Advancement in the Breeding, Biotechnological and Genomic Tools towards Development of Durable Genetic Resistance against the Rice Blast Disease

Rice production needs to be sustained in the coming decades, as the changeable climatic conditions are becoming more conducive to disease outbreaks. The majority of rice diseases cause enormous economic damage and yield instability. Among them, rice blast caused by Magnaportheoryzae is a serious fungal disease and is considered one of the major threats to world rice production. This pathogen can infect the above-ground tissues of rice plants at any growth stage and causes complete crop failure under favorable conditions. Therefore, management of blast disease is essentially required to sustain global food production. When looking at the drawback of chemical management strategy, the development of durable, resistant varieties is one of the most sustainable, economic, and environment-friendly approaches to counter the outbreaks of rice blasts. Interestingly, several blast-resistant rice cultivars have been developed with the help of breeding and biotechnological methods. In addition, 146 R genes have been identified, and 37 among them have been molecularly characterized to date. Further, more than 500 loci have been identified for blast resistance which enhances the resources for developing blast resistance through marker-assisted selection (MAS), marker-assisted backcross breeding (MABB), and genome editing tools. Apart from these, a better understanding of rice blast pathogens, the infection process of the pathogen, and the genetics of the immune response of the host plant are very important for the effective management of the blast disease. Further, high throughput phenotyping and disease screening protocols have played significant roles in easy comprehension of the mechanism of disease spread. The present review critically emphasizes the pathogenesis, pathogenomics, screening techniques, traditional and molecular breeding approaches, and transgenic and genome editing tools to develop a broad spectrum and durable resistance against blast disease in rice. The updated and comprehensive information presented in this review would be definitely helpful for the researchers, breeders, and students in the planning and execution of a resistance breeding program in rice against this pathogen.


Introduction
Rice (Oryza sativa L.) is the primary staple food and main source of nutrition for 2.5 to 3.5 billion people in the World, especially those living in fast-growing low-income countries [1][2][3]. Moreover, it is the main source of income and employment for more (STM) present on conidium helps the spore to attach to hydrophobic cuticle of rice plant. (c). Conidium germinate using food reserve and produce a narrow germ tube. (d). Germ tube elongates and give rise to appressorium. (e). Autophagy occurs in three-celled conidium and it dies in a programmed process. (f). In appressorium turgor pressure increases with the help of melanin layer on cell wall and synthesized glycerol inside. Then penetration peg forms at the base, punctured the cuticle of rice and allows entry into the epidermis of plant. (g). Plant tissue invasion occurs by means of bulbous, invasive hyphae (IH) that invaginate the rice plasma membrane and invade epidermal cells. Penetration peg develops into two primary hyphae and separated from rice cytoplasm by extra-invasive hyphal membrane (EIHM). Primary hyphae develop into invasive hyphae. At the tip of primary IH, a new structure known as biotrophic interfacial complex (BIC) develops which is present within EIHM. (h). Invasive hyphae moves from one cell to another by plasmodesmata. (i). Disease lesions occur on plant and sporulation starts under humid conditions, Spores develops on conidiophores with sympodially manners.

Attachment and Germination
Magnaporthe oryzae pathogenesis starts from a three-celled conidium (Figure 1a), which attaches itself tightly to a hydrophobic, waxy leaf cuticle of rice by an adhesive called spore tip mucilage (STM). STM exists in the periplasmic regions of the conidial tip cell before attachment (Figure 1b) [47]. Upon attachment, three celled conidia germinate and form unbranched germination tubes (also called germ tubes) from the apical cell and grow across the surface of the cuticle. Out of three cells of the spore, only basal and/or apical cells give rise to the germ tube; the middle cell seldom germinates and may function as a reservoir of energy for fungal growth on the host surface [48,49].

Germ Tube Elongation and Recognition of Host
Various chemical and physical cues, such as primary alcohols, cutin monomers, plant surface waxes, and hard and hydrophobic surfaces, trigger appressorium formation [50]. When the germ tube notices physical cues, such as hydrophobic and hard surface, the tip of the germ tube develops a dome-shaped appressorium which helps in pressure generation to penetrate the host surface. For surface recognition of host, pathogen implies several signal transduction pathways such as PMK1 mitogen-activated protein kinase (MAPK), cyclic adenosine monophosphate dependent protein kinase A (cAMP/PKA), and HOG1 signaling pathways, which also help in appressorium formation, infection peg formation, osmoregulation and cell wall integrity (Figure 1c) [51].
The pth11 gene encodes transmembrane protein Pth11, which is a G-protein coupled receptor (GPCRs). GPCRs are transmembrane receptors confined to the cell membrane of fungal spores and are involved in signal transduction from the outside environment to inside the cell, which helps pathogens to synchronize cell metabolism, cell transport, and growth [52]. Mutational disruption of pth11 triggered a failure in the maturation of appressorium, although the growth of germ tube hooks was not affected in the ∆pth11 mutant and confirmed the role of Pth11 in pathogenesis [53]. GPCRs protein is composed of three G alpha (MagA, MagB, and MagC), one G beta (Mgb1), and one G gamma (Mgg1) subunits. Several mutational studies confirmed the role of subunits in pathogenesis [54]. Host cues and signals bind to the GPCR and activate downstream signaling cascade via G-protein, cAMP-PKA, and MAPK signaling pathways that influence metabolism, cellular growth, and morphogenesis of appressorium. A total of eight G protein signaling (RGS) proteins were identified (MoRgs1 through MoRgs8) in Magnoporthe oryzae. Surface hydrophobicity, conidiation, and mating are positively regulated by MoRgs1 and MoRgs4. For germ tube growth and appressorium formation, MoRgs1, MoRgs2, MoRgs3, MoRgs4, MoRgs6, and MoRgs7 are crucial. Although all RGS proteins are involved in the regulation of intracellular cAMP levels, only MoRgs1, MoRgs3, MoRgs4, and MoRgs7 are indispensable for complete virulence [55].

Appressorium Formation and Maturation
Appressorium formation is regulated by a cascade of the gene in Magnoporthe oryzae. When a three-celled conidia land on the host surface, it attaches itself to the hydrophobic surface of the cuticle with the help of mucilage [56]. On germination, it forms a narrow germ tube in which a nucleus migrates and undergoes mitosis 4-6 h after germination. Various receptors present on the cell membrane of the germ tube recognize the host and initiate dome-shaped appressorium formation after the termination of the tip growth of the germ tube. Following two mitotic divisions, a daughter nucleus moves to develop appressorium, and the rest of the three nucleus returns to conidium and is degraded together with other spore contents, leaving a single nucleus in appressorium (Figure 1d) [57]. Turgor pressure generation through glycerol and other polyols synthesis inside appressorium and the formation of melanin layer between the cell membrane and the cell wall is known as appressorium maturation (Figure 1e) [45,58]. In Magnoporthe oryzae, cargo-independent autophagy and cell death were reported in three-celled conidium just before appressorium maturation to help turgor pressure generation in appressoria [59]. A total of sixteen genes were identified in non-selective macroautophagy; the mutation in any one gene leads to loss of pathogenicity in Magnoporthe oryzae [60]. Yin et al. [61] revealed the role of the autophagy-related gene (MoAtg1) of Magnaporthe, which encodes for kinase protein and possibly phosphorylates MoMkk1 to respond to endoplasmic reticulum stress during plant infection. Increased glycerol inside appressorium causes an influx of more water and builds tremendous pressure on the cell wall up to 8 MPa, and the melanin layer on the cell wall act as a barrier to the efflux of solute and helps to create structural rigidity to maintain increasing pressure [45]. Glycoprotein-rich mucilage adhesives glue melanized appressoria to host surfaces. Mucilage secreted around the base of the appressorium from the appressorial pore helps build up pressure on the host cuticle. Appressorium maturation and penetration are regulated by the MAPK signaling pathway mediated by an Mst11-Mst7-Pmk1 cascade [62]. The MAPK signaling cascade Mck1-Mkk1-Mps1 was reported to be involved in appressorium penetration, maintenance of cell wall integrity, and invasive growth of Magnoporthe oryzae [63].

Penetration Peg Formation and Invasion
Turgor pressure generated by glycerol and melanized wall of appressorium is focused onto an unmelanized, thin-walled appressorium base where penetration peg starts to develop and generate tremendous pressure on host cuticle and rupture the rice cell cuticle (Figure 1f) [64]. A small amount of cell wall degrading enzymes (CWDEs) such as cutinase, Poly-galactorunase (PGs) is secreted by growing pathogen cells to weaken hard host surfaces [65]. The CUT2 gene of Magnoporthe oryzae produces a cutinase enzyme to degrade cutin present in the cuticle of rice [66]. Two homologs of PGs were identified in the genome of Magnoporthe oryzae, namely endo-PG (MGG_08938.6) and exo-PG (MGG_08752.6). Then, Magnoporthe oryzae obtains entry forcefully to the host cell by translating turgor force into physical force [67]. Ultra-structural analysis reveals that the appressorium pore of Magnoporthe oryzae is different from the rest of the appressorium, with the absence of melanin and a much thinner cell wall [68,69]. A turgor-sensing protein, Sln1, triggers downstream pathways when turgor pressure reaches a threshold, and the septin ring develops around the pore and acts as a barrier of lateral diffusion and control appressorium repolarization [70].
After penetration peg formation, rapid membrane biogenesis and F-actin polymerization occur at the penetration site [68,71]. Recent studies revealed the role of reactive oxygen species (ROS) burst in the re-modeling of cytoskeletal and rapid polymerization of F-actin. ROS burst is catalyzed by two genes, NOX1 and NOX2, that code for NADPH oxidase [71]. NOX1 gene plays a role in the maintenance of the polarized growth and organization of the toroidal F actin network and NOX2required for septin ring formation at the base of the appressorium during penetration peg formation [71,72]. Later appressorium nucleus migrates into the penetration peg, where it undergoes further rounds of mitosis; later, the penetration peg differentiates into primary infective hyphae, then globular invasive hyphae (IH) (Figure 1g). Fungal cells inside the host are confined by the plant-derived extra-invasive hyphal membrane (EIHM) to protect the host defense mechanism. This creates an enclosed apoplastic space between the pathogens IH and the cytoplasm of rice [72]. Further, the EIHM matrix contains several proteins, such as BAS4, which help plants generate defense mechanisms against the pathogen [72,73]. First invaded host cell filled by growth of IH before pathogen spreads into the neighboring host cell. IH switches back to primary IH and moves into uninfected neighboring cells through the plasmodesmata (Figure 1h) [72]. Later it again converted to IH in the newly infected adjacent cell. At the tip of primary IH, a new structure known as biotrophic interfacial complex (BIC) develops, which is present within EIHM. As the fungus multiplies within the first infected cell, BIC remains behind the bulbous IH and again reappears at the tip of the primary IH that will move into neighboring cells (Figure 1i) [73,74]. The mode of action of the focal BIC is unknown, but several effectors accumulate in the BIC to suppress the host immune responses and the virulence of Magnoporthe oryzae [72,74].

Invasion and Defense Suppression
Magnoporthe oryzae secretes a variety of effector proteins into the host cell to evade the immune response, manipulates host metabolism, and avoids recognition to take advantage during pathogenesis [74]. To date, many effector proteins identified in Magnoporthe oryzae interfere or interact with different target sites of rice. Most effector proteins are produced during the biotrophic phase [73]. Based on the secretion of the effector, there are two kinds of effectors in Magnoporthe oryzae. Apoplastic effectors are secreted into space between IH and EIHM, while Cytoplasmic effectors are secreted into BIC and then translocated into the host cytoplasm with the help of the exocyst complex and t-SNAREs. However, apoplastic effectors are secreted by the conserved ER (endoplasmic reticulum) to the Golgi secretory pathway [75]. Proved by treatment with Brefeldin A that interferes with Golgi-dependent secretion inhibited the secretion of apoplastic effectors such as Bas4 and Slp1 but did not affect the localization of cytoplasmic effectors Pwl2, Bas1, and Bas107 to the BIC [73].

Pathogenomics of Magnaporthe oryzae
Magnaporthe oryzae is a hemibiotrophic fungus that is responsible for developing blast disease in rice. The genus Magnaporthe was created and accommodated in order Diaporthales [102]. It belongs to the phylum ascomycetes, which develop sexual spores known as ascospores for infecting host plants. Magnoporthe oryzae is known to have hundreds of pathotypes (races) that infect paddy. It is capable of causing damage in almost all stages of paddy, starting from the nursery stage up to grain formation [103]. In order to gain a better understanding of the genomics of this fungus, genome sequencing will play a major role in the future in developing varieties that will be resistant to multiple strains over different geographical regions [104]. Moreover, with the help of various advanced techniques such as genome editing and biotechnological innovations, any of the steps in the biological system of a pathogen can be disrupted or terminated to achieve resistance against blast disease [105]. Host-specific strains can be silenced using targeted mutations or RNAi techniques to create sustainable and strong resistance against the attack of Magnoporthe oryzae.
Interestingly, the genome of Magnoporthe oryzae has been sequenced firstly amongst various phytopathogenic fungi and is being utilized commonly as a model system to understand the mechanism of pathogenicity of pathogen and host-pathogen interactions. Magnoporthe oryzae showed huge genome instability due to the availability of recurrent repetitive sequences in its genome [42,106]. This genome instability plays an important role in the genome variation and the fast evolution of a new race of pathogens within the population. Until now, genome sequencing of more than 74 races of Magnoporthe oryzae has been completed. Among them, each strain contained isolated specific genes and genomic regions, which determine their racial evolution, environmental adaptation, chromosomal variability, variation in repeat element distribution, and host range specificity [96,107]. Consequently, Magnoporthe oryzae has a genome size of 40.12 Mb and contains 12,684 genes in the genome [42]. Further, one of the first strains to be completely sequenced was 70-15 using a whole-genome sequencing shotgun approach. It was discovered that this pathotype contained three MPAK pathways, which were associated with virulence [106]. Two strains of Magnoporthe oryzae, P131 and Y34, were sequenced using Sanger (2-fold) and 454 sequencing technologies [104]. Both of these races had approximately 13% unique DNA when compared with the previously sequenced laboratory strain 70-15. The interesting thing to note was that the deletion of a few genes would cause a decrease in the virulence capacity of the pathogen. For example, deletion of P131_scaffold00208-2 from P131 and Y34_scaffold00875-3 from Y34 would lead to a reduction in virulence and conidiation, respectively [104]. Paired-end libraries of FJ81278 and HN19311 strains of Magnoporthe oryzae were generated through Illumina sequencing. This helped in identifying many virulent genes which were different from 70-15, and genome variation was found at both the basic nucleotide level and chromosome level [96]. The whole genome assembly of another pathotype (RMg-Dl) was completed using PacBio Single-molecule and IlluminaHiSeq 2500 techniques. Strain RMg-Dl was isolated from the Swarna variety cultivated in the Bihar region of India [105].

Genetics of Blast Disease Resistance in Rice
Understanding the genetics of the defense system generated by rice plants against the Magnoporthe oryzae is essential to designing a breeding program for developing the disease-resistant variety. A schematic representation of the ice defense system or immunity against blast pathogens is presented in Figure 2. Plants developed two layers of defense; in the first layer, pathogen-associated molecular patterns (PAMPs) are recognized by pattern recognition receptors (PRRs) present on an extracellular membrane or on a transmembrane [108]. If PRRs of a plant can recognize PAMPs molecules of the pathogen, it induces a relatively weak basic immune response, known as PAMP triggered immunity (PTI), that obstructs the establishment of invading pathogen [109]. Further, the pathogen secretes effector protein to avoid or defeat triggered defense response, which is known as effector-triggered susceptibility (ETS) [110]. The second layer of plant defense is governed by resistance (R) proteins that recognize avirulence (Avr) effectors of the pathogen by direct or indirect binding and induce a wide array of defense responses; this response is known as effector-triggered immunity (ETI). A specific R protein binds to a specific AVR; hence, it is race-specific immunity [111].  PAMP molecule chitin present on the cell wall of Magnoporthe oryzae could be recognized by rice CEBiP (a chitin elicitor binding protein), a lysin motif (LysM) containing plasma membrane proteins, LYP4 and LYP6 [112]. In order to overcome this PTI response in rice, Magnoporthe oryzae secretes an effector protein, Secreted LysM Protein1 (Slp1), during the pathogenesis of new rice cells. Effector Slp1 accumulates in space between the rice plasma membrane and fungal cell wall and competes with CEBiP for binding of chitin molecules, and defeats plant defense gene expression and generation of reactive oxygen species [90].
The first studied interaction between AVR and R proteins in the Magnoporthe oryzaerice pathosystem was AVR-Pita and Pi-ta. AVR-Pita is the first identified avirulence gene in Magnoporthe oryzae, encoding a predicted secreted protein that interacts with Pi-ta and triggers resistance [118]. Pi-ta codes a constitutively expressed 928 amino acidcytoplasmic NLR receptor which is an NBS-LRR class of R protein [119]. Effector AVR-Pita binds to the leucine-rich domain (LRD) of Pi-ta protein directly. A recent study reveals that in the absence of Pi-ta in rice, Avr-Pita targets the rice mitochondria and interacts with the OsCOX11 (Oryzae sativa cytochrome c-oxidase) assembly protein. OsCOX11 participates in mitochondrial reactive oxygen species (ROS) metabolism in rice. Avr-Pita enhances COX activity and decreases ROS accumulation in the host cell, and suppresses host innate immunity by perturbing ROS metabolism in the mitochondria [120].
Pi54 (earlier Pi-kh) is a dominant R gene that encodes~43 kD protein and has a unique Zinc finger domain that overlaps with the leucine-rich repeat regions and belongs to the NBS-LRR family of R protein [121]. Unlike Pita, Pi54 is induced only in response to pathogen attack [121,122]. In Magnaporthe oryzae AVR-Pi54 gene encodes a predicted secreted protein with a signal peptide (SP) at the N-terminal region. The molecular docking study revealed that AVR-Pi54 protein physically interacts with Pi54 protein through novel non-LRR domains such as STI1 and RhoGEF. The STI1 and GEF domains that interact with AVR-Pi54 are also components of the rice defense complex [28,123]. Microarray analysis in transgenic rice performed at 72 h post-inoculation of the Magnoporthe oryzae revealed that many defense-related genes, such as PAL, laccase, callose, peroxidase, and enzymatic activities of defense response enzymes viz., phenylalanine ammonia-lyase, polyphenol oxidase, b-1,3-glucanase, peroxidase, chitinase, and b-glucosidase, were significantly Upregulated [124].
Pik is a major R gene located on the long arm of chromosome 11 of rice and requires two NRL receptors, Pik-1 and Pik-2, to trigger cell death upon binding to the AVR-PikD [125]. Effector AVR-PikD interacts with specific rice HMA domain-containing heavy metal-associated isoprenylated plant proteins (OsHIPP19) and heavy metal-associated plant proteins (HPPs) [125]. Both Pik-1 and Pik-2 belong to the coiled-coil nucleotide-binding site leucine-rich repeat (CC-NBS-LRR) class of R proteins [126]. AVR-Pik encodes a secreted protein with a signal peptide at the N-terminus. Pikh-2 initiates host defense response and also physically interacts with the CC domain of Pikh-1 directly. AVRPik-D and Pikh-2 both bind the CC domain of Pikh-1 and form a complex AVR-Pik-Pikh-1-Pikh-2, then a specific signal is transferred from AVR-Pik to Pikh-2 and mediates resistance responses in Rice [83,127].
Pia gene encodes R protein present on chromosome 11 of O. sativa. Pia is composed of two adjacent NLR protein genes, RGA4 and RGA5, and is required for Pia and AVR-Pia interaction [82,128,129]. AVR-Pia encodes a predicted secreted protein with an SP at the N-terminus [81,130]. RGA5 transcripts generate two isoforms by alternative splicing, RGA5-A and RGA5-B. Only RGA5-A is required for Pia-mediated resistance. RGA4 acts as a constitutively active cell death inducer and is inhibited by RGA5 in rice plants without pathogen infection. However, RGA5 is an Avr receptor and has no role in cell death induction. RGA5 and RGA4 form hetero-complexes, and when AVR-Pia or AVR1-CO39 physically binds to the C terminal, non-LRR domain of RGA5, the interaction releases RGA4 and induces hypersensitive cell death in O. sativa [82,129].
AVR-Pii encodes a secreted protein belonging to the pex33 protein family, having four homologs [81]. AVR-Pii first accumulates in the BIC and is then translocated into the host cytoplasm [131]. Avr-Pii form complex with OsExo70-F2 and OsExo70-F3. OsExo70-F2 and OsExo70-F3 are presumably involved in exocytosis, and these proteins stably form homo-and hetero-dimers that incorporate AVR-Pii. R gene Pii encodes a 1025-amino acid protein predicted to be an NLR protein [132]. R protein Pii and AVR-Pii interact indirectly in the host cell [133]. In the absence of R protein Pii, AVR-Pii directly binds to Os-NADP-ME2 (Os nicotinamide adenine dinucleotide phosphate-malic enzyme), inhibits their activity, and thereby leads to the reduction of the PAMP-triggered ROS burst and successful biotrophy [131]. However, in the presence of Pii, OsExo70 acts as a decoy or helper in Pii/AVR-Pii interactions and activates defense response against Magnoporthe oryzae [133].
AvrPiz-t codes for a predicted secreted protein [80] and is first secreted into the BIC before translocation into the rice cell [77]. Effector AvrPiz-t targets 12 APIPs (AvrPiz-t interacting proteins) of rice. Among the twelve APIPs, APIP6 and APIP10 are functional ring E3 ubiquitin ligases, APIP5 is a bZip transcription factor, and APIP12 is a nucleoporin2 domain (Pfam 04096) containing protein [77,134,135]. Piz-t and AvrPiz-t interact indirectly [77,134,135]. Effector AvrPiz-t participates in both PTI and ETI of rice to Magnoporthe oryzae and targets the rice RING E3 ubiquitin ligases APIP6 (AvrPiz-t Interacting Protein 6) and APIP10 by promoting their degradation to suppress PTI in rice [77,136]. APIP10 promotes the degradation of Piz-t via ubiquitination. AvrPiz-t can remove negative regulation of Piz-t by degradation of APIP10 through the AvrPiz-t/APIP10 protein-protein interaction [136]. At the necrotrophic stage, a functional bZip transcription factor, APIP5 interacts with AvrPiz-t directly. APIP5 form homo-dimers and then interact with AvrPiz-t through its bZip DNA-binding domain at the N-terminus. The interaction suppresses APIP5 transcriptional activity and protein accumulation, leading to cell death. Thus, AvrPiz-t promotes effector-triggered necrosis (ETN) in the absence of Piz-t. When Piz-t is present in the rice cell, the N-terminus of Piz-t also interacts with the N-terminus of APIP5 and stabilizes APIP5 accumulation and activity to prevent rice cell necrosis. At the same time, APIP5 promotes the accumulation of Piz-t to maintain its basal level for providing resistance [134].

Disease Screening Protocols for Blast Resistance in Rice
The first step in a resistance breeding program is to rapidly screen all the available genetic stocks, including the local land races, improved cultivars, and exotic germplasms, using empirical techniques in glass houses or by field tests. Efficient, accurate, and reliable methods for screening disease resistance/susceptibility in crop plants are very important in developing resistant crop varieties in a relatively short period of time in a sustainable manner [137]. Based on the accuracy of the screening methods, resistant or susceptible genotypes might be identified for further breeding programs. Standard screening protocols of rice varieties for susceptibility to rice blast are usually carried out by spraying the plant with conidial suspensions under greenhouse and field conditions using local isolates [138]. Details of individual techniques are briefly described hereunder.

Field Screening Technique
A high-throughput and reliable field screening protocol for rice blast resistance are essential for the identification of resistant germplasm/varieties and resistant genes for further breeding programs [139]. In field conditions, artificial leaf blast disease screening usually takes place in a Uniform Blast Nursery (UBN) [138,140,141], which has a 10 m length and 1 m width ( Figure 3). Test entries (30 plants/test entry) are planted in a nursery bed at a spacing of 10 cm plant to plant and 50 cm row to row. Moreover, a mixture of susceptible check entries is planted after every 10 lines of test entry as a spreader line and also planted throughout the border of UBN as a border line to facilitate the even spread of the blast disease. One or more susceptible entries could be used as border/spreader lines. The soil in the UBN is enriched with farm yard manures (FYM) and recommended doses of fertilizers. However, applying an excess rate of nitrogen fertilizer (150 kg N/ha) makes rice more vulnerable to spreading blast infection [138]. Simultaneously, isolation, maintenance, and multiplication of local and highly virulent blast cultures (fungal conidia) should be performed according to the method suggested by Vasudevan et al. [141], Prasad et al. [142], and Chhallagulla et al. [143]. Moreover, artificial inoculation is performed with a local and highly virulent blast race (fungal conidial suspension at a concentration of 1 × 10 5 spores/mL) by spraying on UBN beds at 25-30 days after sowing (DAS). Later, the nursery beds are water sprayed 3-4 times per day and are covered with polythene sheets during the night to maintain a high humidity until disease development and progression are observed in border lines and spreader lines [138,141,143]. The observations on disease resistance or susceptibility are taken from each entry 10-15 days after the artificial inoculation and taken 2-3 times at 5 days intervals using the Standard Evaluation System 2002 (SES) of International Rice Research Institute, Manila, Philippines [144]. In this system, the disease score has ranged from 0-9 based on the severity of blast infestation in the leaves of the plant. The lines with disease scores of 6-9 are considered susceptible lines, 4-5 as moderately resistant, and 0-3 as highly resistant [144]. Several studies have been reported for using standard blast screening techniques and identified a number of novel resistance sources and genes. Vasudevan et al. [141] conducted a large-scale screening of 4246 geographically diverse rice accessions originating from 13 major rice-growing countries to identify a new resistance source for blast resistance. These accessions were selected from over 120,000 accessions based on their annotated rice blast resistance information in the international rice gene bank. The rice lines were screened using a two-step screening protocol which includes natural infection in a rice uniform blast nursery followed by artificial infections with five single rice blast isolates. Systematic screening for rice blast resistance was performed both under field and controlled environmental conditions, and rice cultivars IR72 and CO39 were used as susceptible control lines. Among the lines screened, 289 accessions showed broad-spectrum resistance (BSR) against all five single rice blast isolates. The accessions showing BSR were genotyped for the presence of the Pi2 resistance gene for the identification of promising accessions for the isolation of allelic variants of the resistance gene. Blast monogenic resistant lines for Pi54 (IRBLkh-K3), Piz-t (IRBLzt-T), Pi9 (IRBL9-W), Pita (IRBLta-CP1), and Pi2 (IRBLz5-CA), Pib (IRBLb-B) were used as control lines to isolate specific rice blast resistance genes.
Similarly, Qin et al. [139] developed a high-throughput and reliable blast resistance evaluation system at the field level for the breeding of resistant varieties. This method consists of the following steps (i) pretreatment of diseased straw; (ii) sowing of seeds (iii) initiating seedling blast of the first batch of spreader population (iv) Sowing of seeds (v) and inducing seedling blast of the second batch of spreader population and test materials (vi) induction of seedling blast. Based on this protocol, a total of 730 indica hybrid rice were screened. This procedure enables uniform and consistent infection, which facilitates efficient and accurate assessment of seedling blast resistance for diverse rice materials.

Screening Techniques under Greenhouse/Polyhouse/Controlled Conditions
Under a greenhouse/polyhouse screening procedure, the test entries 15 plants/test entry) are sown in plastic trays (10 rows × 2 columns per tray) in 4-5 batches for inoculation with different individual blast isolates (The number of batches can be increased/decreased based on the availability of individual isolates of the pathogen for screening). Plants are allowed to grow in normal conditions for 10-15 days. Rice blast pathogen cultured on culture medium may be taken for preparation of conidial suspension at 1 × 10 5 conidia/mL of water. All the plants are then inoculated with 50 mL of spore suspension solution with a concentration of 1 × 10 5 spores/mL with 1% tween-20 per tray after 10-15 DAS [141,145].
After inoculation, the plants need to be kept in a moist chamber at 26-28 • C for 24 h to maintain temperature and humidity. Plants should be transferred to the incubation chamber at 25 • C ± 2 for 1 week, and water is sprayed three to four times during day time to maintain high humidity (humidity should be near 100% for the initial 72 h to favor disease initiation [141,145]. Then the disease reaction should be assessed after nine days of inoculation and scored on a zero to nine rating scale as per the Standard Evaluation System 2002 (SES) of the International Rice Research Institute. Manila, Philippines [144].

Molecular-Marker-Based Screening of Rice Genotypes for Blast Resistance
In addition to the phenotypic screening for the identification of blast resistance source, molecular markers linked to the blast resistance genes/genes-specific primers are being utilized to identify the presence of resistance genes in the rice germplasm/line [146]. Tightly linked molecular markers, viz., simple sequence repeats (SSRs), single nucleotide polymorphisms (SNPs), and CAPSs (Cleaved Amplified Polymorphic Sequences), have been widely used for screening rice genotypes for blast-resistant genes [22,[147][148][149][150][151][152][153]. Molecular-markerbased techniques not only allow the identification of resistant lines in a non-destructive manner but also help in the identification of broad-spectrum resistance genes and alleles of different genes and a combination of resistance genes in the germplasm [149]. This method gives fast and accurate results within a short period of time. With the advent of molecular markers paved the path for changing the paradigm of rice breeding by identification of genomic regions controlling several economic traits and their deployment in the elite rice lines using MAS and MABB approaches.
Singh et al. [149] screened rice accessions with allele-specific SSR markers to identify 10 major blast resistance genes (Piz-5, Pi-9, Pitp(t), Pi-1, Pi-5(t), Pi-33, Pi-b, Pi27(t), Pikh, and Pi-ta) in 192 rice germplasm accessions from different ecological regions. They found genetic frequencies of the 10 major rice blast resistance genes varied from 19.79% to 54.69%. Interestingly, they found 17 rice accessions that harbored seven to eight major blast resistance genes indicating their exploitation in further breeding programs for developing blast-resistant cultivars. Furthermore, Imam et al. [150], Shikari et al. [151], Yan et al. [152], and Teerasan et al. [153] also used the linked SSR markers for screening of blast resistance in rice genotypes. Kim et al. [147] employed eight SNP markers (tightly linked with six major genes, Piz, Piz-t, Pik, Pik-m, Pik-p, and Pit) to determine the genetic diversities of blast resistance (R) genes from 86 accessions of aromatic rice and found four accessions of indica type carrying the six major genes. Moreover, Kim et al. [148] used SSR and CAPS markers for blast resistance screening in rice genotypes.

Strategies to Develop Durable Resistance in Rice Genotypes against the Blast Disease
Many agronomical management practices, biological control, chemical control, disease forecasting, etc., have been adopted by the farmers to escape the hazards and reduce the losses caused by blast disease, but none of them has proven to be 100% efficient. Furthermore, the application of excessive fungicides to control the disease may cause severe loss of biodiversity, a threat to ecology, and produce hazardous food material [1,9,42]. Therefore, the development of broad-spectrum and durable resistance in rice varieties for blast disease is the only hope to combat the infestation of Magnoporthe oryzae. Thus, rice breeders should focus on developing durable, resistant rice varieties as resistance genes provide a worthwhile and environmentally safe option for the management of blast disease [19]. Many approaches, viz., traditional breeding methods, molecular-marker-based breeding approaches, transgenic breeding, genome editing-based methods, etc., are being deployed as major weapons to develop durable resistant varieties and are ecologically as well as economically sustainable. Advancement in genomic approaches and bioinformatic tools has led to the foundation for developing blast-resistant rice varieties with more accuracy and precision in a limited time period. A brief account of various breeding and biotechnological tools which can be used for the development of resistant rice varieties are briefly described in subsequent paragraphs and also presented in Figure 4.

Conventional Breeding Strategies for Developing Resistance against Rice Blast
The traditional breeding approaches are robust and oldest methods for developing novel genetic variants for blast resistance. Breeders have developed many blast-resistant rice varieties through traditional breeding methods, viz., pedigree method, backcross breeding, introduction and acclimatization, multiline breeding, pureline selection, recurrent selection, and mutation breeding. Interestingly, conventional breeding has enabled the International Rice Research Institute, Manila, Philippines, to generate elite cultivars with an enormous range of disease-resistant genes [12]. This approach is appropriate for developing durable and sustainable resistant rice varieties; however, its long and time-consuming breeding cycle and laborious nature are the major drawbacks of this method [19].
Introduction and acclimatization are the important, easiest, fastest, and most economically efficient breeding methods used to develop resistant varieties by introducing promising entries into new areas or regions where they have not been cultivated before. Most of the disease-resistant lines developed by IRRI, Manila, Philippines, have been disseminated to various rice-growing countries through the introduction and acclimatization process and used as breeding material for developing new disease-resistant varieties either through pureline selection or through the hybridization method [20]. Rice varieties IR36 and IR64 are the best examples of introduction and utilization in the development of new varieties. IR36 and IR64 contain the Pita gene, and IR64 also has another closely linked gene, Pi20, conferring resistance to blast disease. In addition to these known Pi genes, these varieties have accumulated several defense genes with their complex lineages [154,155], which make them durably resistant to blast disease in most locations. These varieties are used extensively in breeding programs in the southern states of India, where the blast is recognized as a potential threat to increased productivity [156].
Moreover, scientists from Indonesian Center for Rice Research (ICRR), Indonesia, have used more than 30 blast-resistant varieties of traditional and introduced materials for hybridization-based breeding programs. They have conducted the selection for blast resistance in a greenhouse through artificial inoculation of 19 pathogen races available in the ICRR's collection. Interestingly, a total six promising blast-resistant lines, viz., TB490C-TB-1-2-1, TB361B-30-6-2, BP1976B-2-3-7-TB-1-1, TB356BTB-18-3, IR30176, and IR60080-23, with different patterns of resistance to the pathogen races were selected through participatory varietal selection and tested under farmers field [157]. Furthermore, multiline varieties or a mixture of several resistant near-isogenic lines can be used to reduce the outbreaks of blast disease. These lines carry resistance to different races of the same pathogen, which helps in developing durable resistance against the pathogen. Several scientists confirmed the use of multiline varieties for the control of the severity of blast disease [158][159][160][161][162]. The multiline variety "Sasanishiki" has been developed at the Furukawa Agricultural Experiment Research Station, Japan, in 1995 and commercially cultivated in 5,800 hectares of farmers' fields in Miyagi Prefecture of northern Japan in 1997 as a blast-resistant rice variety. It consisted of seven different lines from BL1 to BL7, which carried seven different resistant genes against blast. These lines were developed by continuous backcrossing of Sasanishiki (recurrent parent) with blast-resistant land races of cultivars (Donor parent) [163]. Moreover, Zhu et al. [164] suggested using a cultivar mixture consisting of 80-90% resistant plants and 10-20% susceptible plants of similar varietal background to reduce the rapid evolution and emergence of new virulent Magnoporthe oryzae [160,161].
Simultaneously, the concept of shuttle breeding was also deployed during the 1970s and 1980s for developing blast-resistant rice varieties. Over 3000 rice germplasms were evaluated in 31 countries at 126 test sites from 1975 to 1992 under the International Network for Genetic Evaluation of Rice (INGER) [165,166]. A total of 522 resistant entries of the International Rice Blast Nursery (IRBN) were utilized in the hybridization program across 18 countries from 1984 to 1992 [166].
Furthermore, a few promising donors such as Ram Tulsi, Oryza nivara, Dawn, Tetep, Carreom, Zenith, Gam pai 15, Pankhari 203, and a number of improved plant types resulted from this study and were used on a regular basis as parents in blast-resistant breeding programs by various countries [165,166].
The pedigree method is the most efficient and extensively used breeding method for handling segregating generations from crosses, and a large number of varieties have been developed in crops such as rice, wheat, barley, sorghum, pulses, oilseeds, etc. It is the most appropriate method for improving the disease resistance ability of existing cultivars within a short span of time if the resistance is governed by major genes [20]. It is possible to combine genes for resistance to six or seven major diseases and insects in a short period through the pedigree method [12,39]. Most of the IRRI bred lines viz., IR24, IR34, IR36, IR60, IR56, IR64, IR46, and IR74 having blast-resistant genes have been used extensively in a breeding program for developing the blast-resistant rice varieties through the pedigree method [20,154]. Moreover, the TN-1 rice variety has also been used in breeding for blast resistance as a susceptible parent. Martínez et al. [167] have developed many blast-resistant genotypes from the segregating lines of Fanny (highly susceptible to blast) and 11 cultivars differing in blast resistance through the pedigree method. However, maintenance of accurate pedigree records and long breeding cycles are the major limiting factors for deploying this method. Moreover, this method will not be fruitful when blast resistance is governed by polygenes [12,19]. Backcross breeding is another widely used and common technique in rice breeding for transferring genes and chromosomes from one variety to another and from related species [20,168]. It has been extensively used for transferring disease resistance to popular and widely adapted varieties. For instance, a backcross was made between IR68835-98-2-B-2-1-1 (a broad-spectrum blast resistance variety) and KDML105 (a susceptible variety), and 83 lines from BC3F2 generation were evaluated for resistance against 12 different strains of the blast. All the BILs (Backcross Introgression Lines) displayed a low level of disease score. These lines can be further utilized in breeding programs for developing blast-resistant varieties [169]. Moreover, backcross breeding was integrated with molecular marker-based techniques to make the breeding process more clear, accurate, and authentic and also to reduce the exhaustive breeding exercise of maintaining a huge population.
In addition to the above-mentioned methods, recurrent selection has also been deployed in rice breeding programs for the development of blast-resistant varieties [170]. Recurrent selection is characterized by being a cyclical method in which gains for the trait or traits under selection occur gradually and continuously. Interestingly, this method requires shorter breeding cycles and provides better genetic gains with wider genetic diversity in breeding lines for blast resistance. The durable blast-resistant rice variety CG-91 was developed through recurrent selection [171].
Conventional breeding methods have played a significant role in sustaining food production for burgeoning populations. However, conventional breeding takes more time and effort, which can be reduced nowadays due to advancements in genomic technologies. Conventional breeding and phenotyping are inefficient when several lineages of the pathogen are present, and resistances to multiple lineages are warranted [156]. Moreover, the success in resistance breeding is generally affected by linkage drag, due to which undesired traits closely linked with resistance genes are also transferred in new selections. Therefore, molecular-marker-based breeding approaches are now preferable in resistance breeding towards increasing its efficiency.

Mutation Breeding for Blast Resistance in Rice
A mutation is a rare event, reversible and recessive in nature, and a primary source of all genetic variations existing in any organism, including plants. Mutation breeding in rice is used to complement conventional breeding since this technique is very effective for improving one or a few traits, such as agronomic traits and resistance to pests and diseases, without altering the unique properties of improved variety/germplasm to make them easy acceptable among the farmers [172]. By the year 2022, about 3402 mutant varieties in more than 225 crops have been developed through induced mutagenesis by different countries and registered in the FAO/IAEA Mutant Variety Database (MVD), International Atomic Energy Agency (IAEA), Vienna, Austria [173] which has made significant contributions for food and nutritional security.
Furthermore, many attempts have been made to develop disease resistance in rice against blast through mutation breeding. According to FAO/IAEA MVD [173], a total of 151 rice mutants having blast resistance have been developed across the World and registered in FAO/IAEA MVD. Interestingly, 72 rice mutants were developed through directly induced mutagenesis (Table 1), and 79 varieties were developed by crossing with mutant variety (Supplementary Table S1). Among the 72 rice mutants, four varieties were developed by chemical mutagenesis, whereas 68 were developed by the use of physical mutagens [173]. China, Japan, and India are the top three countries that have developed 56, 54, and 15 rice mutants, respectively [173]. The mutant variety Xiongyue 613 was the first officially approved blast-resistant rice variety, which was developed through mutation breeding (200 Gy of gamma rays) in 1965. The main improved attributes of Xiongyue 613 are moderate resistance to blast, higher yield, and good quality FAO/IAEA MVD, 2022). Interestingly, with the advancement in technologies, China has developed five rice mutants viz., Zhe 101, Hangtian 36, Huahang-simiao, Liangyouhang 2, and Neiyouhang 148 through the use of cosmic rays (treatment of seeds in aerospace). In India, a total of 15 rice mutant varieties that have blast resistance have been developed and registered in FAO/IAEA MVD, among which some mutants were developed through radiation-induced (X-rays, gamma rays) mutation breeding, while others were the results of hybridization with mutant variety. In India, 'Jagannath' is the first rice mutant produced in 1969 by X-rays irradiation of the popular tall variety T141, which was found to be resistant to blast [173]. Recently, a high-yielding and blast-resistant variety Vikram-TCR has been developed through radiation-induced mutation breeding under the joint collaboration of Indira Gandhi Krishi Vishwavidyalaya, Raipur, India, and Bhabha Atomic Research Centre, Mumbai, India. This variety has been developed with the help of gamma radiation (300 Gy) from the Safri-17 landrace, which is susceptible to blast disease [174,175]. When looking at the outcomes of the mutation breeding, it can be said that it is playing a significant role in combating the impact of blast disease in rice.

Molecular-Marker-Based Approaches for Resistance to Rice Blast Disease
Conventional breeding methods are slow and time-consuming, with uncertain results due to environmental impacts. With the advent in molecular techniques, a combination of traditional methods which are aided by new molecular techniques such as gene pyramiding, marker-assisted selection (MAS), marker-assisted back cross (MABC), QTL mapping, genome-wide association studies (GWAS), etc., can help accelerate the process of selection, screening, and development of blast-resistant rice varieties. Consequently, the identification and mapping of these R genes/alleles through advanced genomic approaches will be helpful in modern plant breeding for developing durable, resistant varieties [22,26,176]. Mining and characterization of disease-resistant genes/QTLs and their further deployment for developing resistant cultivars are the most preferred strategies by plant breeders. About 100 years ago, Sasaki [177] had, for the first time, reported the resistant varieties for rice blast fungus, Magnoporthe oryzae, in Japan, and Kiyosawa [178] identified the first rice blast gene Pi-a from a japonica rice variety "Aichi Asahi". Interestingly, until now, about 146 R genes for rice blast resistance have been identified and mapped from both indica and japonica subspecies of rice by various scientists (Table 2). Among the 146 identified R genes, scientists have molecularly characterized and cloned 36 genes so far (Table 3) [15,24,42].
Identification and mapping of the blast resistance R-genes through advanced biotechnological and genomic approaches are essential for their efficient utilization in molecular breeding programs, especially in marker-assisted selection and marker-based gene pyramiding of two or more R-genes for achieving the broad-spectrum and durable resistance [15,22,24,296,297]. In addition, this also gives information about the gene-linked, gene-based, and functional markers, which can enhance the efficiency of conventional resistance breeding programs. Further, this provides an opportunity for molecular characterization and cloning of R-genes [298]. Recent mapping studies, viz., linkage-based QTL mapping and linkage-disequilibrium-based association mapping, are the most widely used methods for identification and mapping of the blast resistance R-genes and have successfully illustrated the acceleration of the breeding program. Linkage-based QTL mapping was proposed as a useful molecular breeding technique for detecting the QTLs as it utilizes the biparental mapping population developed from two contrasting genotypes. In the past few years, more than 350 QTLs and blast resistance genes have been identified and localized on rice chromosomes for blast resistance through a linkage-based QTL mapping strategy [236,244,247,[299][300][301][302][303][304][305][306][307][308][309][310][311][312].
In association mapping, genome-wide association studies (GWAS) detect the genetic variation (marker) polymorphisms of multiple individuals in the whole genome to obtain the genotype associated with the observable traits. In comparison with the linkage-based QTL mapping, it has the ability to map genes with high resolution, cover rich-captured variations, and have high efficiency in locating multiple traits simultaneously. GWAS has been deployed by scientists for the last two decades for the identification and mapping of blast-resistant genes and revealed more than 230 blast-resistant loci in rice which are distributed throughout the genome [15,22,115,176,[313][314][315][316][317][318][319][320][321][322][323][324][325][326]. Among them, five rice blast resistance loci, including the cloned gene Pita, were first identified by GWAS [115,313].
The identified and mapped genes/QTLs could be easily deployed in the breeding program with the help of molecular markers. Hybridization, backcrossing, marker-assisted selections, and marker-assisted backcross breeding (MABB) are the most popular methods for the introgression of resistance (R) genes into the elite cultivar for improving the rice line for disease resistance. Gene pyramiding involves transferring more than one favorable gene/QTL of traits from multiple parents into a single genotype by markerassisted selection (MAS). A number of studies have been made to deploy R genes in rice breeding programs for blast resistance through the gene introgression or pyramiding approach [27,185,[327][328][329][330][331]. A series of improved intermediate materials with various blast resistance gene combinations or improved new varieties were bred to achieve broader and more durable resistance. The detail of rice lines/varieties improved by introgression of single, multiple genes for blast resistance and introgression of genes for multiple biotic stresses and a combination of biotic and abiotic stresses is presented in Table 4.

Transgenic Breeding for Rice Blast Resistance
The most significant advancement in the area of varietal development for disease resistance is the use of the techniques of genetic engineering to develop transgenic rice resistant to diseases. This approach is advantageous for introducing disease resistance into elite rice cultivars since transgenic plants can acquire a single desired trait without any alteration of the original genetic background. Several studies have been performed to confer the disease resistance in rice against the Magnoporthe oryzae [28][29][30][31][363][364][365][366][367][368][369][370], which are presented briefly in Table 5.
Using Agrobacterium-mediated transformation, Nishizawa et al. [363] reintroduced the chitinase gene in Japonica rice varieties Nipponbare and Koshihikari. Two genes, Cht-2, which accumulated chitinase intracellularly, and Cht-3, which generated chitinase extracellularly, were introduced. It was found that even if either of the genes was expressed in the transgenic plants, they showed enhanced resistance to blast fungus. Studies have demonstrated the expression of the Gns1 gene, which is responsible for hydrolyzing glycosidic bonds in cell walls of the Poaceae family. Gns1 gene was introduced in the plants with CaMV35S as its promoter. The over-expression of this gene led to the development of resistance against blast disease, but it also resulted in stunted growth of the plants. The use of this gene for blast resistance is limited as its constant expression results in poor root formation, diminutive growth, and development of certain brown specks [365].
Interestingly, a different approach to rice blast resistance was undertaken by transferring regulatory and structural genes of maize which were responsible for the production of flavonoids. Flavonoids are responsible for generating various levels of stress responses in plants. In this study, anthocyanin production was increased in Tp309, a japonica rice variety, by the transfer of the C2 gene of maize [364]. Transfer of this gene might have resulted in a mutation leading to the generation of CHS protein which provided resistance against blast disease [364]. Furthermore, Coca et al. [366] developed blast-resistant transgenic rice by transferring the ER-CecA gene from the giant silk moth Hyalophora cecropia. This gene was optimized to produce Cecropin A peptides in paddy, which are a member of antimicrobial protein families. This transformation did not account for any pathogenesis-related gene expression, which is a good indicator of the direct effect of a gene on the pathogen [366].
Another promising approach to rice blast fungus resistance was exhibited by transferring an antifreeze glycopeptide gene using Agrobacterium as a medium. This gene was overexpressed in transformed paddy plants, which were able to withstand low temperatures such as −1 • C up to 24 h. Transgenic and control plants were recovered and thawed. Interestingly, transgenic plants displayed fewer symptoms and more resistance against blast fungus as compared to other fungi. This implies that such pathogenesis-related proteins can be identified and used to generate resistance against blast [367]. Similarly, another host defense antimicrobial peptide, thanatin, was transferred into the rice. The transformants displayed a significant level of resistance against blast fungus [368]. Similarly, Chen et al. [369] transferred the Pi-d2 gene with the help of three vectors into nine lines of paddy to generate blast-resistant rice varieties. This gene exhibited broad-spectrum resistance against blast as it displays resistance against 39 strains. The selection was made using the production of crude toxins by fungus. Helliwell et al. [370] suggested that ethylene plays a major role in resistance against rice blasts. They have generated transgenic plants using ACS2 (1-aminocyclopropane-1-carboxylic acid synthase), which produces ethylene. The transformed lines showed resistance against blast with little or no difference between the agronomic traits of transgenics and wild-type plants [370]. Devanna et al. [28] isolated the Pi54 gene from a wild species, Oryza officinalis, and it was renamed Pi54, which confers resistance against blast disease. Interestingly this study demonstrated that the Pi54 of the gene was structurally more stable and provided a higher level of resistance as compared to Pi54. This gene was transferred in two susceptible rice lines, IET16310 (indica) and TP309 (japonica), which transformed them into highly resistant strains against Magnoporthe oryzae.
Moreover, Wang et al. [29] transferred MoHrip1 and MoHrip2 genes into rice through an Agrobacterium-tumefaciens-based method, which resulted in resistance to blast disease. The transgenic paddy plants constrained the growth of fungal hyphae and also had a high water-retention capacity. Furthermore, marker-free transgenic rice was generated using maize's Ac/Ds transposon vectors carrying fluorescent protein (GFP) and red fluorescent protein (mCherry) genetic markers to generate marker-free transgenic plants. Pi21 gene was expressed in these transgenic plants to generate resistance against rice blasts. The transformed lines had good resistance against Magnoporthe oryzae [30]. Moreover, three Pi genes, viz., Pib, Pi25, and Pi54, were transferred together into two rice varieties, the indica variety Kasalath and the japonica variety Zhenghan 10. The transformed varieties exhibited a good level of resistance against blast pathogens, but this gene pyramiding came with its baggage of linkage drag and pleiotropic effects of these genes. The transgenic plants were impairing many gene transcriptions, which ultimately interrupted the normal development of the plants [31].
The genes mentioned in the above studies suggest a potential future for transgenic crops, which will stand against huge threats to the staple food of the world. The genes can be used to generate new varieties or can be used to create strong resistance barriers against major diseases such as blasts. Genes from other organisms or species can be used to achieve this goal without negatively affecting the desired agronomical traits.

Genome Editing Tools for Developing the Blast Resistance in Rice
The emergence of fatal strains of the rice blast disease is imminent, and the existing tools and available resistance genes might not be enough to cope with their pace. Genome editing tools have provided us with new pathways and have given new perspectives for developing blast-resistant rice varieties. The genes or crops can be targeted to direct their characteristics in a particular direction and to create novel variations [372]. Several methods allow insertion, deletion, mutation, or substitution of nucleotides or sequences of nucleotides in specific locations. This can be accomplished by genome editing using highly advanced genetic engineering tools [373], such as endonucleases which are divided into four categories: Zinc Finger Nucleases (ZFNs), Meganucleases (MNs), chemical nucleases [374] TALENs (Transcription Activator-Like Effector Nucleases) and CRISPR Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) are the latest addition [375].

CRISPR Cas9 Based Resistance for Rice Blast
In recent years, there have been a lot of changes in the environment that directly or indirectly affect the growth of important crops. Insect and disease infestations have increased drastically, and constant evolution in the strains resistant to existing defense mechanisms has forced the scientific community to develop new and innovative methods to fight against these odds. Rice blast is one such disease that causes a lot of damage to the crop and is considered among the top 10 fungal diseases that could threaten global food security [17,376]. Biotechnology helped researchers to identify genes that can confer resistance to rice blasts in resilient varieties of rice. Recently developed techniques such as CRISPR Cas9 can be very useful in developing varieties that are resistant to the attack of Magnaporthe oryzae. CRISPR is a very effective sequence-specific nuclease (SSN) that targets particular genes and carries out efficient genome editing.
Wang et al. [97] used CRISPR Cas9 to edit the OsERF922 gene in rice. They have induced mutations in the target sites using the C-ERF922 enzyme, which generated several insertions and deletions. Interestingly, these mutations were successfully inherited in consecutive generations. Six mutated lines viz., KS2-12-1-3, KS2-27-4-1, KS2-45-6-1, KS2-70-1-2, KS2-75-1-11, KS2-144-1-2 were selected, which displayed a drastically reduced number of blast lesions and were at par with the agronomic traits when compared with the wild types. Similarly, Zhou et al. [377] attempted to mutate three broad-spectrum blast-resistant genes, i.e., Bsr-d1, Pi21, and ERF922, using CRISPR-based technology in Longke638S (LK638S) rice cultivar which is a TGMS line. They observed that single and triple mutants had improved resistance against blast. Furthermore, the improved varieties also had similar kinds of desirable superior agronomic characteristics. In another experiment, CRISPR Cas9 was used to target S genes, Pi21 and Bsr-d1 in rice which was responsible for the susceptible reaction with rice blast pathogen Magnoporthe oryzae [378]. Due to gene editing, resistance was conferred by two methods, firstly by generating a loss of function mutants and secondly by generating knock of mutants [378]. Similarly, the OsSEC3A gene mutated using targeted mutagenesis, which enhanced resistance to rice blast disease [379]. Many scientists have also utilized the CRISPR Cas9-based gene editing approach to develop resistance in rice against the blast disease [64,379,380], which is presented in Table 6.

Transcription Activator-like Effector Nucleases (TALENs)
TALENs induce site-specific double-strand breaks (DSBs) in DNA [381]. These breaks can be amended by homologous recombination (HR) or non-homologous end-joining (NHEJ). The bacteria of Xanthomonas species secrete proteins known as TALEs (Transcription activator-like effectors) which display pathogenic activity in crops such as rice, tomato, etc. [382]. Further research revealed that these proteins could recognize and bind to specific sequences in DNA [383]. This was followed by the discovery of code which recognized specific regions of DNA [384]. The one monomer nucleotide property of TALE fascinated many researchers to work with it. This technique was used to combat filamentous fungus such as Magnoporthe oryzae, and an HR detection-based PtFg TALEN plasmid was constructed. This highly efficient nuclease can improve the efficiency of HR-mediated gene editing up to 100% and can prove to be very important for developing resistant varieties against various strains of Magnoporthe oryzae [385].

Meganucleases (MNs)
These are also known as homing endonucleases, which are divided into five types based on their sequence and structure motifs [386], from which the LAGLIDADG family proteins have the capacity to act as endonucleases that can bind to specific sites on the DNA [387]. They can recognize exon-or intron-free regions and bind with longer DNA sequences (14 to 40 bp), and induce double-strand breaks (DSBs) in DNA [388]. MNs are not commonly used because naturally occurring endonucleases are very limited and can recognize only a few sites on the genome. Artificial MNs require a huge input of time and money which requires a lot of sustained effort and is practically not feasible. Despite having limited usage, this technique can be kept as an alternative to generate novel genetic changes to develop resistance against various strains of Magnoporthe oryzae. ZFNs are counterfeit for natural endonucleases known as Fokl and are derived from Flavobacterium okeanokoites [389]. They consist of two terminal ends, the N terminal, which binds with DNA, and the C terminal, which has cleavage activity. Each ZFN is capable of identifying a sequence of 3-4 nucleotides. Different ZFNs can be combined to recognize a longer sequence and can be used to induce DSBs in DNA for genome editing [390]. Later, using NHEJ or HR repairs, the required nucleotide sequence or gene can be inserted to achieve targeted results. ZFNs can be introduced into hosts using viral or non-viral vectors and have the capacity to incorporate themselves into any genome, including that of mitochondria [391]. ZFNs have been successfully used to induce mutations or edit genomes in various crops. An experiment was conducted on rice to identify safe sites to introduce new genes using three different ZFN constructs (pZFN1, pZFN2, pZFN3), which were delivered using Agrobacterium and β-glucuronidase (GUS) as a reporter gene. Gene expression was measured using TAIL PCR which identified 28 presumed safe sites which can be used to harbor genes of interest for improving resistance against diseases such as rice blasts [392].

Problems Associated with Breeding for Resistance to Rice Blast
Boosting the yield potential and productivity of rice is the primary objective for the plant breeders; however, in complementation, resistance breeding for biotic and abiotic stress has been considered a major issue due to changing climatic conditions [43]. Conventional breeding methods for developing blast-resistant rice varieties are tedious and time-consuming as it takes about 8-10 years to develop one resistant variety. In parallel, Magnoporthe oryzae is mutating continuously and evolving new races against the resistant genes available in the existing cultivars within 2-3 years. In such cases, the resistant cultivar becomes susceptible, leading to discouragement to the breeders as their long efforts have been destroyed quickly. Moreover, low-yielding cultivars that have blast resistance have not been preferred by the farmers and farming community; therefore, breeding for high-yielding rice varieties with multiple durable R-genes for blast resistance is the need of the hour. Advancement in genomic approaches has enabled the breeders to accurately introgress or pyramid multiple R-genes in the high-yielding desirable genotypes within a short period with the help of molecular markers and genomic selection [126,317]. However, linkage drag is the major issue in the case of gene introgression and pyramiding. A number of undesirable changes due to linkage drag in the recipient genotype led to their poor yield performance, and, fortunately, that cannot be compromised by the farmers [19,20,347]. In the current era of genome editing, several scientists have developed blast-resistant and high-yielding rice varieties using genetic engineering and cisgenesis [12,372,378]. However, they have not been accepted worldwide due to the imposition of regulations on genetic modification in many rice-producing and rice-importing countries. Nonetheless, no genetically modified disease-resistant rice is currently commercially available for production worldwide [43]. While these techniques have been proven successful in experimental settings, they have not been made commercially available in rice due to regulatory protocols put in place by many rice-importing countries.

Conclusions and Future Outlooks
It is well understood that combining the multiple race-specific R genes in elite cultivars is the most effective and appropriate strategy for developing broad-spectrum and durable resistance to blast disease. However, multiple R-genes in cultivars may promote the evolution of several new races of the pathogen, and even super races could arise, which may cause severe blast epidemics by overcoming the multiple major R-genes. Therefore, it is crucial to rationally utilize the race-specific R-genes in breeding programs to sustain the blast resistance of rice cultivars which is still poorly understood. This opens many avenues for researchers to explore blast resistance in rice. A diagrammatic representation of the possible aspects for further improvement in blast resistance is represented in Figure 5.
The major obstacle in managing rice blasts is the durability of the genetic resistance. Thus, the right combination of the major R gene and minor QTLs is necessary to confer broad-spectrum and durable resistance with the help of conventional integrated breeding, advanced genomic approaches, and genome editing tools. Interestingly, of the 36 molecularly characterized and cloned R genes, several broadspectrum genes are available for further use in breeding programs. This indicates that along with the mining of novel genes, their cloning and characterization are also important. Furthermore, high-quality Pi genes, which are less prone to evasion by the pathogen and impose fitness penalties in mutations of the pathogen, are expected to be effective and durable. Work on these genes could be accelerated. As neck blast is more damaging to yield and grain quality, screening of neck blast resistance should be incorporated in key evaluation sites. A rice variety, 'Mahamaya' of Chhattisgarh, India, is very popular for flaked rice. However, it is susceptible to neck blasts; therefore, there is a need to incorporate resistant genes against the neck blast. Until now, only a few genes have been identified showing the true resistance against the panicle and leaf blast; therefore, mining the Rgenes/QTLs imparting equal effectiveness against panicle blast and leaf blast diseases would enhance the resources for the breeders. Moreover, disease screening protocols can be improvised through high throughput phenotyping approaches which could help in better monitoring and management of the disease. With the available genome sequence of the blast fungus, comprehensive profiling of secreted proteins of the fungus is now possible. These proteins may confer effector functions and can act as a diagnostic tool for determining the virulence/avirulence spectrum of a given pathogen population.
Furthermore, the ability to evaluate genetic materials at multiple sites is essential to assess the spectrum of resistance of breeding lines and gain a glimpse of the potential durability. Performance records are organized by the International Network Genetic Evaluation of Rice (INGER) and are used as an indicator of the durability of resistance in varieties. This will require extensive sharing of genetic materials between countries, perhaps through the facilitation of INGER. Gene stacking involving major R genes with overlapping resistance spectra involving superior alleles would confer durable resistance. Integrating advanced breeding methods, modern molecular approaches along with nextgeneration sequencing (NGS) based methods, and bioinformatic tools will effectively direct in achieving rice blast resistance. High-throughput whole genome sequence (WGS) or target gene sequencing in the elite rice cultivars or core resistant germplasm will provide useful technological means for breeding selection.
In addition, genomic selection should be actively utilized in breeding for blast disease resistance as it exploits the genomic-estimated breeding values of individuals obtained from genome-wide markers to choose candidates for the next breeding cycle. The availability of a standard phenotyping approach and genome-wide high-throughput, cost-effective and flexible markers, especially with the emergence of NGS techniques, has enabled the plant breeders to exploit genomic selection (GS) for crop improvement. The NGS-based genotyping approaches, such as genotyping by sequencing, have significantly improved the prediction level of genomic-estimated breeding values in cereals and other crop species as compared to the other established marker platform and helped in deploying the GS in breeding programs. Furthermore, GS is the most suited approach for breeding for quantitative resistance conferred by minor effect genes, or a combination of minor and major genes tends to produce a more durable resistance in breeding lines (BLs) because it relies on multi-resistant alleles. In addition to increasing the accuracy of selection, GS is expected to reduce rates of inbreeding because the increased accuracy of Mendelian sampling terms in GS allows for the identification and selection of elite breeding candidates from more families, with lower co-selection of sibs. Improved statistical models that leverage genomic information to increase prediction accuracies are critical for the effectiveness of GS-enabled breeding programs.
More studies towards a deeper comprehension of defense responses and signal transduction leading to defense responses are required. NILs (Near Isogenic Lines), Multi-parent advanced generation inter-cross (MAGIC) populations, and Nested Association Mapping (NAM) are useful genetic resources for genetic analysis and, eventually, gene cloning. More NILs, MAGIC, and NAM populations for diverse and durable Pi-genes and QTLs could be developed primarily for genetic analysis and for monitoring pathogen populations. Marker-aided foreground and background selection can be used to accelerate NIL development. More expertise could be involved in performing recently developed transgenic and genome editing tools for its better exploitation to develop blast resistance in rice through specific genetic modifications. Broad spectrum resistance may arise due to the mutations in susceptibility genes in plants. Such loss of function mutations in susceptibility genes indirectly imparts disease resistance without yield penalty. Future studies must orient towards the mining of such S genes in rice in order to utilize them through genome modification techniques for developing blast-resistant varieties.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants11182386/s1, Table S1: List of blast disease resistant rice varieties developed through crossing with mutant varieties.