEJ causes indel (red) formation at the DSB and results in frameshift of the coding sequence knocking out the gene activity. Although extremely low in efficiency, HDR uses homologous sequence to precisely repair the DSB. If artificial homologous sequence (donor) (green) containing desired nucleotide alteration (blue) is supplied in the vicinity of DSB, HDR could incorporate the change (blue) in the targeted genomic locus

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3.2 CRISPR/Cas9 System for Biotic Stress Tolerance in Rice

CRISPR/Cas9 system has been utilized to install mutation in OsSWEET13 gene to prevent its neutralization by the TAL effector gene pthXo2, leading to improved tolerance against bacterial blight disease (Zhou et al. 2015). A recent study has been demonstrated that CRISPR/Cas9-targeted knockout of an ERF transcription factor gene OsERF922 showed enhanced resistance against rice blast fungus (Wang et al. 2016). Targeted mutagenesis with insertion or deletion at the target site and the frequency of mutation was up to 42% in T0 plant lines. Phenotypic assessment of six T2 homozygous mutant lines demonstrated that there was a significant reduction in the number of blast lesions in mutant lines as compared to wild-type plants. A recent study demonstrated editing of promoters of multiple SWEET genes in rice to develop broad spectrum bacterial blight resistance (Oliva et al. 2019). This result revealed that CRISPR/Cas9 is a powerful tool for enhancing blast resistance in rice. A brief summary of studies on CRISPR/Cas-mediated attempts to develop disease-resistant rice plants is given in Table 3.

Table 3 Use of CRISPR/Cas technology for developing disease-resistant rice
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4 Future Prospects

In the cases where defense manipulation is achieved by expression of a single or multiple protein from microbial origin or phytoalexins, the resistance in transgenic rice is not absolute, and majority of them only show partial or moderate resistance against a particular disease. Surprisingly, a number of disease resistance genes have been isolated from rice, and few have been shown to provide broad-spectrum disease resistance against diverse groups of pathogens.

Engineering of rice varieties with durable and broad-spectrum resistance would be only achieved probably through genetic manipulation of regulatory mechanisms and signaling network controlling activation of multiple defense-responsive genes. Extensive and through studies of rice disease resistance, using approaches such as genomics and proteomics, will lead to identification of novel candidate genes that are involved in the defense signaling as well as subsequent metabolic pathways. Functional genomics aided by new genome editing technologies would play a significant role toward that direction. These identified novel genes will be helpful in the generation of new superior rice varieties with high level of durable resistance against broad range of disease caused by diverse pathogens.

Knowledge of molecular mechanisms of host-pathogen interaction is crucial to utilize the full potential of the advance technologies like genome editing. Versatile technologies like CRISPR/Cas would assist us to decipher the mechanism in one hand and could be utilized to develop disease-resistant plants utilizing that knowledge on the other hand. Most simplified way is to knock out or knock down any known negative regulator or susceptibility genes for a disease. However, it needs to keep in mind that many susceptibility genes play pleiotropic roles and knocking out may have some unknown consequences. The RVD of bacterial TAL proteins has specific binding sequences in the promoter of susceptibility genes to increase their expression. Instead of knocking out by conventional CRISPR, the nucleotide/s of the TALE binding site in the susceptibility gene promoters can be mutated utilizing CRISPR/Cas base editing technologies to enhance resistance without pleiotropic effects (Molla and Yang 2019). Base editing permits C to T and A to G transitions mutations in plants. This editing tool has tremendous potential in installing precise mutation in the genome. However, changing a susceptible allele to a resistant allele through genome editing may need to perform transversion mutation, specific addition, deletion, or replacement of sequences. Homology directed repair (HDR) (Fig. 3) is the only available way to achieve those kinds of changes in the genome. The matter of concern is that HDR is extremely low in efficiency in plants. However, a recently developed technology, prime editing, can perform all kinds of precise editing up to 40 bp with much higher efficiency than HDR (Anzalone et al. 2019). Rapid advancements in technologies would ease genome modification and subsequently aid in developing disease-resistant rice plants.