**7.3 Genome editing approaches**

During evolution, plants have acquired the ability to recognize the invading pathogen and fight against it. In general, the surface proteins of the plants are called pattern recognition receptors to recognize the pathogen-associated molecular patterns (PAMPs) and trigger an array of defense reactions. While this way of recognizing the pathogens is the first step in the plant defense system, several downstream proteins and plant hormones also play an important role in mediating the plant's fight against the pathogen. Like any gene regulatory system, these mediators of plant defense system may impact the defense system. Recently, the availability of sequence-specific nucleases (SSNs) based genome editing tools, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 (CRISPR/Cas9) have made the possibilities of genome editing to regulate gene expression without any genetic modification. Hence, these SSNs-based genome editing technologies may be used in the years ahead to alter the expression of the genes involved in the plant immune system and achieve resistance against the invading pathogen. In a recent study involving

#### **Figure 4.**

*Developing disease-resistant rice: Comparison of conventional breeding, genetic engineering and genome editing [194].*

CRISPR/Cas9, resistance against *M. oryzae* was achieved by knocking out the expression of OsERF922, a plant ethylene-responsive factors (ERF) gene and a key negative regulator of plant immunity [200]. Similarly, *OsSEC3A* gene has been disrupted using CRISPR/Cas9 SSN to explore its role in plant immunity [201]. Proline-rich motif of *Pi*21 was edited to induce resistance against *M. oryzae* [202]. Invention of CRISPR/Cas9 technology in Indian scenario to edit genes mapped or identified against *M. oryzae* is in infancy stage. However, couple of researchers have begun to edit negative regulators of blast resistance genes and other defense related genes identified through RNA sequencing [203]. Rice and other crops genome editing using TALENs produced disease resistance against diverse pathogens [204, 205]. CRISPR-Cas9 is generally limited to perform genome editing at sites with canonical NGG PAMs. Much effort has focused on overcoming this restriction. Numerous Cas9 orthologs have been developed with altered PAM specificities, such as *Staphylococcus aureus* Cas9 (SaCas9) and Cas9-VQR (D1135V/R1335Q/T1337R). The CRISPR-SaCas9 toolset was recently re-optimized by introducing three key mutations, and its activity was analyzed in rice. The newly optimized system performed genome editing with a mutagenesis efficiency of up to 77%. Other versions of Cas9 have also been tested in rice, including expanded PAM SpCas9 (xCas9) and Cas9 that can recognize relaxed NG PAMs (Cas9-NG) [206]. Comparison of conventional breeding, genetic engineering, and genome editing is illustrated in **Figure 4**.

#### **8. Future perspectives and strategies**

In the recent past, rapid development in biotechnology and genomics has aided deep understanding of both host and pathogen. In this view, there are a handful of innovative tools and strategies available for developing rice varieties with effective and durable resistance against several races.

#### **8.1 Allele mining**

Allele mining is the most widely used method in identifying naturally occurring novel alleles or allelic variants of a gene in a set of germplasm. The allele mining

*Rice Blast Disease in India: Present Status and Future Challenges DOI: http://dx.doi.org/10.5772/intechopen.98847*

mainly involves two different approaches, *i.e*., EcoTilling and sequence-based or PCR-based allele mining. Compared to EcoTilling, sequence-based allele mining is reported to be simple and cheaper [163, 207]. The sequence-based allele mining involves PCR amplification of a particular gene and sequencing the PCR product to look for different gene versions. Hence, the host's sequencing information is an essential prerequisite of any allele mining program. The alleles arise in a population due to natural mutations such as transition, transversion, and InDels. Hence, allele mining has to be regularly performed to identify any valuable alleles originating in the germplasm. In a recent report, sequence-based allele mining was performed to amplify and sequence the allelic variants of the major rice blast resistance genes at the *Pi2*/*Pi9* locus of chromosome 6 from the 361 blast-resistant varieties. Thirteen novel *Pi9* alleles (named *Pi9*-Type1 to *Pi9*-Type13) were identified in these 107 varieties. These could potentially serve as a genetic resource for molecular breeding resistance to rice blast [176].

### **8.2 Identification of SNPs for fast-tracking of MAS**

Owing to the evolution of next-generation sequencing technology, genome-wide association mapping (GWAS) has found its way as an efficient tool for mapping genes. Using this method, several QTLs and loci have been identified to be associated with a set of different traits of agronomical importance [208–210]. Similarly, GWAS can identify functional SNPs associated with resistance to rice blast fungus, and MAS can be made much faster and robust. Further, GWAS can also be used to fast-track the background selection of a MAS program by collecting SNPs distributed evenly on the whole genome. The use of these high-density markers and high-resolution genome scans can identify the genomic content contributed by each parent in a breeding program involving multiple parents [181].

#### **8.3 Host induced gene silencing**

Induction of host resistance to several pathogenic fungi following the expression of the fungal genes in the host plant has been demonstrated in several cases [211]. Similarly, genes encoding a set of proteins that are very crucial in the initial establishment of *M. oryzae* in rice can be silenced by their expression in the host system. This approach holds considerable potential in breeding the next-generation rice varieties and seeks more research. However, one of the main drawbacks of this approach is that it requires genetic transformation and expression of the foreign genes in the plants.

#### **8.4 Modification of host genes targeted by blast pathogen**

The infection of the host by the rice blast pathogen requires recognition of some host proteins for establishing the infection. Hence, articulating the host target proteins by genome editing technologies fails the pathogen to recognize the host targets, limiting the infection. This approach is novel and different as the focus is on articulating the host susceptibility genes rather than R genes. A susceptibility gene refers to genes that render the host susceptible to a pathogen. This approach is now facilitated and made practical with the availability of biotechnology tools such as TALENs and CRISPR/Cas9 technologies. In this direction, the proof of concept has been demonstrated by modifying a specific target gene recognized by *Xanthomonas oryzae pv. oryzae* using TALEN technology [205].

Here, we report the identification and functional characterization of a new member of the miR812 family in rice (named as miR812w) involved in disease

resistance. miR812w is present in cultivated Oryza species, both japonica and indica subspecies, and wild rice species within the Oryza genus, but not in dicotyledonous species. miR812w is a 24 nt-long that requires DCL3 for its biogenesis and is loaded into AGO4 proteins. Whereas overexpression of miR812w increased resistance to infection by the rice blast fungus Magnaporthe oryzae, CRISPR/Cas9-mediated MIR812w editing enhances disease susceptibility, supporting that miR812w plays a role in blast resistance.

One recent report showed the identification and functional characterization of a new member of the miR812 family in rice (named as miR812w) involved in disease resistance. miR812w is present in cultivated *Oryza* species, both japonica and indica subspecies. miR812w is a 24 nt-long that requires DCL3 for its biogenesis and is loaded into AGO4 proteins. Overexpression of miR812w in rice increased resistance to infection by *M. oryzae*, CRISPR/Cas9-mediated MIR812w editing enhances disease susceptibility, supporting that miR812w plays a role in blast resistance [212].

#### **8.5 Race dependent deployment of R genes**

The success of any resistance breeding program mainly depends on the precise identification of the Avr genes prevailing in a particular location. This challenge can be met by identification and characterization of different races of *M. oryzae* of a location. While the irrational deployment of R genes to address blast disease incidence will not lead to the expected outcome, it adds additional burden to the host in expressing a specific R gene for which the Avr gene is absent and finally results in yield penalty. Furthermore, this exercise has to be continued due to the shift of the avirulence composition in *M. oryzae* populations. To date, a significant number of Avr genes have been identified and cloned. Hence, a simple PCR can be used to ascertain the frequencies of Avr genes and further planning of the breeding program.

#### **9. Conclusion**

The management of rice blast fungus is complex due to the continuous evolution of new pathotypes worldwide and India. Although several fungicides, cultural and biological control measures of blast disease are employed at the field level, the use of durable host plant resistance has shown great potential. In addition to being cost-effective, resistance breeding is environmentally friendly and demands less attention and intervention by illiterate farmers. Most of the resistance breeding programs in India were primarily based on single-gene resistance through conventional breeding approaches. However, blast pathogen has successfully overcome the single-gene resistance in a short period and rendered several varieties susceptible to blast, which was otherwise intended to be resistant. Some of the blast endemic areas of India are characterized by the existence of a mixture of more than one race of the blast pathogen, making the situation more challenging. However, the recent technological advancements, including genomics, gene editing, and pyramiding of more than one resistance gene assisted by genetic markers, hold huge promise in counteracting *M. grisea*. Hence, future resistance breeding programs should exploit the modern biotechnology tools and conventional breeding approaches in developing durable blast resistance varieties harboring multiple R genes. The harmonious blending of the bio-control approaches, cultural management practices, and modern breeding methods is the key to successfully addressing blast disease in rice cultivation ecosystems. Further, the effectiveness of the blast-resistant varieties developed for a location can only be achieved when the gene deployed is based on

the Avr genes prevalent in that area. Therefore, more efforts are needed to conduct the basic research pertaining to a specific location.
